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ACTIVATION MECHANISMS FOR ZYMOGENS
BELONGING TO THE PAPAIN FAMILY OF CYSTEINE
PROTEASES
OMAR QURAISHI
Biochemistry Department
McGill University, Montreal
September 1999
A thesis submitted to the Faculty of Graduate Studies and Research in
partial fulfilment of the requirements of the degree of Doctor of
Philosophy
O Omar Quraishi, 1999
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PAGE
Acknowiedgments
Abstract
Résumé
Introduction and Literature Review
- Serine Proteases
- Activation of Trypsinogen and Chymo trypsinogen - Prothrombin Activation - Proprotein Convertases
- Aspartic Proteases
- Zinc Metailoproteases
- Procarboxypeptidase A and B - Prostromelysin-1
- Cysteine Proteases
- Caspase Family - Pap ain Family
Chapter 1: The Occluding Loop in Cathepsin B Defines the pH Dependence of Inhibition by Its Propeptide
- Connecting Text for Chapters 1 and 2
C h a ~ t e r 2: Identification of Interna1 Autoproteolytic Cleavage Sites Within the Prosegments of Recombinant Procathepsin B and Procathepsin S
- Connecting Text for Chapters 2 and 3
Chapter 3: Functional Expression of Human Procathepsin H in Pichiapastoris and Attempts at its Correct Processing
Summary
References
LIST OF FIGURES AND TABLES FOLLOWING PAGE
WTRODUCTION AND LITERATURE REVIEW
FIGURE 1: Activation of Chyrnotrypsinogen
IFIGURIF 2: Prothrombinase Cornplex
FIGURE 3 : Progastrïcsin Activation
FIGURE 4: Sequence Alignment of Cathepsin L-like Prodomains
FIGURE 5: Prosegments of Cathepsin L and Cathepsin B
FIGURE 6: Structures of Procathepsin L and Procathepsin B
FIGURE 7: Structure of Procathepsin B
FIGURE 8: Model of Cystatins in Cornplex with Papain
ILLUSTRATIONS FOR CHAPTER 1
FIGURE 1 : Prosequences of Rat and Human Cathepsin B
FIGURE 2: Conformations Adopted by the Occluding Loop
FIGURE 3: Autocatalytic Processing of Procathepsin B
TABLE 1: Propeptide Inhibition of Cathepsin B Mutants
TABLE 2: Role of Aspartic Acid Residues in Propeptide
TABLE 3: Activity of Cathepsin B Mutants Towards 2-Phe-Arg-MCA
ILLUSTRATIONS FOR CHAPTER 2
FIGURE 1: Am3-stained SDS-PAGE of Procathepsin B Processing
FIGURE 2: PVDF-membranes of Procathepsins B and S
FIGURE 3: Autoproteolytic Cleavage Sites in Procathepsins B and S
FIGURE 4: Active Site Cleft in Rat Cys29Ser Procathepsin B
FIGURE 5: Plots of k,b, versus Proenzyme Concentration
FXGURE 6: Western Blots of Wild-Type and Arg8Ala Propapain
ILLUSTRATIONS FOR CHAPTER 3
FIGURE 1: Homology Mode1 of Procathepsin H 11 1
FLGURE 2 (A,B):Non-Reducing SDS-PAGE of Glycosylated Procathepsin H 111
FIGURE 3 : Structural Alignment of Prosegments Composed of Cys82p 111
FIGURE 4: Progress Curves of Aminopeptidase Activity for Cathepsin H Isoforms 11 1
The research was fûnded in part by the Govemment of Canada's Network of
Centres of Excellence Program supported by the Medical Research Council of Canada
and the Natural Sciences and Engineering Research Council of Canada through Pence
Inc. (the Protein Engineering Network of Centres of Excellence).
The author gratefully acknowledges the mentorship and encouragement
provided by Dr. Andrew C- Storer who made sure that he left the laboratory with the
ability to 'think independently' in order to be prepared to take on firture challenges.
The author also wishes to thank the encouragement and advice provided by several
members of the laboratory and other employees of the Biotechnology Research
Institute, National Research Council Canada @ast and present) : Drs. Marko Pregel,
Dorit K. Nagler, Robert Ménard, Edmund Ziomek, Richard Hrabal, J. Sivaraman,
Shahul NiIar, and Dr- John S. Mort (Joint Diseases Laboratory, Shnners Hospital for
Children). The author wishes to thank Mr. Robert Dupras for introducing him to the
hockey and softball games which enabled him to interact with the fiendly employees
of the BRI.
The author would also like to reserve special mention for his wife, Dr.
Katharine A. Cacpenter, who demonstrated tremendous patience during the course of
these studies. To his beloved parents, (late) Abdul Mateen Quraishi and Yolande
Quraishi, for instilhg the values of obtaining a higher education fiom a very early age.
Finally, the author is grateful to the Almighty God : The Creator, Ruler, and Sustainer,
Cherisher of Al1 Worlds, The Most Gracious, and The Most Mercifil.
ABSTRACT
The activation mechanisms for zymogens belonging to the papain family of
cysteine proteases were investigated. This was accomplished using site-directed
mutagenesis, kinetic measurements, the identification of processing intermediates, and
the analysis of the various X-ray crystal structures reported to date. Procathepsin B is a
unique precursor of papain-like enzymes in that it is composed of a shorter prodomain ;
i-e., 62 residues versus 290 residues for those belonging to the cathepsin L-subfamily,
and the mature enzyme is composed of a twenty residue insertion termed the occluding
loop. In this study, the pH dependence of cathepsin B inhibition by its propeptide was
shown to be eliminated upon the removal of this enzyme's occluding loop.
Furthemore, variants of cathepsin B c-g the mutation Asp22Ala or His 1 1 OAla
also displayed a loss of pH dependence for their affinity to the propeptide inhibitor.
Sirnilarly, the overall rate of autoprocessing for full-length procathepsin B was shown
to be affected by the occluding loop mutations. These results suggest a possible
influence of the pH-dependent stability of the occluding loop on the overall rate of
processing for this precursor.
The addition of the protein-protehase inhibitor, cystatin C, impeded the overall
rate of autoactivation for procathepsin B and procathepsin S and caused the
accumulation of processing intermediates for these precursors which were subsequently
identified by autornated Edrnan degradation. The N-terminal sequences o f these
processing intermediates correspond to an area of the prodomains which binds through
the substrate-binding clefts of these enzymes, thus suggesting a plausible
intramolecular step of processing for this family of zymogens. This unimolecular
mechanism was found to rely on the confoxmational mobility of prosegment residues
(Le., at the C-terminal end). Furthemore, in contrast to what has been observed for
zymogens belonging to the aspartic protease family, it was determined that charged
residues located at the N-temiuius of the mature segment found in propapain do not
contribute to the overall pH-triggering mechanism of activation for this precursor.
Procathepsin H was determined to be an unusual mammalian member of the
cathepsin L-subfamily due to its inability to autoprocess, and the aminopeptidase
activiw of mature cathepsin H was found to be incapable of converting its own
precursor. Furthemore, prosegment residues located near the pro/rnature junction of
procathepsin H are highly resistant to the proteolytic action of secondary proteases.
These findings are consistent with the pre-formation of a disulfide bond within the
cathepsin H precursor which links the prodomain to the enzyme using Cys82p and
Cys214. Consistent with the fïndings for procathepsins B and S, the unrestncted
conformational mobility at the C-terminal end of the prosegment (i-e., near the
pro/rnature junction) is an important prerequisite for efficient autoactivation to occur
among zymogens of papain-like enzymes.
RÉsU1MÉ
Les mécanismes d'activation de zymogènes des protéases à cystéine de la
famille de la papaine ont été étudiés. Ceci a été accompli en utilisant la mutagénèse
dirigée, des mesures de cinétique, l'identification d'intermédiaires de maturation, et
l'analyse des diverses structures crystallines connues a ce jour. La procathepsine B est
un précurseur unique dans le groupe d'enzymes similaires à la papaine d u au fait
qu'elle est composée d'une prorégion plus courte, contenant seulement 62 résidus,
comparativement aux 90 et plus résidus rencontrés dans les précurseurs appartenant au
sous-groupe d'enzymes similaires à la cathepsine L. De plus, la cathepsine B mature
contient une insertion de vingt acides amines qui constituent une boucle d'occlusion.
Dans cette étude, il a été démontré que l'influence du pH sur l'inhibition de la
cathepsine B par son propeptide est éliminée lorsque cette boucle d'occlusion n'est pas
présente. De plus, des variantes de la cathepsine B contenant la mutation Asp22Ala ou
Hisl lOAla ont aussi perdu cette dépendence du pH pour l'affinité de l'inhibiteur
propeptide. Similairement, la vitesse globale d'automaturation de la procathepsine B
est affectée par les mutations de la boucle d'insertion. Ces résultats suggèrent que la
stabilité de cette boucle d'insertion, qui est dépendante du pH, influence la vitesse de
maturation du précurseur de la cathepsine B.
L'addition d'un inhibiteur protéique des protéases à cystéine, la cystatine C,
cause une forte diminution de la vitesse de maturation autocatalytique de précurseurs
tels la procathepsine B et la procathepsine S, et en conséquence conduit à une
accumulation d'intermédiaires de maturation pour ces précurseurs. Ces intermédiaires
ont été identifiés par l'analyse de la séquence N-terminale par la méthode dYEdrnan.
Les séquences N-terminales de ces intermédiaires permettent de localiser Ies sites
d'hydrolyse à une région des prodomaines située au site de liaison de subtrats de ces
enzymes, ce qui nous permet de suggérer une étape intramoléculaire dans la maturation
de cette famille de zymogènes. Ce mécanisme unimoléculaires dépend de la mobilité
conformationelle de résidus de la prorégion dans la partie C-terminale. De plus,
contrairement à ce qui a été observé pour les zyrnogènes de la famille des protéases
aspartyle, il a été démontré que des résidus chargés situés en N-terminal du domaine
correspondant à l'enzyme mature dans la propaine ne contribuent pas au mécanisme
pH-dépendant de déclenchement de la maturation pour ce précurseur.
Il a été déterminé que la procathepsine H est un membre particulier du sous-
groupe d'enzymes similaires à la cathepsine L, du au fait qu'elle ne peut procéder à une
maturation autocatalytique, et à l'incapacité de la cathepsine H de convertir son
précurseur en enzyme mature via son activité aminopeptidase. De plus, les résidus du
prodomaine situés près de la jonction de la prorégion et de l'enzyme mature de la
procathepsine H sont très resistants à l'action protéolytique de protéases exogènes. Ces
résultats peuvent être expliqués par la présence d'un pont disulfùe dans le précurseur
de la cathepsine H (Cys82p-Cys214) qui relie le prodomaine au reste de l'enzyme. En
accord avec les résultat obtenus pour les procathepsines B et S, la mobilité
conformationelIe dans la partie C-terminale de la prorégion (près de Ia jonction
prodomaine-enzyme mature) est un facteur déterminant pour la maturation
autocatalytique des zymogènes des enzymes de la farnille de la papaine.
lNTRODUCTION AND LITERATURE REVIEW
Enzymes have been specifically designed to be proficient in catalyzing a diverse
array of chernical reactions. These reactions may involve the proteolysis of peptide
bonds (serine, cyst eine, aspartyl, or zn2+-coordinated pro teases), fo lding/unfo lding of
polypeptides or large proteins (caInexin (1,2)), shifting the cis/trans isomenzation
equilibrium of proline residues within proteins (cyclophilins (3)),
phosphorylation~dephosphorylation of proteins (kinases/phosp hatases (43)) or
mediating protein-protein or protein-DNA interactions, etc,. Enzymes have the
capacity of accelerating the rates of reactions by many orders of magnitude using
several methods. For exarnple, enzymes are capable of increasing the effective
concentrations and optimizing the relative orientations of reactants. Enzymes also
provide a unique chemical environment which lowers the energy of activation for the
reaction (E,) and possess catalytic residues with enhanced nucleophilicity. The
diversity of chemical reactions needed for the survival of living organisms requires a
similar diversity arnong enzymes. In humans, enzymes have been documented to
partake in many physiological functions such as general protein degradation, antigen
processing, bone resorption, cartilage proteoglycan breakdown, blood coagulation, and
cellular apoptosis, etc. As central components to a vanety of biological processes,
factors which lead to deregulated enzymatic activity inevitably lead to several disease
states. Thrombin, for exarnple, is critical for activating the blood coagulation cascade.
Disorders in regdating thrornbin activity may, therefore, lead to heart attacks or
strokes. Furtherrnore, deregulated activity of the matrix metalloproteases (MMPs) have
been implicated in tumor metastasis (6) and the lysosornal cysteine proteases be lonmg
to the papain family have been associated with arthritis (7) and osteoporosis (8).
Hence, enzymes are increasingly being viewed as important targets for therapeutic
intervention.
In order to regulate the activity of these proteases, al1 known ceIIu1a.r and
bacterial proteolytic proteases are expressed as latent higher molecular weight
proprotein precursors. Ttiese proenzymes possess extensions of various lengths at the
N-terminus of the enzyme. For example, the precursor of cathepsin C is composed of
over 200 residues (9,10) and that of trypsin consists of only six amino-acids (1 1). The
prosegments usually display their inhibitory activity by blocking access of natural
substrates to the enzyme's substrate-binding cl&. Proregions have also been shown to
promote proper protein folding of the enzyme, chaperone the enzyme to its appropriate
destination, and stabilize the enzyme until it is transported to either the stomach,
vacuoles, golgi, lysosome, or extracellular matrix, etc,. Once the zymogens arrive at
their final destination, however, a mechanism is then needed to activate them so as to
allow the active enzymes to perfonn their catalytic duties. in order to make this
conversion possible, most (but not dl) proenzymes contain the structural information
necessq to produce active protein ; i.e. a preformed and functional catalytic ion-pair
and a mature substrate-binding cleft. The proenzymes which are competent in
performing autoactivation rnay do so using intra- or intermolecular pathways. The
proenzymes which are not capable of 'self activation, however, must rely either on the
activity of secondary proteases, the binding of accessory protein molecules, or a
combination of the two in order to complete the maturation process. Proenzymes
capable of autoprocessing usually require the destabilization of the prodornain/enzyme
complex. This 'loosening' rnay be achieved by simply varying the pH environment to
which the precursor is subjected. The difficulties in elucidating the rnolecular
mechanisms involved in proenzyme activation arise due to the fact that many events
may occur simultaneously, such as limited proteolysis of the precursor and
conformational changes within the prosegments and/or catalytic domains. Clearly, a
detailed characterization of the rnethods utilized by the prosegments to inhibit their
parent enzymes is an important step in understanding the events which lead to
precursor activation.
In this thesis, zymogens of proteolytic enzymes and their mechanisms of
activation will be the main focus of discussion. Proteases have evolved several
methods of cleavïng peptide (amide) bonds. Some proteolytic enzymes utilize the side-
chain of either a serine or a cysteine residue as a catalytic nucleophile. For these
proteases, the formation of a hydrogen bond between the catalytic nucleophile and an
irnidazole (often located at a distant position relative to the catalytic residue within the
prirnary structure of the protein) is required to enhance the nucleophilicity and
reactivity of the enzyme by acting as a general base and serving to lower the pK, of the
catalytic nucleophile. Other proteases may utilize metal CO factors or the surrounding
solvent to carry out their proteolytic activity. In addition, proteolytic enzymes possess
an electrophilic pocket, temed the oxyanion hole, which accepts and stabilizes the
generated negative charge on the carbonyl oxygen of the tetrahedral intermediate. For
some farnilies of enzymes, such as those of the serine and aspartic proteases, their
mechanisms of activation have been well characterized due to the fact that much
progress has been made in eludidating the three-dimensional structures of their
zymogens. However, for families of zymogens which have been identified only
recently such as those of the caspase farnily of cysteine proteases or the proprotein
convertase farnily of serine proteases, there is a corresponding lack of structural
information available. Hence, much progress remains to be made in the
characterization of processing mechanisms for several families of precursors.
The main body of the thesis will address the structural and mechanistic features
of autoactivation among zymogens belonging to the papain family of cysteine
proteases. In order to appreciate the similarities and differences arnong the different
families of proproteins, the introduction of this thesis reviews the available literature
whkh focusses on the mechanisms of processuig ; Le., structural requirements,
identification of cleavage sites, etc., for families of proteases other than the papain
family. The classes of enzymes will be divided upon the nature of the catalytic
nucleophile used to carry out hydrolysis. Therefore, al1 enzymes will be classified as
either serine, aspartic, 2n2+-coordinated, or cysteine proteases. Each group will also be
divided into sub-families since enzymes using the same type of nucIeophilic residue
may be composed of different three-dimensional folds, or have a different ceIlular or
physiological localization and fimction.
SERINE PROTEASES
To date, zymogens belonging to the senne family of proteases have been the
best charactenzed as a result of over 45 years of accumulated data using kinetic,
chernical, and physical techniques. Chyrnotrypsin and trypsin are biosynthesized as
larger inactive precursors by the pancreatic acinar cells. Using the pancreatic duct,
these proenzymes are then channelled into the duodenum where they are subseqiiently
activated due to the low pH environment of the small intestine. Upon their activation,
they utilize a senne residue as the catalytic nucleophile and serve as catalysts for the
digestion of peptide (amide) bonds with varying specificities for the side-chains located
adjacent to the peptide bond to be cleaved. Acute pancreatitis is a senous medical
condition which rnay arise fiom the premature activation of these zyrnogens within the
pancreatic tissues.
ACTIVATION OF TRYPSINOGEN AND CHYMOTRYPSTNOGEN
The zymogen of trypsin, termed ûypsinogen, is activated (processed) by the
proteolytic action of a second s e ~ e protease, enteropeptidase, which is secreted by the
duodenal mucosa. Enteropeptidase recognizes the high concentration oE negative
charge formed by 4 consecutive aspartate residues (Asp 1 1 p-Asp l4p) within
trypsinogen's N-terminal hexapeptide extension, and serves to cleave between Lys l5p
and Ile16 to yield the active enzyme and cause the permanent removal of the
prosegment (Note : Suffix 'p' refers to residues located in the prodomain). This site of
proteolysis rnay be recognized by the small amount of trypsin produced by
enteropeptidase activity, and therefore, the extent of trypsinogen activation is arnplified
by the activity of the mature form of the protein. Hence, trypsinogen activation is
autocatalytic but requires the initial activity of a secondary protease. Crystallograp hic
studies of trypsinogen (12,13,14) indicate that the catalytic triad is indistinguishable
fiom that of the mature enzyme, but its inactivity is due to a partially obstructed
substrate-binding cleft and immature oxyanion hole arising Erom a disordered loop
(residues 186-294) located within the active site. These observations are consistent
with the necessity for structural changes during activation of this farnily of zymogens
(1%
The situation for chymotrypsinogen activation is similar to that for trypsinogen.
The active site center in mature (active) chyrnotrypsin is composed of Ser195 and
His57. In addition, AsplO2 is situated in the vicinity of the catalytic histidine to
stabilize the required conformation of the imidazoliurn side-chain (16,17)- The
oxyanion hole, stabilizing the developing negative charge on the carbony l ox ygen atom
of the tetrahedral intermediate, is forrned by the backbone amide protons of Gly 193 and
Ser195 (18). Within the mature protein, therefore, the ability of these amides to donate
their protons to the carbonyl oxygen of the Pi residue (notation of Schechter & Berger,
1967 (1 9)) requires a specific backbone conformation for residues 190- 1 95. S imilarly
to trypsinogen, chyrnotrypsinogen activation is initiated by the activity of a secondary
protease. The activity of mature trypsin (or that of enteropeptidase) is required to
cleave between residues Argl Sp-Ilel6 of the 15-residue proregion found in
chymotrypsinogen (Figure 1 on next page). Residues lp- 15p of the prosegment,
however, remain covalently attached to the main body of the enzyme via a disul fide
bond linking Cys lp and Cys122. The liberated N-terminus at IIe 16 then forms an ion-
pair with the carboxylate side-chain of Asp 194. This interaction is a prerequisite to
form an active enzyme. Following these events leads to the autocatalytic release of the
dipeptides Serf4p-ArglSp and Thr147-Asn148. Direct cornparison of the crystaI
structures of bovine chymotrypsinogen (20) and mature cc- and y-chymotrypsin ( 2 1,22)
reveals that their overall fold are identical and that only a small segment of residues
undergo conformational changes during conversion. Significantly, it has been
determined that the substrate-binding cleft is only partially forrned in the chymotrypsin
precursor. The peptide bond between Met1 92-Gly193 is in the wrong orientation to
allow the backbone amide atom to contribute a proton to the oxyanion hole. As has
been observed for trypsinogen, chyrnotrypsinogen consists of a preformed catalytic
triad but an immature oxyanion hole, thus leading to a substrate-binding clef? which is
partially obstmcted. In the chyrnotrypsin precursor, the Aspl94 carboxylate (located in
Chymotrypsinogcn . 1 (inactive)
245
I I I I
Trypsin L
Conformationd change
K-Chymotrypsin
d3yrnotryptin 1 7 1 116 245
I K 1 I
Conformationai change
a-aiymotrypsin
S S
Chymotrypsin
FIGURE 1
S S
Ser 14 -Arg 15 Thr 147 -Am 148
the active site clefi) is H-bonded to His40. In the mature protein, however, Aspl 94
interacts with the new N-terminus at IIel6. This new interaction causes Aspl94 to
rotate approximately 180" about its own Ca-C bond. This conformational change
necessitates the backbone amide nitrogens of Glyl93 and Ser195 to protrude more into
the substrate-binding clef? and contribute to the oxyanion hole as hydrogen donors to
the substrate carbonyl oxygen. In addition, the side-chah of Met192 changes its
position fiom a buried position in the zymogen to that of a solvent exposed residue in
the mature enzyme. In sumrnary, the activation of trypsinogen and chymotrypsinogen
hvolves the maturation of a partially forrned substrate-binding clefi and oxyanion hole
rnediated by limited proteolysis at the pro/mature junction due the activity of a
secondary protease.
PROTFrROMBm ACTWATION
In order to illustrate the diversity of activation mechanisms found among serine
proteases, the process of converting prothrombin to mature thrornbin is also discussed.
Thrombin is the central protease which triggers the blood coagulation cascade,
activates the conversion of soluble fibrinogen of blood plasma to the insoluble fibnn
clot (23), and is known to activate blood platelets (24). Hence, the conversion of
prothrombin to active thrombin must be under tight regdatory control in order to
prevent the production of unwanted bIood dots which could dtimately lead to strokes
or heart attacks.
Active thrombin is generated via formation of the prothrombinase complex (25)
(Figure 2 on next page). Upon vascular injury, soluble prothrombin will associate with
the surface of blood vessicles through its fiagment 1 (or laingle 1 (KI)) domain at the
u-) Throm bin
PROTH.ROMBINASE COMPLEX
FIGURE 2
site of haernomhage with the assistance of ca2+ ions. Circulating factor Va will then
recognize and associate with the fragment 2 (or krhgle 2 (K2)) domain of the
prothrombin molecule. The complex of prothrombin and factor Va exposes two sites
with the sequence, Ile-Glu-Gly-Arg, which are then recognized and cleaved by the
active serine protease, factor Xa (26). These sites, however, are not accessible in the
absence of factor Va and major conformational changes are, therefore, required within
the thrombin precursor. Following this limited proteolysis, m e r autocatalytic
hydrolysis take place to produce active thrombin. Hence, the efficient conversion of
prothrombin to active thrombin requires the sirnultaneous presence of membrane, ca2+
ions, factor Va, and the activity of a secondary protease, factor Xa.
PROPROTEIN CONVERTASES (PCs)
Recently, seven marnmalian senne proteases homo logous to the yeast kexin and
bacterial subtilisins, referred to as proprotein convertases (PCs), have been identified
and shown to be implicated in the maturation of a diverse array of prohormone
polypeptides and proprotein precursors (27). Located mainly within the ~runs-Golgi
network, these enzymes cleave a variety of precursors at the consensus (Arg/Lys)-
(xaa),-&? sequence, where Xaa is any amino-acid except Cys and n = 0, 2, 4, or 6
(28). Common examples of substrates for the PCs include a- and y-endorphin (29), the
metalloprotease ADAM-10 (30), as well as the arnyloidogenic peptides AP40, -42, and
-43 (3 1). Al1 zymogens belonging to the PC family of senne protease are composed of
an N-terminal signal peptide, followed by a prosegment, a catalytic domain, a P-
domain (whose function is not well understood), and an enzyme-specific C-terminal
segment. Although PCs are specifically designed to process other precursors, it is
interesting to note that PCs are themselves biosynthesized as proproteins in order to
reguiate their hydrolytic activity. The prosegment is thought to act as a molecular
chaperone serving to promote proper folding of the convertases in the endoplasmic
reticulum. The prosegment is cleaved by an autocatalytic mechanism (the molecular
basis for this conversion is not well understood) and acts as a non-covalent inhibitor
until the enzyme is safely channelIed into the @ans Golgi network.
ASPARTTC PROTEASES
The physiological roles of aspartic proteases include ; the digestion of dietary
proteins in the stomach of mammals, the regulation of blood pressure (e-g. renin (32)),
and maturation of the retroviral Gag polyprotein to the structural, regulatory and
enzymatic proteins irnplicated in the stability and replication of the HIV virion (33).
Cornmon to al1 members of the aspartic protease farnily is the dimerization of a
compact P-barre1 domain to forrn mature enzyme. The active site is composed of two
aspartate residues (Asp32 and Asp215 in pepsin ; Asp32 and Asp217 in gastricsin)
which reside in close proximity to one another within a hydrophobic binding clef?
formed between the two P-barre1 domains. hterestingly, a water molecule is
hydrogen-bonded to both aspartate residues. These proteases perforrn their catalytic
activity using a single displacement mechanism whereby the Hz0 rnolecule
(deprotonated) acts as a nucleophile and is able to attack the carbonyl carbon of the
scissile amide bond to be cleaved (34,35). This is in contrast to the serine proteases
which utilize a double displacement reaction rnechanism. This process involves
nucleophilic catalysis perfoxmed by a residue belonging to the enzyme leading to the
formation of a covalent complex with their substrates. Furthemore, senne proteases
use a &O molecule to assist in the deacylation step. The X-ray crystal structures of
porcine pepsinogen (36), hurnan pepsinogen A (37), and human progastricsin (38) have
been detennined. Within the farnily of aspartic proteases there exists an excellent
example of an intermediate of progastricsin processing (39) which may be stabilized by
adjusting the pH to neutral conditions. Zymogens belonging to this family typically
possess a positively charged N-temiinal prosegment (> 40 residues) that interacts with
the negatively charged catalytic domain using several salt bridges. In hurnan
progastricsin, Lys37p forms a H-bonded salt bridge to both aspartate residues located in
the active site. These salt bridges allow the prosegment residues Pro34p to Arg39p to
block access of natural substrates to the preformed active site. Similar to the precursors
belonging to the papain family (described later), activation of the aspartic protease
zymogens requires the destabilization of salt bridges caused by their exposure to low
pH followed by lirnited proteolysis of the prosegment. Unlike the precursors of the
papah family (described later), however, zyrnogens of the aspartic protease h i r y are
disthguished by a major conformational rearrangement (irreversible) within the N-
temiinus of the mature enzyme segment which occurs as a consequence of activation.
Upon exposure of the gasûicsin precursor to low pH, for exarnple, there is
loosening of the prosegment/enzyme complex involving the unfolding of residues
Phe26p-Leu43p which bind through the active site cleft, and thus leading to the
formation of interrnediate 1 (40) (Figure 3 on next page). The first proteoIytic event
involves intrarnolecular autocatalytic cleavage of the peptide bond between Phe26p and
Leu27p followed by intermolecular autocatalytic cleavage at the pro/mature junction
FIGURE 3
(Leu43p-Serl), resdting in the formation of intermediate 2. Residues AIalp-Phe26p
remain temporarily associated non-covalently to the mature segment (40). Intermediate
2 is unique in that it may be stabilized by increasing the pH to neutral conditions which
serves to inhibit the catalytic activity of these enzymes and preventing fûrther
processulg to f o m mature protein. Interestingly, the catalytic HzO molecule was
observed to bind to the two aspartate residues in Intermediate 2. In summary, the
catalytic centers and substrate-binding clefts found among the precursors of aspartic
proteases are prefonned and functional, and lowering the pH environment to which
they are exposed results in the protonation of salt bridges forrned by the carboxylate
side chains of aspartate and glutamate residues which stabilize the association behveen
the prosegment and enzyme.
ZINC WTALLOPROTEASES
PROCARBOXYPEPTIDASE A AND B
Zinc metalloproteases such as carboxypeptidase A and carboxypeptidase B are
found in the pancreas and serve to degrade proteins in the alimentary tract of marnrnals
(41) by cleaving peptide bonds at the C-terrnini of polypeptide substrates. These
enzymes consist of a zn2' at the active site that is directly involved in the catalytic
mechanism. The 2n2' ion is coordinated by three residues located within the substrate-
binding cleft of these enzymes ; His69, Glu72, and His196. Sirnilar to the aspartyI 6
proteases, the mechanism of peptide hydrolysis for the pancreatic zinc metalloproteases
involves the activation of a water molecule by Glu270 (Procarboxypeptidase A
nurnbering) followed by nucleophilic attack of the scissile amide bond by the
hydroxide (42,43). The positively charged environment fonned by the residues located
within the substrate-binding cleft, including the znZf ion itself, assists with the
hydro lytic reactions b y neutralizing the developing negative charge O f the tetrahedral
intermediate.
The prosegments of the zinc carboxypeptidases are approximately 95 residues
in length. Similarly to chymotrypsinogen, activation of procarboxypeptidases is
initiated by limited proteolysis at the pro/mature junction by active trypsin. The
pro/mature junction in procarboxypeptidase A (Arg99p-Aial) is accessible to direct
recognition and proteolysis since it is localized to a conformationally fkee loop (Le.
with high B-factors) (44,45). The prosegment is m e r degraded by trypsin and
mature carboxypeptidase A at interna1 sites that are not accessible in the zymogcn (at
Arg74p-Tyr75p in procarboxypeptidase A; and at Arg83p-Ser84p in
procarboxypeptidase B). Therefore, major conformational changes must occur within
the prosegment during conversion of these zyrnogens but none have been observed
within the catalytic domain.
PRO-STROMELYSIN-1
As opposed to carboxypeptidases A and B, strornelysin-1 is a 2n2'
endopeptidase ; i.e., cleaves at intemd sites within a polypeptide substrate, and is a
mernber of the family of matrix metalloproteases (MMPs). MMPs function at neutral
pH and are involved in the degradation of connective tissue for the purpose of
regulating tissue homeostasis (6,46). The deregulated activity of this farnily of
enzymes has been associated with tumor metastasis (6) . The prosegment of this
zymogen is composed of 82 residues and the C-terminus of the enzyme has been shown
to be composed of a domain that has been proposed to mediate interactions with
rnacromolecular substrates and inhibitors (47). The X-ray crystal structure of pro-
stromelysin-1 (47) reveals that the catalytic zn2+ is directly coordinated by a residue
fiom the prosegment, Cys75p. This interaction utilizes the sulfur atom from the side-
chain of Cys75p and thereby prevents catalytic activity for this zyrnogen. Sîmilar to
the prosegments found m o n g precursors of the papain farnily (discussed later), the
prosegment of pro-stromelysin-1 binds through the enzyme's active site cleft in the
reverse substrate-binding mode.
The conversion of pro-strornelysin-l to its mature protein involves lirnited
proteolysis at the pro/rnature junction (HisSZp-Phel) using the activity of other
proteolytic enzymes, and cleavage sites within the prosegment (e-g., Glu68p-Va169p,
a-helix 3p) have also been identified (48). The only significant conformational
changes which take place is in the loop consisting of the pro/mature junction.
Following cleavage, the loop undergoes a conformational rearrangement that results in
salt bridge formation between the newly liberated N-terminus at Phel and Asp237 in a
rnanner similar to that observed for mature serine proteases. This salt bridge, however,
is 12 A away fi-om the catalytic 2n2+ and is not expected to affect the active site or the
catalytic activity of the enzyme. In contrast, the newIy fonned salt bridge formed
among serine proteases (trypsin, chymotrypsin ; discussed previously) is crucial for the
maturation of the oxyanion hole.
CYSTEM PROTEASES
CASPASE FAMILY
Apoptosis is a form of ce11 death that is vital for morphogenesis, tissue
homeostasis, and host defense (49). Disruptions in the apoptotic program are
associated with neurodegenerative disorders, where there is excessive ce11 death, and
cancer, where there is insufficient ce11 death. Key mediators that initiate and execute
the apoptotic program are members of the aspartate-specific family of cytoplasmic
cysteine proteases, known as caspases (50,51)- In order to regulate their activities,
these apoptotic catalysts are synthesized as inactive precursors. This family of
precursors are divided into two classes ; Class I (apical ; initiator enzymes) such as
caspases (-2, -8, -9, and -10) are composed of long amino-terminal prodomains in
excess of 100 residues and Class II (executioner enzymes) such as caspases (-3, -6, and
-7) consist of relatively short prodomains (52,53). The zymogens of apical caspases
may be recruited by specific adaptor molecules. For exarnple, procaspase-8 and - 10 are
recruited at the cytosolic side of death receptors (e-g. Fas receptor) via their interaction
with the adaptor molecule FADD (54), whereas procaspase-9 has been associated with
the rnitochondria in the signaling pathway leading to apoptosis (55,56). Processing of
procaspases into the mature heterotetramer product, (p20p10)2, requires specific
cleavage after aspartic acid residues located within the interdomain linkers OF the
protein (57). Furthermore, recent studies suggest that the long prodomains in some
class 1 caspases are able to mediate dimenzation of procaspase molecules, thereby
promoting autoprocessing (5 8). To date, no three-dimensional structure has been
solved for any of these zymogens and the mechanism of activation of procaspases in a
particular apoptotic pathway is poorly understood. The catalytic activity of mature
initiator enzymes are subsequently used to process downstream executioner enzymes in
trans. Hence, there exists an elaborate hierarchy of zymogen activation arnong the
caspase family of cysteine proteases. Furthermore, accessory proteins are also utilized
as death stimuli. For exarnple, a regulator of apoptosis termed Apaf-1 (a marnrnalian
homolog of C. elegans CED-4 (59)), is capable of binding to and activating procaspase-
9 in the presence of cytochrome c and dATP, thus leading to the activation of caspase-
3. Furthemore, the serine protease granzyme B derived Erom cytotoxic T cells,
triggers activation of both procaspase-3 and -7 (59). Much progress remains to be
made, however, in elucidating the molecular mechanisms of processing utilized by the
caspase family of zymogens.
PAPAIN F A m Y
Papain, derived from the dried latex of the tropical papaya h i t , is the canonical
enzyme of a family of cysteine proteases which continuously increases in its
rnembership. Much interest has been placed on papain as a mode1 for protein structure
and fiinction studies. Papain has been known to be a cornmon ingredient in meat
tenderizers since it is an efficient endopeptidase and is safely digested by the stomach.
This enzyme has also been used to prevent chi11 haze during beer production. Papain-
like enzymes may be found in bacteria, yeast, plants, and mammals. In addition, a
papain-like gene has also been identified in the baculovinis Azrtographa califarnica
Nuclear Polyhedrosis Virus genorne (60). The rnamrnalian homologs of papain include
cathepsins B (61), C (9,10), H (62), K (63), L (64), S (65), W (66) and X (67). With
some notable exceptions, these enzymes are targetted to the mature lysosome using the
mannose-6-phosphate receptor (68) and have been shown to be responsible for general
intracellular protein turnover (69), bone resorption (70), cartilage proteoglycan
breakdown (71), and antigen processing within the endosomal system (72). Recently,
cathepsin L has been found to be a critical protease for invariant chain degradation in
thymic epithelium (73,74). Degradation of the invariant chain was found to be
perturbed in cathepsin L-deficient mice, thus leading to impairment of positive
selection by C D ~ + T cells caused by a reduced repertoire of major
histocornpatibili~-foreign peptide complexes on the cortical epithelial cells.
Cathepsin S is an enzyme which fiilfills a sirniIar role to that of cathepsin L but
mediates negative selection within peripheraI bone mmow-derived antigen-presenting
cells in the thymic medulla (74). Furthemore, cathepsin K has been found to be very
highly expressed in osteoclasts, thus suggesting a role for this enzyme in bone
remodelling (75). Deficiency in the cathepsin K enzyme has been linked to a disease
h o w n as pycnodysostosis (or Pycno) which is a rare recessive trait characterized by
bone fiagility and short stature (76). Due to their ability to cleave type 1-collagen,
elastin, and proteoglycans, enzymes such as cathepsin B are also beIieved to have a role
in tumor progression (77). Therefore, these enzymes constitute interesting targets for
therapeutic intervention. Despite the availability of inhibitors with sufficient potency,
however, they often lack the required selectivity needed for therapeutic applications.
Enzymes belonging to the papain family perform their duties by utilizing a
catalytic thiolate-imidazolium ion-pair. Nucleophiiic attack on the carbonyl carbon of
the peptide bond to be cleaved is performed by the sulfur atorn of the catalytic cysteine,
Cys25 (papain numbering ; Cys29 in cathepsin B). The resulting tetrahedral
intermediate carries a forma1 negative charge on the oxygen which is then stabilized by
the oxyanion hole formed by Gln 19 (papain numbering ; G W 3 in cathepsin B). The
departure of the leaving group leads to the formation of a covalent acyl-enzyme
intermediate. The catalytic unidmole, HislS9 (papain nurnbering ; His 199 in cathepsin
B) acts as a weak base and serves to deprotonate a water molecule. The covalent
intemediate is then attacked by the hydroxyl group causing the formation of the final
product and the regeneration of free enzyme (78). In addition to the thiolate-
imidazolium ion-pair, each enzyme possesses an extended substrate-binding clef3
capable of accomodating several substrate residues simultaneously and accounting for a
very modest degree of substrate selectivity. For example, cathepsin B prefers a basic
amino-acid residue in PI such as an Arg and a hydrophobie Phe group in the Pz position
(79). In general, however, designing an inhibitor which is highly selective for a given
enzyme within this farnily constitutes a challenge due to their relatively broad substrate
specificity.
Most members of the papain fâmily function as endoproteases. Cathepsins B,
C, and H, however, may exhibit unique exopeptidase activities. Cathepsin C is known
to homodimerize into a tetramer and function as an arninodipeptidase (9)- whereas
cathepsin H functions as a mono-aminopeptidase (80,81). Cathepsin B is capabte of
carboxydipeptidase activity ; i-e., the removal of dipeptides fiom the C-terminus of an
extended polypeptide substrate (79). The X-ray crystal structure of cathepsin B (82)-
the most abundant lysosomal cysteine protease, reveals the molecular basis for the
unique exopeptidase activity of this enzyme. Cathepsin B shares a common three-
dimensional fold as other papain-like enzymes but also contains a novel insertion of
over twenty residues. Within this insertion is fbund an exposed disulfide Ioop (residues
Cysl08-Cysllg), refefred to as the occluding loop, which partiaIly 'occludes' the
portion of the substrate-binding cleft that would otherwise bind substrate residues to the
C-terminal side of the scissile amide bond (discussed in Chapter 1). Two specialized
histidines located within the occluding loop, His 1 l O and His 1 I l , are strategically
positioned such that both of their side-chains are directed into the substrate-binding
clef3 facing the S2' subsite. Therefore, the orientation of the two histidines allows them
to accept the negatively charged Pz' carboxylate of the bound substrate. Conversely,
the unusual mono-aminopeptidase activity docurnented for cathepsin H is attributable
to the exclusion of natural substrates fiorn the unprimed subsites of this enzyme's
substrate-binding cleft (83) (discussed in Chapter 3).
To protect themselves from unwanted digestion, eukaryotic cells synthes ize
these proteases as latent proproteins. These proenzymes consist of polypeptides of
various lengths at the N-terminus of the protease that act as potent pH-dependent
inhibitors of the parent enzymes. The prosegments have also been shown to be stnctly
required for the expression of native proteases and promote correct protein folding in
vivo and in vitro (84). These proregions may also act as intrarnolecular chaperones and
aIso heip to stabilize the enzymes upon exposure to neutral or alkaline pH
environments (84). Similar to the caspase farnily, the papain-like enzymes have also
been divided into sub-families based on the lengths of their prosegments. For example,
most precursors belonging to the cathepsin L-subfamily possess over 90 residues within
their prosegments (refer to Figure 4 on next page). Upon their conversion to mature
protein, they generally display endopeptidase activity with varying substrate specifrcity.
Sequence identity arnong the cathepsin L-like proregions is lower than that observed
for the mature enzyme domains. However, cathepsin L-like prosegments display a
higher level of sequence homology within the central region composed of residues 2 1 p-
77p, including the ERFNIN rno tif (Glu27p-X3-Arg-X3-Phe-X2-Asn-X3-Ile-X3-Asn46p)
which foxms part of an extended a-helix (called cc2p), and the (G1A)NFD segment
(Gly59p-XI-Asn-XI-Phe-Xi-Asp65p) which binds between the primed subsites of the
enzyme's substrate-binding cleft and the hydrophobic prosegrnent-binding loop (also
called exosite) (85-88). The second subfmily is that of cathepsin B to which this
enzyme is the only member. The cathepsin B precursor is distinguished by a shorter
prosegment of only 62 residues (refer to Figure 5) which lacks the ERFNIN motif, yet
contains a motif (Gly27p-Xi-Asn-Xi-Tyr-XZ-Asp34p ; cathepsin B prosegment
numbering) which binds between the primed subsites of the active site cleft and the
prosegment-binding loop of cathepsin B (89-91); i.e., reminiscent of the GNFD
segment within the cathepsin L-like prodomains. Furthemore, another unique Feature
associated to cathepsin B is an insertion of over twenty residues located within the
catalytic domain, termed the occluding loop, which has been implicated in the
dipeptidyl carboxypeptidase activity of this enzyme (discussed previously).
The X-ray crystal structures of precursors beIonging to the papain- famil y (85-
91) reveal a conserved mode of prosegrnent binding, and a conserved mechanism of
inhibiting the activities of this family of cysteine proteases (refer to Figures 6 and 7).
The prosegments tend to be composed of more secondary (a-helical) structure within
their N-terminal ends (residues 5p-75p) than their C-terminal ends (residues 76p-96p)
(cathepsin L prosegrnent numbering). The bound prodomain is composed of a four-
turn cc-helix (cclp : 5p-19p) followed by a long arnphipathic a-helix consisting of 7.5
turns (a2p : 25p-51p). Furtherrnore, the end of helix a2p turns and folds back into an
extended conformation fonning a hairpin structure (P lp) where residues 56p-59p reside
along the surface of the enzyme. Prior to its way to the substrate-binding cleft of the
enzyme, the prodomain follows into a third a-helix composed of only two turns (u3p :
68p-75p). In general, residues 76p-96p stretch along the surface of the enzyme in an
extended conformation fkom the substrate-binding cleft to the pro/mature junction.
Enzyme residues in contact with the prosegments are located within three major
surfaces. The first major contact region is the active site def i of the enzyme which
FIGURE 7
accomodates residues 75p-8lp that adopt an extended confôrmation and bind in a
direction opposite to that expected for natural substrates ; i-e., reminiscent of the 2n2+
endopeptidase prostrornelysin-1 discussed previously. Hence, the activities of these
enzymes are inbibited by the ability of the prosegments to block the access of natural
substrates to the catalytic center. The second enzyme interface for prosegrnent
interactions is the primed subsites of the substrate-binding clefi which is required for
accomodating residues 63p-75p. The third major contact region is the hydrophobic
prosegment-binding loop (also referred to as the exosite) which interacts with residues
54p-58p. Key prodomain residues at the enzyrne interface are Phe56p (hornologous to
Trp24p in procathepsin B) and Gly77p (Gly43p ; cathepsin B prosegment numbering)
(92). The residue in position 56p is composed of an aromatic residue (Phe or Tyr ; Trp
in procathepsin B) which nestles into the hydrophobic prosegment-binding loop
(exosite) of the enzyme and plays a critical role in stabilizing the N-terminal cap of the
prodomain to the surface of the enzyme. The residue in position 77p is usually a small
uncharged residue (Gly or AIa) which allows deep penetration of the prose,ment into
the substrate-binding cleft of the enzymes ; i.e., in the reverse substrate-binding mode.
The stability of the prosegment-enzyme complex in cathepsin L-like precursors also
relies heavily on the formation of salt bridges involving highly conserved residues of
the prodomain ; Le. between GIu27p and Arg3lp with Glu70p as well as between
Arg3lp and Asp65p, which help helices a l p and cc2p to fold into close proximity to
helix a3p (85-88).
The possibility of having mistargetted enzymes ; Le. enzymes misrouted to the
extracellular matrix rather than ta the lysosome, may lead to severe pathological
conditions. As stated previously, aberrant proteolytic activity for cysteine proteases
belonging to the papain farnily has been implicated in several disease States such as
rheumatoid arthritis (6), osteoporosis (76), and cancer metastasis (77). Therefore,
organisms are required to possess 'self' defense mechanisms kom the potentially
destructive activities of these enzymes. Cysteine proteases of the papain family are
strongly but reversibly inhibited by the cystatins (dissociation constants in the
femtomolar+picomolar range), a superfardy of proteins composed of 120 residues
and hornologous to chicken cystatin (93). For example, cystatin C is present in al1
human body fluids but is most abundant in cerebrospinal fluid and seminal plasma (94).
The molecular basis for the interactions of cystatins with members of the papain fkmily
is distinct fiom the strategies utilized by the prodomains in the zymogen structures.
Similarly to the prosegrnents, cystatins inhibit these enzymes by blocking access of the
substrate-binding cleft to incoming subs trates. Ho wever, as opposed to the prodomains
which extend through the active site cleft in the reverse substrate-binding mode,
cystatins utilize three exposed loops (a tripartite interaction) which combine to form a
wedge-shaped hydrophobic edge that is highly cornplimentary in shape to the active-
site cleft of enzymes belonging to the papain farnily (95) (refer to Figure 8).
Significantly, the protrusion of Gly 1 1 (arnino-terminal segment) into the Sz subsite is
critical for the stability of this 1 :1 complex (95). Furthermore, the first hairpin loop is
composed of a highly conserved Q W A G region (Q55-IVAG in human cystatin C)
which is fianked on both sides by the amino-terminal segrnent (composed of Glyl 1 )
and a second hairpin loop. Curiously, the QVVAG region is adjacent to another
conserved motif found in the prïmary arnino-acid sequence of the protein, Gly59-X-
Asn-X-Phe-X-Asp, which is rerniniscent to the GNFD segments found within the
prodomains of the papain family (96).
FIGURE 8
The majority of precursors belonging to the papain family are capable of
autoactivating upon their exposure to acidic pH environments as is the case within the
mature lysosome, which results in the production of mature (active) enzyme
corresponding to an N-terminal sequence which begins at or near the pro/mature
junction ; i-e., the area of the prosegment with the least secondary structure.
Furthemore, subjecting inactive propapain (Cys25Ser) (97), procathepsin B
(Cys29Ser) (98), and procathepsin L (CysZSSer) (99) to catalytic quantities of their
corresponding mature enzyme leads to correct cleavage of the precursor to form mature
protein ; Le., mature enzymes belonging to the papain Family are capable of maturing
their own precursors via intermolecular cleavage (processing in tram). Furthemore,
kinetic analyses are suggestive of an event which is independent of proenzyme
concentration ; Le., a unimolecular mechanism of processing (97-100). Chemically
synthesized peptides (termed propeptides) with a sequence identical to that of the
proregions of rat cathepsin B (102), human cathepsin L (103), and hurnan cathepsin S
(104) are much weaker inhibitors towards the parent enzyme at low pH @H 4.0) than at
neutral pH values (pH 6.0). Hence, the weaker affinities of the enzymes for their
propeptides correlates with the observation that this farnily of zyrnogens autoprocess
faster at low pH than at neutral pH (97-100). These results suggest that the influence of
the ionkation of one or more carboxylic groups is important in regulating the stability
of the prosegmentlenzyrne complex dong with the concomitant release of the
prodomain due to proteolytic processing at or near the pro/mature junction. The partial
or complete digestion of the f?ee propeptide is also necessary in order to ensure that
activation of these enzymes is irreversible.
This thesis addresses some of the structural requirements for efficient
processing to occur among zyrnogens belonging to the papain family. This has been
achieved by studying unique members of the p apain superfamily, namely procathepsin
B and procathepsin H. Initially, this project was highIighted by the finding of
Ouraishi. O. that the chemically synthesized propeptide of cathepsin B (residues lp -
56p) was a more potent inhibitor of this enzyme upon the deletion of the exposed
occluding loop (ACys 108-Cys 1 19 ; termed the M 1 mutant). (Please refer to
reference (105), entitled 'Role of the Occluding Loop in Cathepsin B Activity' by
Illy, C., Quraishi. O., Wang, J., Purisima, E., Vernet, T., and Mort, J.S. (1997) J.
BioL Clrem. 272, 1197-1202). In Table IV of this articie, a 50-fold improvernent in the
affinity of the propeptide for the surface of the M l mutant was observed at pH 6.0 in
cornparison to the wild-type enzyme. A similar improvement was determined by I&
C. for the binding affinity of cystatin C to the M l mutant. Therefore, in addition to -
'occluding' the active site cleft in cathepsin B to extended polypeptide substrates, the
occluding loop was also found to obstruct the binding of cathepsin B inhibitors (the
cathepsin B prodomain and cystatin C). In addition, it was observed that the M l
mutant autoprocessed much slower than Sie wiId-type precursor; Le,, complete
maturation of the M l precursor required 5 days at pH 5.0 and 4°C as opposed to
several hours for wild-type procathepsin B under the sarne conditions. This
preliminary data lead to fürther investigating the role of the occluding loop in
regulating the pH dependence of propeptide binding to cathepsin B as well as
procathepsin B processing. Chapter 1 of this thesis entitled 'The Occluding Loop in
Cathepsin B Defines the pH Dependence of Inhibition by Its Propeptide' has been
accepted for publication (refer to reference (106)). This chapter identifies a critical salt
bridge on the enzyme (Asp22-HisllO), which stabilizes the occluding loop to the
surface of cathepsin B- As a consequence, perturbation of this salt bridge through site-
directed mutagenesis essentially eliminates the pH dependence of propeptide binding
and also has a profound effect on the overall rate of procathepsin B processing.
Chapter 2 of this thesis entitled 'Identification of Interna1 Autoproteolytic Cleavage
Sites Within the Prosegments of Recombinant Procathepsin B and Procathepsin
S' provides evidence for a slow unirnolecular rnechanism of processing for this farnily
of zymogens. This was achieved using the ability of human cystatin C to impede the
rapid intennolecular cascade of autoproteolysis, and thus favoring the observation of
intramolecular reactions. Inspection of the X-ray crystal structures for this farnily of
zymogens (85-91) indicates that the N-termini of the novel processing intermediates
identified in this study correspond to an area of the prosegments which bind through
the substrate-binding clefts of these enzymes, and that the sites of intrarnolecular
hydrolysis occur within a sketch of prosegment residues which are in close proximity
to the catalytic center. Finally, Chapter 3 entitled 'Functional Expression of Human
Procathepsin H in Piclria pastoris and Attempts at its Correct Processing'
highlights a unique member of the cathepsin L-subfamily (approximately 40 %
sequence hornology to procathepsin L) which displays mono-aminopeptidase activity
upon its maturation. The exopeptidase activity of this enzyrne has been attributed to an
octapeptide mini-chain, denved fiom the C-terminus of the cathepsin H prodomain,
which remains attached to the main body of the enzyme via Cys82p and Cys214
foliowing activation of the cathepsin H precursor. Therefore, in addition to the
covalent attachent at the pro/mature junction, procathepsin H is an unusual
mammalian homolog of papain-like enzymes in that it is also composed of a second
covalent at tachent which Iinks the prosegment to the enzyme, thus restricting the
conformational mobility of prosegment residues near the prohature junction. The
results of this chapter illustrate that, unlike procathepsin L, the cathepsin H precursor is
incapable of autoprocessing and that mature cathepsin H is not independently
responsible for the conversion of its own precursor. It has also been established that
the prohature junction in procathepsin H is highly resistant to proteolysis by the
activities of mature cathepsins B, D, H, K, L, and S. Furthemore, it is shown that the
mature rnini-chah (derived fiom the C-terminus of the cathepsin H prosegment)
optimizes but is not strictly required for the aminopeptidase activity displayed by this
enzyme.
CHAPTER 1
Cha~ter 1 : Contributions of Authors other than Omar Quraishi
Dorit K. Na~ler- Provided the P. pastoris transformed with hurnan wild-type and
occluding loop mutants of cathepsin B.
Ted Fox- Performed experirneots which lead to the data provided in Table 2 of this
c hap ter.
J. Sivaraman and Miroslaw Cvder- Provided the coordinates of the crystal structnre
of rat procathepsin B and assisted with the preparation of Figure 2 of this chapter.
John S. Mort - Provided the original cDNA of procathepsin B
Andrew C. Storer - Provided funding and mentorship for this project.
The Occluding Loop in Cathepsin B Defines the pH
Dependence of Inhibition by Its propeptide?
' W C Publication No. 00000. The research was funded in part by the Governrnent of
Canada's Network of Centres of Excellence Program supported by the Medical
Research CounciI of Canada and the Natural Sciences and Engineering Research
Council of Canada through PENCE Inc, (the Protein Engineering Network of Centres
of Excellence).
Omar ~uraishi) Dorit K. ~agler)"ed FOX? J. sivaramant
Miroslaw ~ygler:~ John S. MO& and Andrew C. ~ t o r e r * ~ * ~ ~
*protein Engineering Network of Centres of Excellence and Department of
Biochemisw, McGill Universiw, 3655 Dnimmond Street, Montreal, Quebec, Canada
H3G 1Y6, '~harmaceutical Biotechnology Sector, Biotechnology Research Institute,
National Research Council Canada, 6 100 Royalrnount Avenue, Montreal, Quebec,
Canada H4P 2R2, l ~ o i n t Diseases Laboratory, Shriners Hospital for Children, 1529
Cedar Avenue, Montreal, Quebec, Canada H3G 1A6, and Protein Engineering Network
of Centres of Excellence and Department of Surgery, McGill University, Montreal,
Quebec, Canada.
' Address correspondence to this author at the Biotechnology Research Institute, 6100
Royalrnount Avenue, Montreal, Canada H4P 2R2. E-mail: andrew.storer@nrc.ca.
Present address: Vertex Pharmaceuticals Inc. 130 Waverly St., Cambridge,
Massachusetts, USA 02 1 3 9.
RUNNING TITLE: Propeptide Inhibition of Cathepsin B Mutants
ABBREVIATIONS:
Residue numbering relates to mature human cathepsin B.
The abbreviations used are: 2-Phe-Arg-MCA, benzyioxycarbonyl-L-phenylalanyl-L-
a r m e 4-rnethylcoumarinyl-7-amide; SDS-PAGE, polyacrylamide gel
electrophoresis; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; A(Cys 108-Cys 1 19)
corresponds to the occluding loop deletion mutant as reported in Illy, C , Quraishi, O.. et
al., Mort, J.S. (Ref. 105).
In the text, the words "proregion" and "prosegment" refer to the polypeptide stretch
located N-terminal to the mature enzyme in the proenzyme, while the word
"propeptide" refers to the chemically synthesized polypeptide corresponding to the
proregion sequence but without the mature enzyme.
Abstract :
Papain-like proenzymes are prone to autoprocess under acidic pH conditions.
Similarly, peptides derived fiom the proregion of cathepsin B are potent pH dependent
inhibitors of that enzyme ; Le., at pH 6.0 the inhibition of human cathepsin B by its
propeptide is d e h e d by slow binding kinetics with a Ki of 3.7 n M and at pH 4.0 by
classical kinetics with a Ki of 82 nM. This pH dependency is essentially elirninated by
either the removal of a portion of the enzyme's occluding Ioop through deletion
mutagenesis or by the mutation of one of either residue Asp22 or Hisl 10 to alanine ;
e-g., the mutant enzyme Hisl lOAla is inhibited by its propeptide with Ki's of 2.0 i 0.3
nM at pH 4.0 and 1.1 0.2 nM at pH 6.0. For the Hisl lOAla mutant the inhibition also
displays slow binding kinetics at both pH 4.0 and pH 6.0. As shown by the crystal
structure of mature cathepsin B usi il, D., et al. (1991) Embo J. 10, 2321-2330 (82)]
Asp22 and HisllO form a salt bridge in the mature enzyme and it has been shown that
this bridge stabilizes the occluding loop in its closed position pagler, D.K., el al.
(1997) Biochemisv 36, 12608-12615 (107)]. Thus the pH dependency of propeptide
binding can be explained on the basis of a competitive binding between the occluding
loop and the propeptide. At low pH, when the Asp22-Hisl lO pair forms a salt bridge
stabilizing the occluding loop in its closed conformation, the loop more effectively
cornpetes with the propeptide than at higher pH where deprotonation of His 1 10 and the
concomitant destruction of the Asp22-HisllO salt bridge results in a destabilization of
the closed form of the loop. The rate of autocatalytic processing of procathepsin B to
cathepsin B correlates with the aanity of the enzyrne for its propeptide rather than with
its catalytic activity, thus suggesting a possible influence of occluding loop stability on
the rate of processîng.
Cathepsin B (EC 3.4.22.1) is the most abundant lysosomal cysteine protease and
is a unique member of the papain superfamily (108) in that it exhibits both
endopeptidase and dipeptidyl carboxypeptidase (exopeptidase) activity. The X-ray
crystal structure of mature cathepsin B reveals the molecular basis for this duaiity (82).
Unlike other papain-like proteases, cathepsin B possesses an extra structural element
referred to as the 'occluding loop' which conîributes to the prïmed subsites of the
substrate binding cleft- Two cntical residues of the occluding loop, HisllO and
HisZ I l , are strategicaily positioned to 'occlude' extended substrates from binding to
the enzyme and to accept the negatively charged carboxylate of the Pz7 residue at the C-
terminus of the substrates. Removal of this exposed disulfide loop (residues C ys 1 08-
Cysl19) in cathepsin B was shown to increase affbity for the protein inhibitor cystatin
C and the cathepsin B propeptide due to unrestricted access of these inhibitors to the
active site (105). In addition, this variant of procathepsin B was shown to autoprocess
much more slowly than the wild-type enzyme (105). Cornparison OF the recently
deterrnined three-dimensional structures of rat (89) and human (90,91) procathepsin B
with mature cathepsin B (82) reveals that the occluding loop is a highly flexible
segment of the protein. The backbone of the occluding loop is able to undergo a
conformational transition in which the tip of the loop moves by as much as 10Â. The
largest movernent is that of the side chah of His l l l which is displaced by over 14A
with a sirnultaneous large rnovement of Kisl 10. The X-ray crystal structure of
procathepsin B (89-91) also reveals that the 62-residue proregion adopts an extended
conformation along the surface of cathepsin B with the majority of close contacts
provided by residues 22p-47p of the prosegment (Figure 1 of Chapter 1).
Cathepsin B residues in contact with the proregion are located within three
major areas. The first major contact region is the substrate-binding cleft of the enzyme,
which accornodates residues 41p to 47p of the prosegment (Region 1). In this region,
the prosegment adopts an extended conformation and binds in a direction opposite to
that expected for natural substrates. The second major site is the pnmed subsite of the
active site cleft, tenned the occluding loop crevice, which becomes exposed upon
movement of the occiuding loop. Exposure of this surface on cathepsin B is required
for residues 29p to 40p of the proregion to bind (Region 2). The third major contact
region is the hydrophobie prosegment-binding loop (exosite) which interacts with
residues 2Zp to 26p of the prosegment (Region 3). The ability of the proregion to
utilize al1 possible interactions with the surface of cathepsin B, namely with the active
site, the occluding loop crevice and the exosite, is therefore highly dependent on the
conformational mobility of the occluding loop. Analysis of the observed
conformations which are adopted by the occluding loop (Figure 2 of Chapter 1) shows
that specific contacts between His 1 10 and His 1 1 1 of the occluding loop and the rest of
the protein change as the occluding loop shifts fiom the 'open' conformation found in
the proenzyme structure (89-9 1) to the 'closed' one f o n d in the structure of the mature
enzyme (82). When the loop is in the 'open' conformation, the side chains of residues
HisllO and As11222 as well as His l l l and Asp224 are in close proximity to one
another. Following the disposal of the proregion to form the mature enzyme, these
contacts are destroyed and new ones are formed. In mature cathepsin B, the side chains
of Hisl 10 stacks against Trp221 and its EN fonns a salt bridge with Asp22, located in
the primed subsite of the active site cleft. The side chah of His l l l is closest to
Leu181, Vall12, and with the backbone of Asp224 and Trp225, but no salt bridges are
present. Furthemore, Argll6 forms a hydrogen bond with Asp224.
It has been suggested that the expected effect of pH on the stability of the
occluding loop ; Le., due to protonation/deprotonation of the stabilizing salt bridges,
could in part define the pH dependencies observed for the exo-and endo-peptidase
activities of cathepsin B (107). In addition, it has been observed that the processing of
procathepsin B and the inhi'bition of mature cathepsin B by its corresponding
propeptide exhibit similar pH dependencies ; Le., at neutral pH, which can be expected
to favor the 'open' conformation of the loop, procathepsin B is less prone to
autoprocessing (109) and the propeptide is a tight binding inhibitor (102) whereas at
acidic pH where the 'closed' conformation of the loop is expected to be favored, the
propeptide binds less tightly and the proenzyme autoprocesses more rapidly. Thus it is
the goal of this present study to explore the possible links between the pH dependency
of : the cathepsin B occluding loop conformational flexibility; the propeptide binding
afnnity; and procathepsin B autoprocessing.
MATERIALS AND METHODS
The substrate Z-Phe-Arg-MCA was purchased fiom IAF Biochem International Inc.,
Laval, Québec. The synthesis and purification of the rat cathepsin B propeptides was
as described previously (102). The pepsin-agarose resin was purchased £tom the Sigma
Chernical Company, Recombinant human cystatin C was a generous gifi fiorn Dr.
Irena Ekiel (Biotechnology Research Institute).
Synthesis and Purification of Human PCBI. Peptide synthesis of the human
cathepsin B propeptide (residues l p to 56p) was carried out using standard Fmoc
chemistry on an Advanced Chemtech M P S 396 solid-phase synthesizer. Cnide human
peptide W C B I ) was partially purified by HPLC on a Vydac C4 (300 A) column (5 x
25 cm) using a linear 20-60% acetonitrile gradient at a flow rate of 33 mumin for 120
min (A = O. 1 % TFA in HPLC grade water; B = 0.1 % TFA in HPLC grade acetonitrile).
Human PCB I was rechromatographed with a Vydac C 18 column (0.46 x 25 cm) using
a linear 10-70% acetonitrile gradient (l%/min) at a flow rate of 1 mumin and stored
lyophilized at 4OC. Amino acid analysis was performed using a Beckrnan mode1 6300
arnino-acid analyzer. Electrospray mass spectral analy sis using a Perkin Elmer SCIEX
API III spectrometer operated in the positive mode for detection of protonated species
confirmed the expected molecular mass of 65 12 daltons.
Expvession of Wild-Type and Occhding Loop Variants of Numan Procathepsin B. In
vitro site-directed mutagenesis was performed as descnbed previously (1 05,107). The
oligonucleotide 5'-TGT GAG CAC GCT GTG AAC GGC GCC C-3' (mutated bases
are underlined) was used for the Hisl l lAla mutation which introduced a new DraIII
site. These cDNA constructs, consisting of wild-type and occluding loop variants of
human procathepsin B as a fusion with the preproregion of yeast a-factor, were
digested with Xho 1 and Not 1 and the proenzyme nagments were subcloned into the
pPIC9 vector (Invitrogen Inc., San Diego, California) and expressed in the yeast Pichia
pastons. For integration into the Pichia genome, the pPIC9 based constructs were
linearized by cleavage with Bgl II and purified. The P. pasruris host strain GS115
(Invitrogen Inc.) was then transfomed with the Linearized constructs by
electroporation. Positive transformants were grown for 2 days in medium containing
glycerol as the carbon source followed by incubation in the presence of methanol for a
fûrther 3 days to induce expression of recombinant protein. The consensus sequence
for oligosaccharide substitution in the mature protein had been removed by the
substitution, SerllSAla, for al1 variants of cathepsin B. The site for oligosaccharide
substitution within the proregion (Asn2lp) was left unaltered. For the purpose of
clarity on SDS-PAGE (12 % gels), recombinant proenzyrnes were deglycosylated using
endoglycosidase H prior to their purification.
Purification of Procathepsin B. The recombinant proenzyrnes were purified fiom the
culture supernatant using a hydropho bic resin under non-acidic conditions. The culture
supernatant (250 ml) was concentrated to 40 ml using an Amicon stirred-ce11 (YM-10
membrane). D m g concentration, the buffer was exchanged to 50 mM Tris (pH 7.4)
containing 1.2 M (N&)2S04. Concenhated proenzyme samples were then applied to a
10 ml column of butyl-sepharose (Pharmacia Inc.) resin. Proenzyme fiactions eluted
fiom the column by applying a linear gradient of decreasing ammonium sulfate
concentration. Procathepsin B (wild-type and mutants) eluted at 0.6-0.8 (NK&SO4
and were stored at 4°C.
Procathepsin B Activation. Both wild-type and His 1 l l Ala procathepsin B
autoprocessed efficiently against 50 rnM sodium acetate (pH 5.0) to form mature
enzyme. The occluding loop deIetion mutant, A(Cys 108-Cys 1 i 9), and variants
carrying the mutation Asp22Ala or HisllOAIa autoprocessed slowly under these
conditions. For this reason, these variants of procathepsin B were readily converted to
mature enzyme at pH 4.7 using 50 U/mL of pepsin Unmobilized on agarose resin.
Imrnobilized pepsin was removed by filtration following two hours of incubation with
proenzyme. The processed enzymes were purified and their kJKM values for 2-Phe-
AG-MCA were obtained as described previously (lOS,lO7).
Procathepsin B Autoprocessing. Autoprocessing of punfied procathepsin B to form
mature enzyme was monitored using SDS-PAGE (12% gels). Wild-type @PM),
His 1 1 1 Ala (3 PM), His 1 1 OAla (7pM), and Asp22Ala (6pMJ procathepsin B were
subjected to acidic pH conditions, i.e. at 50 rnM sodium acetate buffer (pH 5.0) with 1
mM DTT.
Kirzetic Measzrrernents. Kinetic fluorescence measurements were carried out using a
Perkin Elmer LS-SB luminescence spectrometer which monitored MCA formation
using an excitation wavelength of 380 nm and a detection wavelength of 440 m.
Since the KM of wild-type human cathepsin B for the substrate 2-Phe-Arg-MCA was
estirnated to be 0.100 mM under these conditions (los), a substrate concentration of 10
pM was used for slow-binding kinetics ([SI cc KM). 2-Phe-Arg-MCA concentrations
of 10, 20, 40, and 80 pM were used for plots of l/v vs [inhibitor] (1 10). The final
concentration of cathepsin B mutants was 0.1 nM for each assay, except for the
A(Cys 108-Cys 1 19) mutant ai pH 4.0 where a final concentration of 1.5 nM was used
due to its low activïty under these conditions (105). Unless otherwise stated assays
were performed at 2S°C and conditions were 50 mM phosphate (pH 6.0) or acetate (pH
4.0-5.0) buffer containing 0.2 mM EDTA, 1mM DTT, and 3% DMSO. The enzymes
studied were sufficiently stable under the assay conditions used for the tirne required.
Analysis of Data. Under the experirnental conditions used, progress curves for the
inhibition of the occluding toop mutants by propeptides at pH 6.0 (and at pHc4.7 for
A(Cysl08-Cys 1 19), His 1 1 OAla, and Asp22Ala) followed typical one-step slow-binding
kinetics as defined by the equations (1 11-1 15).
where p] is the concentration of fkee MCA formed, vi and v, are the initial and steady-
state velocities, respectively, t is the time, and kob, is the rate constant for inhibition.
Nonlinear regression using the program Enzfitter (published by Elsevier-Biosoft,
Cambridge, U.K.) gave the individual parameters (vi, v,, and hb,) for each progress
curve. For each data set, the enzyme-inhibitor dissociation constant (Ki) values were
obtained fkom the relationship vilvS - 1 = mlKi (1 16), where the vi represents the initial
rate for substrate hydrolysis in the absence of inhibitor. A plot of kobr vs [inhibitor]
remains linear over the range of inhibitor concentrations studied (4-40 n.), confkning
that inhibition of the occluding loop mutants by both hPCBl and rPCBl occurs by a
one-step process (102). Due to the near-zero intercept, kff values were calculated using
the relationship Ki = k,dkon. Inhibition of wild-type and Hisl 1 1 Ala cathepsin B at pH
4.0-4.5 gave linear plots of [Pl vs tirne, and Ki values were obtained firom plots of l lv
vs [inhibitor] (1 10) at the four substrate concentrations.
REMJLTS AND DISCUSSION
As shown in Table 1, the afnnity of cathepsin B for a peptide derived from its
proregion is increased approximately 40 fold at pH 6.0 and 200 fold at pH 4.0 by the
partial deletion of the occluding loop. In addition, the affullty of cathepsin B for the
same propeptide is increased significantly at both pH 4.0 and 6.0, although to a lesser
degree, by the mutation of either or both of residues Hisl10 and Asp22 to alanines. As
shown by Nagler et al. (107) these mutations result in the destabilization of the closed
conformation of the occluding loop found in the mature enzyme thus decreasing its
ability to compete with the propeptide for binding to the active site cleft.
It has been previously shown that a synthetic peptide with a sequence identical
to that of the proregion of rat cathepsin B is a much weaker inhibitor (160 fold) of that
enzyme at pH 4.0 than at pH 6.0 (102). At pH 6.0 the rat cathepsin B propeptide
displays slow binding inhibition kinetics whereas at the lower pH it behaves as a
classical cornpetitive inhibitor. This pH dependent switch in inhibition type c m be
accounted for by the lower a i t y of the enzyme for the propeptide at pH 4.0 coupled
with a more rapid rate of dissociation at the lower pH. As can be seen fiom Table 1 the
human cathepsin B-propeptide interaction displays a sirnilar though smaller (22 fold)
pH dependency. The weaker binding and faster off rates observed for both the rat and
human propeptides correlates with the observation that the processing of procathepsin
B is considerably faster at pH 4.0 than at pH 6.0. Fox et al. (102) reported that the pH
dependency of the for the inhibition of rat cathepsin B by its propeptide is consistent
with the influence of multiple ionisations in the pH range 4.0-6.0 and it is suggested
that the ionisation of one or more carboxylic groups is important for binding.
However, the question of whether these carboxylic acid residues reside on the mature
enzyme, the propeptide or both was not addressed. Frorn the present study, based on
the results discussed below, it is evident that it is residues within the mature enzyme
that are responsible for this effect.
On comparing the sequences of the rat and human propeptides (Figure 1 of this
chapter) it c m be seen that only three acidic side chains are conserved, Le. aspartic acid
residues at positions 1 l p and 34p, and at position 12p aspartic acid (rat) and glutarnic
acid (human). In order to explore the possibility that one or more of these acid groups
contributes to the pH dependency of propeptide binding, peptides with a 5-residue
deletion at the N-temiinus were synthesized in which each of the three aspartic acid
residues found in the rat propeptide were sequentially substituted by asparagine
residues (the deletion of 5 residues fiom the N-terminus results in a 3-fold increase in
Ki(pH 4.0)/KiCpH 6.0) versus the fùll length propeptide but does not adversely affect
the overall pH dependency of binding, whereas it facilitates synthesis). The
dissociation constants obtained for each of the modified peptides at pH 4.0 and 6.0 are
given in Table 2. It can be concluded fkom Table 2 (following this chapter) that the
carboxylic acid side chahs found within the propeptides do not contribute significantly
to the pH dependency of propeptide binding. In addition, the presence of negative
charges within the propeptide does not have a large influence on the overall strength of
peptide binding. A recent study (107) indicates that pH dependent conformational
changes occur in the occluding loop of cathepsin B in the pH range of 3.0 to 8.0. It was
suggested that at the higher pH the loop becomes more flexible and as such less able to
compete with extended endopeptidase substrates for the S' subsites. Conversely at
lower pH values the more rigid loop binds more tightly to those sites thus occluding
them and giving rise to a higher level of exopeptidase activity. That this pH
dependence of the conformation of the occluding loop also plays a major role in the pH
dependency of the propeptide binding is evidenced by the effect of partial deletion of
the loop- In Table 1 it can be seen that for this mutant, A(Cysl08-Cysl l9), the effect
of pH on the K; of the propeptide is essentially elirninated. Also fiom Table 1, it can be
seen that removal of the ionic interactions between residues Asp22 and His 1 10 through
the mutation of either or both residues to alanine also largely eliminates the pH
dependency of the inhibition of cathepsin B by its propeptide. Essentially, as with the
occluding loop deletion mutant, the high affuiity of the propeptide is maintained
through the pH range 4.0-6.0. As discussed above the salt bridge between residues
Asp22 and Hisl10 is impoaant for maintaining the loop in a closedrigid conformation.
These results again support the view that at low pH (4.0) as opposed to pH 6.0 the
occluding loop is able to compete more effectively with the propeptide for the binding
site on the enzyme. Clearly, any change to the overall charge of the Asp22-Hisl10 ion
pair c m be expected to infiuence the conformational stability of the occluding loop and
as a consequence the measured Ki of the propeptide. Thus the deprotonation of Hisl10
or the protonation of Asp22 can be expected to idluence propeptide binding. Despite
the fact that Hisl l O and H i s l l l of the occluding loop reside adjacent to one another,
there is significant selectivity observed for the Hisl 10 residue to regulating the pH
dependence of propeptide binding, This selectivity can be accounted for by their
difference in chernical environment since the HisllO residue anchors the occluding
loop to the rest of the enzyme. In order for the Asp22-His 1 10 interaction to iufluence
the propeptide binding it is necessary that the pK, of either of these two residues be
within or very close to the range 4.0-6.0. Given that the stabilization effect decreases at
higher pH it is necessary to conclude that it is the deprotonation of the EN of Hisl10
that is responsible for the observed pH dependency.
Since the pH dependency of propeptide binding in the range of pH 4.0-6.0
appears to be determined by the integrity of the occluding loop and since mutation of
either residue Asp22 or Kisl 10 (but not Hisll 1) to ahnine eliminates the pH
dependency (Table l), as discussed above, it is possible to speculate that it is the
protonation state of the Asp22-HisllO ion pair that directly determines the pH
dependency of Ki. In this case the relationship of measured Ki (Ki meas) to the intrinsic
Ki and pH is given by the equation:
Ki rneas = Ki (l+ H/K3) / (l+H/&)
where Ki = dissociation constant for the propeptide binding to the enzyme when His 1 10
is deprotonated and K3 and K4 are the pK,'s of HisllO in the fiee enzyme and enzyme-
propeptide complex, respectively. It is interesting to note (Table 1) that the effect of
the aspartic acid and histidine mutations to alanines are not additive, i.e. the double
mutation, AspZZAla/Hisl lOAla, has the same effect as the individual mutations. This
implies that the two single mutations have equivalent effects, i.e. the end result of the
two mutations is the same and does not reflect interactions of these side chains with
other groups. For example, in the open state Hisl IO interacts with residue Asn222. If
the effect of the Hisl lOAla mutation was due, in part, to a loss of the Hisl10-Asn222
interaction it is expected that the effect of the individual mutations Hisl lONa and
Asp22Ala should show a significant degree of additivity for the double mutant.
In the study of Fox et al. (102) it was reported that the pH dependency of Ki is
due to the influence of pH on kotr rather than k,,. Since both kOn and k,, for the mutants
Asp22Ala and Hisl lOAla are largely pH independent (Table 1) it follows that for the
wild-type enzyme the pH dependency of kOrr is a reflection of the pH dependency of the
aspartate-histidine interaction. How is this possible if, as discussed above and as
demonstrated by the crystal structure of procathepsin B, the aspartate-histidine
interaction is broken when the proregion and presumably the propeptide is bound? One
possible explanation is that for the enzyme-propeptide complex protonation of HisllO
serves to actively displace the propeptide, i.e. rather than the sequence, dissociation of
the propeptide followed by formation of the aspartate-histidine ion-pair, the ion-pair is
either partially or fûlly formed prior to the dissociation of the propeptide. Thus
displacement of the propeptide would take place as a two step process: an initial
displacement fiom the occluding loop binding site as a result of the closing of the
occluding loop and formation of the Asp22-Hisl lO ion-pair, followed by dissociation
of the propeptide fiom the active site cleft. Clearly, competing mechanisrns could
involve initial displacement fiom the active site followed by dissociation fkom the
occluding loop binding site and the direct one step simultaneous displacement f?om
both sites. The pH dependency results would suggest a significant role for the two step
process involving the earlier displacement from the occluding loop site. Conversely,
the lower pH-sensitivity of k,, implies that for the binding process, the binding of
propeptide to the open f o m of the enzyme (Asp22-His110 ion-pair broken)
predominates.
The rat and human cathepsin B propeptides (residues l p to 56p) share an overall
sequence homology of 71% (Figure 1). Furtherniore, fiagrnent analysis and alanine
scannïng studies revealed that the segment of polypeptide between the motif NTTW
(21p to 24p) which binds to the hydrophobic prosegment-binding loop (exosite) on the
enzyrne and the CGT (42p to 44p) motif which binds through the active site clefi of
cathepsin B is crucial for the binding afflnity of the fiee peptide to cathepsin B
(92,117). The homology between these polypeptides within the segment 2 1 p to 50p
increases to 86%. Therefore, it is not surprishg that both the rat and human
propeptides display similar inhibitory activify towards mature human cathepsin B. In
fact, the & values for the human propeptide did not show any significant differences
over those of the rat (Table 1).
Procathepsin B, in vivo, is synthesized as a glycoprotein consisting of two
solvent exposed sites of N-linked oligosaccharide substitution, one at A s d l p of the
prosegment which is located near the hydrophobic prosegment binding loop (exosite)
and a second at Asnll3 located on the occiuding Ioop of the enzyme. Due to the fact
that Asn2lp is not in direct contact with the sur£ace of the enzyme (89-91) it c m
reasonably be concluded that the absence of glycosylation on Asn2lp of the chemically
synthesized propeptides will not significantly affect their afEn.ity for the enzyrne. This
is supported by alanine scanning studies (92) which did not link any unique importance
of Asn2lp to propeptide binding. Conversely, the absence of glycosylation on Am113
of the occluding loop could affect the conformational stability of the occluding loop
and as a consequence the values of the inhibition constants obtained for the
propeptides. However, similar pH dependencies of autoprocessing of the glycosylated
and non-glycosylated proenzymes are observed (data not shown) implying that the
influence of glycosylation may be small.
There is an interesting correlation between the rate of autoprocessing of purified
fiill-Iength variants of procathepsin B (Figure 3 of this chapter) and the decrease in the
mature enzyme's affinity for the propeptides at pH 4.0 (Table 1). For example, the
f i t i e s of the propeptides towards mature wild-type and His I l 1 Ala cathepsin B are
similarly afZected by pH ; Le., there is a marked decrease in a f f i t y at low pH.
Similady, exposwe of the Hisll lAla procathepsin B to acidic pH conditions leads to a
rate of maturation comparable to wild-type procathepsin B ; Le., under the conditions
given for Figure 3 processing of these enzymes is complete within minutes rather than
days. Conversely, there is a maintenance of potent inhibition of the A(Cysl08-
Cysl l9), Asp22Ala, and Hisl 1 OAla forms of the enzyme by the propeptides at low pH
and the proenzymes of these three variants process much slower than either wild-type
or Hisl 1 1 Ala procathepsin B (Figure 3). The A(Cysl08-Cys 1 1 9) proenzyme requires 5
days incubation at pH 5.0 (105)' and both Asp22Ala and HisllOAla procathepsin B
require over 7 days under the same conditions. Full length procathepsin B is stable in
neutral pH environments when the occIuding loop favors an 'open' state and is prone to
autoprocess under acidic pH to form mature cathepsin B where the occluding loop
favors a 'closed' state. It is interesting to note, therefore, that the gating equilibrïum of
the occluding loop and autoprocessing of procathepsin B are both pH dependent. The
Asp22Ala and HisllOAla procathepsin B mutants shared much slower rates of
autoactivation at pH 5.0 compared to wild-type and HislllAla procathepsin B
(Figure 3). Once the occluding loop mutants have been matured using immobilized
pepsin, however, they are still able to cleave small synthetic substrates (Table 3).
Hence, the catalytic capability of these mutants has not been disr-upted but the
autoprocessing machinery has been greatly perturbed- It is possible, therefore, that the
closure of the occluding loop plays an important and unique step in the elimination of
the proregion in procathepsin B. Such a processing mechanism would explain the
observation that perturbation of the pH-dependent gating equilibrium of the occluding
Ioop has such a profound effect on the affinity of the free propeptide binding at low pH
(Tables 1 and 2) as well as the rates of procathepsin B autoprocessing (Figure 3).
The absence of an occluding loop and the presence of longer proregions in other
papain-like proenzymes (>90 residues versus 62 residues in procathepsin B) would
suggest that the pH-triggering mechanism of autoprocessing in procathepsin B is as
unique as the enzyme's dual exopeptidase and endopeptidase character. S equence
alignment reveals that Asp22 in cathepsin B is not entirely conserved throughout the
papain superfamily. It is repIaced by an asparagine residue in rnany other cysteine
proteases such as papain, cathepsin L and cathepsin H, and substituted by a tyrosine
residue in cathepsin S. ~urthermore, stnictural alignrnent indicates that the residue
located in this position is not in close proximity to the bound prosegment (85-91).
Therefore, other enzymes would not share the same pH dependence of prosegrnent
binding as that observed for cathepsin B. In addition, the negative charge of a highly
conserved aspartate residue in the proregion of propapain (Asp65p, papain nurnbering)
was shown to be important in maintaining the papain precursor in a latent f o m and to
participate in an electrostatic triggering mechanism of propapain processing (100).
Despite the difficulty in aligning the prosegments of propapain and that of procathepsin
B, the closest match to Asp65p in propapain is Asp34p in procathepsin B (cathepsin B
numbering, Figure 1). As noted earlier, replacement of Asp34p in the cathepsin B
propeptide to an asparagine did not alter the pH dependence of cathepsin B inhibition.
Curiously, structural alÏgnment reveals that the consewed Asp6Sp resides within an
area of propapain which is homologous to the position of the occluding loop in
procathepsin B (85-91). Furthemore, only the koff value of the propeptide in cathepsin
B was found to be pH dependent in the pH range 4.7-6.0 (102) and not that of k,, as is
the case with the cathepsin L propeptide (103). Interestingly, the modest increase in Ki
values of the propeptides for the A(Cys 108-Cys 1 19) mutant of cathepsin B as the pH is
dropped fiom 6.0 to 4.0 corresponds to a sirnilady modest decrease in the km value
only. This suggests that the presence of the occluding loop in wild-type cathepsin B is
responsible for the observed increase in the off-rate of the propeptide upon exposure to
low pH. It has already been established that maturation of procathepsin B proceeds by
an autoactivation mechanism (98,101,109) and that the main role of cathepsin B in its
natural environment, the acidified lysosome, is to act as an exopeptidase (105) which
relies on the 'closed' configuration of the occluding loop.
In sumrnary, this study confims a link between the pH dependencies of the
cathepsin B occluding loop conformation and the propeptide binding affinity, and
supports a direct correlation of this link with the in vitro rates of procathepsin B
autoprocessing.
ACKNOUZEDGMENT
The authors would Iîke to thank Jean Lefebvre for his technical assistance in the
synthesis and purification of the human propeptide of cathepsin B, and Dr. Robert
Ménard for many valuable discussions.
FIGURE 1
RAT RmAN
HDK V DM1 1 R 1 K V L E G RSRPSFHPLS DELVNYWKQ. NT'I'WQAGHNF YNVDMSYLKR LCGTFLGGPK PPQRVM 1P OP OP 3 0 ~ 4 OP 5 OP
Region 3 Region 2 Region 1
3
Prosequences of Rat and Human Cathepsin B showing the primary sequence of the proregions which were chemically synthesked (residues I p to 56p). Consensus of these tyo sequences is 86% within the 21p - 50p segment.
Figure 2: Superimposition of the confornations adopted by the occluding loop in
procathepsin B @lue) and mature cathepsin B (brown). Note the positions of both His
0 1 10 and His 1 1 l with respect to the main body of the enzyme and the orientations
adopteci - by the disulnde bridge- Linking Cys 1 O8 to Cys 1 19 (green). single ietter codes
used,
Figure 3: Monitoring procathepsin B (36 kDa) autoprocessing to mature cathepsin B (30
kDa) in 50 mM acetate buffer pH 5.0 with ImM DTT using SDS-PAGE (12% gels).
Gels A, B. C, D correspond to Wild-Type @FM), Hisll lAia (3pM), Hisl lOALa (7pM),
and Asp22Ala (6pM) procathepsin B, respectively. Initial proenyme concentrations are
indicated 'in brackets. Note the incubation times.
MIN
MIN
DAY
DAY -
Table 1: Equilibrium and kinetic data for the inhibition of human cathepsin B mutants* by both human and rat cathepsin B propeptides at pH 4.0 and 6.0
Hurnan 11
I
II
I
I
Rat
I
I
I
l 4
11
Wild type
&Cl 0861 19)
Asp22Aa
Hlsl1 OAla
Asp22AlaMlsll OAla 1 .4 f 0.2 2.1
Hls111Ala 86.0 f - Wild type 85.0 f 6.0' - &ci oscr i 9) 0g6 0.1 4-2
Asp22Ala 5.1 f 0.5 0.5
Hls11OAla 4.1 f 0.3 0.4
Asp22AleMlsl lOAla 1.8 0.2 1
Hlsl 1 1 Ala 87.0 f 8.0' - aPropeptidss comprise of reslduer l p56 of the correspondlng cathepsln B proreglon., he 4 values are g b n as the averages of 4 detemha~lons wlth the calculetecf standard devlatlons. CClasslcal lnhlbltlon obseived, Ys delermlned ;mm plots of 1 k vs [II (Dlxon, 1953).
Table 2: Rote of rat propeptlde aspartic acid residues In the Inhibition of rat cathepsin B by its propeptlde at pH 4.0 and 6.0'
' Assay conditions were: 0.1M phosphate (pH 6.0) or acetate (pH 4.0) buffer containing 1 mM EDTA, I mM DTT, 0.025% Brij-35 and 3% DMSO. ~ubstituted propeptide residues are underlined and in boldface. Classical inhibition observed, Ki's determined from plots of I lv vs [Il (Dixon, 1953)
Table 3: Activity of Caîhepsin B Towards ZPhe-Arg-MCA &/KM (M''s-')
Wild-Type 180,000 380,000 430,000 His111Ala 680,000 1,150,000 1,225,000 His 1 lOAia 190,000 320,000 400,000 Asp22Ala 550,000 900,000 1,200,000 A(Cys 108-Cys 1 19) 75,000 330,000 261,000
CONNECTTNG TEXT FOR CHAPTERS 1 AND 2
The importance of the occluding loop, particularly salt bridge formation
between HisllO and Asp22, in definhg the pH dependence of cathepsin B inhibition
by its propeptide and affecting the overall rate of procathepsin B processing was
discussed in Chapter 1. It may be reasonably concluded that the pH dependent mobility
of the occluding loop constitutes a unimolecular process within the zyrnogen. Not
addressed in Chapter 1, however, is whether this process contributes to an
intrarnolecular autoproteolytic event Perturbation of the closed form of the occluding
loop using site-directed mutagenesis; Le., destruction of the His 1 1 O/Asp22 salt bridge,
was shown to lead to variants of procathepsin B which could no longer autoprocess. In
Chapter 2, intemal cleavage sites within the prosegment of cathepsin B (as well within
the prosegment of cathepsin S) were identified using the inhibitory capacity of cystatin
C . These autoproteolytic reactions occur within an area ofthe prosegments which bind
through the substrate-binding clefts of these enzymes. Given the kinetic data and that
these intermediates of processing are observed at al1 concentrations of proenzyme (even
nanomolar concentrations), suggests that these reactions correspond to intramolecular
proteolytic events.
Furthemore, it has been postulated that the N-terminus of the mature segment
in propapain rnay play a role in a unimolecular autoproteolytic event (118). This
possibility has been investigated by disrupting the formation of a highly conserved salt
bridge between Asp6 and k g 8 through site-directed mutagenesis. In this study, the
ability of Arg8Ala propapain to autoprocess is compared to that of the wild-type
precursor.
CHAPTER 2
Contributions of Authors other than Omar Ouraishi:
Andrew C. Storer: Provided h d i n g and mentorship for this project.
Note : Both wild-type and Arg8Ala propapain (and the antibodies for Western blot
analysis) were provided by the laboratory of Dr. D. Y. Thomas (BRI/NRC).
Xdentification of Interna1 Autoproteolytic Cleavage Sites
Within the Prosegments of Recombinant Procathepsin B and
Procathepsin S
CONTRlBUTION OF A PLAUSIBLE UNIMOLECULAR AUTOPROTEOLYTIC EVENT FOR THE PROCESSING OF ZYMOGENS BELONGING TO THE PAPAIN FAMILY*
'NRCC Publication No. 00000. The research was fünded in part by the Government of
Canada's Network of Centres of Excellence Program supported by the MedicaI
Research Council of Canada and the NaturaI Sciences and Engineering Research
Council of Canada through PENCE Inc. (the Protein Engineering Network of Centres
of Exceilence).
Omar Quraishiz and Andrew C . Storer$§T
From the $Protein Engineering Network of Centres of Excellence and Department of
Biochemistry, McGilI University, 3655 Dnimmond Street, Montreal, Quebec, Canada
H3 G 1Y6, and the §Pharrnaceutical Biotechnology Sector, Biotechnology Research
Institute, National Research Council Canada, 6 100 Royalmount Avenue, Montreal,
Quebec, Canada H4P 2R2-
To whom correspondence should be addressed: Pharmaceutical BiotechnoIogy
Sector, Biotechnology Research Institute, National Research Council of Canada, G LOO
Royalmount Ave., Montreal, Quebec, Canada H4P 2R2. Tel.: 5 14-496-6256; Fax: 5 14-
496- 1629; E-mail: Andrew.Storer@nrc.ca.
RUNNING TITLE: Processing Intermediates in Procathepsins B and S
ABBrnrnTIONS
1 As indicated in the text, residue numbering relates to that of cathepsin B for
recombinant human cathepsin B, and to that of cathepsin L for recombinant
human cathepsin S. Residues in the proregion are identified with the suffix p.
2 The abbreviations used are: E-64, trans-epoxysuccinyl-L-leucyl-amido-(4-
guanidino)butane; 2-Phe-Arg-MCA, benzyloxycarbonyl-L-phenyIalany1-L-
arginine 4-methylcoumarinyl-7-amide; SDS-PAGE, sodium dodecyl sulfate-
polyacrylamide gel electrophoresis; DMSO, dimethyl sulfoxide; EDTA,
ethylene-diamine tetraacetic acid; DTT, dithiothreitol.
3 In the text, the words 'prosegment', 'proregion', 'prosequence', or 'prodomain'
refer to the polypeptide stretch located N-terminal to the mature enzyme in the
proenzyme, while the word 'propeptide' refers to the chemically synthesized
polypeptide corresponding to the proregion sequence but without the mature
enzyme.
4 In the text, the terms 'autoprocessing', 'autoactivation', or 'maturation' relate to
the ability of a zymogen to convert itself to a mature protein resulting in
cleavage at or near the pro/mature junction.
Abstract:
Autoprocessing, is a mechanism by which a latent higher molecular weight
zyrnogen rnay convert itseif to form active enzyme. Autoprocessing of proenzymes
belonging to the papain family of cysteine proteases, namely the identification of
intermediate events, bas been difficult to characterize. Processing near the pro/mature
junction, due either to the catalytic activity of the mature enzyme or to secondary
proteases, has been documented for this family of proenzymes. Furthermore, kinetic
studies are suggestive that a slow rnechanism occurs during autoactivation which is
independent of proenzy-me concentration. However, inspection of the recently
detemined X-ray crystal structures (85-91) does not support this evidence. This is due
prirnarily to the extensive distances between the catalytic thiol-irnidazolium ion-pair
and the putative site of proteolysis required to form mature protein. Furthermore, the
prosegments have been shown to bind through the substrate-binding clefts in a
direction opposite to that expected for natural substrates. Previous to this work, the
recent study of a non-homology knowledge based prediction of propapain activation
(118) proposed that an intrarnolecular proteolytic event rnay involve major
conformational rearrangernent at the N-terminus of the mature enzyme domain, We
report, using the cystatin C inhibitor and N-terminal sequencing, novel autoproteolytic
intermediates of processing for recombinant procathepsin B and procathepsin S in
vitro. The crystal structures (85-91) indicate that these reactions occur within a
segment of the proregion which binds through the substrate-binding clefts of the
enzymes, thus suggestive that these reactions are o c c e g as unimolecuIar processes.
Using site-directed mutagenesis, we also show that charged residues located at the N-
tenninus of the mature enzyme domain of propapain do not participate to the overall
pH-triggering mechanism of autoprocessing for this precursor. These results provide
the molecular basis for a plausible unimolecular step of processing among zymogens of
papain-like enzymes.
Prior to being shuttled to the mature lysosome, cysteine proteases of the papain
family are first synthesized as latent precursors of higher molecular weight. Zymogens
of papain-like enzymes are composed of polypeptide extensions of various lengths at
the N-terminus of the mature enzyme domain that act as potent and selective inhibitors
towards the cognate enzyme (102-104). The stability of the prosegment/enzyme
complex is believed to rely m a d y on electrostatic interactions since most precursors of
the papain superfarnily are susceptible to autoactivation upon their exposure to acidic
pH environments (97-101,109). The crystal structures of rat (89) and hurnan (9O,9 1)
procathepsin B, hurnan procathepsin L (85), procarkain (86), and human procathepsin
K (87,88) have been reported recently. The crystal structures reveal that each enzyme
is inhibited by a small segment of the proregion binding through the substrate-binding
cleft in a direction opposite to that expected for natural substrates. In order to protect
cells from unregulated digestion, this reverse configuration is believed to help ensure
proenzyme stability at neutral pH as the zyrnogen is passed fiom the endoplasmic
reticulum to its final destination; Le., the acidified lysosomal compartment of the cell.
Progress still remains to be made in elucidating the molecular mechanisms
involved in the conversion of many types of zymogens to their catalytically active
forms. For instance, autoproteolytic cleavage of the serine precursor prosubtilisin E
has been suggested to occur at the identical site (near the pro/mature junction) in either
an intemolecular or intramolecular manner and that the mechanism which
predominates is dependent mainly on the starting concentration of the proenzyme
(119,120). The molecular basis for the unimolecular mechanism of prosubtilisin E,
however, has yet to be elucidated. Similarly, precursors belonging to the papain family
of cysteine proteases have been proposed to undergo a non-exclusive unimolecular step
(97-100). It is not clear, however, whether this step could represent a process other
than an intramolecular cleavage reaction such as a rate-limiting activation process that
exposes the active-site and renders the proenzyme active. If a unimolecular cleavage
reaction is involved, it is also important to determine whether the cleavage site for the
intramolecular event is identical to that observed via intermolecular processing (Le. in
mm) as has been proposed for prosubtilisin E (1 I9,120), or if it occurs elsewhere in
the proregion generating a catalytically competent processing intermediate followed by
its m e r conversion to yield mature enzyme.
In general, zymogens of all families of proteolytic enzymes rnay undergo
maturation via interrnolecular or intramolecular non-exclusive pathways. Kinetic
studies of the conversion of propapain (97), procathepsin B (98) and procathepsin L
(99) to form mature enzyme have revealed both an interrnolecular and intrarnolecular
component to processing. Precursors belonging to the papain family may, therefore,
utillize different pathways to convert thernselves into a mature protein. For example,
these precursors may participate in direct bimolecular cleavage to form mature enzyme.
Furthemore, these proenzymes may utilize an intramolecuIar process to form
catalytically competent processing intemediates followed by M e r intermolecular
proteolysis to fonn mature enzyme. However, the expedient interrnolecular reactions
which contribute to proenzyme processing make it difficult to 'trap' and identiQ any
processing intermediates. For exarnple, intermediates corresponding to N-tennini
located elsewhere in the proregion may be unstable and difficult to detect under normal
experimental conditions. Significantly, kinetic studies (97-99) have also revealed that
the molecularity; Le., the concentration of proenzyme at which the rate of the
intermolecular events equals that of the unimolecular process, is in the range of only
1 o - ~ - 1 O-' M.
The occurrence of a unimolecular step of maturation, however, is inconsistent to
what is observed in the three-dimensional structures for this family of precursors (85-
91). For example, the crystal structure of procathepsin B reveals that the pro/mature
junction ; Le., the putative site for proteolytic processing, is approximately 28A fkom
the catdytic nucleophile. In addition, direct cornparison of the crystal structures of
procathepsin B (89-91) with that of mature cathepsin B (82) reveals no evidence for
major N-terminal rearrangement within the mature segment following maturation as is
found in zymogens belonging to other families (36,12 1,122). Independently, the
crystal structures have not provided any ches with regards to the possible existence of
a unimolecular step. Despite this, a non-homology knowledge-based strategy predicted
that a plausible unimolecular proteolytic step in propapain processing could involve the
adjustment of a single p-turn that rearranges the &st 12 residues of the enzyme domain
and allows the mature N-terminus to reach the active-site in a cleavable direction (1 18).
Within the mature N-terminus are Asp6 and Arg8 which are highly conserved residues
arnong cysteine proteases of the papain farnily (108). Interestingly, al1 X-ray crystal
structures of papain-like enzymes (pro- and mature) reported to date reveal that the
side-chahs of Asp6 and Arg8 contribute to the formation of a saft bridge (82,83,85-9 1).
Here, we atternpt to identie novel intermediates of processing for the
precursors of cathepsin B and cathepsin S in vitro. This has been achieved by
monitoring using SDS-PAGE the processing of purified procathepsin B and
procathepsin S in the presence of the endogenous inhibitor, cystatin C. Cystatin C has
been shown to be a substrate-binding cleft-directed protein inhibitor of papain-like
enzymes with Ki values in the sub-picomolar range (96,123). Since the formation of a
tight-binding complex with cystatin C requires that the substrate-binding cleft of
papain-like enzymes be accessible ; i.e., fÏee fiom natural substrates or the prodomain,
it rnay be reasonably assumed that the affkity of cystatin C for the mature enzyme
would be superior to that for either the proenzyme or any intermediates generated
during autoproteolysis ; e . hierarchy of cystatin C affinity for mature
enzyme>intemediates>proenzyme. In excess, there fore, cystatin C rnay provide the
desired effect of inhibiting the rate of the intermolecular proteolytic cascade caused
r n d y by the activity of mature enzyme, and thereby favoring intrarnolecular events
which would otherwise go undetected. Furthemore, we investigate any role the N-
terminus of the mature enzyme domain may play in processing. This has been
achieved by perforrning site-directed mutagenesis of Arg8 to an alanine residue in
propapain ; i.e., destruction of the Asp6/Arg8 salt bridge, and monitoring the overall
effect of this mutation on the ability of propapain to autoactivate.
EXPERIMENTAL PROCEDURES
Matenals - Human wild-type and GIyl ZGIu cystatin C were a generous gift from Dr.
Irena Ekiel (l3iotechnology Research Institute). Recombinant human wild-type
procathepsin B was expressed and purified as described previously (106). The vector
(pPIC9) and P. pastoris strain GS 1 1 5 were purchased kom Invitrogen Corp. (San
Diego, CA). The substrate benzyloxycarbonyl-L-p henylalanyl-L-arginine 4-
methylcoumarinyl-7-amide hydrochloride (Z-Phe-Arg-MCA) and the covalent inhibitor
E-64 (1 -[[(L-h-ans-epoxysuccinyl)-L-Zeucyl]amino]-4-guanidino)butane) were
purchased fkom IAF Biochem International Inc. (Laval, Canada).
Eqression of Procathepsin B and Procathepsin S - A cDNA construcf consisting of
human wild-type procathepsin B or procathepsin S as a fusion with the preproregion of
yeast a-factor, was digested with XhoI and NotI and the proenzyme fragment was
subcloned into the pPIC9 vector (Invitrogen Inc., San Diego, California). For
integration into the Pichia genome, the pPIC9 based constructs were linearized by
cleavage with BgZII and purified. The Pichia pastoris host strain GS 1 15 (Invitrogen
Inc.) was then transformed with the linearized constructs by electroporation. Positive
transformants were grown for 2 days in medium containhg glycerol as the carbon
source followed by incubation in the presence of methanol for a further 3 days to
induce expression of recombinant protein. The consensus sequence for oligosaccharide
substitution located in the occluding loop of mature cathepsin B had been removed by
the substitution, Serl lSAla. Al1 other sites for oligosaccharide substitution within
procathepsin B and procathepsin S were lefi unaltered. Protein secreted into the culture
supernatants was analyzed by SDS-polyacrylarnide gel electrophoresis (12% gels).
Expression of Wild-Type and R8A Propapain - Propapain was produced as
described previously (97,100). Briefly, the Saccharomyces cerevisiae strain B J3 50 1
was transformed with the expression vector derived fkom pVTIOO-U in which the
propapain gene is under the control of the a-factor promoter. Yeast cells were k s t
grown under selective conditions to ensure plasrnid maintenance and then transferred
into a rich medium. The ceils were lysed using a French Press where the soluble
fraction of the lysate included propapain. Complete processing in cis was achieved by
incubating soluble cellular extracts with 50 mM sodium acetate (pH 3.8), 20 mM
cysteine at 65OC for 30 min. Sarnples were analyzed by Western blot following
separation of the proteins using SDS-PAGE.
PuriJcntion of Procathepsin B and Procathepsin S - The proenzymes were purified
fiom the culture supernatant using a hydrophobic resin under non-acidic conditions.
The culture supernatant (250 ml) was concentrated to 50 ml using an Arnicon stirred-
ce11 (YM-10 membrane). During concentration, the supernatant was exchanged to 50
mM Tris (pH 7-4) containhg 1.6 M (NH&S04. Concentrafed recombinant proenzyme
was then pwified on an FPLC system (Pharmacia) using a butyl-sepharose fast fiow
column. Proenzyme fractions eluted from the column b y applying a linear gradient of
decreasing ammonium sulfate concentration. Giycosylated procathepsin B and
procathepsin S eluted at 0.6-0.8 M and 0.3-0.5 M (NH&S04, respectively, and samples
were stored at 4OC.
In Vitro Processing of Procathepsin B and Procatheps Zn S - Puri fied procathepsin B
(20 PM) and procathepsin S (4 pMJ samples were dialysed against 50 mM sodium
acetate (pH 5.0), 1 mM dithiothreitol at 4°C overnight in the presence (or absence) of
5-fold excess human wild-type cystatin C. Each sample was then treated with excess
E-64 followed by SDS-PAGE (12% gels).
N-Teminal Idenfz$cation of Protein Ban& - After electrophoresis, protein bands
were blotted onto hydrophobie polyvinylidene difluoride membranes using the method
as described previously (124). The membranes were then stained with Coomassie
Brilliant Blue R250 (Bio-Rad Laboratories) and each protein band of interest was
subjected to a minimum of five cycles of automated solid-phase Edman degradation.
Fluorogenic Assay for Monitoring Proenzyme Processing - Processing of human
wild-type procathepsin B and procathepsin S was followed in a continuous manner by
carrying out the reaction in a 3-ml quartz cuvette in the presence of the substrate Z-Phe-
Arg-MCA (1OpM) and measuring fluorescence as a h c t i o n of time. The conversion
of procathepsins B and S to active enzyme Ieads to hydrolysis of the substrate, and
fluorescence of the MCA product was monitored using excitation and emission
wavelengths of 380 and 440 nm, respectively. Processing was initiated by lowering the
pH firom 7.4 (pH of the stock solution of procathepsins B and S) to 5.0 (pH of the
assay). Reactions were carried out at 25OC in the presence of 50 rnM sodium acetate
buffer, 0.2 M NaCl, 2 rnM EDTA, 2 rnM dithiothreitol, and 3% DMSO. The reaction
mixture was stirred continuously in the cuvette during the reaction. The product versus
time curves were fitted to the following equation (1 1 1 - 1 15),
@?] = VPE f - ((VE - VPE) (1 - exp(-kobd>)) 1 kobs
where P is the MCA product formed, v p ~ represents the initial rate of product release
(which shouId refiect activity of the proenzyme, if any), v~ corresponds to the rate for
mature cathepsin B or cathepsin S, and kbs is a first order rate constant.
RESUltTS AND DISCUSSION
Exposing proenzymes of the papain family such as procathepsin B and
procathepsin S to acidic pH environments Ieads to their rapid autocatalytic conversion
to form mature protein. Exceptions to this general phenornenon includes the precursor
of papaya proteinase N which is incapable of autoprocessing (125) despite its
demonstrated enzymatic activity (1 26). This incapacity has been attributed to the
resûicted specificity of this enzyme for substrates containing glycine in the Pi position.
Furthemore, we have recently determined that the inability of procathepsin H to 'self
activate is due to the formation of a disulfide bond ((127) ; Chapter 3 of this thesis),
featured only in the precursors of cathepsin H (mamrnalian), aleurain (barley), and
orizain y (rice seeds), which links the prosegment to the catalytic domain via residues
Cys82p and Cys2 14 (cathepsin L numbering) (1 28). The occurrence of a unimolecular
processing step for precursors of papain-like enzymes has been postulated based on
kinetic data (97-99) where a rate of autocatalysis was extrapolated for proenzyme
concentrations approaching zero. The nature of this step, however, has remained
elusive. Monitoring the autocatalytic processing of purified precursors belonging to the
papain farnily (no mature enzyme present) using SDS-PAGE typically reveals protein
bands which correspond only to the full-length precursor of higher molecular weight
and/or to the mature enzyme of lower molecular weight ; Le., processing intermediates
are usually not observed. In the study of Vernet et al. (100), however, an intermediate
protein band of 30 kDa was observed for propapain using Western blots analysis, yet
insufficient quantities led to the inability to identifjr this species using Edman
degradation.
Monitoring the autoprocessing of puified recombinant procathepsin B and
procathepsin S in vitro in the presence of excess amounts of the protein inhibitor,
cystatin C , effectively increases the population of intermediate species which would
otherwise be difficutt to identiS using SDS-PAGE. Cystatin C is an endogenous active
site-directed inhibitor of papain-like enzymes. Three exposed loops on cystatin C, with
Glyll playing a key role, are necessary for the inhibitory activity of this protein (129)
as is its unobstructed access to the substrate-binding cleR of the enzyme (105).
Therefore, the prosequence of any precursor of papain-like enzymes must be removed
fkom the active site clefl in order for this tripartite interaction to take place. The
presence of excess arnounts of cystatin C introduces a cornpetition (between the
prodomains and cystatin C) for the substrate-binding clef? of the enzyme which results
in significantly slower rates of activation due rnainly to its eEect on the intennoIecular
proteolytic cascade. Since stable complex formation between cystatin C and papain-
like enzymes requires that access to the substrate-binding cleft of the enzyme be
unobstructed, inhibition by cystatin C of the different isoforms of the enzyme is
expected to be of the order : mature enzyme>intemediate>proenzyme. Therefore,
intermolecular reactions will be more strongly inhibited than intramolecular processes.
As s h o w in Figures 1 and 2 of Chapter 2, the presence of cystatin C successfully
retards proenzyme processing and allows for the time-dependent accmuIation of an
intermediate protein band at 32 kDa for cathepsùi B and 29 kDa for cathepsin S.
Identzpcation of Intemediate Cleavage Sites in Procathepsin B using the Cystatin C
Assay - With the addition of cystatin C to the reaction mixture, an intermediate species
of cathepsin B is observable even at nanomolar proenzyme concentrations as
determined by AgN03-stained SDS-PAGE (Figure 1) as weIl as in micromolar
quantities (PVDF membranes ; Coomassie-Blue staining ; Figure 2A). Direct N-
terminal sequencing of the intermediate band of cathepsin B processing indicates a
mixture of species following cleavage at ~ y s 4 2 ~ ? ~ 4 3 ~ (70%) and A.rg40p'f'~eu4 1
(30%) (Figure 3, cathepsin B prosegment numbering). FolIowing several weeks of
incubation, both full-length procathepsin B and the processing intermediate disappear
and only the protein band corresponding to mature cathepsin B is observed. Therefore,
it may be concluded that these are true intermediates of processing and not dead-end
(side product) reactions. The crystal structure of procathepsin B (89-9 1) indicates that
this segment of the cathepsin B proregion binds through the substrate-binding cleft of
the enzyme in the reverse mode and that the carbonyl carbon of Cys42p is in closest
proximity to the catalytic residue, Cys29 (Figure 4). Based on structural analysis of
this region of the cathepsin B precursor, the carbonyl carbon of Cys42p is located
approximately 4A fiom the catalytic nucleophile and the potential bond angle between
the catalytic nucleophile and the carbonyl oxygen of Cys42p is 127 degrees ; i.e.,
conducive to fonning a tetrahedral intermediate (Figure 4). For the ~ r g 4 0 ~ f ~ e u 4 l ~
cleavage site, the carbonyl carbon of Arg40p is approximately 6.7 A fiom Cys29 and
the potential bond angle between the carbonyl oxygen of Arg40p and the catalytic
nucleophile is 36.6 degrees. Hence, the ability of the carbonyl carbon of Arg40p to
reach the catalytic center of the enzyme for hydrolysis requires significant
conformational mobility of the small a-helix composed of residues Asp34p-tLeu4lp
which interact with the occluding loop crevice and the primed subsites of the substrate-
binding cleft of cathepsin B.
Given the proximity of these carbonyl carbons (that of Cys42p and Arg40p) to
the catdytic center, it is tempting to speculate that these reactions occur in an
intramolecular manner. As discussed previously, kinetic studies are suggestive of a
unimolecular step among members of the papah family of precursors whose
molecularïty is unusually low ( L O - ~ - ~ O ~ M) yet still rapid when compared to
uncatalyzed (spontaneous) peptide hydrolysis (10-''M& HOW is this possible if, many
(but not all) intramolecular enzymatic reactions have molecularities in the range 102-
104 M due to an increased effective concentration of reacting species and favorable
entropic effects? This may be accounted for by the reverse binding mode adopted by
the prodomain in its interaction with the substrate-binding cleft of the enzyme. As a
consequence, the formation of a tetrahedral intemediate at the carbonyl carbon of
Cys42p or Arg40p would not be stabilized by the oxyanion hole formed by Gln23.
This structural incompatibility has, therefore, lead to the suggestion that it would not be
possible for the enzyme to perform such reactions (91). Previous work with oxyanion
hole mutants of papain (130) and cathepsin B (131), however, indicate that the
specialized glutamine is not an absolute requirement to hydrolyse small synthetic
substrates but rather is a feature which may improve the catalytic efficiency of these
enzymes by only 10 to 100-fold depending on the substrate under study. Alternatively,
the sIow rate of these unimolecular reactions may also be due to the fact that the
reverse complementarity between the bound proregion and the enzyme's substrate-
binding cleft causes the distance between the 6N of the catalytic His199 and the
primary NH (leaving) group of Gly43p to be larger than would be the case for natural
substrates (132). Hence, the donation of a proton fiom the 6N of Hisl99 to the leaving
amide group of Gly43p may be less efficient to that observed for natural substrates, and
thus, resulting in a reversible nucleophilic reaction which has difficulty going to
completion. Since the catalytic thiol among cysteine proteases is more nucleophilic
and simultaneously constitutes a better leaving group than the catalytic hydroxyl found
among serine proteases, it has been postulated that proton transfers effectuated by the
SN of catalytic histidines found among cysteine proteases would need to be more
efficient than those found among serine proteases (133); i.e., the catalytic histidine
found in cysteine proteases must compensate for the lower pK, of the catalytic thiol
group. This compensation may involve the rotation of the catalytic imidazole side-
chain about its CP-Cy bond as has been observed in the crystal structure of cathepsin B
in complex with a pyridyl disulfide inhibitor (134). In this complex, fis199 was
shown to be rotated 120 degrees relative to its position found in other crystal structures
of cathepsin B (82,89-91). In surnmary, it is advantageous for eukaryotic ceIls to
require that the conversion of these zymogens to be under strict regulatory control,
namely the pH environment in which they are found. Typically, precursors of papain-
like enzymes are stable at neutral pH and are prone to autoprocess more efficiently
under acidic pH conditions (97-101,109), as is the case in the acidified lysosomal
cornpartment of the cell.
The ability to detect cleavage at the carbonyl carbon of Arg40p indicates a
significant degree of conformational mobility that exists for the proregion within the
prosegrnent/substrate-binding cleft interface. This mobility rnay be accounted for by
the pH-dependent stability of the enzyme's occluding loop which consequently defines
the pH-dependence of propeptide binding as well as the overall rate of procathepsin B
processing (106). Cornpetition between the prosegment and the occluding loop for the
surface of the enzyme termed the 'occluding loop crevice' was shown to be regulated
by the formation of a salt bridge between HisllO of the ocduding loop and Asp22
located on the main body of the enzyme. Site-directed mutagenesis of either one of
Hisl 10 or Asp22 to an alanine residue produces a variant of procathepsin B which is
stable and incapable of autoactivation. Remaining elusive (fiorn the data presented in
Chapter l), however, was whether these mutations caused the perturbation of a
unimolecular event involving proteolysis of the prosegment. As procathepsin B is
exposed to acidic pH conditions, it is possible that salt bridge formation between
HisllO and Asp22 promotes cornpetition between the occluding doop and the N-
terminal cap of the proregion. From this cornpetition, it follows that the rernaining C-
terminal residues of the proregion; i-e., prosegrnent residues which stretch fiom the
substrate-binding cleft to the pro/mature junction, would have reduced affinity for the
surface of the enzyme and increased conformational mobility. In agreement with this
proposa1 is the consistent lack of secondary or tertiary structure found within the C-
terminal end of papain-like prosegments when bound to the cognate enzyme (85-91).
Furthemore, truncated propep tides composed only of these C-terminal residues display
weaker affinity for the enzyme than the full-length propeptide (92,117). Additional
evidence for mobility within the prosegment has been shown for the propeptide of
cathepsin L which loses most of its tertiary structure yet almost none of its secondary
structure at low pH (135). It is believed the high B-factors corresponding to the C-
temiinal end of papain-like prosegments facilitates the autocatalytic conversion of these
zyrnogens upon their exposwe to acidic pH. That the conformational mobility within
the C-terminal end of papain-like prosegments is important for autoprocessing is
evidenced by the results obtained for procathepsin H (127); presented in Chapter 3 of
this thesis).
Identification of Intemediate Cleavage Sites in Procathepsin S using fie Cystatin C
Assay - In the case of procathepsin S , the presence of cystatin C is not stnctly required
to observe an intermediate processing band on SDS-PAGE which is approximately 2
kDa heavier than the mature enzyme (data not shown). In order to ensure the
accumulation of sufficient quantities of this species for N-terminal identification,
cystatin C was added to procathepsin S under processing conditions to inhibit
bimolecular reactions as has been discussed previously for procathepsin B (Figure 2B).
Based on their migration on SDS-PAGE, it is assumed that the intermediate species
formed in the presence of cystatin C are identical to those formed in the absence of the
inhibitor. This band corresponds to a mixture of species containing N-termini of
? ~ e r 7 7 ~ - ~ e u 7 8 ~ - ~ r ~ 7 9 ~ (50%) and ? ~ e r 7 3 ~ - ~ e u 7 4 p - ~ e t 7 5 ~ (50%) (Figure 3 ;
cathepsin L prosegrnent numbenng). Despite the low primary arnino-acid sequence
hornology between the prosequences found in cathepsin B and cathepsin S and that the
three-dimensional structure of procathepsin S has yet to be detennined, the fold of the
prosegments and the mechanism by which they inhibit enzyrnatic activity is comrnon to
al1 precursors of the papain family reported to date (85-91). The structural homology
between the prosegments shown in Figure 3 reveals that cleavage at the carbonyl
carbon of residues in position 42p (procathepsin B) and 76p (procathepsin S) are
perfectly aiigned. This conservation is observed despite the great di fference in length
for these two prodomains ; i.e., 62 residues for procathepsin B and >90 residues for
procathepsin S. It follows, therefore, that Ser76p is predicted to bind through the
substrate-binding cleft of cathepsin S in the reverse binding mode ; Le., as is the case
for Cys42p in procathepsin B, and that its carbonyl carbon is located closest to the
catalytic residue, Cys25.
It is interesting to note that cathepsin S prefers to cleave at interna1 sites within
its prosegment where serine residues are l~cated in the Si' position, namely at the
putative ~ ~ s 9 1 ~ T ~ e r 9 2 ~ site near the pro/mature junction to form mature cathepsin S
as well as at the ~ e t 7 2 ~ T ~ e r 7 3 ~ and ~ e r 7 6 ~ ? ~ e r 7 7 ~ sites identified using the cystatin
C assay (Figure 3). Located within the prosequence of cathepsin S is found two
adjacent serine residues, Ser76p and Ser77p. Curiously, proteolysis is only observed at
the carbonyl carbon of Ser76p and not at the carbonyl carbon of Met75p. This is
indicative that cleavage at the carbonyl carbon of Ser76p may be selective. Cleavage at
the carbonyl carbon of Met72p indicates a significant arnount of conformational
mobility among residues of the proregion which bind through the substrate-binding
cleft of cathepsin S ; Le., as has been observed for procathepsin B.
The conversion of procathepsin H to its mature form (discussed in Chapter 3)
has been proposed to involve cleavage at the carbonyl carbon of Ser77p (83) (Figure 3 ;
cathepsin L prosegment numbering) which is located adjacent to the cleavage sites
identified in this study for procathepsin B and procathepsin S using the cystatin C
assay. This cleavage site has been proposed to be a prelude to the formation of an
octapeptide of prosegrnent residues (Glu78p+Thr85p), called the 'mini-chain', which
rernains attached to mature cathepsin H via a disulfide bond (136). Similarly to
proaleurain (137), we have recently determined that procathepsin H is an unusual
zymogen of the cathepsin L-subfamily which is incapable of autoactivation (127;
Chapter 3 of this thesis). This incornpatibility has been attributed to the pre-formation
of a disulfide bridge linking the prosegment to the enzyme domain within the
precursor. This additional covalent attachment is believed to restnct the
confornational mobility at the C-terminal end of the cathepsin H prosegment. It
follows, therefore, that procathepsin H was found to be unable of performing cleavage
at the carbonyl carbon of Ser77p (or Trp76p) in an intra- or intermolecuIar manner,
thus requiring the action of secondary proteases (127; Chapter 3 of this thesis).
Continuous Monitoring of WiZd-type Procathepsin B and Procathepsin S
Autocatalytic Processing - A continuous assay based on the hydrolysis of the substrate
2-Phe-Arg-MCA by the active enzyme generated in the autocatalytic process was used,
The rate of substrate hydrolysis increases with tirne due to time-dependent release of
active enzyme fiom the precursor until a constant rate is obtained that corresponds to
the activity of fuily processed enzyme. The curves c m be fitted to a mode1 which
assumes a first order increase in rate fkom an initial rate vf i corresponding to the
activity of the precursors (if any), to a final rate VE, corresponding to the activity of
h l ly processed enzyme. Based on the results of non-linear regression analysis, no
significant activity of the precursors against the 2-Phe-Arg-MCA substrate could be
detected and the first-order rate for autocatalytic processing, kob,, increases linearly
with proenzyme concentration (Figure 5). The direct link between the rates of
autoprocessing and precursor concentration confirms the occurrence of a bimolecular
reaction ; i.e., intermolecular processing of proenzyme by fulfy or partially processed
(active) enzyme. In support of the unirnolecular autoproteolytic reactions discovered
using the cystatin C assay (discussed above), the extrapolated rate constants as the
concentration of either procathepsin B or procathepsin S tend toward zero were not
null. This kinetic data suggests the occurrence of an intrarnolecular event; Le.,
independent of the concentration of proenzyme, in the processing of these precursors.
Role of the N-teminus of the Mature Segment in the Autocatalyhc Processing of
Precursors BeZonging to the Papain Family - Previous to this work, a non-hornology
knowledge based prediction of propapain activation proposed that an intrarnolecular
event may involve the N-terminus of the mature enzyme domain moving towards the
active site cleft and thus facilitating the release of the prosegment (1 18). Using the
structure of mature papain as a template ; Le., the effect of the prosegment was not
considered, it was proposed that the adjustrnent of a single p-turn extends the first 12
residues at the N-terminus of the enzyme and is capable of allowing the prohature
junction to reach the active site in the substrate-binding mode. For this rearrangement
to be made possible, it would be expected that the salt bridge formed between Asp6 and
Arg8 found in al1 papain-like enzymes reported to date (82,83,85-91) ; i-e., residues
which contribute to the p-turn, wodd also contribute to the overall pH-triggering
mechanism of propapain processing. In order to test this hypothesis, site-directed
mutagenesis aimed at removing this salt bridge was performed. In this study, we show
that Arg8Ala propapain remains competent to autoactivate to form mature protein
(Figure 6). These results collaborate with the X-ray crystal structures of papain-like
enzymes (pro- and mature enzymes) which demonstrate hi& resolution arnong residue
side-chains located at the N-terminus of the mature segment, thus corresponding to a
region of the molecule which is conformationally constrained (low B-factors).
Furthermore, the N-terminus of the mature segment within the precursors (85-91) is
essentially in the identical conformation to that found in the crystal structures of mature
enzyme (82,83), thus suggesting that no major N-terminal rearrangernent is observed
during precursor activation. In addition, the overall assurnption that the putative site of
proteolysis to f o m mature protein near the prohature junction is the only possible
cleavage site ; i.e., as has been proposed for prosubtilisin E (119,120), remains
speculative as an unidentified processing intermediate was observed for propapain at 30
kDa (100). For exarnple, autoprocessing of procathepsin B predominantly yields
mature cathepsin B composed of six residues derived fiom its prosegment
(Phe57p+Lys62p) rather than starting at Leu1 (98,lO 1,109) despite the presence of the
salt bridge formed by Asp6 and Arg8 within this precursor (89-91). In summary, it is
not clear how the N-terminus of the mature segment within this family of precursors
could ; (a) undergo major rearrangernent f?om a low energy state consisting of a
conserved salt bridge fonned by Asp6 and k g 8 ; (b) to a stretched conformation
consisting of little secondary structure with the concomitant destruction of the
AspG/Arg8 salt bridge; (c) followed by its eventual r e m to the position shown in the
X-ray crystal structures (85-91 ). High-resolution nuclear magnetic resonance studies
may provide M e r insight into the possible dynarnical role that the N-terminus of the
enzyme plays in pH-triggered precursor activation.
Nature of the Steps Irzvolved in the Autocat~lytic Processing of Procathepsin B and
Procathepsin S - Previous studies have established that the reactivity of the catalytic
cysteine residue found within the precursors of papain-like enzymes is responsible for
the maturation of this family of zymogens (97- 1 0 1,109). Hence, auto activation of
zymogens belonging to the papain family require that the precursors be composed of a
preformed and functional catalytic center and substrate-binding clef?. In this study, we
have described the identification of novel processing intermediates for procathepsin B
and procathepsin S. The intermediates identified for cathepsin B are observable only in
the presence of cystatin C at either very low starting concentrations of proenzyme (low
nM) as determïned by Am3-stained SDS-PAGE (Figure 1) or at higher
concentrations of the precursor (pMJ (Figure 2). In the case of cathepsin S, processing
intermediates are observable on SDS-PAGE in the absence of cystatin C (data not
shown). Due to their low quantities, however, their identification is facilitated only by
the addition of cystatin C to the reaction, This addition has the desired effect of
significantIy increasing the population (half-life) of the cathepsin S intemediates. That
these novel cleavage products are observed at al1 starting concentrations of proenzyme,
including very low concentrations, suggests that these reactions are occuring as
unimolecular processes and that they may be important. The crystal structures of
precursors of the papain family (85-9 1) demonstrate that these cleavage reactions are
taking place within a segment of the proregion which binds through the substrate-
binding clefis of the enzyme in the reverse binding mode. Following the completion of
these proteolytic steps, over 20 residues derived from the C-terminal end of the
prosegment remain covalently attached to the mature segment at the pro/matue
junction. Intuitively, these intermediate species would be as catalytically competent as
the mature enzyme since the C-terminal end of papain-like prosegments possess low
inhibitory activity as compared to that of the full-length prodomain (92,lO3,ll7).
Given the kinetic and structural data presented here, it is tempting to specdate
that these reactions are occurring as slow unùnolecular steps which may be necessary
for tnggering the intermolecdar proteolytic cascade. It is also possible, however, that
these intermediates merely represent birnolecular side-reactions which are more easily
isolated using the cystatin C inhibitor. The incubation of inactive procathepsin B
(Cys29Ser) with wild-type mature cathepsin B, however, leads onIy to the formation of
mature enzyme as proteolysis elsewhere in the cathepsin B proregion was not observed
(98,lOlJOg). A similar experiment for the inactive procathepsin S (Cys25Ser) has yet
to be performed. It is interesting to note that these processing intermediates in
cathepsin B (Figure 1) and cathepsin S (data not shown) are also observed at nanomolar
concentrations of the precursors. As has been suggested for propapain (97,100), the
conversion of procathepsin B and procathepsin S appears to involve mutualIy non-
exclusive processes. The first step may involve the slow intrarnolecular cleavage
reactions presented here, followed by the rapid intermolecuIar proteolytic cascade
performed by the catalytic activity of mature or semi-mature species whose quantities
accumulate with tirne.
The cystatin C assay described above was attempted with other precursors such
as propapain, procathepsin K and procathepsin L. Due to the supenor affinity of
cystatin C (10~'~-10- '~ M) for these enzymes as compared to their prosegments (IO-'
M), the capacity of these precursors to autoprocess was essentially eliminated even
after prolonged periods (data not shown). Following approxirnately two weeks of
incubation, cystatin C was gradually degraded; Le., due either to its own instability or
to proteolysis by catalytic arnounts of mature enzyme, followed by the rapid maturation
of the proenzymes. How is it possible that the inhibition of procathepsin B activation
by cystatin C produces a processing intermediate that is not observed for other
precursors using SDS-PAGE? One possible explanation is the decreased affiniiy of
cystatin C for the cathepsin B substrate-binding cleft due to the obstruction caused by
this enzyme's occluduig loop (105). Therefore, the presence of the occluding loop in
cathepsin B decreases the affinity of cystatin C for this enzyme to that found for the
full-length propeptide ; Le., low nanomolar versus picomolar range (1 05). In an
attempt to decrease the disparity between the afEnïties of cystatin C and the
prodomains of cathepsin K and cathepsin L for these enzymes, a variant of human
cystatin C carrying the mutation, Glyl lGlu, was also used (96,129). It was determined
that Gly 1 l Glu cystatin C inhibited the activation of procathepsin K and procathepsin L
as effectively as the wild-type inhibitor and did not lead to the accumulation of
processing intermediates (data not shown).
The pH dependence of propeptide binding which has been observed for several
enymes belonging to the papain family (102-104) suggests that charged residues
located within the prosequences such as the highly conserved Asp65p and Glu70p
found within prosegments of the cathepsin L-subfamily (85-88), and not those found on
the enzyme, are responsible for the pH-triggering activation of these precursors.
However, we have recently determined that autoactivation in procathepsin B is as
unique as its three-dimensional structure (106). The prosequence of cathepsin B is over
3 0 residues shorter than those of cathepsin L-like prosegments and the enzyme
possesses a unique insertion of 20 residues, tenned the occluding loop, which
contributes to the p h e d subsites of the substrate-binding cleft. Tt was determined that
the formation of a salt bridge within cathepsin B, and not its prosequence, stabilizes the
occluding loop to the surface of the enzyme which consequently affects the pH
dependence of propeptide binding as well as the overall rate of procathepsin B
processing.
It has been proposed that deregulated secretion of papain-like enzymes to the
extracellular matrix may serve as a catalyst for propagating turnour metastasis (138) as
well as rheumatoid arthritis (7). Since the prodomains are known to act as
intrarnolecular chaperones (84) and serve to stabilize the enzyme at neutral pH
conditions, it may be reasonably concluded that these enzymes are targetted to the
extracellular maûix in their precursor form. Despite the neutral pH environment
generally associated to the extracellular matrix, it has been postulated that
rnicroenvironments of acidic pH, cailed resorption pits, may be the Iocation where low
concentrations of zymogens of papain-like enzymes are converted to their mature
forms. The discovery of a unirnoIecular mechanism of processing for this family of
precursors may help to explain how lysosomal enzymes (even at low concentrations)
are implicated in a number of degradative and invasive pathological conditions
extracellularly (7,69-7 1,138). Although inhibitors with sufficient potency are available
for this class of enzymes, they often lack the required selectivity needed for therapeutic
applications. In addition to the traditional approach of designing substrate-binding
cleft-directed inhibitors, an improved understanding into the mo lecuIar basis of
autoprocessing for zymogens of the papain family rnay lead to novel therapeutic
strategies where the conversion of proenzyme is intervened. This would have
significant consequences given that the prosegments of papain-like c ysteine pro teases
are intrinsic appendices which are both potent and selective inhibitors of the enzyme
fkom which they originate.
ACKNOWLEDGMENTS
We thank France Dumas for the N-terminal sequence determinations and Dr. Irena
Ekiel for her generous gift of human wild-type and GlyllGlu cystatin C. We also
thank Dr. J. Sivaraman for his assistance in preparing Figure 4. We also thank Daniel
Tessier, Dr. Thierry Vernet and Dr. Dave Thomas for providing the constructs of wild-
type and Arg8Ala propapain. We are also grateful to Dr. John S. Mort for many
valuable discussions,
Figure 1 : SDS-PAGE stained with AgN03. Procathepsin B (37kDa) at low
concentrations (2 nM) exposed to 50 mM acetate buffer (pH 5-0) and ImM DTT for 12
hours in the presence of 10 n M (lane 1) and 50 n M (Iane 2) hurnan wild-type cystatin C.
In lane 3 is s h o w the autocatalytic processing of 2 p M procathepsin B in the absence of
cystatin C (heavy band at 30 kDa corresponding to W y processed protein). Note that
the proportion of processing intermediate (32 kDa) to mature cathepsin B (30 kDa) is
improved with increasing concentration of cystatin C.
Figure 2 : Polyvinylidene difluoride membranes (Applied Biosystems, Problott TM
Membranes) stained with Coomassie Brilliant Blue R-250 (Bio-Rad Laboratones)
containing immobilized procathepsin B (A), and a mixture of glycosylated and
deglycosylated procathepsin S (B). Lane 1 and lane 2 of each membrane represent the
conversion of proenzyme to mature enzyme in the absence and presence of cystatin C
(M, = 12,500), respectively. The processing intexmediates migrate at 32kDa and 29 kDa
for procathepsin B and procathepsin S, respectively. The 29kDa species for procathepsin
S is detectable in low quantities using SDS-PAGE in the absence of cystatin C (data not
shown).
7 2 ~ OP 85P 9 0 ~ 9 6 ~ H H K Y L W S E P Q N C S A T K S N Y L R G T G P Y
fi fi fi fi
S M S L M S S L R V P S Q W Q R N I T Y K S N P N R
't' 't' fi
3 8 ~ 45P OP 5 5 ~ 6 2 ~ B L K R L C G T F L G G P K P P Q R V M F T E D L K
'P + fi
Figure 3 : Structural alignment of the C-terminal ends of the prosegments of cathepsin B,
cathepsin S, and cathepsin K. Cathepsin B prosegment numbering was iised for
cathepsin B, and cathepsin L prosegment numbering was used for cathepsin S and
cathepsin H. The established cleavage sites to form mature protein near the pro/mature
junction are represented as (fi). The sites of autoproteolytic processing detected using
the cystatin C assay are denoted as (+).
Figure 4: The Active Site Cleft in Rat Procathepsin B (Cys29Ser) (89). The
cathepsin B prosegment (red residues) binds through the substrate-binding cleft of
cathepsin B @lue residues) in the reverse N+C direction to that taken by natural
substrates. The carbonyl carbon of Cys42p is in closest proximity to the catalytic
nucleophile (approx. 4-41 and the potential bond angle between the catalytic nucleophile
and the carbonyl oxygen of Cys42p is 127 degrees. The carbonyl carbon of Arg4ûp is
not shown.
Figure 5: Continuous Assay for the Autocatalytic Processing of Wild-Type - Procathepsin B (A) and Procathepsin S (B). Plots of the first-order rates of processing
(kbs) obtained by nonlinear regression of the data to the equation discussed in
EXperimental Procedures as a fùnction of precursor concentration (determined by active
site titration with the E-64 inhibitor). The data are in agreement with a fmt-order rate of
processing. For both procathepsins B and S, the rate of processing, br, increases
linearly with proenyme concentration, indicative of a bimoIecular reaction which most
likely corresponds to intermolecular processing of proenzyme by mature or semi-mature
protein. In addition, there is a corresponding rate constant of precursor activation (2.4 x -4 1 10 s' for procathepsin B and 8.1 x 10-~ s-l for procathepsin S) as the concentration of
proenyme is extrapolated to zero, indicative of an activation event which is independent
of the concentration of precursor.
(A) PROCATHEPSIN B AUTOPROCESSING
0.00 0.05 0.10 0-15 020 0 2 5 0.30 0.35
[ Procathepsin 6 ] (nM)
(B) PROCATHEPSIN S AUTOPROCESSING
0.00 0.05 0.10 0.15 020 0 2 5 0.30 0.35
[ Procathepsin S ] (nM)
Figure 6 : Monitoring the autocatalytic processing o f wild-type propapain (iane 1) and
Arg8Ala propapain ( h e 3) using Western blot analysis following their incubation at 65
degrees in 50 mM acetate buffer @H 3.8) and 20 mM cysteine for 30 min (iane 2, wild-
type mature papain; lane 4, Arg8Ala mature papain). Molecular weights of the papain
precursor and mature form are 37 kDa and 25 kDa, respectively. The Arg8Ala variant of
propapain was observed to autoprocess as efficiently as the wild-type precursor.
COhWCTIIVG TEXT FOR CHAPTERS 2 AND 3
In chapter 2, unimolecuIar mechanisms of autoproteolytic processing for
procathepsin B and procathepsin S were identi fied. These reactions involve cleavage at
interna1 peptide bonds within the prosegments which bind through the substrate-
binding clefts of these enzymes. Cuiously, the maturation of procathepsin H to its
mature form has been proposed to involve proteolysis at ~ e r 7 7 ~ f ~ 1 ~ 7 8 ~ . This reaction
is a prelude to the production of an octapeptide of prosegment residues, termed the
rnini-chain, which remains covalently attached to the main body of cathepsin H using a
stable disulfide bond formed by Cys82p and Cys214. Based on structural analysis, it is
interesthg to note that Ser77p and Glu78p reside within an area o f the cathepsin H
prosegrnent which is homologous to the stretch of amino-acid residues believed to
interact with the active site cleft of the enzyme. Similarly to what was identified for
procathepsins B and S in chapter 2 using the cystatin C inhibitor, it was hypothesized in
chapter 3 of this thesis that procathepsin H would be capable of performing
unimolecular autoproteolytic processing at ~ e r 7 7 ~ f ~ l u 7 8 ~ . Instead, it has been
determined that procathepsin H is an unusual mammalian member of the cathepsin L-
subfamily which is incapable of autoactivation. This incapacity has been attributed to
the pre-formation of a disulfide bond linking Cys82p and Cys214 and the presence of a
unique tryptophan residue within the structure of the cathepsin H precursor. This
additional covalent attachrnent was found to limit the conformational mobility of the C-
terminal end of the prosegment, and thus rendering the pro/mature junction within the
cathepsin H precursor highly resistant to proteolysis.
Contributions of authors other than Omar Quraishi :
Mort, J.S. - Provided the cDNA constructs (procathepsin H integrated into the pPIC9
vector), t r d o m e d P. pastoris, wild-type human cathepsin H, and wild-type human
cathepsin D.
S torer, AC. - Provided fùnding and mentorship for this project.
Functional Expression of Human Procathepsin H in Pichia
Pastoris and Attempts at its Correct Processing*
CATHEPSN H LACKING THE MINI-CHAN EETAINS STGNIFICANT
AMINOPEPTIDASE ACTIVITY
*NRCC Publication No. 00000. The research was fûnded in part by the Govemrnent of
Canada's Network of Centres of Excellence Program supported by the Medical
Research Council of Canada and the Natural Sciences and E n g i n e e ~ g Research
Council of Canada through PENCE Inc. (the Protein Engineering Network of Centres
of Excellence).
Omar Quraishi$§, John S. M o w , and Andrew C. Storer$II*
From the $Protein Engineering Network of Centres of Excellence and Department of
Biochemistry, McGill University, 3655 Dnimmond Street, Montreal, Quebec, Canada
H3G 1Y6, the YJoint Diseases Laboratory, Shriners Hospital for Children, 1529 Cedar
Avenue, Montreal, Quebec, Canada H3G lA6, and Protein Engineering Network of
Centres of Excellence and Department of Surgery, McGill University, Montreal,
Quebec, Canada, and the llPharmaceutica1 Biotechnology S ector, Biotechnology
Research Institute, National Research Council of Canada, 6100 Royalmount Avenue,
Montreal, Quebec, Canada H4P 2R2.
* To whom correspondence should be addressed: Pharmaceutical Biotechnology
Sector, Biotechnology Research Institute, National Research Council of Canada, 6100
Royalmount Ave., Montreal, Quebec, Canada H4P 2R2. Tel.: 5 14-496-6256; Fax: 5 14-
496- 1629; E-mail: andrew.storer@nrc.ca.
§ Present Address : B iochem Therapeutic Inc., 275 band-Frappier Blvd., Laval,
Québec, Canada H7V 4A7
RUNNING TITLE: Cathepsin H Expression, Processing, and Activity
ABBREVIATIONS
* Unless stated otherwise, residue nurnbering in the text relates to that of cathepsin L as
presented in Coulombe, R et al. (85). Residues in the proregion are identified with the
suffix p.
2 In the text, the words 'proregion', 'prosegment', 'prodomain', or 'prosequence' refer
to the polypeptide stretch located N-terminal to the mature enzyme in the proenzyme,
while the word "propeptide" refers to the chemically synthesized polypeptide
corresponding to the proregion sequence but without the mature enzyme.
In the text, the ternis 'autoactivation', 'autoprocessing', or 'maturation', relate to the
ability of an enzyme (pro- or mature) to convert its own precursor to a mature protein
by cleaving at or near the proIrnature junction.
The abbreviations used are: E-64, tans-eporysuccinyl-L-leucyl-arnido-(4-
guanidino)butane; Arg-MCA, L-ar-oinine 4-methylcouma~inyl-7-amide; Z-Phe-Arg-
MCA, benzyloxycarbonyl-L-phenylalanyl-L-arginine 4-methylcournarinyl-7-amide;
SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 'DMSO,
dimethyl sulfoxide; EDTA, ethylene-diamine tetraacetic acid; DTT, dithiothreitol.
Abstract :
Within the papain family of cysteine proteases, cathepsin H is unusud in that it
displays mono-aminopeptidase activity. This capacity has been iinked mainly to an
octapeptide, termed the mini-chain, that blocks the unprimed subsites of this enzyme's
substrate-binding cleft. The mini-chain, derived fiom residues located within the C-
terminal end of the cathepsin H proregion, rernains covalently linked to the main body
of the enzyme following activation of the precursor using a disulfide bond formed by
Cys82p and Cys214 (83). The mechanisms leading to the formation of mature
cathepsin H, however, have yet to be elucidated. To date, al1 reported attempts to
isolate cathepsin H have required that it be purified fkom natural sources. Here, we
report that a cDNA encoding the hurnan cathepsin H precursor has been expressed as
* an a-factor fusion construct in the methanotropic yeast Pichia pastoris. Unlike most
proenzymes belonging to the papain farnily, procathepsin H was determined to be
incapable of autoprocessing under acidic pH conditions in vitro and mature cathepsin H
does not independently contribute to the conversion of its own precursor. The
conversion of procathepsin H to its mature form, therefore, requires the action of other
proteases. For example, cathepsin D has been found to cleave at the N-teminus of the
rnini-chah in the presence of SDS detergent. Furthemore, prosegment residues
located near the pro/mature junction were found to be resistant to proteolysis.
Homology modelling suggests that the unusual stability of the cathepsin H precursor to
autocatalysis and cleavage near the pro/mature junction is attributable to the pre-
formation of a disulfide bond linking the prodomain to the enzyme. This feature,
unequaled in other mammalian precursors of the papain family, serves to restrict the
conformational mobility at the C-terminal end of the proregion. Proteolysis near the
pro/mature junction of procathepsin H at may only be achieved uskg
a secondary protease such as cathepsin L in the simultaneous presence of DTT and
SDS detergent. hterestingly, this isoform of cathepsin H retains its aminopeptidase
activity towards the Arg-MCA substrate; Le., with a /&,/KM = 1,950 M%' veTsus
-1 1 11,700 M s' and 4.2 M-'s" for human wild-type cathepsin H and cathepsin L,
respectively. These results suggest that the mini-chain serves to optimize, yet is not
strictly required, for the arninopep tidase (exopeptidase) activity of cathepsin H.
Therefore, an additional role for the rnini-chain is to Iirnit the endopeptidase activity of
cathepsin H.
Cathepsin H (EC 3.4.22.16) is a ubiquitously expressed lysosornal enzyme
belonging to the papain superfafnily of cysteine proteases (1 08) which includes the
rnammalian cathepsins B, K, L, and S. The identification of downstrearn targets and
the precise physiological roles of cathepsin H have yet to be determined. Increased
cathepsin H activity, however, have been correlated with human glioma ce11 invasion
(139). Furthemore, the expression level of cathepsin H or cathepsin H-like enzymes
(140) has been shown to be increased in other disease States such as melanoma and
tumor metastasis (141) as well as breast carcinoma (142). Cathepsins B (107), C (9)' H
(62) and bleomycin hydrolase (143) are the o d y known exopeptidases of the papain
family. Cathepsin B is primarily a carboxydipeptidase, while cathepsin C is an
arninodipeptidase. The carboxydipeptidase activity of cathepsin B has been linked to a
unique insertion of 20 residues, caIled the occluding loop, which contributes to the
primed subsites of this enzyme's substrate-binding cleft. Conversely, a feature which
distinguishes bleomycin hydrolase and cathepsin H fiom al1 other marnrnalian rnernbers
of the papain family is their mono-aminopeptidase activity; Le., cleavùig a single
residue fiom the N-terminus of an extended polypeptide substrate. The arninopeptidase
activity of bleomycin hydrolase is accounted for by the protmsion of the C-teminal
end of the enzyme into the active site cleft (144). Similar to the role of the occluding
loop for the carboxydipeptidase activity found in cathepsin B, it has been proposed that
the aminopeptidase activity of cathepsin H is due mainly to the presence of an
octapeptide, termed the mini-chain, that blocks the unprimed subsites of this enzyme's
substrate-binding cleft (83). Cornparison of the X-ray crystat structures for this farnily
of zymogens (85-91) to the recently determined c~ystal structure of mature porcine
cathepsin H (83) has raised many questions concerning the maturation mechanism for
the cathepsin H precursor in particular, and that for zyrnogens of the papain family in
general.
Cathepsin H isolated fkom subcellular fiactions has been shown to consist of
different N-terminal sequences. Typically, this enzyme contains the N-terminal
sequence corresponding to the pro/mature junction, namely Gly(-2)Pro(- 1)Tyr 1 -Pro2-
Pro3 and Tyrl-Pro2-Pro2 (136). The major isofonn of cathepsin H has also been
shown to possess an additional N-terrninal sequence corresponding to a glycosylated
octapeptide (Glu78p-Pro-Gln-Asn-Cys-Ser-Ala-ThrWp), referred to as the mini-chain,
which is composed of residues located at the C-terminal end of the cathepsin H
proregion, Following maturation of the cathepsin H precursor, the mini-chah has been
shown to remain attached to the enzyme domain using a disulfide bridge. The crystal
structure of mature porcine cathepsin H (83) reveals the method by which the mini-
chah interacts with the main body of the enzyme. Significantly, the disulfide bridge
linking the mini-chain to the catalytic domain is composed of two cysteine residues
which are unique to cathepsin H (rnammalian), aleurain (barley), and orizain y (rice
seeds), Cys82p of the prodomain and Cys214 Iocated on the enzyme (128). Contrary to
full-length proregions which have been reported to bind in the reverse substrate-binding
mode through the active site clefts of their cognate enzymes (85-91), the mini-chain
interacts with the enzyme in the same direction as that taken by natural substrates (83).
Since the fold of the prosegments and their mechanism of inhibiting enzyrnatic activity
are conserved arnong the zymogen structures reported to date (85-91), it follows that
the terminal residues of the mini-chah; Le., Glu78p and ThrSSp, essentially exchange
places with one another during the activation of procathepsin H. The 'flipping' of the
mini-chah enables Thr85p to be strategically positioned within the S2 subsite of the
enzyme's active site cleft with its fiee carboxyl group facing the Si subsite (83) ; i.e.,
the carboxyl group of Thr85p serves to accept the positively charged N-terminus of the
bound substrate. Based on the structure of the mature protein, it has been proposed that
maturation of procathepsin H involves 'cLippingY at the N-terminus of the mini-chain
(carbonyl carbon of Ser77p) as a first step, followed by a second cleavage near the
prolmature junction with the disulfide bond linking Cys82p and Cys214 remaining
intact (83). In addition, it was proposed that the remaining prosegment residues (C-
terminal end of the prosegment) 'flip' such that they bind to the enzyme in the
substrate-binding mode folIowed by intramolecular 'trimming' reactions leading to the
mature mini-chah composed of Thr85p at its C-temiinus (83). Here, we attempt to
elucidate the sequence of proteolytic events leading to the formation of mature
cathepsin EI in vitro as well as to identify the maturase(s) responsible for these
processes. Interestingly, mode1 building of procathepsin H (Figure 1 of Chapter 3)
predicts that residue Glu78p of the cathepsin H proregion binds through the substrate-
binding cleft in the reverse mode and that the carbonyl carbon of Ser77p is in closest
proximity to the catalytic center. Evidence for the capacity of cathepsin H to perform
proteolysis at ~ e r 7 7 ~ ' ? ~ l u 7 8 ~ would be of significant interest since a unimolecular
rnechanism of processing for procathepsin B and procathepsin S has been identified
((145) ; Chapter 2 of this thesis). These observations, therefore, are suggestive that a
unimolecular proteolytic event at the ~ e r 7 7 ~ T ~ l u 7 8 ~ site is a plausible mechanism
leading to procathepsin H maturation. Confirmation of such a process would have
important implications for other zymogens belonging to the papain family.
Previous to this work, nahiral sources such as rabbit liver (146) and lung (1 47),
rat skin (148), human liver (149,150), placenta (15 1) and kidney (1 52), porcine (83)
and bovine spleen (153), as well as bovine and human brain (154), have been used to
isolate cathepsin H. Moreover, the activity yields in al1 of these cases were very poor.
The expression of recombinant proenzymes belonging to the papain family of cysteine
proteases has been achieved as an a-factor fùsion constmct in the methanotropic yeast
Pichia pastoris (85,88,89,99,105-107). We have used this system to produce human
procathepsin H which may be easily purified, and thus, facilitate the monitoring of
procathepsin H processing in vitro. The ability of procathepsin H to perfom
intramolecular 'self cleavage at the N-terminus of the mini-chain ; i.e., at the carbonyl
carbon of Ser77p, is also investigated. Furthemore, we also present a structure-activity
relationship for different isoforms of cathepsin H towards the exopeptidase substrate,
AG-MCA.
EXPERfMENTAL PROCEDURES
Production of Active Cathepsins B, D, K, K, L, and S - Active human cathepsin H
and cathepsin D were obtained from the Iaboratory of Dr. John S - Mort (Joint Diseases
Laboratory ;Shriners Hospital for Children) and Arg-MCA substrate was purchased
fi-om Bachem Bioscience Inc. For cathepsins B, K, L, and S, each enzyme was
similady expressed as a precursor in P. pastoris as descnbed in this study for
procathepsin H. Each precursor was activated by subjecting them to acidic pH and
rnildly reducing conditions. Each processed enzyme was independently purified with a
fast-flow column of SP-sepharose resin (Pharrnacia) using 50 mM sodium acetate (pH
5.0). Each enzyme was eluted from the column using a linear gradient of increasing
NaCl concentration. To each enzyme was added Hg2C12 to a final concentration of 0.1
mM and al1 sarnples were stored at 4OC.
Hornology Model of Procathepszk H - The crystal structure of procathepsin L (85)
(over 40% sequence homology to procathepsin H) was used as a template to construct a
hornology model of procathepsin H. The aromatic side-chains extending from the
substrate-binding cleft to the prosegment-binding loop (exosite) are highly conserved
among proenzymes belonging to the papain farnily. The side-chains of residues found
in the prosegrnent of procathepsin L were sequentially substituted to those found in the
cathepsin H precursor using ON0 version 5.10. The prosegment of cathepsin H was
also oriented such that the side-chah of Cys82p is in closest proximity to Cys214; Le.,
conducive to fonning a disulfide bond. Finally, Insight II was used to present the
model shown in Figure 1 of Chapter 3.
Construction of WiZd-Type Human Procathepsin H - A cDNA constmct consisting of
human wild-type procathepsin H as a fusion with the preproregion of yeast cc-factor
had been prepared (obtained from the laboratory of Dr. John S. Mort, Joint Diseases
Laboratory, Shriners Hospital for Children). AL1 sites for glycosylation, AsnSOp,
Asn8lp and Asnl il, were kept intact. The n o 1 and NotI restriction sites were then
introduced into the C-temiinal segment of the a-factor preproregion and the 3'-
untranslated region of procathepsin H, respectively, to allow subcloning into the Pichia
pastoris expression vector pPIC9 (Invitrogen Inc., San Diego, California).
Expression of Procathepsin H in P. pastoris - For integration into the Pichia
genome, the pPIC9 based constructs were linearized by cleavage with Bgm and
purified. The P. pastoris host strain GS115 (hvitrogen) was then transformed with the
linearized constnicts by electroporation. Positive transformants were grown at 30°C
for 2 days in medium containhg glycerol as the carbon source followed by incubation
in the presence of methanol for a M e r 3 days to induce expression of recombinant
protein. Protein secreted into the culture supernatant was analyzed by non-reducing
SDS-PAGE.
Purij5cation of Procathepsin H - The culture supernatant (250 ml) was concentrated
to 40 ml using an Amicon stirred-ce11 (YM-IO membrane). During concentration, the
supernatant was dialyzed against 50 mM Tris (pH 7.4) containhg 1.6 M (NH&S03.
Concentrated recombinant proenzyme was then purified on an FPLC system using a
butyl-sepharose fast flow column (Pharmacia Inc.). Proenzyme fractions eluted fkom
the coiumn by applying a linear gradient of decreasing ammonium sulfate
concentration. Both glycosylated and deglycosy lated procathepsin H eluted at 0.3 -0.6
M w ) 2 S o 4 and the samples were stored at 4OC.
In Vitro Processing of Procathepsin H - For autoprocessing assays, purified sarnp les
of wild-type human procathepsin H were dialyzed against 50 mM acetate (pH 4.0-5.0)
and 1mM DTT at various temperatures (25, 37, and 60°C). For bimoIecu1ar
processing (in tram) assays, catalytic arnounts of lysosomal cysteine proteases such as
human cathepsins B, H, K, L, and S were each added to procathepsin H (molar ratio of
procathepsin Wexogeneous protease > 250/1) under mildly reducing conditions
(+DTT-SDS). For the addition of the aspartic proteases, 50 units/ml of imrnobilized
pepsin (Sigma-Aldrich Canada Ltd.) was incubated with procathepsin H at pH 4.7 for
two hours at 37°C followed by its removal by filtration. The addition of human
cathepsin D was canied out under the sarne conditions as those for pepsin (-DTT-
SDS). Following incubation with the various proteases, each reaction was then treated
with 10 pM of either E-64 or pepstatin. Furthemore, processing reactions were also
attempted in the presence of O. 1 mM SDS using either cathepsin L or cathepsin D as
catalys t.
N-Terminal Identijkation of the Cathepsin H Isoforms - Iso fonns of cathepsin H
imrnobilized in non-reducing polyacrylarnide gels were electoblotted onto hydrophobic
polyvinylidene difluoride membranes using the method as descnbed previously (124).
The membranes were then stained with Coomassie Brilliant Blue R250 (Bio-Rad
Laboratories) and each protein band of interest was subjected to a minimum of five
cycles of automated solid-phase Edrnan degradation. In some cases, aqueous sarnples
of cathepsin H were applied directly fur N-temiinal sequence analysis without pnor
purification.
Active Site Titration - Due to the inability of procathepsin H to autoactivate,
exogeneous proteases were needed to produce various isoforms of active cathepsin H
with an exposed active site. To procathepsin H was added catalytic arnounts of a pre-
determined quantity of cathepsin L in the presence of 0.1 rnM SDS and this reaction
was incubated at 37°C for 1 hr. The active site titrant, E-64, was determined to be a
slow-binding inhibitor to cathepsin H as compared to cathepsin L. For this reason, the
reaction mix of cathepsin H and cathepsin L was first treated with a pre-determined
excess of E-64 ([--641 where no activity is detected for either enzyme ; Le.,
Arg-MCA for cathepsin H and Z-Phe-Arg-MCA for cathepsin L. Finally, aliquots of
cathepsin L were then added to this mixture d l its activity towards the 2-Phe-Arg-
MCA substrate was detected. With volume corrections taken into account, the onginal
concentration of cathepsin H was calculated as being equal to the difference of p-
64ITOTAL and [cathepsin LITOTAL when fluorescence generated by z-Phe-Arg-MCA
cleavage was recovered.
Enzyme Assays - Kinetic fluorescence rneasurements were carried out using a SPEX
Fluorolog-2 spectrofluorometer which monitored MCA formation using an excitation
wavelength of 380 nm and a detection wavelength of 440 nrn. A final concentration of
1.0 nM cathepsin H was used for each assay. The KM of wild-type hwnan cathepsin H
for the exopeptidase Arg-MCA substrate was determined by the non-linear fitting of
measured initial velocities at different concentrations of substrate to the Michaelis-
Menten equation. Since the KM was estimated to be 0.282 rnM, a substrate
concentration of 10 pM was used for each assay to estimate kcat/KM values ([SI KM).
Previous studies on cathepsin H purified fkom cow brain (154) resulted in KM values of
0.169 mM and 0.195 mM at pH 7.0 and 37OC for Arg-2-naphthylamide and Arg-p-
nitroanilide, respectively. In the present study, assays were performed at 25°C using 50
mM phosphate (pH 6.0) buffer containhg 0.2 rnM EDTA, ImM DTT, and 3% DMSO.
Despite the pre-incubation with 0.1 mM SDS in some assays, each isoform of cathepsin
H was sufficiently stable under the assay conditions used for the time required. As a
control, no activiiy of cathepsins B, D, K, L, or S towards the Arg-MCA substrate was
detected during the time required for all assays (5 lhr), thus c o n h n h g that production
of fkee MCA (fiom Arg-MCA) was due strictly to the aminopeptidase activity of the
various cathepsin H isoforms. From these assays, values of kcaL/Ki were obtained
using the equation v = [E][S]k&t/KM at [SI<<&.
Non-Redzrcing SDS-PAGE Analysis - Proteins were treated with 10 pM of E-64 or
pepstatin (for reactions using pepsin and cathepsin D). When necessary,
deglycosylated procathepsin H was obtained by incubating 1 unit of endoglycosidase H
Poehringer Mannheim) for 1 hr at 37°C. In order to obtain accurate determinations of
the sites of proteolysis due to the activities of cathepsins D, K, L, and S ; i-e., in order
to ensure the integrity of the disulfide bond linking Cys82p and Cys2 14, the cathepsin
H isoforms were purified using non-reducing SDS-PAGE (12% gels). This was
followed by blotting the proteins onto polyvinylidene difluoride membranes for direct
Edman degradation. It should be noted, however, that the apparent molecular weights
of the various cathepsin H isoforms is based on their migrations in non-reducing SDS-
PAGE (Figure 2A and 2B) rather than standard (reducing) SDS-PAGE.
Modelling of Human Procathepsin H - The primary amino-acid sequence homology
between procathepsin H and procathepsin L is over 40% and the structural homology
among proenzymes belonging to the papain family; Le., the fold of the prosegments
and their mechanism of inhibiting enzymatic activity, is ais0 conserved (85-91). In an
attempt to construct a mode1 of procathepsin H, the tertiary fold of procathepsin L was
used as a template. Depicted in Figure 1 are the unique cysteine residues, Cys82p and
Cys214, found only in procathepsin H among the mammalian members of papain-like
enzymes as well as in proaleurain (barley) and pro-orizain y (rice seeds). From Figure
1, it is revealed that the two cysteines are in close proximity to one another and located
within the unprimed segment of this enzyme's substrate-binding cleft. Furthemore,
the scaffold of residues located on the enzyme; Phe143, Tyr146, Tyr15 1, Trp189, and
Trp193, are also highlighted. Each of these residues contributes to an intncate ladder
of hydrophobie interactions which are highly conserved among members of the papain
family. These residues extend from the hydrop hobic prosegment-binding loop
(exosite) composed of Tyr146 and TyrlS1, to Trp189 located within the prïmed
subsites of the enzyme's substrate-binding cleft. It has also been documented that
residue Phe56p of the proregion (not shown in Figure 1) binds to the pr~se~gnent-
binding loop in procathepsin L (85-88) and is positioned perpendicularly to Tyr15 1.
This position within the prosequence consists typically of an arornatic residue. For
example, Phe56p is replaced by a tyrosine in procathepsin K and procathepsin S, and a
tryptophan (Trp24p) in procathepsin B (85-9 1). The insertion of these hydrophobic
residues into the exosite contributes to the hydrophobic ladder and provides added
stability to the prosegment-enzyme complex. The prosegment of cathepsin H is
unusual in that, in addition to possessïng a phenylalanine residue in the 56p position, it
is also composed of a unique tryptophan residue, Trp76p, which is predicted to bind in
close proximity to the S i 7 subsite of the substrate-binding cleft in cathepsin H.
Interestingly, as the prosegment is oriented such that the side chains of Cys82p and
Cys214 are capable of forming a disulfide bond, the side chah of Trp76p is shown to
stack against Trp189 and is in close proximity to the catalytic Hisl63. Although the
thiolate-imidazolium ion-pair has been shown to be preformed, and presumably
functional, in the structures of zymogens belonging to the papain family as is found in
the corresponding mature enzymes, the presence of Trp76p in procathepsin H may
induce the catalytic Es163 to stack against this unequaled aromatic side chain rather
than to form a stable ion-pair with the catalytic cysteine, Cys25.
Expression and Purification of Procathepsin H - The recombinant cathepsin H
precursor was expressed at far lower levels in Pichia pastoris as compared to other
precursors reported previously; Le., approximately 1.0 mghter of culture medium as
opposed to 10-20 mg/liter for rat procathepsin B (89), hurnan procathepsin L (85), and
human procathepsin K (88). The proenzyme was purified using hydrophobic
interactions (butyl-sepharose resin, Pharmacia Inc.) under neutral pH conditions. The
cathepsin H proenzyme was heterogeneous due to modification of the N-linked
oligosaccharide moieties on the proregion and enzyme domain and rnigrated with an
apparent molecular mass ranging fiom 45 to 60 kDa with a dominant band at 50 kDa in
non-reducing SDS-PAGE (Figure 2). Following enzymatic deglycosylation with
endoglycosidase H (Boehringer Mannheim), procathepsin H migrated as a single band
with the expected size of 37 kDa (data not shown).
Processing of Procathepsin H ln Vitro - Exposwe of procathepsin H to similar
conditions used for most other zymogens of the papain farnily; Le., acidic pH and
rnildly reducing conditions, was insufficient to prornote autoactivation of either
glycosylated or deglycosylated foms of the proenzyme. The addition of active
cathepsins D, K, L, and S, but not cathepsin H (Figure 2A) or cathepsin B, to the
cathepsin H precursor (+DTT-SDS) yielded intermediate species (Figure 2B and
Figure 3) with N-termini corresponding to a confined area of the pradomain which is
solvent exposed and accessible to proteolytic processing in trans. This segment of the
prosequence is composed of Asp65p and Glu70p which are highly conserved residues
among the prosegments of precursors belonging to the cathepsin L subfamily (>90
residues) (Figure 3). The crystal structure of human procathepsin L (85) reveals that
Asp65p forms a salt bridge with Arg31p, and Glu70p contributes to salt bridge
formation with both Arg3lp and Glu27p. These conserved interactions serve to fold
helices n l p (residues 6p-19p) and a2p (residues 25p-51p) in close proximity to helix
a3p (residues 68p-75p). Cunously, it was proposed by Vernet et al. (100) that the N-
terminal sequence of an intermediate band of propapain processing corresponded to a
sequence residing in proximity to the segment, Gly/Aia59p-Xxx-Asn-Xxx-Phe-Xxx-
Asp65p. This conclusion was based on the apparent molecular weight of the
intermediate as detennined by its migration using Western blot analysis (100).
Production of Active Cathepsin H - In order to produce cathepsin H lacking the
majority of prosegment residues, and consequently the mini-chain, the precursor must
be incubated with catalytic amounts of a secondary protease, such as cathepsin L, with
the simultaneous presence of DTT and SDS detergent (+DTT+SDS). Under these
conditions, proteolysis at ~ r ~ 9 2 ~ ? ~ l ~ 9 3 ~ located near the pro/mature junction was
observed (Figure 3). In the absence of SDS detergent, however, this site is not
hydrolysed by cathepsin L. Under these conditions (i-e., +DTT-SDS), the major
isoform of cathepsin H produced corresponded to an N-terminal sequence of t ~ l u 7 0 ~ -
Ile7lp-Lys72p (Figure 3), thus suggesting that over 20 residues 6om the prosegrnent
continue to remain covalently attached to the enzyme via the pro/mature junction and
the disulfide bridge formed by Cys82p and Cys214. Furthemore, incubation of
procathepsin H with active cathepsin K produces an identical N-terminal sequence of
cathepsin H to that observed via cathepsin L, and cathepsin S cleaves at T ~ h e 6 8 ~ -
Ala69p-Glu70p. Conversely, procathepsin H was resistant to cleavage by the
exopeptidase activities of cathepsin H and cathepsin B.
Incubation of procathepsin H with pepsin Ieads to the hydrolysis at the
t ~ s n 6 1 p - ~ l n 6 2 p - ~ he63p site. Following incubation with cathepsin D (-DTT-SDS),
the major cathepsin H isoform produced was detennined to have the N-terminal
sequence ?69p~la-70p~lu-~1e71p. Upon the addition of SDS detergent (+DTT+SDS),
however, cathepsin D was capable of cleaving at the N-termimal end of the mini-chah,
? ~ l u 7 9 ~ - ~ r o 8 0 ~ - ~ l n 8 1 ~ (25% of signal), in addition to the ?69p~la-70p~lu-~le71p
site (75% of signal).
Activiîy of Procuthepsin H - The progress curves shown in Figure 4 indicate that
cathepsin H is capable of cleaving the Arg-MCA substrate, regardless of whether it is
composed of the mature mini-chain. For example, most intemediates of processing
continue to be composed of over 20 residues derived fiom the C-terminal end of the
cathepsin H proregion (attached covalently at two sites to the enzyme), yet they
demonstrate significant exopeptidase activity with respect to cathepsin H containing the
mature octapeptide. The &/KM value obtained for mature cathepsin H (composed of
the mini-chain) was determined to be 11,700 lK1s-l. For the cathepsin H intermediate
produced following incubation with cathepsin L (+DTT-SDS; ?~ lu70~-1 le71~-
Lys72p), the value was determined to be 889 M-'s". Finally, production of cathepsin H
lacking the majonty of prosegment residues following the incubation of the zymogen
with cathepsin L in the simultaneous presence of DTT and SDS detergent
(+DTT+SDS ; ? ~ 1 ~ 9 3 ~ - ~ h r 9 4 ~ - ~ 1 ~ 9 5 ~ ) was determined to have a kcal/KM = 1,950 M-
l -1 s . Interestingly, this isofonn of cathepsin H is highly homologous in sequence and
three-dimensional structure to mature hurnan cathepsin L. Similady to mature human
cathepsins B, D, K, and S (data not shown), however, cathepsin L is shown to display
poor activity towards the Arg-MCA substrate (Figure 4).
DISCUSSION
The ability of zymogens belonging to the papain family to undergo autocatalytic
maturation is predicated on them containing an intact active-site machinery ; i.e., a
preformed catalytic ion-pair and substrate-binding cleft, sirnilar to that found in the
mature enzyme. Proenzymes of the papain family are more stable at neutral pH and are
prone to autoprocess under acidic conditions (97401,209) such as that found in the
mature lysosornai cornpartment of the cell. This observation irnplies that electrostatic
interactions are critical in regulating the stabilization or destabilization of these
prosegment/e;izyme complexes and correlates with the demonstrated pH dependence of
inhibition by the propeptides of cathepsin B (102,106), cathepsin L (103), and
cathepsin S (104) towards their parent enzyme. Recently, we have deterrnined that the
occluding loop of cathepsin B d e h e s the pH dependence of inhibition by its
propeptide, and consequently, regulates the moiecular switch for procathepsin B
autoactivation (106). The formation of a critical salt bridge on cathepsin B, invoIving
HisllO of the occluding loop and Asp22 located near the Sz' pocket of this enzyme's
substrate-binding cleft, helps to stabilize the closed form of the loop at low pH and
allows it to compete with the propeptide for the surface of the enzyme (106).
Interestingly, the absence of an occluding loop and the presence of longer proregions in
most other precursors of the papain family such as those of the cathepsin L-subfarnily
( ~ 9 0 residues versus 62 residues in procathepsin B), implies that the pH-tnggering
mechanism of autoprocessing for procathepsin B may be unique to that enzyme, and
that the important 'activating' salt bridges arnong cathepsin L-like precursors are likely
to reside predominantly within the prodomains. In marnrnals, procathepsin H is a
unique member of the cathepsin L-subfamily of zymogens in that it is composed of a
conformational constraint at the C-terminal end of its prosegment. This constraint is
the pre-formation of a disulfide bond linking Cys82p of the proregion to Cys214
located on the enzyme which has been shown to remain intact in mature porcine
cathepsin H (83). Therefore, a detailed characterization of the mechanism of
processing for procathepsin H in vitro would provide unique insights into the
conformational requirements of the prodomain for efficient autoprocessing to occur
among zymogens of the papain farnily. Here, we attempt to elucidate the mechanism
of processing for the cathepsin H precursor whose prosegment shares the same length
(>90 residues) and high identity (>40%) to that of cathepsin L (Figure 3), but consists
of amino-acid residues which are unequaled among the marnmalian homologs of
papain ; Le., Trp76p and Cys82p within the proregion and Cys214 located on the main
body of the enzyme.
Previous to this work, the only precursors of the papain family which were
found to be incapable of autoprocessing were those for papaya proteinase IV (PPIV)
(126) and the plant equivalent of cathepsin H, a barley vacuolar thiol protease known as
aleurain (137,155). The inability of pro-PPIV to autoprocess has been attributed
mainly to its crowded active-site cleft consisting of the unique residues, G133 and
Arg65 (papain nurnbering) which confers strict'specificity of this enzyme for substrates
with a glycine residue in the Pl position. In the case of proaleurain, it was deterrnined
that this precursor was not capable of autoprocessing in the absence of aIeurone ce11
extracts and that mature (active) aleurain did not participate in the processing of its own
precursor (137). Curiously, the prosequence and catalytic domain of aleurain share the
identical residues Cys82p and Cys214, respectively, to those found in cathepsin H and
orizain y (128) (Figure 3). Similar to what was observed for proaleurain, we report that
procathepsin H is a stable prosegment/enzyme complex and incapable of
autoprocessing in vitro. On the basis that the cathepsin H proregion inhibits the
enzyme using the reverse substrate-binding mode as has been reported for other
proregions (85-91), the disulfide bridge linking Cys82p to Cys214 would significantly
reduce the degrees of conformational freedom at the C-terminal end of the
prosequence; Le., frorn prosegment residues which bind through the substrate-binding
cleft of cathepsin H to those near the pro/mature junction (Ser77p-Glu78p-Pro79 +
Tyr1 -Pro2-Pro3). Therefore, regardless of the pH conditions to which procathepsin H
is subjected, the disulfide bond may serve to eliminate the pH dependence of
prosegment binding as has been demonstrated for non-covalent propeptide/enzyme
complexes (1 02-1 04,106). The negative charge of a highly conserved aspartate residue
in the proregion of propapain (Asp65p) was shown to be important in maintainhg the
papain precursor in a latent f o m and to participate in an electrostatic triggering
rnechanism of propapain processing (100). Despite the conservation of both Asp65p
and Glu70p within the cathepsin H proregion (Figure 3), it may be reasonably assumed
that the Cys82p/Cys214 disulfide bridge impedes the prodomain ffom dissociating
fiom the surface of the enzyme.
Full-length procathepsin H is voici of catalytic activity towards synthetic
substrates suc11 as 2-Phe-Arg-MCA or kg-MCA (data not shown). This observation
suggests that the prosegmentfenzyme complex is stable and that access of small
substrates to the enzyme's substrate-binding cleft is restricted- Structural alignment
and mode1 building indicates that the prosegment of cathepsin H contains a unique
tryptophan residue, Trp76p, which is predicted to bind near the Si' subsite of the
enzyme's substrate-binding clef3 (Figure 1). Curiously, there exists no other papain-
Iike prosegment which contains a bullcy aromatic group in this position. For example,
the proregions of human procathepsins K, L, and S consist of threonine, asparagine,
and serine, respectively, in this position and those for proaleurain and pro-onzain y
consist of a leucine (85). The codonnation of the bound prosegrnent as illustrated in
Figure 1 is such that the side- chahs of Cys82p and Cys214 are at the closest possible
distance to one another; Le., conducive to fomiing a disulfide bond, This orientation
also causes Trp76p to stack against Trp189 and contribute to a highly conserved and
intricate ladder of hydrophobie residues that extend fiom the prosegment-binding loop
(exosite) to the substrate-binding cleft of the enzyme. In precursors belonging to the
cathepsin L-subfamily (85-88), it has been established that the side ch& of Phe56p
(structurally homologous to Trp24p in procathepsin B) pmtrudes into the cavity of the
exosite formed by residues Tyr146 and Tyrl51. In procathepsin H, therefore, the
ladder is composed of seven aromatic residues compared to only six in other precursors
of papain-like enzymes and may, therefore, contribute to the increased stability of the
cathepsin H precursor. Furthennore, due to the unique location of Trp77p, it is possible
that this novel aromatic group interferes with proper formation of the catalytic thiolate-
imidazolium ion-pair in the full-length cathepsin H precursor. It is predicted that
Trp76p is in close proxirnity to the catalytic imidazole, His163 (Figure 1). Ln order to
assist in the transfer of protons to the leaving amino group, it has been proposed that
the catalytic histidine of senne proteases has the inherent capacity to adopt various
conformations via rotation about its Ca-CP axis (156). As an illustration of this
capacity arnong cysteine proteases, the crystal structure o f cathepsin B in cornplex with
the pyridyl disulfide inhibitor revealed a rotation of 120" and 6S0 for the side-chahs of
the catalytic His199 and Cys29 residues (134), respectively, cornpared to their
orientations observed in other crystal structures of cathepsin B (82,89-91).
Furthennore, the crystal structure of porcine cathepsin H (83) revealed dirnerization of
the enzyme caused by crystal packing. This dimerization was shown to induce salt
bridge formation between the EN of the catalytic His163 and the carboxyl group of the
C-terminal residue, Va1222, from a neighboring rnolecule in the crystal. Consequently,
this salt bridge causes the catalytic histidine to rotate 80° about its Ca-CP bond.
Hence, the positioning of the buky Trp76p aromatic group in the Si' subsite rnay
influence the catalytic histidine to rotate its side-chain perpendicular to Trp76p rather
than to form a stable ion pair with the catalytic cysteine, and thus, may help to explain
how the full-length cathepsin H precursor is void of catalytic activity towards small
synthetic substrates.
In the case of proaleurain, correct processing of this zymogen was only
observed following its incubation with barley ce11 extracts (137). Both 'clipping' (loss
of 9 kDa) followed by 'trimmïng' (loss of 1 kDa) proteolytic events were necessary for
the complete maturation of proaleurain and were shown to be due to the activity of two
independent maturases. The inhibition of the trimrning reactions by E-64 suggested
that this event was mediated by the activity of a thioI protease, but the barley enzyme
needed to perform the 'clipping' reaction was not identified. Sùnilarly to proaleurain,
the conversion of the cathepsin H precursor has been proposed to occur in a multi-step
fashion (83) involving cleavage at the pro/mature junction and at both termini o f the
mini-chain. Most notable is the proposed cleavage site at the N-terminus of the mini-
chah, ~er77~ 'T '~lu78~, since this segment of the proregion is predicted to bind in the
reverse substrate-binding mode through the enzyme's substrate-binding cleft and to be
in close proxunity to the catalytic ion-pair (Figure 1). However, the sequence of
proteolytic events and the protease(s) responsible for these reactions had yet to be
determined. Previous to this work, procathepsin H had been shown to be processed
following its incorporation into the lysosome and that this conversion was inhibited by
pepstatin, a potent inhibitor of aspartic proteases such as cathepsin D (157-159). It was
also shown that processing of the cathepsin H precursor displayed significantly slower
kinetics when compared to that of cathepsin B (157-159). As has been docurnented for
proaleurain, we report that conversion of the cathepsin H precursor requires the
catalytic activity of a secondary protease and not that of cathepsin H itself (Figure 2A).
Based on the proposed sites of proteolysis discussed previously, it is reasonable to
assume that the activiq of an endopeptidase would be more efficient to perform these
reactions as comp&ed to that of an exopeptidase. This is evidenced by the resistance of
procathepsin H to cleavage by either mature cathepsin B which functions primarily as a
carboxydipeptidase or by mature cathepsin H. Conversely, the various endopeptidases
used in this study such as cathepsins D, K, L, S, and pepsin, were each capable of
converthg procathepsin H to a lower molecular weight species. Curiously, N-terrninal
identification of each cathepsin H intermediate indicated that the cysteine
endopeptidases were only capable of cleaving within a finite sketch of proregion
residues which includes the highly conserved Asp65p and Glu70p (Fi3we 3). Upon the
addition of SDS detergent and cathepsin D, however, a minor signal (25% of total)
corresponding to the N-terminus of the mature rnini-chain, 'T '~lu78~-~ro79~, was
detected in addition to the f ~ l a 6 9 ~ - ~ l u 7 0 ~ site (75% of total signal). It is not clear
whether cathepsin D may cleave directly at the N-terminus of the mini-chain or if this
reaction is tirne-dependent and biphasic in nature ; Le., that cathepsin D needs to cleave
upstream at ? 6 9 ~ ~ l a - ~ l u 7 0 ~ before being capable of cleaving at the N-terminus of the
mini-ch&. Conversely, no proteolysis was observed at the C-terminus of the mature
mini-chain ( ? ~ h r 8 5 ~ - ~ y s 8 6 ~ ) . Clearly, the cathepsin H intermediates composed of the
disulfide bridge linking Cys82p and Cys214 display remarkable stability under the
conditions used in this study which ultimately facilitates their identification by Edrnan
degradation. Conversely, if the corresponding processing intermediates among other
papain precursors are produced, their identification would be severely irnpeded by their
rapid degradation to form mature protein. The enhanced stability of the cathepsin H
intermediates may be due in part to the presence of the disulfide bridge luiking Cys82p
and Cys214. Cleavage of procathepsin H by endopeptidases such as cathepsin D and
cathepsin L remove over 60 residues at the N-terminal cap of the proregion (clipping),
which includes the removal of Phe56p fiom the exosite surface of the enzyme-
Presurnably, this 'clipping' reaction has the effect of partially exposing the substrate-
binding cleft of cathepsin H as well as to destabilize the hydrophobie interactions
between Trp76p and Trp 189, and between Trp76p and the catalytic His 163 residue.
The lack of catalytic activity for the full-length cathepsin H precursor towards
either Arg-MCA or 2-Phe-Arg-MCA suggests that the enzyme's substrate-binding cleFt
is inaccessible to small substrates and/or the catalytic ion-pair is not optimally formed
to partake in chernical reactions. It is interesting to note, however, that the intermediate
of cathepsin H processing produced by the activity of cathepsin L ( ~ l a 6 9 ~ ? ~ l u 7 0 ~ )
demonstrates significant aminopeptidase activity; i.e., kCat/KM = 889 M-'s-' as compared
to 11,700 M' for wild-type cathepsin H cornposed of the mature mini-chain (Figure 4).
How is this possible if, as discussed above and as demonstrated by the homology mode1
(Figure 1), the intermediate of cathepsin H lacks a mature mini-chain but rather is
composed of over 20 residues derived fkom the C-terminal end of the prosegment?
First, removal of the N-terminal a-helical cap of the prodomain (approximately 70
residues) will cause the active site cl& of the enzyme to be more exposed to incoming
substrates than is the case for the full-length precursor ; i-e., the remaùiing prosegrnent
residues will bind less tightly to the surface of the enzyme. Second, the substrate-
binding cleft of cathepsin H may be uniquely designed to accomodate aminopeptidase
activity independently of the mature mini-chah For exarnple, the crystai structure of
porcine cathepsin H reveaIs that the unprimed region of this enzyme's active site cIeft
is narrower than those of other related structures (83). Significantly, the backbone
carbonyl oxygens surrounding GIy65-Gly66 have been shown to be positioned closer to
the SZ pocket ; Le., the putative location of the positively charged N-terminus of a
bound substrate. Furthemore, cathepsin H contains an insertion loop within the R-
domain consisting of Lysl55A-Thrl55B-Pro 155C-Asp 155D located between Serl55-
Ser156 (papain numbering). This insertion is positioned adjacent to the unprimed
pockets of the active site cleft as well as to the bound mini-chah. When the mature
mini-chain is bound to the enzyme, the side-chain of Asp155D is directed away fiom
the substrate-binding cleft of the enzyme and faces the surrounding solvent. In the
absence of the mini-chah, however, the orientation of the Asp155D side-chain may
change such that it faces the positively charged N-terminus of the Arg-MCA substrate ;
Le., in a rnanner analogous to Thr85p of the mature mini-chah Conversely, the
catalytic activity of the cathepsin H processing intermediates may be due instead to the
production of trace amounts of cathepsin H composed of the mature mini-chain. Based
on the N-terminal sequence analyses of aqueous sarnples of cathepsin H, however, no
such species was detected.
Processing in pans among papain-like precursors usually results in proteolysis
at or near the pro/mature junction. Papain-like prosegments strategicaliy possess the
least secondary structure near the pro/mature junction when bound to their cognate .
enzyme (85-91) ; Le., the most conformationally disordered segment of the proregions.
Consequently, this conformational keedom faciliates proteolysis near the pro/mature
junction to form mature protein. Similady, tnincated propeptides composed only of the
C-temiinal end of the full-Iength propeptide display poor inhibitory activity towards the
parent enzyme (92,103,117). In this study, we report that the C-terminal end of the
cathepsin H prodomain is unusually resistant to cleavage by endopeptidases despite
consisting of a Leu-Arg motif at positions -6 and -5, respectively; Le., a sequence
compatible for recognition by many thiol proteases such as active cathepsins B, L, S
(160) and K (161). Furthemore, the C-terminal end of the mature mini-chah (Thr85p)
is also resistant to proteolysis- As discussed above, the homology model of
procathepsin H predicts that Cys82p and Cys214 f o m a disuEde bridge near the S4
subsite linking the C-terminai end of the proregion to the main body of the enzyme.
Intuitively, this additional covalent attachrnent would significantly lirnit the
confornational rnobility at the C-terminal end of the prosegment, thereby providing
resistance to proteolysis in an area of the proregion where processing in tram normally
occurs. Therefore, susceptibility of the cathepsin H precursor to proteolysis near the
pro/mature junction may improve only upon the complete or partial denaturation of the
bound prosegment. This is evidenced by the ability of cathepsin L to cleave near the
pro/rnature junction of procathepsin H with the sirnultaneous presence of DTT and SDS
detergent (Figure 3). This reaction leads to the production of an isoform of cathepsin H
(?~1~93~-Thr94p) which lacks the rnajority of prosegment residues, and consequently
the mini-chah, and is therefore highly homologous in sequence and structure to mature
human cathepsin L. Similar to the cathepsin H processing intermediates discussed
above, this isoform retains significant aminopeptidase activity (kcat/KM = 1,950 M%')
towards kg-MCA compared to mature hurnan cathepsin H (kcat/KM = 1 1,700 M%' )
and possesses enhanced aminopeptidase activity compared to mature human cathepsin
L (kat/& = 4.2 ~ ' ' s - l ) . Given this data, it is necessary to conclude that the mùii-chain
serves to optirnize, yet is not stnctly required, for the rnono-aminopeptidase activity of
cathepsin K. It is aIso important to note that intramolecular proteolysis at the
~ e r 7 7 p T ~ l u 7 8 ~ site was not obsewed. The ability to perform such a unïmolecular step
arnong other precursors of the papain family which lack the disuIfide attachent,
however, has been demonstrated for procathepsin B and procathepsin S ((145) ;
Chapter 2 of this thesis). In agreement with previous work (157-159), mature cathepsin
H composed of the mature rnini-chah is incapable of processing its own precursor and
cleavage at the ~ e r 7 7 ~ î ~ l u 7 8 p site may be performed instead by another enzyme such
as cathepsin D. Therefore, in order to produce active cathepsin H composed of the
mature mini-chah, it rnay be necessary to subject procathepsin H to a cocktail of
proteases as is the case within the mature lysosome.
Cathepsïns B; C, H, aleurain, onzain y, and bleomycin hydrolase have each
evolved from an ancestral papain-like cysteine protease (108). In the case of cathepsin
B, the addition of a 20-residue insertion, called the occluding loop, contributes to the
p b e d subsites of the substrate-binding clef3 and enables this enzyme to fwiction as a
carboxydipeptidase. The shorter prosegment of cathepsin B (62 residues) also contains
a cysteine residue, Cys42p (cathepsin B numbering), yet procathepsin B is capable of
autoprocessing (98,lOl,lO9). The crystal structure of procathepsin B reveals that
Cys42p binds close to the SI' pocket and is in proximity to the catalytic Cys29 residue,
yet has no covalent partner (89-91). However, alanine scanning studies have revealed
that Cys42p is a critical residue for the inhibitory activity of the hl-length cathepsin B
propeptide (92). In contrast, the unprimed subsites found in cathepsin H are more
narrowed and a disufide bond, formed by Cys82p and Cys214, links the prosegment to
the main body of the enzyme. We propose that this additional covalent attachent
causes procathepsin H, and consequently proaleurain, to be incompatible to "self'
activate and thus requiring the action of other maturases. As has been shown for
proaleurain (137), it remains possible that procathepsin H requires the activity of more
than one endopeptidase for complete maturation to take place. This is evidenced by the
inability of either one of the lysosomal protease used in this study to produce mature
cathepsin H (+ or - mini-chah) without the partial or complete denaturation of the
prosegment by the simultaneous addition of DTT and SDS detergent. Furthemore, we
have shown that the additional disulfide bond inhibits direct proteolysis at the
pro/mature junction by secondary proteases, presumably by impeding the
conformational mobility of the C-termina1 end of the prosegment. Therefore, the
simultaneous addition of SDS detergent and DTT to procathepsin H denatures the C-
terminal end of the proregion sufficiently by abrogating the disulfide bond and thus
rendering it more susceptible to proteolysis. In addition to the involvement of other
maturases, these fïndings suggest that zyrnogen-membrane interactions mediated by the
prosegment of cathepsin H may be important during lysosomal targeting and activation
of procathepsin H as has been observed for mouse procathepsin L (162).
ACKNOWLEDGMENTS
The authors thank France Dumas for N-terminai sequence identifications of the various
cathepsin H isofoms and Dr. J. Sivaraman for his assistance in preparing Figure 1. We
also thank Dr. Robert Ménard and Dr. Dorit K. Nagler for many valuable discussions.
Figure 1 : Homology mode1 of procathepsin 8. View of the backbone trace (brown)
of human Cys2SSer Procathepsin L as reported in Coulombe, R et al. (85). The
prosegment residues (green) were sequentially mutated to those found in procathepsin H
and the conformation adopted by the bound prosegment reflects the highty conserved
mode of inhibition observed in the crystal structures reported to date of precursors of
papain-like enymes (85-91). A ladder of highly conserved aromatic residues are
highlighted (pi&) as well as the catdytic residues, Cys25(Ser) and His163 (white).
Located near the unprimeci subsites are residues Cys82p and Cys214 (yellow), found
only in the precursors of cathepsin H, aleurain, and orizain y, linking the prosegment to
the main body of the enzyme. Residue Trp76p, unequaled in other prosegments of the
papain family, is also illustrateci and shown to stack against Trp189 (and perhaps the
catalytic His163) near the Si' subsite of the substrate-binding cleft. Residue Phe56p (not
shown), is laiown to stack aga% Tyr151 located within the hydrophobie prosegment-
binding loop (exosite) of the enzyme (equivalent to the position of Trp24p in
procathepsin B). Prodomain residues which are predicted to bind through the substrate-
binding cleft of cathepsin H in the reverse mode contains the following sequence:
Leu75p-Trp-Ser-Glu-Pro-GIn-Asn-Cys82p with the carbonyl carbon of Ser77p predicted
to be in closest proximity to the catalytic center.
Figure 2 : Non-Reducing SDS-PAGE of purified glycosylated procathepsin H (Coornassie
BLue staining). Gel A : (Iane 1) ptuified procathepsin H ; Oane 2) procathepsin H foiIowing
treatment with mature human cathepsin H composed of the mini-chain (28 kDa). Gel B :
procathepsin H incubatexi with active cathepsins B, D, K, L, and S. Positions of molecular
mass standards are indicated on the le&
Figure3 : Cornparison of primary prosequences of human cathepsin L, aleurain
(barley), orizain y (rice seeds) and human cathepsin H. The Cys82p residue found in
the prosequences of aleurain, orizain y and cathepsin H are underlined and io boldface as
is the unique Trp76p residue located in the prosegment of cathepsin H. The mature
rnini-chain is composed of residues Glu78p+Thr85p. Both Asp65p and Glu70p are
highly conserved among prosegments of the papain family. The various N-tenninal
sequences detected for cathepsin K following treatment of the precursor with active
cathepsins D, K, L, and S as well as pepsin (P) are indicated by arrows. The treatment of
procathepsin H with cathepsin L with the simultaneous addition of DTT and SDS
detergent leads to an N-terminal sequence located near the prohature junction indicated
as L(SDS). The treatment of procathepsin H with cathepsin D in the presence of SDS
detergent leads to a major sequence at ?A.ka69p-~lu70p (75% of signal) and a minor
sequence correspondhg to the N-terminus of the mature minichain f ' ~ l u 7 8 ~ ~ r o 7 9 p
(25% of signal, indicated as D(SDS)).
CATL TLTFDHSLE A QWlXWKAM HN * -RLYO - MNEEOW RRAVWEKNM K MI ELHNQEYR ALEU OALGRTRHAL RFARFAVRYO --KSYESAAEVRR R F R I F S E S L E E V R S T M - - ORiZy AALGRTROAL ' RFARF A V R HO - - KRYGDAAEV Q R RF R i F SE S L E LVRST NRR -2
CATH E LSVNSL EKF HF KS WMS KHR. - - KTY STE - EYHH RLQT F AS NWR K1 NAHN - NGN
CAïL EGKHSF T MAM NAFO DMTS E E FR Q -VM NOFQ NRKPR KOKVF QE PLFYE ALEU - - GLPYR L O I M FS DMSW EE FQ A - T R LOAA QTDATLAON HLMRDAAA ORET - - GLPYRL GI NRFA DMSW EE FQA - SR LOAA QNCSATLAGN HRMRDAPA
Figure 4: Structure-Activity Relationship of the Various Cathepsin H Isoforms.
Curves 1, 2, 3, and 4 correspond to the cataiytic activities of mature human wild-type
cathepsin H composed of the mature mini-chin (&'/KM = 1 1,700 MIS-'), cathepsin H
composed of the N-terminus ? ~ 1 ~ 9 3 ~ - ~ h r 9 4 ~ - ~ 1 ~ 9 5 ~ (&,/KM = 1950 MIS-') formed
following treatment with cathepsin L i n the presence of SDS detergent (+DTT+SDS),
cathepsin H composed of ?~lu70pIIe7 1 p ~ ~ s 7 2 ~ (kt/KM = 8 89 MIS-') following
treatment with cathepsin L in the absence of SDS detergent, and mature human cathepsin
L (Lt/KM = 4.2 MIS-'), respectively, towards the Arg-MCA substrate which measures
mono-arninopeptidase (exopeptidase) activïty. Cathepsins B, D, K, L, and S did not
display any appreciable hydrolytic activity towards Arg-MCA (5 1 hour of reaction
Cathepsin H Activity Towards Arg-MCA
SUMMARY
The living ceIl possesses a variety of degradative enzymes. The mamrnalian
homologs of papain are diverse in terms of their substrate specificity despite the fact
that theu overall three-dimensional folds are quite sirnilar. This diversity rnay be
accounted for by subtle differences in their primary arnino-acid sequences and other
structural features located within the substrate-binding clefts of the enzymes. Insights
into the proteolytic pathways to which enzymes such as cathepsin L (73) and cathepsin
S (74) participate have only been elucidated recently, thus leading to their consideration
as important targets for therapeutic intervention.
Papain-like enzymes are first synthesized as zymogens consisting of long
extensions at the N-terminus of the enzyme which serve to inhibit, stabilize, and target
the enzymes until they reach their final destination ; i.e. the mature lysosome. Upon
their arriva1 to the acidified lysosomal cornpartment of the cell, these proenzymes
undergo maturation. Due to the high local concentration of proteases within the mature
lysosome, activation of these proenzymes may take place through autolysis or by the
action of other proteases.
Procathepsin B, is capable of autoactivation in vitro (98,101,109) and is a
unique member of the papain farnily of zymogens in that it is composed of a shorter
proregion (62 residues) than those found among cathepsin L-like precursors (>90
residues). Furthemore, cathepsin B is composed of an exposed disulfide loop, terrned
the occluding loop, which contributes to the primed subsites of this enzyme's substrate-
binding clefi. Kinetic assays using small fluorogenic substrates have indicated that the
rate of autocatalytic processing of procathepsin B (and other rnernbers of this family)
correlate with the affinity of the enzyme for its propeptide rather than with its catalytic
activity (102-104,106) ; i.e. peptides derived from the sequence of proregion residues
display potent inhibition of the enzyme at neutral pH where full-length precursors are
more stable, but display weaker a.fEnity for the enzyme under acidic pH conditions
where zymogens of papain-like enzymes autoprocess more efficiently.
In Chapter 1 of this thesis, it has been demonstrated that the affuiity of cathepsin
B for its propeptide may be improved significantly upon the deletion of the occluding
loop (1 05,106). Hence, the formation of a stable propeptidekathepsin B complex
requires unobstmcted access of the inhibitor to the enzyme's substrate-binding cleft.
This finduig collaborates with the recently determined X-ray crystal structure of
procathepsin B (89-91) which indicates that the occluding loop is a flexible motif
capable of undergoing major conformational changes. In mature cathepsin B, the
occluding loop is in a closed position (82) and shown to be stabilized by the formation
of a saIt bridge between Asp22 located within the primed subsites of the substrate-
binding cleft of cathepsin B and His 1 10 located within the occluding loop (106,107).
In this study, the occluding loop was s h o w to compete with the cathepsin B propeptide
for the surface of the enzyme (termed the occluding loop crevice). This cornpetition
was shown to be maximized at Iow pH and regulated by the interactions of Hisl10, but
not Hisl l l , with the main body of the enzyme. Therefore, major conformational
changes during the autocatalytic processing of procathepsin B involves the
intramolecular pH-dependent movernent of the occluding loop, thus suggestive that the
pH-triggering rnechanism of autoprocessing in procathepsin B is unique to this
zymogen ody. In summary, the critical electrostatic interactions which regulate
procathepsin B processing reside within the enzyme domain and not the cathepsin B
prosegment. Conversely, for members of the cathepsin L-subfamily ; i.e., prodomain
composed of over 90 residues and no occluding loop insertion within the mature
protein, the pH triggering mechanian is likely to reside within the prosegment. In
particular, the protonation of salt bridges which bring helices orlp and a2p in close
proximity to helix a 3 p ; i.e., involving the highly conserved GI37p and Arg3 1 p to
Glu70p, and k g 3 l p to Asp65p, are likely to contribute to the maturation process. In
the study of Vernet et al. (100), it was demonstrated that the negative charge of Asp65p
participates in the control of intrarnolecular processing of the papain precursor.
In Chapter 2 of this thesis, interna1 cleavage sites within the prosegments of
cathepsin B and cathepsin S were identified during the autocatalytic conversion of their
zymogens. This was accomplished by adding to the precursors excess amounts of the
protein-proteinase inhibitor, cystatin C, prior to their exposure to acidic pH
environments. The addition of cystatin C was successfbl in trapping these precursors
into a slower cascade of autoproteolytic processing ; Le., a E t y of cystatin C for the
mature enzyme>intemediate>full-length precursor. Hence, the addition of cystatin C
irnpedes the rate of intermolecular processing caused by the activity of mature protein
and thus facilitates the detection of unimolecular reactions. Based on structural
analysis of the precursors (85-92), the newly identified sites of proteolytic processing
take place within a stetch of prosegment residues which are known to bind in the
reverse mode through the substrate-binding clefis of these enzymes. That these
autoproteolytic processing reactions are observed at al1 concentrations of proenzyme
suggests that they occur as unirnolecular events and that they may be important. The
unusually low molecularity of these unimolecular events may be accounted for by the
fact that the prosequence binds through the active site cleft in the reverse direction to
that taken by natural substrates. Significantly, this reverse complementarity causes the
distance between the 6N of the catalytic histidine and the teaving backbone amide
group to be greater than would be the case for natural substrates, thus leading to highly
reversible nucleophilic reactions at the carbonyl carbon of the scissile amide bond
(within the prosegment) which have difficulty going to completion ; Le., protonation of
the leaving backbone amide group, and consequently, formation of the covalent acyl-
enzyme intermediate are inefficient processes.
The N-terminus of the mature segment h a . been shown to undergo major
conformational changes during the autoactivation of zymogens belonging to the
aspartic protease farnily (e.g. progastricsin, discussed in Literature Review and
Introduction). Conversely, it has been detemined that highly conserved charged
residues (Asp6 and Arg8) which contribute to the formation of a salt bridge within the
catalytic domain of propapain were not found to be involved in regulating the pH-
triggering mechanism of this precursor.
In Chapter 3 of this thesis, the precursor of cathepsin H was s h o w to display
unusual stability to autoactivation as well as to proteolysis near the prolrnature junction.
These fbdings have been attributed to the pre-formation of a disulfide bond, using
Cys82p and Cys214, which serves to link the C-terminal end of the prosegment to the
enzyme domain and is known to remain intact following maturation of the precursor
(83). Procathepsin H is the only marnmalian precursor of papain-like enzymes to be
composed of Cys82p and Cys214. Other zymogens which contain these cysteine
residues are proaleurain (barley) and pro-onzain y (rice seeds) (128). Following the
incubation of procathepsin H with various proteases, it was demonstrated that the
various isoforms of cathepsin H lacking the mature mini-chain retain significant
aminopeptidase activity towards Arg-MCA as compared to other members of this
family (e-g., cathepsins B, D, K, L, and S ) as well as to mature wild-type cathepsin H
which is composed of the octapeptide. This may be accounted for by novel insertions
of amino-acid residues which narrow the substrate-binding cleft (Le., unprimed portion)
found in cathepsin H.
In the case of the caspase family of cytoplasmic cysteine proteases involved in
cellular apoptosis (discussed in the introduction), there exists a sophisticated hierarchy
of intermolecular processing. Among lysosomal cysteine proteases belonging to the
papain family, however, no such hierarchy of proteolytic events has been reported. The
activation of most lysosomal enzymes appear to be mutually non-exclusive since most
of these enzymes are competent to autoactivate upon exposure to acidic pH
environments. Based on the results fiom this study, however, the inability of
procathepsin H to autoactivate and its dependence on the activity of other proteases
suggests that this precursor may be an important exception.
Sirnilar to what has been observed for zymogens of other farnilies of proteases
(serine, aspartic, ~n-coordinated), precursors of papain-Iike enzymes possess a pre-
fonned and functional catalytic triad and a mature substrate-binding cleft ; Le., a
prerequisite for the capability to 'self activate. These zymogens rnay also be activated
due to the action of secondary proteases in vivo, yet they do not need to bind to adaptor
molecules as has been reported for some precursors belonging to the caspase family of
cysteine proteases (e.g. the association of Apaf-1 with procaspase-9) (58). In addition,
pH-induced conformational rearrangements are necessary for the efficient conversion
of these zymogens. In this study, the pH-dependent conformational stability of the
occluding loop was shown to be critical in regulating the overall rate of procathepsin B
processing. Furthermore, the conformational mobility of residues at the C-terminal end
of the prosegments ; i-e., residues which stretch fkom the substrate-binding cleft to the
pro/mature junction, was also found to be important. Intemal cleavage sites within the
prosegments have been identified which take place while the prosegment is bound in
the reverse substrate-binding mode. Therefore, the maturation of zymogens belonging
to the papain family c m proceed via intermolecular or intramo Iecular non-exclusive
events. The discovery of a unimolecular step of autoactivation may help to explain
how this family of enzymes, when even present at low concentrations, may be
implicated in several invasive and pathological conditions extracellularly.
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