plAN~CiENCEELSEVIER Plant Science 142 (!999) 115-121

Spontaneous processing of peptides during coagulation of latex from

Carica papaya

v. Moutim a, L.O. Silva a, M.T.P. Lopes b, O. Wilson Fernandes c, C.E. Salas a,*

a Departament of Bioquímica, Farmacologia, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, 11270-901, Belo Horizonte,

MG, Brazilb [nstituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, 31270-901, Belo Horizonte, MG, Brazil

"Biologia Geral, [nstituto de Ciências Biológ.icas, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627,31270-901, Belo Horizonte,

MG. Brazil

Received 16 July 1998; received in revised form 10 November 1998; accepted 19 November 1998

~

Ah"t.r9ct.

Latex is actively secreted on wounded unripe fruits from Carica papaya. We describe the changes in peptide composition bySDS-PAGE analysis of latex from C. papaya collected at various times after incision of the unripe fruit. Non random changes inthe relative amount of several peptidesoccur during latex coagulation. The measurement of amidase activity of coagulating latexshows three peaks of activity between 2\and 1200 s. The major activity is found at 1000 s, which is when coagulation is underway. The data from in situ proteolyticêtivity experiments confirm the presence of active enzyme(s) when latex begins to flowfrom damaged fruits. @ 1999 Eisevier S'cience Ireland Ltd. Ali rights reserved.

Keywords: Carica papaya; Latex coagulation; Endopeptidase; Cysteine proteinase; Papain

1. Introduction brasiliensis L. (rubber tree) contains isoprenemonomers [4]. During tapping lacticifers are

.severed, and the latex flows out of the tree, WhlChupon polymerisation and subsequent processingbecomes natural rubber .

Instead, latex of Carica papaya L., contains amixture of cysteine endopeptidases [5], such aspapain (EC 3.4.22.2) [6], chymopapains A and B

(3.4.22.6) [7], papaya endopeptidase III, papayaendopeptidase IV [8,9] and a recently identifiedone designated as endopeptidase .Q (caricain) [10].Fresh or dried latex obtained by injuring leaves orfruits of C. papaya contains at least some of theseproteolytic enzymes. The latex bleeding proceedsfor a few minutes until a clot forms around thewounded area. The coagulation process is vital forplant defence against possible pathogen attack.The coagulation in the rubber tree involves aggre-

Plants store a variety of fluids, including latexes,resins, gums and mucilages within secretory celIscavities and canals [I ]. In latex-containing plantsthere is strong evidence that this fluid is part of aninduced defence mechanism [2,3]. Latex is typi-cally contained within lacticifer cells, which areinterconnected by ramified structures forming acomplex array of tubes throughout the plant. Inmost species, damage to the canals elicits anabrupt release of secretion.

Latex contains severaI compounds, whose bio-logical properties confer protection against envi-ronmental damage. For instance, latex from Hevea

.Corresponding author.E-mail address: cesbufmg~mono.icb.ufmg.br (C.E. Salas)

0168-9452/99/$ -see front matter @ 1999 Eisevier Science Ireland Ltd. Ali rights reserved.

PTT. ~Ol f\R-Q4,?(QR)OO??f\-Y

16 Moutim et al. / Plant Science 142 (1999) 115-12.

gation of rubber particles, whereas in latex fromC. papaya, the major component is protein andthe subsequent clot is mainly made up of proteins.The latter example strongly resembles blood coag-ulation and clot formation during wounding inmammals [II]. We have proposed that these twophenomena bear some similarities and that latexproteinases may play a role akin to blood coagula-tion factors endowed with proteolytic activity [12].We provide here further evidence that cysteineproteinases from c. papaya become activated dur-ing clot formation.

tachi laser densitometer supplied with CS 9301PCsoftware. Each latex sample corresponding to thevarious collection times was run and analysed onthe same day to minimise variations within gels.The intensity of a single band on each lane wasrelated to a standard peptide (internal standard)whose concentration remained constant at the var-ious intervals studied. The relative mass of eachpeptide in the samples was determined by regres-sion analysis of a mix of protein standards runtogether with the samples.

2- In situ proteolytic activiry

2. Material and methods The in situ proteolytic activity was determinedby 12% SDS-PAGE or acid PAGE following elec-trophoresis [17]. Protein samples (60 Jlg) were notdenatured by heat before applying onto gels.Briefly, after electrophoresis the gel was rinsedtwice with 50 ml buffer containing 0.4 M Tris-CIpH 8.0 and then a 0.5% agarose solution, contain-ing 0.1 M Tris pH 8.0 and 1.8% casein was appliedonto the gel surface. The gel was then incubated at37°C between 12-48 h to visualise the proteolyticactivity. The gel was then photographed with Po-laroid film 665 and the negative densitometricallyscanned as previously described.

2.1. Latex collection

Latex was col1ected at various interva1s (1-1200s) fol1owing sing1e wounding of unripe fruits at thep1ant with a stee1 razor b1ade. The samp1e dup1i-cates obtained from different fruits (100-200 J.11sample- I) were col1ected in eppendorf tubes con-taining 150 J.11 water, or 5 mM MMS (Methy1methane thiosulfonate) and d(opped into a dry-icebath, fol1owed by storage at -70°C, until used.Samp1e dup1icates col1ected in , water withoutMMS were used as contro1s tQ .determine theinhibitory effect exerted by MMS on proteo1ysis.Fol1owing protein dosage of each samp1e, equiva-,lent amounts of protein from each' dup1icate werepoo1ed for e1ectrophoresis ana1ysis.

2.4. Latex amidase activitv

This procedure measures the enzymatic hydroly-sis of BAPNA fo1lowing incubation 30-40 min at37°C [18]. Briefly, a proteinase sample (15 J.ll) wasincubated with a solution containing 4 mM cys-teine, 25 mM Tris-Cl, 2 mM EDT A pH 8.0 and 4mM BAPNA in a final volume of 750 J.ll. Thereaction was stopped by addition of 60 J.ll 60%acetic acid. The release of p-nitroanilide was mea-sured spectrophotometrically at 405 nm.

2.2. Electrophoretic of proteins from latex

3. Results

A complex pàttern of 5-20 bands of differentintensities was observed on each lane of the gel(Fig. I). To analyse this complex peptide pattern adensitometric scanning of the stained gel was re-quired. To facilitate analysis, bands were groupedinto four classes (a, b, c, and d), based on theirelectrophoretic migration. Group a contained thelargest peptides ( > 38.5 kDa). group b consisted

Protein detemlination was carried out by Brad-ford [13]. The samp1es (4 ~g) were e1ectrophoresedaccording to Laemmli [14] for 2 h at 110 V, and 15mA at 8°C. MMS was added to a final concentra-tion of 2.5 mM before sample heat denaturationto prevent degradation during electrophoresis.Acid PAGE was done according to the techniquepreviously described [15]. Protein bands were visu-alised by silver staining as previously described[16]. The molecular weight standards were bovinealbumin (66000), lactic dehydrogenase (36500),carbonic anhydrase (29 000), trypsinogen (24000),and (J.-lactalbumin (14200).

For a comparative analysis of the proportion ofeach peptide in the mix, we scanned each lane bytransmission densitometrv at 430 nm usin!!: a Hi-

v. Mou!im e! al. / Plan! Science 142 (1999) 115-121

kDaô6 -

36.5-29 -

24 -

14 -

300 600 1200sec.

2 3 4 5 6

Fig. 1. SDS-PAGE of 1atex fractions obtai1)ed at different intervals after fruit tapping. Latex samples were collected at varioustimes following an incision of the unripe fruit as described in Section 2. Aliquots (4 J.lg) were heat-denatured for 1 min with MMSand loaded on each lane of the gel. The run was performed at 8°C followed by silver staining. Densitometry was done with staineddried gels. A profile similar to that shown in the figure was obtained in trip1icate experiments, each using different latex stocks.The first lane of each gel contains the molecular weight standards.

of peptides with a size between 26-38 kDa, groupc, contained peptides of intermediate size (22-25kDa), whi1e group d contained the sma11est pep-tides (12-21 kDa). Samp1es co11ected and stored inwater showed fewer bands than those co11ected inMMS, confirming the notion that the su1fuydryl

modifying reagent efficient1y prevents proteindegradation before and/or during e1ectrophoresis.Consequently, in contro1s without MMS, a1mostno protein remained in the ge1 after electrophore-sis (Table 1). MMS also afforde8.better inhibitionof proteinases during electrophoresis when com-pared to mercaptoethanol or dithiothreitol (notshown). These are reducing agentscommonly usedin e1ectrophoresis buffers ([14)).

The re1ative changes in content of selected pep-tides. during the interva1 1-1200 s was obtained byplotting the relative areas of each band against thelatex collection times. The summary of these re-sults is shown in Table 1. The data obtained foreach peptide were fitted into curves by regressionanalysis using Sigmap1ot software and furtherc1assified into six categories: (1) peptides showingan increase that peaked before 2 min, fol1owed bya decrease and stabilisation throughout the re-maining period; (II) peptides whose relativeamounts showed two to three peaks during theinterval 1-1200 s; (III) peptides that decreaseduntil reaching a p1ateau; (IV) peptides that in-creased unti1 reaching a plateau; (V) peptides re-maining unchanged during the interval; and (VI)peptides that increased their relative amount in alinear or non-linear mode. In addition, other pep-tides not included in Table 1 disp1ayed variations

in their content that could not be fitted into acurve by regression analysis, and therefore theywere excluded from further analysis. Based onthese data, we concluded that the changes in rela-tive peptide profile observed are non;.stochastic.

Next, we evaluated the changes in specific activ-ity of proteolytic enzymes during the period oflatex coagulation. The amidase activity of latexsamples co11ected at different intervals was dosed(Fig. 2). Three major peaks (350, 700, and 1000 s)

.were detected during the period surveyed. Linear

extrapolation of the specific activity to zero time inFig. 2 suggests that the enzyme is already presentwhen latex begins flowing out from the fruit. Itsvalue was estimated to be 54.6 x 10 -6 M xmin- IX mg- 1 by linear regression analysis.

To verify the presence of proteolytic enzymes inactive form at early times fo11owing co11ection, wecarried in situ proteolytic activity experiments fol-lowing electrophoresis. Both acid PAGE and SDS-PAGE gels were screened for enzyme activity. Fig.3 shows the in situ activity data of SDS-PAGEgels containing samples co11ected between 2-15 s.Although the enzyme activity was present at eachtime point, a gradual reduction in caseinolyticactivity was observed during the interval analysed.By using molecular weight markers we estimatedthe relative mass of this activity to be 24-26 kDa.The densitometric analysis of the band shown onFig. 3 was analysed by densitometry as before.The peak area obtained on each lane was plottedagainst the time of co11ection. The result on Fig. 3reveals an exponentiallinear decrease of the activ-ity during the period between 2 and 15 s.

v. Mnutim et al. / Plant Science 142 (1999) 115-121118

Table 1Changes in the relative content of some peptides during latex coagulationa

MMS+MMS H2O+MMS H2O

-MMS

Peptide groups Variation

(I)

(II)

(III)

(IV)

(V)(VI)

l'\. --+

l'\./,"'--+l'--+

d17; d1s

aS7; aSS; a46; b37; b3o; C2S; C23; d16

a43

a61; b36

bJ4; bJJ; C22

a43; a42; b33-

a52:..~ b2R; C24

a The relative variation of each band was deterrnined by densitometry and analysed with CS9301PC software (Hitachi)

using the less variant peak as standard: Thêestimated molecular mass (kDa) fJr each bana designated with letters (a-d) isshown as subscript. Samples were collected in H2O or MMS. MMS was added ( + MMS) or not ( -MMS) to the samplebuffer. The orientation of the arrows shows the direction of peptide change.

4. Discussion

While a variety of non-defensive functions havebeen proposed for latex canals and their fluid,including conduction and storage of food [19], itsdefensive role is supported by many lines of evi-dence. Plant varieties or individuals with reducedcanal systems or decreased titers of chemicals intheir latex are more vulnerable to herbivore attackthan are plants with normally sized canals and or,.latex content [2,20]. The presen~, of secondarymetabolites known to be toxic to' insects or ani-mals or the presence of bacteriolytic, proteolyticenzymes [21], furanocumarins [22J~ vanillic acid[23], aliphatic ketones [24] in various latexes confer

protection against pathogens.In Caricaceae, latex appears to protect by sani:.

tising 'and sealing the wounded area, thus wesearched for biochemical ground to support thisnotion. We provide evidence that during latexcoagulation a number of peptides are being pro-cessed in a non-random manner. Many of thechanges involve transient increases that after re-turning to a basal level, further decrease or in-crease depending on the peptide. In contrast, therelative amount of other peptides increase or re-main constant during the period analysed(a61, b36). In same cases, groups of consecutivepeptides adopt the same processing profile, such isthe case for peptides a57, a55, and a46. It could beargued that an overlapping profile of a57 and a55 isdue to crossover contamination between the twopeptides in the gel, but this could not be theexplanation for the similar profiles observed be-tween as, and a41i in which a 11 kDa difference

separate them. We attribute this simi1arity inprofile between neighbouring bands, to a1ternativeprocessing of these peptides, thus for instancepeptide a57 or its a1ternate form a55 undergo thesame processing pathway. A similar situationmight occur with peptides d17/d15' b34/b33, and C25/C23. It is known that severa1 proteo1ytic enzymesare components of 1atex from C. papaya. How-ever, their presence has been genera11y afforded innon-fresh 1atex preparations, therefore, we carriedthis ana1ysis to probe the amidase activity in fresh1atex preparation. Unexpected1y, an initial ami-dase activity was a1ways detected in fresh 1atexsamp1es (Fig. 2). The zero-time extrapo1ated spe-cific activity was about (0.25 nkat mg protein- I)1/3 of the maxima1 va1ue measured at 1000 s (0.75nkat mg- I). These figures are in agreement withthe data from [10], who reported 1.68 and 1.98nkat mg- 1 for purified proteinase n and chymo-

papain, respective1y.Fo11owing a transient decrease in activity, the

data show two moderate peaks at 350 and 700 s,fo1lowed by a third major peak at about 1000 s.Similar resu1ts were obtained with 1atex co11ectedat different times during the day, different fruitsize and before or after p1ant watering (notshown). AlI three peaks occur when 1atex is coagu-1ating, that is to say, when dripping from the fruitis no 1onger observed. The observed peaks maycorrespond to a sequerItia1 activation of the vari-ous proteinases in 1atex from C. papaya.

The amidase activity at 1ater interva1s (1800-2500 s) gradua11y decreases to attain 40% of theactivity detected at 2 s (not shown). Based onthese resu1ts it is conc1uded that the proteo1ytic

v. Moutim et al. / Plant Science 142 (1999) 115~121 119

activity in commercia1 1atex preparations of c.papaya represents the remaining activity fo11owingthese changes. The presence of active proteo1yticenzymes in fresh 1atex suggests thai these enzymesmust be kept 'iso1ated' to prevent undesired prote-olysis within the p1ant.

To confirm the presence of active enzyme(s) atear1y times fo11owing fruit tapping, we dosed the insitu proteolytic activity after SDS-PAGE. Thedata presented in Fig. 3 confirm the presence of anactive enzyme(s) as ear1y as 2 s after fruit injuring.Moreover, in agreement with Fig. 2, the datashown on Fig. 3 confirm the initia1 steady decreaseof this initial activity, suggesting the inactivationof this enzyme(s). The estimated re1ative mass ofthis activity is 24-26 kDa. This mobi1ity is sharedby papain, chymopapain, papaya proteinase IVand proteinase .o. on SDS-e1ectrophoresis. Alterna-tive1y, by running native acid P AGE which dis-criminate by charge instead of size, it is possib1e todistinguish between papain, chymopapain andproteinase .o. [10]. By app1ying this e1ectrophoreticcondition the initia1 proteo1ytic activity behaves1ike chymopapain (not shown).

While the primary sequence of chymopapainhas been disc1osed [7], many reports describe thevarious isoforms of this protein in latex [25,26,8].

~.

';-O)Ex

't:

"Ex

~

b.-x

i?:-.>'8(I]u

I;=.(3Q)o.(1)><Q)ro-.J

It is possible that the various isoforms representthe different processing steps for chymopapaindâring latex coagulation.

The presence of papain precursors between 39-24 kDa in c. papaya was also demonstrated[27,28]. Molecular cloning of papain, papayaproteinase IV and proteinase Q confirmed thatthese enzymes are synthesised as pro-enzymes, andthat removal of the pro-region which is essentialfor correct enzyme folding, is also required forenzyme activation. Interestingly, the size of thesepro-enzymes previously reported are like some ofthe peptides described in Table 1.

The removal of the pro-region can be achievedin vitro by autocatalytic cleavage at pH 4 to yieldthe active mature enzyme. The significance of thisreaction in vivo remains to be demonstrated, sincethe pH of latex is closer to 6 rather than 4. Also,previous attempts to produce active papain follow-ing in vitro translation of larger precursors wereunsuccessful [28].

If the pro-region remains in contact with theenzyme after cleavage, it inhibits enzyme activity[29]. This mechanism has not yet been demon-strated in the case of chymopapain, although thepro-regions from papain and proteinase IV cross-react with chymopapain. Molecular cloning of thechymopapain precursor will show whether thisenzyme(s) has its own inhibitory pro-region. Ac-cording to our preliminary data, a chymopapain-like enzyme appears to be active at the time whenlatex begins flowing out of thé truit. If this is true,chymopapain or an isoform is kept uninhibited inthe plant. It is also possible that the activity ofchymopapain is controlled by a different mecha-nism from that dbscribed for I ihe other enzymes.

Based on these results it is concluded that asequential processing of some peptides takes p1aceduring latex clotting, along'\\iith the formatiori 6fother peptides and the transiént activation of pro-teolytic enzymes. ,

It is tempting to establish a paralleí betweenlatex and blood coagulation pathways, the látterprocess evolved by mammals with participation ofserine proteinases [11]. The analogies betweenthese two processes are outlined: both phéhomenaoccur as part of a defence mechanism, in eithercase, a solid clot is produced and proteolytic en-zymes are activated while clot is being formed(Table 1 and Fig. 2 in this work), in mammalsfibrin is the major component of clot, while ourunDublished data reveal that latex clot is com-

Fig. 2. The amidase activity of latex fraction collected duringcoagulation. Aliquots of latex collected at various intervals asdescribed in the Section 2 were dosed for their specific activ-ity. The protein concentration of each sample was measuredby Bradford ([13]). The data represent mean of three separatelatex samples co11ected separately with their standard devia-tion.

1?0 v. Moutim et ai. /Piant Science 142 (1999) 115-121

220D

cu~«~mD..

1600

1400

1?00

1000

800

600

2 5 10 15

Time. sec

Fig. 3. The in situ proteolytic activity of latex after SDS-PAGE. Latex samples (60 ~g) were applied onto SDS-PAGE gels asdescribed in Section 2. After electrophoresis the gel was rinsed and an overlay of agarose containing casein was applied onto thegel and allowed to polymerise. After incubation for 12 h at 37°C the gel was photographed with Polaroid film 665 (inset) and thefilm negative scanned densitometrically. The area corresponding to each point determined as in Fig. 1 with software CS-9301PC,,was plotted against the time of collectioi: .

called 'hevein', the major protein of vacuolarstructures in the latex of the rubber tree, is in-volved in the coagulation of latex by polymeriz-ing rubber monomers [4]. Interestingly, theC-terminal region of 'hevein' shares homologywith wound-inducible genes. Wound-induciblegenes are part of a systemically induced defensemechanism found in leaves from Lycopersiconesculentum and Solanum tuberosum [31]. Thesegenes encode serine proteinase inhibitors that areexpressed in response to leaf wounding. It is notknown whether hevein from H. brasiliensis is in-volved in the wound inducible function as well.It will be interesting to find out if the expressionof wound-inducible genes involving proteinaseinhibitors also occurs in Caricaceae.

posed by 1- 2 peptides and that regenerative pro-cesses leading to healing of damaged tissue areevident after clot formation, in plants as well asin mammals. In addition, preliminary evidence(not shown) suggests that mitogenic substancespresent in latex from Caricaceae stimulate celldivision when added to mamma~ian cultured ep-ithelial cells. The fact that latex proteolytic en-zymes are cysteine proteinases does not challengethese analogies, as cysteine enzymes might be theproducts of independent evolution. In this re-gard, it is worth to note that a group of pep-tides was isolated in seeds from squash thatinhibit the serine proteinases from the b1ood co-agulation system [30], while our attempts to in-terfere with the blood coagulation process usinglatex fractions were unsuccessful (unpublishedobservations). In spite of these analogies, furtherwork is required to establish the role of process-ing proteinases from latex on clot formation.

A quite different defense mechanism occurs inlatex from H. hra.çilien.çi.ç. A lectin-Iike nrotein

Acknowled2ements

This research was supported by FAPEMIGHnd C:NPO-

v. Moutim et al./Plant Science 142 (1999) 115-121

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