THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 263, No. 30, Issue of October 25, pp. 15528-15534,1988 Printed in U.S.A.

Characterization of the Gene and Protein of the al-Antitrypsin “Deficiency” Allele Mprocida *

(Received for publication, May 25, 1988)

Hideki Takahashi$#, Toshihiro NukiwaQ, Ken SatohQ, Fumitaka OgushiQ, Mark BrantlyQT, Gerald Fells#, Larue Stierg, Michael Courtney11 , and Ronald G. Crystal# From the §Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 and the 11 Transgene, SA, Strasbourg 67000, France

The “deficiency” group of al-antitrypsin (alAT) al- leles is characterized by a lAT genes that code for a lAT present in serum but in amounts insufficient to protect the lower respiratory tract from progressive destruction by its burden of neutrophil elastase. Mproeih, a rare a lAT allele associated with a1AT serum levels less than 10 mg/dl (normal 150-350 mg/dl), codes for an a1AT molecule that focuses on immobi- lized pH gradient isoelectric gels slightly cathodal to the common normal M1(Va1213) protein. On a per mol- ecule basis, Mprocida has a mildly reduced function as an inhibitor, with an association rate constant for human neutrophil elastase of 7.0 2 0.1 x lo6 M” s” (normal M1(Val2I3) 9.3 f 0.8 X lo’, p c 0.01). The Mproeih molecule behaves normally in vivo with a half-life similar to normal M1 a lAT molecules. Restriction endonuclease mapping demonstrates that the cloned Mproeida gene was grossly intact. Sequencing of all the exons, exon-intron junctions, and the major promoter region demonstrated Mproeida to be identical to the M1(Va121S) gene except for a single base substitution in exon I1 coding for amino acid 41 of the mature protein

the coding sequence of the a1AT residues 40-41 is recognized by the restriction endonuclease PuuII so that using a probe corresponding to this region of exon 11, the Mproeih mutation can be rapidly identified by Southern analysis. Evaluation of the crystallographic structure of a lAT suggests the Leu4’ to Pro“ mutation may disrupt a-helix A in the region of P r ~ ~ ~ - S e r ~ ~ , suggesting the possibility that the a1AT Mproeida mole- cule is unstable and degraded intracellularly prior to secretion.

(M1(Va1213) Leu4’ CTG+Mprooida Pro4’ CCG). usefully,

al-Antitrypsin (a1AT) is a 52-kDa glycoprotein that func- tions as the major inhibitor of human neutrophil elastase, a potent serine protease capable of destroying most connective tissue matrices (1-3). The a1AT gene encompasses seven exons and six introns over 12.2 kb’ of chromosome 14 at q31- 32.3 (4-8). The gene is highly pleomorphic; approximately 75 alleles have been identified. The two parental alleles are codominantly expressed and dictate the serum alAT levels

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 To whom reprint requests should be addressed Bldg. 10, Room 6D03, National Institutes of Health, Bethesda, MD 20892.

7 Francis B. Parker Fellow in Pulmonary Research. The abbreviations used are: kb, kilobase; IEF, isoelectric focusing;

bp, basepairs.

(1, 9, 10). The most common “normal” alAT alleles are M1(Va1213) (allelic frequency in United States Caucasians 44- 49%), M1(Ala213) (20-23%), M2 (14-19%), and M3 (10-11%) (13-15). Individuals inheriting any homozygous or heterozy- gous combination of these alleles have serum a1AT levels sufficient to provide anti-elastase protection to the fragile structures of the lower respiratory tract (1, 16, 18).

In contrast to these normal a1AT alleles, there are “defi- cient” alleles that are associated with detectable but low serum levels (1, 10, 12, 16, 17). The most common of these are Z (allelic frequency 1-2%) and S (3-4%) (10, 13, 14, 19); rarer deficient alleles include I (21). Mmalton (22, 23), Mdua* (24),

When inherited in certain combinations (e.g. ZZ, Mdua*Z), the serum alAT levels are sufficiently deficient to put the affected individual at risk for the development of emphysema, usually by ages 30-40 (1, 9, 10, 12, 16, 17). In the context of the knowledge that alAT provides 95% of the anti-elastase protection to the lower respiratory tract (30, 31), the emphy- sema associated with this deficiency state is thought to de- velop secondary to insufficient protection of the alveolar wall from the low level burden of neutrophil elastase to which the lower respiratory tract is chronically exposed (1, 30-33).

The basis for two of these deficiency alleles, Z and S, has been defined (see Refs. 12, 17, and 34 for reviews). The purpose of the present study is to characterize the gene and protein of a newly recognized a1AT deficiency allele, “Mprocide.”

Mheerlen (25), Make (26), M,,,, (27), P (28), and Zaugsburg (29).

MATERIALS AND METHODS

Study Population-The Mpmcid allele was identified in a family that also carries a Null a1AT gene. One individual, a 44-year-old male (identified as the index case), inherited both the Mpmeida and Null genes, allowing characterization of the Mpmib protein. This individual developed increasing dyspnea on exertion 5 years prior to evaluation. The individual had a 15-pack/year smoking history. The presence of emphysema was documented by physical examination showing a barrel chest and distant breath sounds; chest x-ray dem- onstrating hyperinflation and a loss of vascular markings in the lower lung fields; 133Xe ventilation scan showing abnormal retention in the bases; ssTc-macroaggregated albumin perfusion scan demonstrating decreased vascularity in the same regions; and pulmonary function tests (35) revealing a vital capacity of 82% predicted, total lung capacity 123% predicted, forced expiratory volume in 1 s (FEVl.0) 23% predicted; FEVl.,Jforced vital capacity 42% predicted, and dif- fusing capacity (corrected for volume and hemoglobin) 49% predicted. Evaluation of serum revealed an a1AT level of 8 mg/dl and isoelectric focusing (IEF) using an immobilized pH gradient demonstrated an M pattern (see below). There was no evidence of liver disease on physical examination and liver function tests and histological exam- ination of the liver demonstrated no accumulation of a1AT in hepa- tocytes.

The family members of the index case available for evaluation included his mother, five siblings, spouse, and a son (Fig. 1). Using

15528

a lAT Allele Mprocda 15529

serum IEF and serum alAT levels, evaluation of the pedigree dem- onstrated that the index case was a heterozygote for the Mpmcib and Null alAT alleles. The deficient M-type allele was referred to as Mproeih using the birth place (Monte di Procida, Italy) of the index case as the designator (11).

alAT Phenotype Identification-Serum alAT phenotypes were determined by a combination of IEF evaluation of serum, serum alAT levels, and family analysis. The IEF of serum was carried out to achieve maximum separation of alAT “subtypes with a modifi- cation of the method of Constans et al. (361, combined with an immunofixation print using rabbit anti-human alAT antibody (Ac- curate Chemical and Scientific) as an enhancing technique (37). IEF was also performed using an immobilized pH gradient as described by Gorg et al. (38). Serum levels of a lAT were determined by radial immunodiffusion (Behring Diagnostics). The quantification of a lAT is complicated by the fact that the commonly used commercially available standards (e.g. Behring Diagnostics) yield values for amounts of alAT that are approximately 40% higher than the true values (18). Because most clinical studies have used the commercially available standards, we have based our serum values on both the commercial standard and a true laboratory standard (18). Values expressed as mg/dl are based on the commercial standard, and those expressed using micromoler units are based on the true laboratory standard (multiplying the commercial standard values by 0.71 cor- rects them to the true values).

Eualuation of the Function of Mpmeda Type alAT-Because the Mproeih allele was inherited with a Null allele in the index case, the only alAT protein in the serum was Mprmih, thus permitting char- acterization of the function of this rare a lAT variant. Purification of the alAT was accomplished by positive selection affinity chromatog- raphy, followed by a molecular sieve column, and then negative selection affinity chromatography (39). All assays were carried out using the most common form of a lAT (M1(Va1213) purified from M1(Va1213) homozygotes, n = 4), as a control. The ability of Mprocib type alAT to inhibit human neutrophil elastase was evaluated using two methods (39): 1) titrating increasing amounts of alAT against a fixed amount of human neutrophil elastase and incubating under conditions such that time and concentration are irrelevant (2 h, 23 “C) (this approach assesses what proportion of a1AT molecules in the purified preparation are functional, but does not evaluate the time and concentration dependence of the alAT-neutrophil elastase interaction); and 2) quantifying the association rate constant (KO) of the interaction (this approach assesses, for those alAT molecules capable of inhibiting neutrophil elastase, the kinetics of the interac- tion that are dependent on concentration and time). Titration of a lAT Mprmih against a fixed amount of neutrophil elastase was assessed as described by Ogushi et al. (39) while the KO of the interaction was measured using minor modifications of the method of Beatty et al. (40) as described previously (39).

Eualuation of the in Vivo Half-life of Mp,&-The serum half-life of the Mpmib-type alAT was compared to normal M1(Va1213) type alAT in mice as described previously (41). Both the Mpmeih and the M1(Va1213) types of purified alAT were lZ5I-labeled by the chloramine- T method (42). To determine the serum half-life of each preparation, 6-8 week-old BALB/c mice weighing 15-20 g were injected intrave- nously in the tail vein with 8 X lo4 dpm/g body weight. Blood was obtained at 5 min, 4 h, 8 h, 1 day, 3 days, 4 days, and 5 days after injection. The amount of a lAT remaining in the serum at each time point was determined in triplicate (one value/mouse). To accomplish this, trichloroacetic acid was used to precipitate the proteins from 100 p1 of serum using glass microfiber filters (Whatman GF/C), the filters were washed, and radioactivity on each filter measured by liquid scintillation spectroscopy (41).

Isolation of the MPmh alAT Gene-Until recently, it was thought that the alAT gene was comprised of five exons and four introns (4). However, recent studies have revealed two additional exons 5’ to what was thought to be exon “I” (5-7). Thus, as it is now understood, the alAT gene is comprised of seven exons and six introns. To avoid confusion with the past nomenclature concerning alAT gene struc- ture, we have maintained the nomenclature of Long et al. (4), referring to the last four exons as exon “11-V,” and refer to the exons 5’ to exon I1 as exons ‘‘IA,’’ ‘‘IB,” and ‘‘IC,” i.e. “IC” includes exon I of Long et al. (4) plus an additional 5’ 50 bp, while IA and Ie are two exons newly identified by Perlino et al. (5).

The Mprmib alAT gene was cloned from a genomic DNA library of the index case, using methods described previously (43). In brief, high molecular weight genomic DNA was obtained from blood leukocytes by the method of Jeffreys and Flavell (44). Genomic DNA (100 pg)

was partially digested with Sau3AI and size fractionated using a 4- 20% NaCl density gradient. Fractions containing fragments larger than 15 kb were ligated into the X-phage EMBL3 (Stratagene). Starting with lo6 X-phage plaque-forming units, the library was screened with 32P-labeled 1.9-kb StuI fragment of the alAT gene encompassing the exon Ig and IC area, a 1.1-kb PstI fragment encom- passing the exon V area, and 1.4-kb full length alAT cDNA as the probes. Five full length alAT clones were obtained and one of these clones (18 kb in length, designated at XATMpmi&3.0) was chosen for further analysis.

Evaluation of the Overall Structure of the Mpmeda alAT Gene-The overall structure of the cloned Mpmib alAT gene was evaluated using restriction endonuclease mapping and Southern blotting with 32P- labeled full length cDNA as the probe (45). For comparison, a cloned M1(Va1213) alAT gene (15) was analyzed in parallel. Seven restriction endonucleases, AuaI, BamHI, BglI, EcoRI, PstI, PuuII (all from Bethesda Research Laboratories) and BstEII (New England Biolabs) were used under the recommended conditions.

Inheritance of the Mpm& alAT Gene-Evaluation of Mpmih a1AT clone with restriction endonuclease mapping demonstrated that the Mproeih gene had a mutation in exon I1 resulting in a loss of a restriction site of endonuclease PuuII (see “Results”). Genomic DNA from family members was evaluated using this restriction fragment length polymorphism. To accomplish this, genomic DNA was digested with the restriction endonucleases PuuII and PstI and analyzed with Southern blotting using 32P-labeled exon I1 (PstI fragment, 1.64 kb) of the normal alAT gene as the probe.

Sequencing of the Mpmda alAT Gene-Sequencing of the major promoter region and its associated noncoding exon (Ic), all coding exons and the associated exon-intron junctions of the Mpmeib alAT gene, was carried out by digesting XATMp,,id.18.0 into four fragments with PstI (0.5-, 1.6-, 2.4-, and 1.1-kb fragments, encompassing exon I,, exon 11, exon I11 and IV, and exon V, respectively, see represen- tation of the gene in Fig. 5B). The fragments were subcloned into pUC13 and sequencing was carried out in both directions by the dideoxy chain termination method with synthetic bidirectional oli- gonucleotide primers (46).

RESULTS

Identification of the alAT Allele Mp,~a-A family study demonstrated that the a l A T phenotype of the index case was that of a heterozygote with an Mprocida and a Null gene (Fig. 1). The Mprocida protein migrated to a position cathodal to M1- type a l A T but anodal to the M3 allele on an immobilized gradient at pH 4.45-4.90 (Fig. 2). Immunofixation of the immobilized pH gradient gel confirmed the identity of the alAT bands (not shown). The presence of a Null allele in the family was identified in one sibling of the index case who exhibited an IEF pattern of an M2 but had a serum level much lower than an M2 homozygote. Consistent with this, a

M2M2 M2M2 M2M2 M2M2 MPNull / M1 M3

MlNuII Unknown

FIG. 1. Pedigree of a family with the a lAT MPrw,h allele. The phenotype of each individual was determined by a combination of isoelectric focusing of the alAT protein in serum, serum alAT levels, and analysis of the pedigree. The serum a1AT levels (mg/dl) are shown within the symbols (0 males; 0 females), and the pheno- types are shown below the symbols. The index case (indicated with an arrow) inherited the Mpmih gene along with a Null a lAT gene; the Null gene in this family resulted from a deletion of the entire a lAT gene (see text). The phenotype of the deceased father of the index case (in brackets) was derived from analysis of the pedigree. One son of the index case was also deceased; the phenotype is unknown.

15530 alAT Allele MDrocdn Index

M2M2 M1M3 Case

\ I I I .

+.- procida *, 2

7 .

1 2 3

FIG. 2. Demonstration of the IEF pattern of a lAT type Mpraid. in serum. IEF was carried out using an immobilized pH gradient to achieve maximum separation of a lAT "M" subtypes (38). The anode (+) is at the top and cathode (-) at the bottom. The brackets indicate the two major IEF bands for M-type a lAT alleles (referred to as the M, and Me bands, see Refs. 1,9, 10). IEF pattern of the standards are indicated at the top of each lane. Lune I , M2M2; lane 2, M1(Va1213)M3; lane 3, index case. Note that the 4 and 6 bands of the index case (lane 3) are more cathodal than Ml(Val*13) and more anodal than M2 and M3, thus defining the IEF phenotype as "M."

son of the index case exhibited the IEF pattern of an M1 homozygote but actually had the phenotype M1 Null, i.e. the individual inherited the M1 allele from his mother, inherited a Null allele, but not the Mpmib allele from the index case, and had a serum a l A T below the range for an M1 homozygote. Thus, the profound deficiency state of the index case must result from the heterozygous inheritance of Mpmib (from the mother) and Null (presumably from the deceased father).

Functional Activity of M,,,aa-Titration of increasing amounts of Mpmida serum against a fixed amount of neutrophil elastase for a sufficient amount of time for all functional nlAT molecules present to interact with neutrophil elastase demonstrated that, given enough time, Mpmib appeared to function in a fashion similar to that of a normal M1(Va1213) homozygote Mpmida: 92 k 1% of the a lAT molecules were active; M1(Val2I3): 95 k 1% active; p > 0.2): However, when evaluated in a time-dependent fashion, it was apparent that the Mproeida molecule had a mild, but significant, reduction in its activity to function normally as an inhibitor of neutrophil elastase. In this regard, quantification of the K association of purified Mproeih a l A T for neutrophil elastase was less than that of normal M1(Va1213) type a l A T (Mpmih 7.0 f 0.1 X lo6

s-', M1(VaP3) 9.3 k 0.8 X lo6 M" s-'; p < 0.01) (Fig. 3). In Vivo Half-life of the Mpmaa Protein-Intravenous admin-

istration of the '251-labeled Mpmid protein resulted in a grad- ual time-dependent reduction in the serum levels of the pro- tein (Fig. 4). This behavior was typical of a1AT and was similar to that of 12sI-labeled M1 normal a1AT protein ad- ministered to mice in parallel (T1/2Mpmi~ 1.1 days; M1 1.1 days).

Cloning and Overall Gene Structure of MprOcan a l A T Gene- Five full length a1AT gene clones were obtained from the genomic DNA library of the index case. To identify the Mpmib gene, restriction mapping of all clones was evaluated in com- parison to the normal M1(Val2I3) gene using the restriction endonucleases, AuaI, BarnHI, BglI, BstEII, EcoRI, PstI, and PouII together with a full length cDNA as the probe (Fig. 5). In the analysis with the enzyme PvuII, while the normal

' All data are expressed as mean k S.E. of the mean; all statistical comparisons were made using the two-tailed student t test.

z 20

I I 5 10 1'5 '*

Time (min)

FIG. 3. Time-dependent inhibition of neutrophil elastase by alAT-type Mproel,+.. Purified alAT type Ml(VaIz1') (0) or alAT type Mpmib (0) was incubated with an equivalent amount of neutro- phil elastase for the times indicated and the residual elastase activity quantified. Shown for each time point is the mean k standard error of the mean for triplicate determinations.

FIG. 4. Evaluation of the in vivo half-life of Mpmel&. Purified a1AT type M1 and Mpmib was labeled with 12sII, administered intra- venously to mice, and the serum levels of T - a l A T quantified. The half-life of the two molecules are similar.

M1(Va1213) gene contained 2.7- and 0.2-kb fragments in the region 5' to and including part of exon 11, all five clones of the index case had instead a 2.9-kb fragment (Fig. 5A, lanes 13 and 14, Fig. 5B, line labeled PvuII, "*" identifies the PvuII site missing in the five clones). To determine whether this loss of a PvuII recognition site in exon I1 was characteristic of the Mpmib gene or the Null gene of the index case, total genomic DNA obtained from the mother (phenotype M2Mpmih), a sibling (M2Null) and the son (MlNull) was analyzed by restriction mapping with double digestion of PvuII and PstI (Fig. 6: PvuII polymorphism is best visualized using both enzymes, see figure for description). The polymor- phism was present in the mother of the index case but not in his sibling or son. Thus, all clones obtained from the index case had to be derived from the Mpmih alAT gene and could not have been derived from the Null gene.

Evaluation of the restriction mapping of one of the Mpmih clones (XATMpmi&3.0) demonstrated that the overall struc- ture of the Mpmih a l A T gene was similar to that of M1(Val2I3) except for the PuuII polymorphism (Fig. 5A). Importantly, the BglI pattern (lanes 5 and 6) showed that all the major 5' encoding exon (IC) and all four coding exons were present and

15531

111.

2.6- " 2.0- - 0.7-

1

B.

5.8-

0.8-

7 6 . 0 - h m 7.6-- "- FF 1o.e I r .- -77

4.0- 4.1- - -

2.0- 1.5- 1.3' 0.9" -

0.2" I

2 3 4 5 6 7 8 9 10

2.4- m 0

1.6- a 1.1- 6 "

2.93 2.4- 1 .E-

l l 12 13 14

IV v

2.7

0.2

ATG(starl) TAA(stop)

-2 0 2 4 6 8 10 12 14 16 kb I . I . I . I . I , I , I . I

grossly intact (BgZI cuts in the introns 5' to exon Ig, 11, 111, IV, and V of the a lAT gene and does not cut in any exon). In addition, analysis with the enzyme BstEII suggested that the Mpmeida a l A T gene was on the background of the M1(Va1213) allele (BstEII cleaves in the sequence coding for residue 213 in the mature M1(Ala2I3) gene; all a l A T alleles identified to date are either VaP3 or Ala2I3, see Ref. 15).

Sequence of the Promoter Region, Coding Exons, and Exon- Intron Junctions of the Mpmao Gene-Sequencing the major promoter region (the region 5' to exon IC) and all coding

exons together with their respective exon-intron junctions of the Mpmih a1AT gene demonstrated that it was identical to those regions of the normal M1(Va1213) a1AT gene except for a single nucleotide substitution in exon I1 (Fig. 7). Of the total 1938 bp sequenced (including 201 bp flanking and in- cluding exon IC, 798 bp flanking and including exon 11, 361 bp flanking and including exon 111, 300 bp flanking and including exon IV, and 278 bp flanking and including exon V), all were identical to the normal M1(Val2I3) a1AT gene except for the single base substitution (T+C) in exon 11. This

15532 a l A T Allele MPmcido

A. 39 40 41 42 43

A. 0.14 Leu Ala HIS

Mpoclaa C G C C A G C C G G C A C A C Arg Gln Pro Ala His 39 40 41 42 43

+*+ Exon II P” P” I

1 2 3

FIG. 6. Demonstration of the transmission of the MPmid. mutation using the exon I1 PuuII restriction fragment length polymorphism. A , the exon I1 of a l A T M l ( V a P ) gene showing the restriction sites for the endonucleases PuuII (CAG/CTG, blunt end, indicated as Pu) and PstI (indicated as Ps). As a result of the mutation in exon I1 of the Mpmida gene (CTG in M1(VaP3) to CCG in Mprocib), a PuuII restriction site is lost. Although PuuII specifically identifies the exon I1 MPmih mutation, the difference is best visualized by double digestion with PuuII and PstI. After digestion with these two enzymes and hybridization with a 1.64-kb exon I1 genomic probe, 3 fragments (5’ to 3‘: 0.97,0.21, 0.46 kb) should be seen in M1(VaIzL3), while Mpmida would only reveal 2 fragments (5’ to 3’: 1.18, 0.46 kb), a pattern that can be easily distinguished by Southern analysis. B, genomic DNA (5 pg/lane) of each family member was digested with PuuII and PstI, and analyzed by the Southern technique using a 1.64- kb exon I1 probe as indicated in panel A. hne 1, genomic DNA from the index case; note the presence of 1.18-kb band and loss of the 0.97- kb band. Lune 2, genomic DNA from the mother of the index case, both the 1.18 and 0.97-kb bands are present, consistent with the heterozygous state M2Mp,ib. Lane 3, genomic DNA from the son of the index case; the presence of the 0.97-kb fragment and absence of the 1.18-kb fragment are consistent with the MlNull state. The band with faint density a t 0.85 kb (indicated by an arrow) is due to an incompletely digested fragment that is not relevant to the point mutation. The expected fragments of 0.46 and 0.21 kb presumably do not bind to a nitrocellulose filter efficiently.

single base change results in the replacement of leucine (C_TG) at residue 41 by proline (CCG). In addition, this single base substitution (CAGC_TG-&AGCCG) explains the loss of the restriction site for the endonuclease PuuII (CAG/CTG, blunt end) in exon 11, that causes the restriction fragment length polymorphism with PuuII (Figs. 5 and 6). Since the restriction mapping with BstEII and the sequence data demonstrated that the sequence surrounding residue 213 was identical to that of the M1(Va1213) gene, the identity of the “background” allele of the Mpmids gene was that of normal M1(VaP3) gene. Thus, taken together, the sequence data demonstrated that the coding region of the Mpmida gene can be formulated as M(M1(Va1213), Leu4’+Pro4’).

DISCUSSION

The a1AT deficiency genes code for a1AT protein that is detectable in serum but in amounts insufficient to protect the

0.21 0.10 0.15 0.27 0.21 1.40 t 1, 5.31 0.65 1.45 0.27 1.26 (0.82 f

1-H- -“I

ATG TAA (start) (stop) - - - - ,Regions

sequenced B.

40 C f Gln 40

42 Ala C A

C Ala 42 A C A His 43 \: Gln 44

FIG. 7. Identification of a mutation in the a lAT Mpmcid. gene causing a single amino acid replacement. A , the overall structure of the Mpnrida gene showing the size of the exons and introns and the major promoter region. The exons are shown as rectangles (Ia to V) and introns as lines between the exons. The size of each exon and intron are indicated above the gene. The regions sequenced (indicated by solid bars) were included in four PstI fragments subcloned into pUC13. B, the sequence of the a1AT Mpmi* gene in the region different from the normal a l A T M1(Va1213) gene. Shown are the autoradiograms of the sequencing gels of the region in exon I1 con- taining the single-base substitution in Mprocib. Left, the normal Ml(Valz13) sequence around Leu4’ in exon 11. Right, the sequence of the same region of the Mpmib gene. Shown for each gene are the four lanes representing the nucleotides C, T, A, and G, respectively. The nucleotide sequences are indicated with the corresponding amino acids and residue numbers. In the Mpmcib gene, nucleotide “T” (in- dicated by “*”) is substituted by “C” (indicated by “*”) in the codon for Leu“(CTG), causing a single amino acid change to Pro4‘(CCG).

lower respiratory tract from progressive destruction by its burden of neutrophil elastase (1, 10, 12, 16, 17). Although the two parental a1AT genes are codominantly expressed, these deficiency alleles have clinical importance only when inher- ited in a homozygous fashion with another deficiency allele, or with a Null allele (1,lO-12,16, 17). Mpmid, the a lAT allele that is the subject of the present study, is a new member of the family of a l A T deficiency alleles.

Like all other deficiency alleles described to date, Mpmid is identical to a normal a1AT allele except for a single-base substitution in a coding exon (see Table I for a summary of the deficiency alleles). Several lines of evidence suggest that this mutation (CTG Leu41 in the normal a1AT gene+CCG Pro41 in Mpmib) is the cause of the deficiency state associated with this allele. First, in the major promoter region and all coding exons with their corresponding exon-intron junctions, the T+C substitution in the codon for residue 41 was the only difference between Mpmida and the normal M1(Val2I3). Second, except for the restriction fragment length polymor- phism recognized by PuuII which identifies the single nucleo- tide substitution in exon 11, evaluation of the overall structure approximately 18 kb encompassing the Mpmih gene by restric- tion endonuclease mapping revealed no major deletions, ad- ditions, or rearrangements. Third, the Leu4’-Pro4’ substitu- tion involves two uncharged amino acids, a fact that is con- sistent with the observation of a very small difference in electrophoretic mobility between Mpmida and the common normal M1(Va1213) protein, suggesting no other amino acid

d A T Allele Mprocida 15533

TABLE I al-Antitrypsin "deficiency" alleles

alAT alleles that code for alAT detectable in serum, but in amounts insufficient to protect the lower respiratory tract from progressive destruction from its burden of neutrophil elastase. See text for references to each deficiency allele.

Allelic Function as

"Base" Difference an inhibitor Half-life Accumulation

elastase

Likely Subgroup *'lele frequency" normal allele f~~~~~ of neutrophil in uiuob in hepatocytes deficiency mechanism state of

Common Z 0.01-0.02 M1(Ala213) G l ~ ~ ~ ' + L y s Decreased Normal Yes Loss of salt bridge, intracellular aggregation

S 0.02-0.03 M1(Va1213) G1u2"+Val Decreased Normal No Loss of salt bridge, intracellular degradation

"family Mpmid rare M1(Va1213) Leu"+Pro Decreased Normal No Intracellular degradation

degradation Mheerlen rare M1(Ala2I3) Pr~~~ '+Leu ? ? No Intracellular

Mrnalton rare ? ? ? Normal Yes ? Mduarte rare ? ? ? ? Yes ?

M,,., rare ? ? ? ? No ? M i k e rare ? ? ? ? ? ?

Others P rare ? ? ? ? ? ? Zaueburg rare ? ? ? ? ? ? I rare ? ? ? ? ? ? For Caucasians of Northern European descent.

* See Refs. 55 and 56.

substitution is involved. Fourth, evaluation of the crystallo- graphic structure of alAT (47) suggests the single amino acid substitution of proline for leucine at residue 41 likely disrupts a-helix A comprised of the sequence P r ~ ~ l + S e r ~ ~ . Since pro- line does not support a helix (48), this mutation likely changes the tertiary structure of the alAT molecule. In this context, and with knowledge that the in vivo half-life of Mpmid is similar to the normal M1(Va1213) molecule, the most likely explanation for the profound deficiency state associated with Mproeih is that the molecule is degraded intracellularly prior to secretion. Consistent with this concept, analysis of the liver of the index case demonstrated no intracellular accumulation of alAT in hepatocytes. Thus, although we cannot definitely rule out small deletions, additions, or rearrangements in in- trons or 5' regions of the Mproeib gene not sequenced, it is not necessary to hypothesize any additional mutations to explain the alAT deficiency state.

Interestingly, the single amino acid substitution in the Mpmid protein causes a mild reduction in the association rate constant of Mpmih for human neutrophil elastase. While this difference (normal 9.3 f 0.8 X lo6 M" s-', Mprocid 7.0 f 0.1 X lo6) seems to be mild, when put in the context of the markedly reduced levels of Mprocih, the "in uiuo" inhibition time (for the actual in uiuo concentration of a protease inhibitor, the cal- culated time it would take to inhibit >95% of an equal concentration of its target protease) would be markedly in- creased. In this regard, although the index case was too ill to permit sampling of lower respiratory tract epithelial lining fluid, it is reasonable (18, 30, 31) to assume that the lung levels of Mprocida were approximately 0.11 FM. In that case, with an association rate constant in the range of 7.0 X lo6 M-' s-', the in vivo inhibition time would be approximately 7.1 s, 60-fold longer than that of alAT in the lungs of normal individuals (0.12 k 0.04 s) (49). Thus, together with the profound deficiency state associated with Mpracida, the muta- tion in this form of a1AT also renders the molecule relatively impotent in providing a protective anti-neutrophil elastase protective shield for the lung.

In the context of this conceptualization of the pathogenesis of the deficiency state associated with the Mpmih gene, the reduced serum levels of alAT associated with Mpmid likely occurs by mechanisms different from that associated with the common Z type of alAT deficiency. In this regard, crystallo- graphic analysis suggests that the Z gene G l ~ ~ ~ ~ + L y s muta- tion prevents the formation of a critical internal salt bridge ( G l ~ ~ ~ ~ - L y s ~ ~ ) (47), although recent evidence (50) suggests that this does not explain the entire molecular pathogenesis of the deficiency state. However, whatever the exact mecha- nism, the Z-type mutation clearly causes the newly synthe- sized a1AT protein to aggregate in the rough endoplasmic reticulum, probably through interactions of hydrophobic res- idues that are normally hidden in the three-dimensional form of the protein (47, 51). Consequently, there is reduced trans- location to the Golgi, resulting in reduced secretion of the protein and the low serum a1AT levels (see Refs. 34, 43, and 51 for reviews). In addition, for those Z-type molecules that are secreted, like Mpmih but even more so, the Z-type alAT molecules do not function normally as inhibitors of neutrophil elastase (39), likely because of the differences in the tertiary structure of the molecule.

In contrast to the differences in the pathogenesis of the deficiency state associated with the Z gene, the pathogenesis of the deficiency that characterizes Mpmih shares some mech- anisms with the deficiency associated with the common S deficiency gene and the rare Mheerlen gene. Like Mpmih, the S mutation is not associated with intracellular accumulation of a1AT in alAT producing cells (1, 16, 52), probably because the S protein (M1(Va1213) G&4 G1uZa+S GTA Val264) is less stable and more susceptible to intracellular degradation (52). Furthermore, similarly to Mprocih, the S protein has a mildly reduced function as an inhibitor of neutrophil elastase (49). The Mheerlen variant results from a single-base substitution of the normal M1(Ala213) gene (M1(Ala213) C_CC Pro369+Mheerlen CTC Led6') (25, 53). Like Mpmih and S variants, there is no apparent accumulation of a1AT protein in the hepatocytes of

affected individuals, and it is probable that the Pro369+Le~369 substitution in Mheerlen also affects the intracellular stability of the alAT molecule.

For the other “rare” deficiency variants, including I (21), Mmalton (22,231, Mduarte (241, Mlik. (26), M,,, (271, P (281, and Zeugsburg (29), the molecular basis of their association with alAT deficiency is unknown. Furthermore, for the rare M- family deficiency alleles Mmaltan, Mduarte, MLike, and MrOuen, all of which represent only one or two reported cases and all of which migrate on IEF gels in closely related positions, it is conceivable that some are identical to Mpmi& or Mheerlen.

In comparison to all of the alAT deficiency variants that

15534 d A T Allele Mpmcda 13. Kueppers, F., and Christopherson, M. J. (1978) Am. J. Hum. Genet. 3 0 ,

14. Dykes, D. D., Miller, S. A., and Polesky, H. F. (1984) Hum. Hered. 3 4 ,

15. Nukiwa, T., Brantly, M. Ogushi, F., Fells, G., Satoh, K., Stier, L., Courtney,

16. Morse, J. 0. (1978) N. EngL J. Med. 299,1045-1048,1099-1105

18. Wewers, M. D., Casolaro, M. A., Sellers, S. E., Swayze S. C., McPhaul, K. 17. Carrell, R. W., and Owen, M. C. (1979) Essays Med. Biochem. 4,83-119

M., Wittes, J. T., and Crystal, R. G. (1987) N . Engl. >. Med. 316.1055-

359-365

308-310

M., and Crystal, R. G: (1987) Biochemistry 26,5259-5267

I ne2 19. Evan& H. E., Bognacki, N. S., Perrott, L. M., and Glass, L. (1977) J.

20. Pierce, J. A., Eradio, B., and Dew, T. A. (1975) JAMA 231,609-612 21. Arnaud, P., Colette, C.-C., Vittoz, P., and Fudenberg, H. H. (1978) J. Lab.

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have been characterized at the gene level, the newly recog- nized Mpmi& allele is the only mutation that can be identified at the genomic DNA level with a restriction fragment length polymorphism localized at the site of mutation. Although the overall frequency of this newly identified allele is unknown, the polymorphism permits rapid detection for epidemiologic purposes as well as for prenatal diagnoses.

From the viewpoint of alAT gene evolution, alAT variants can be categorized into two groups; the variants derived from the “oldest” human alAT gene ‘‘M1(Ala213)’’, such as Z, Mheerlen, and Nullg,enite falls, and those derived from the “newer” alAT gene “M1(Va1213)”, such as M3, M2, s, and Nullhellingham (12, 15, 54). In this regard, since the Mpmi& gene was derived from the allele M(Va1213), it is reasonable to conclude that it represents a mutation that occurred, in evolutionary terms, relatively recently.

Acknowledgments-We would like to thank P. Arnaud, University of South Carolina, for his helpful suggestions relating to the isoelectric focusing identification of a lAT variants. We also thank L. Sichert for excellent editorial assistance.

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