Vol. 10, No. 1MOLECULAR AND CELLULAR BIOLOGY, Jan. 1990, p. 47-560270-7306/90/010047-10$02.00/0Copyright © 1990, American Society for Microbiology

Molecular Basis of otl-Antitrypsin Deficiency and EmphysemaAssociated with the ot-Antitrypsin Mminerai sprngs Allele

DAVID T. CURIEL, CLAUS VOGELMEIER, RICHARD C. HUBBARD,LARUE E. STIER, AND RONALD G. CRYSTAL*

Pulmonary Branch, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892

Received 12 June 1989/Accepted 26 September 1989

The Mjj,wr spr, al-antitrypsin (alAT) allele, causing alAT deficiency and emphysema, is unique amongthe alAT-deficiency alleles in that it was observed in a black family, whereas most mutations causing alATdeficiency are confined to Caucasian populations of European descent. Immobilized pH gradient analysis ofserum demonstrated that calAT M,,,,,ea, prig migrated cathodal to the normal M2 allele. Evaluation ofM,n,,.r.a sprigs alAT as an inhibitor of neutrophil elastase, its natural substrate, demonstrated markedly lowerthan normal function. Characterization of the alAT M,inj,ter sprin, gene demonstrated that it differed from thecommon normal Ml(Ma213) allele by a single-base substitution causing the amino acid substitution Gly-67(GGG)->Glu-67 (GAG). Capitalizing on the fact that this mutation creates a polymorphism for the restrictionendonuclease AvaIl, family analysis demonstrated that the M,,,...I springs alAT allele was transmitted in anautosomal-codominant fashion. Evaluation of genomic DNA showed that the index case was homozygous forthe alAT Mnarwsp. ins allele. Cytoplasmic blot analysis of blood monocytes of theM . ng homozygotedemonstrated levels of alAT mRNA transcripts comparable to those in cells of a normal Ml(Val2_3)homozygote control. Evaluation of in vitro translation of M,,i,r spigs alAT mRNA transcripts demon-strated a normal capacity to direct the translation of alAT. Evaluation of secretion of alAT by the bloodmonocytes by pulse-chase labeling with [35S]methionine, however, demonstrated less secretion by theMn,jn,e sprigs cells than normal cells. To characterize the posttranslational events causing the alAT-secretorydefect associated with the alAT M,,i,,er, pi gene, retroviral gene transfer was used to establish polyclonalpopulations of murine fibroblasts containing either a normal human Ml alAT cDNA or an Mnjn.,w sp..ngsalAT cDNA and expressing comparable levels of human alAT mRNA transcripts. Pulse-chase labeling ofthese cells with [35S]methionine demonstrated less secretion of human alAT from the Mnin,a spr.igs cells thanfrom the Ml cells, and evaluation of cell lysates also demonstrated lower amounts of intracellular human alATin the Mmineral springs cells than in the normal Ml control cells. Thus, the Gly-67--Glu mutation thatcharacterizes Mn,..,* spring. causes reduced alAT secretion on the basis of aberrant posttranslational alATbiosynthesis by a mechanism distinct from that associated with the alAT Z allele, whereby intracellularaggregation of the mutant protein is etiologic of the alAT-secretory defect. Furthermore, for the alAT proteinthat does reach the circulation, this mutation markedly affects the ability of the molecule to inhibit neutrophilelastase; i.e., the alAT Mnan spng allele predisposes to emphysema on the basis of serum alAT deficiencycoupled with alAT dysfunction.

al-Antitrypsin (alAT) is a 52-kilodalton (kDa) serumglycoprotein produced by hepatocytes, mononuclear phago-cytes, and neutrophils that functions as the principal inhib-itor of neutrophil elastase (NE) in the lower respiratory tract(6, 7, 8, 12, 25, 26, 30). In alAT deficiency, an autosomalrecessive hereditary disorder, insufficient amounts of alATallow unimpeded elastolytic destruction of the pulmonaryparenchyma, with the consequent development of emphy-sema in the third to fourth decades (6, 7, 8, 12, 25).The alAT gene is 12.2 kilobases long and is located on

chromosome 14 at q31 to q32.2 (36, 50). It consists of sevenexons: three 5' noncoding exons (IA to Ic) that are involvedwith initiation of transcription and four protein-encodingexons (II to V) (6, 36, 45). The oalAT gene codes for a418-amino-acid primary translation product that includes a24-amino-acid signal peptide cleaved during biosynthesis asthe protein moves into the rough endoplasmic reticulum,where oligosaccharide side chains are cotranslationallyadded (8, 18, 29, 34, 36, 48). The protein is then translocatedto the Golgi, where the oligosaccharide side chains are

* Corresponding author.

processed to the complex type and the mature alAT proteinis secreted (18, 34).The alAT gene is characterized by marked pleomor-

phism, with approximately 75 variants known (6, 12). TheoalAT M family of alleles includes those that code forproteins which migrate to the M region by isoelectric focus-ing (IEF) of serum at pH 4.2 to 4.6 (6, 21, 24, 32). Thecommon normal alAT M-type alleles include M1(Ala213),Mj(Val213), M2, and M3 (12, 40, 41). Homozygous inheri-tance of any of these alleles is associated with normal serumoalAT levels of 20 to 48 ,uM (6, 12, 57). Together, thesenormal M-type alleles comprise more than 95% of the normalotlAT alleles (6, 10, 12, 17, 21, 33, 49). There are at least 9deficiency-type otlAT alleles, i.e., alAT genes that code foralAT protein detectable in serum but in reduced amounts (6,12). When these alleles are inherited with other deficiencyalleles or with a null allele (alAT genes associated with nooalAT in serum), the affected individual has insufficientamounts of alAT to protect the lung against NE, andemphysema results (6, 12, 25, 57).

Extensive epidemiologic studies have demonstrated thatthe deficiency class of alAT alleles occurs almost ex-clusively in Caucasians of European descent (17, 21, 33,

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48 CURIEL ET AL.

M1Mmineralsprings21 ,M

II

1

Mmineral springsMmineral springs

9 ,uMFIG. 1. Inheritance of the alAT Mmineral sprngs allele. The phe-

notype of each family member was determined by a combination ofIEF of the alAT protein in serum, determination of serum oalATlevels, and analysis of the pedigree. The alAT phenotypes and thealAT serum levels are shown below the kindred members. Theindex case (II1) is an Mminerul sprngs homozygote. The serum levelsindicated are the lowest levels detected (see Results). The pheno-type of the deceased father of the index case (I,) is unknown. Thehomozygosity of the index case was proven by molecular analysisand quantification of alAT gene number (see text).

49). The purpose of this study was to characterize theMrninersi spings allele, an alAT variant associated with re-duced serum levels of alAT as well as reduced function ofthe molecule as an inhibitor, found in homozygous form inan American black individual with marked alAT deficiencyand emphysema.

MATERIALS AND METHODS

Study population. The alAT Mmineral spnngs allele wasdemonstrated in two members of a black kindred (Fig. 1). Noother family members were available for analysis. The indexcase, a 49-year-old male, had a 10-year history of dyspnea onexertion. He had a 15-pack-per-year smoking history andhad stopped smoking 10 years before evaluation. Severeemphysema was documented by physical examination dem-onstrating hyperresonant chest and distant breath sounds;chest X ray demonstrating hyperinflation, flattened hemi-diaphragms, and marked loss of vascularity at both bases;xenon-127 ventilation scan showing abnormal retention ofgas in the lower lobes and a technetium-99m perfusion scandepicting loss of vascularity in the same regions; pulmonaryfunction testing demonstrating a vital capacity of 47% pre-dicted, total lung capacity (body plethysmography) of 127%predicted, forced expiratory volume in 1 s (FEV1) of 31%predicted, FEV1/forced vital capacity of 86% predicted, anddiffusing capacity (corrected for volume and hemoglobin) of30% predicted. There was no evidence of liver disease.

Characterization of the alAT M ral.p s protein byIEF. The alAT phenotype of members of the study kindredwas characterized by a combination of IEF of serum,determination of serum alAT levels, and family analysis (10,21). The IEF analysis was carried out to achieve a maximumseparation of alAT M subtypes by a modification (40) of themethod of Constans et al. (9). Serum alAT levels weredetermined by nephelometry (Calbiochem-Behring nephe-lometry analyzer), using a true alAT laboratory standard(57).

Evaluation of the function of M ,a spigs alAT. Toevaluate the alAT Mmineral sp variant as an inhibitor ofits natural substrate, NE, t[heMminerai springs protein waspurified from plasma of the index case as previously de-scribed (44). To quantify the time-dependent NE-inhibitoryactivity of MminerW springs, a fixed amount of NE (2 nM) was

incubated with an equal amount of Misneraispngs alAT, andthe residual NE activity after various time intervals (0.5 to120 min) was measured. In addition, the association rateconstant of Mmineral spnngs for NE was determined by themethod of Beatty et al. (4) as modified by Ogushi et al. (44).Purified alAT from a normal Ml(Val213) homozygote wasused as a control.

Cloning and sequencing of the alAT Mmieralsping gene.The alAT Mminerai springs gene was cloned from genomicDNA of the index case by using a cosmid vector (2). Afterisolation, subclones containing individual exons plus flank-ing regions were generated and used as templates for se-quencing by the dideoxynucleotide-chain terminationmethod, using bidirectional primers of 15-mer oligonucleo-tides (55). Sequencing included 150 base pairs (bp) 5' to exonIc, exon Ic-V together with the intron-exon junctions, and 40bp 3' to exon V.

Demonstration of inheritance of the clAT Mnj,.rai spigsgene. Analysis of the otlAT Mminera springs mutation demon-strated that it generated a polymorphism in exon II of thealAT gene for the restriction endonuclease Avall, permit-ting a simple method of evaluating the presence of this allele.To accomplish this, genomic DNA was subjected to ampli-fication of exon II of the alAT gene plus flanking regions bymeans of the polymerase chain reaction (15). The amplifiedDNA was purified by gel electrophoresis with electroelution,followed by digestion with the restriction endonucleaseAvaII under recommended conditions; the results wereevaluated by agarose gel electrophoresis with ethidium bro-mide staining.

Evaluation of alAT gene copy number. Since the father ofthe index case was not available for analysis, the alATphenotype of the index case was consistent not only withhomozygosity for the alAT Mminera springs allele but alsowith hemizygosity of the Mminerad spings allele together withdeletion of the other parental alAT gene. To distinguishbetween these possibilities, the alAT gene copy number ofthe index case was determined comparing genomic DNA ofthe index case with that of a normal control containing twocopies of the alAT gene per diploid genome. To accomplishthis, genomic DNA of the index case and of a normal controlwith the phenotype M1M2 was subjected to digestion withthe restriction endonuclease PstI under recommended con-ditions. The digested DNA was denatured (0.4 N NaOH),various dilutions (2.5, 1.25, and 0.625 ,ug) were blotted ontoZeta-Probe membranes (Bio-Rad Laboratories), and theblots were hybridized with a 32P-labeled alAT probe encom-passing exon II of the alAT gene plus flanking regions (42).The resulting autoradiograms were quantified by densitom-etry (16).

Quantification of alAT mRNA transcript levels associatedwith the Mmijerai s gene. alAT gene expression associ-ated with the alAT Mminera springs allele was evaluated bydetermining the steady-state level of cytoplasmic alATtranscripts in blood monocytes, cells that normally expressthe alAT gene (39, 46). Blood monocytes were obtained bymonocytapheresis from the index case and an Ml(Val213)homozygote control, and cytoblot analysis was carried outwith hybridization using a 32P-labeled alAT cDNA probeand densitometric quantification of the resulting autoradio-grams (58).

In vitro translation of alAT Min sp. mRNA tran-scripts. The capacity of oalAT Mmineral springs mRNA tran-scripts to direct the translation of an alAT protein wasevaluated by preparing alAT mRNA transcripts ofMl(Val213) or Mmineral springs type, using the SP6 polymerase

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al-ANTITRYPSIN MrineXal spngS ALLELE 49

M4 3 3V=2 1

M6 33

1 2 3 4

.mineral springs

_ mineral springs

5

FIG. 2. IEF pattern of the otlAT Mirne< s allele in serum.IEF was performed by using an immobilized pH gradient to achievemaximum separation of aLlAT M subtypes. Positions of the anode(+) and cathode (-) are marked. Brackets indicate the regions of thetwo major IEF bands (M4 and M6) for M-type alAT alleles.Migration positions of the two major bands of the alATMminera springs allele are indicated on the right. Lanes: 1, MlMl; 2,M1M2; 3, M1M3; 4, family member I1i, Mminemi spinngsMminera springs;5, family member I2, MlMmin,eral springs,

in vitro transcription system (Promega Biotec) and plasmidSP64(polyA), containing an otlAT cDNA of Ml(Val213) orMmineral springs type, respectively, as the template (31, 38).Purified alAT mRNA transcripts (1.0 ,ug) were used todirect the synthesis of alAT, using reticulocyte lysate(Promega Biotec) and [35S]methionine as the label. Evalua-tion of the translation products was by sodium dodecylsulfate (SDS)-acrylamide gel electrophoresis, followed byfluorography and densitometric quantification.

Evaluation of secretion of alAT by blood monocytes. Thelevel of secretion of otlAT associated with the oalATMmineral springs gene compared with the normal level wasdetermined by [35S]methionine pulse-chase labeling of blood

monocytes of the index case and an Ml(Val213) homozygotecontrol. The supernatant of labeled cells was evaluated forlabeled, secreted alAT by immunoprecipitation, SDS-acryl-amide gel electrophoresis, and fluorography as previouslydescribed (39).

Evaluation of expression of human alAT Mmlijeri spgcDNA in murine fibroblasts. To reproduce the abnormalitiesof alAT biosynthesis associated with the alATMmineral springs gene, a human otlAT Mminerj springs cDNAwas transferred to murine fibroblasts by using the N2 retro-viral vector as previously described (1, 14, 27). To achievethis, human oalAT cDNAs ofMl or Mminera springs type werecombined into the N2 retroviral vector. These constructswere transfected into the 42 helper virus-free packaging cellline to generate recombinant ecotropic retrovirus capable oftransferring the human alAT cDNAs. The recombinantretroviruses were used to infect NIH 3T3 cells to generatepolyclonal populations of cells containing the Ml orM,niner-M spriotsalAT cDNA and expressing comparableamounts of human oalAT mRNA transcripts. Synthesis andsecretion of human alAT by the modified murine fibroblastsand the intracellular human aLlAT in the same cells wasevaluated by immunoprecipitation, SDS-acrylamide gel elec-trophoresis, and fluorography as previously described (14,27).

Statistical evaluation. Where indicated, error estimates aregiven as plus or minus standard error of the mean, and allcomparisons were made by using the two-tailed Student's ttest.

RESULTS

Identification of the alAT Ma,,,,,.p,h, allele. Familyanalysis demonstrated the presence of the oalATMmineral springs allele segregating in the study kindred (Fig.1). Evaluation of serum samples of the index case obtainedat various intervals over a 5-year period demonstrated oalATlevels of 9, 13, and 15 ,uM. Thus, the lowest level measured

0)n

CUCU

._

0o

C0

X-

C

0

L--

Ml (Val2l3)

Mmineral springs

5 10 15 30 60 120Time (min)

FIG. 3. Time-dependent inhibition ofNE by Mi,w r alAT.Equal concentrations (2 nM) of purified Mminer alAT and normalM1(Val213) alAT were incubated with an equivalent amount of active NE for the indicated times, and the residual NE activity was quantified.Shown for each time point is the mean and standard error for quadruplicate determinations.

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50 CURIEL ET AL.

was below the 11 ,uM threshold needed to protect the lung (6,12, 26, 57); analysis at other times revealed serum alATlevels above this level. Analysis using an immobilized gra-dient at pH 4.2 to 4.6 demonstrated that the Mmineral springsprotein migrated to a position cathodal to the M2-type alAT(Fig. 2). However, the migration of Mmineral springs wasanodal to the rare Mmalton-type alAT (not shown), thusdefining it as an M type of alAT, not an N type. Immuno-fixation of the immobilized pH gradient gel confirmed theidentity of the alAT bands (not shown).

Functional activity of M spner n oalAT. Evaluation ofthe Mminerai springs alAT as an inhibitor ofNE demonstratedthat it was markedly dysfunctional. In this regard, compar-ison of inhibition ofNE by equal amounts ofalAT over timerevealed that the Mminer spnngs alAT was much less effec-tive than normal Ml(Val ) alAT (Fig. 3). Evaluation of theinhibition curve revealed two features. First, as noted in theearly phase of the inhibition curve, the time-dependent rateof inhibition of NE by Mmineral springs was slower than thenormal control rate; when evaluated with equal amounts ofactive alAT and NE (2 nM), this value translates into anassociation rate constant of M neral sprngs alAT for NEmuch lower than that of Ml(Val2") alAT [5.8 x 106 ± 0.3 x106 M-1 s-1 for Mmneral springs versus 8.9 x 106 + 0.6 x 106M-1 s-1 for Ml(Val ' );p < 0.011. Second, even at 120 min(essentially infinity for the reaction), Mmineral springs was ableto inhibit only 14 ± 1% of the NE molecules, compared with92 ± 1% for the Ml(Val213) control (P < 0.001).

Identification of the alAT Mmineral spgs mutation. Cloningof the oLlAT Mnineral springs gene from genomic DNA of theindex case and sequence analysis of exons Ic-V, all intron-exon junctions, and the 5'- and 3'-flanking regions demon-strated identity to the normal Ml(Ala213) alAT gene exceptfor a single-substitution mutation in exon II that caused achange in the coded protein (Gly-67 [G_iG}-+Glu-67[GAG])(Fig. 4). This predicted amino acid change results in thesubstitution of the bulky, negatively charged glutamic acidfor the small, uncharged glycine in a helical region (helix B)in the hydrophobic core of the molecule (35).

Inheritance of the alAT Mm,i,eral springs allele. The alATMminerai springs mutation results in ablation of a recognitionsequence for the restriction endonuclease AvaIl in exon II ofthe alAT gene, providing a means for direct detection of themutation in genomic DNA (Fig. 5A). In this regard, ampli-fication of the exon II region of the alAT gene followed bydigestion of the amplified DNA fragment with AvaII demon-strated that the alAT Mmineial springs gene was inherited, asexpected from the serum analysis. In this regard, genomicDNA of an Ml homozygote control analyzed in this fashionyielded a restriction pattern characterized by DNA frag-ments of 400, 220, and 120 bp (Fig. 5B, lane 1). In contrast,analysis of the mother of the index case, phenotypicallyMlMmineral springs' demonstrated these same fragments plusa novel fragment of 620 bp (lane 2), resulting from the loss ofan AvaII restriction site. The index case showed only therestriction pattern expected from analysis of theMmineral springs gene (lane 3), demonstrating that an alATallele with a normal sequence at the Mmineral springs mutationsite was not present; this finding suggested that inheritanceof the alAT Mmineral springs gene was in an autosomal fashionfrom both parents.

Determination of alAT gene copy number. The alATphenotype of the index case, Mmineral springs' together withthe analysis of the genomic DNA demonstrating only thealAT Mmineral springs mutation was consistent with homozy-gosity for this mutant allele but also with hemizygosity and

IAA

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IA -LYI[3isigAaAA A'AAA

ehrlT x--.

m - _ m _BW rsA { _

D ts_

_t

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t_ > W z J_

riTt,- - r-

FIG. 4. Identification of alAT Mmineral springs mutation by se-quence analysis. (A) Schematic of the alAT gene indicating regionssequenced (a). Indicated are the start codon (ATG) and regioncoding for the signal peptide in exon II as well as the stop codon(TAA) and polyadenylation signal (ATTAAA) in exon V. (B)Evaluation of exon II of the alAT gene, coding for residues 65 to 69of a normal M1(Ala213) allele and the Mmineral springs allele. Substitu-tion of an adenosine for a guanosine (*) results in a change of theencoded protein of Gly-67--Glu-67, defining the Mmineral springs mu-tation. The remainder of the sequence of the Mmineral springs allelewas identical to that of the M1(Ala213) allele.

deletion of the alternate alAT allele. To distinguish betweenthese possibilities, the alAT gene copy number of the indexcase was compared with that of a normal control containingtwo copies of the alAT gene per diploid genome. By thisanalysis, the index case had a number of alAT gene copiesper diploid genome comparable to that of the M1M2 hetero-zygote control (P > 0.5) (Fig. 6). Thus, the index case mustbe an Mmineral springs homozygote, likely having inherited acopy of the mutant allele from both parents.

Evaluation of alAT mRNA transcripts associated with theatlAT Mminera spngs gene. To examine the effects of thealAT Mmineral springs mutation at the transcriptional level ofalAT gene expression, alAT mRNA levels were quantifiedin blood monocytes, cells that normally express the alATgene, from the index case and an Ml(Val2l3) homozygotecontrol. Cytoplasmic blot analysis of blood monocytes of theMmineral springs homozygote demonstrated steady-state cyto-plasmic levels of alAT mRNA transcripts similar to thatdirected by normal M1(Val213) cells (P > 0.5) (Fig. 7).The alAT Mminera, sprin mRNA transcripts were compa-

rable to normal Ml(Val 3) alAT mRNA transcripts inability to direct translation of alAT. In this context, in vitrotranslation of synthetic OalAT Mminerai springs mRNA tran-scripts directed the synthesis of a 47-kDa alAT proteinspecies corresponding to the nonglycosylated alAT primarytranslation product, similar to that directed by normalMl(Val213) alAT mRNA (3, 19, 54). Furthermore, theamount of alAT Mmineral springs protein generated was com-parable to the amount of alAT Ml(Val213) protein generatedfor the same amount of input alAT mRNA (P > 0.5) (Fig. 8).Thus, the abnormality of alAT biosynthesis associated with

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al-ANTITRYPSIN Mrnnnera sprngs ALLELE 51

Avail

MlI(Ala213)Leu56 GI 6 Thr68 Lys69..C TGGi CCA AAC.

M,neraisorras .CTGGG GACCAAG...Leu66 Glu67 Thr6 Lys69

l-o 740 bptl exon 11

---4~~~ If.- }

.4

1

3' 5'exon 113II _

Avall Avail

-1 12 2-* --- 2202o 400 - |--1 20 1j-I 620 a1

B.

bp

620 *

400 -0-

220 -

120

1 2 3FIG. 5. Demonstration of transmission of the atlAT MsXineral sprngs gene by capitalizing on an AvaII polymorphism located at the site of the

Mmineral springs mutation. (A) Strategy for detecting AvaII polymorphism in the Mmmer,w sprigS alAT gene by using polymerase chain reactionamplification of exon II of the alAT gene. Amplification primers flanking exon II of the alAT gene [exon II 5' (+) and exon II 3'(-)] wereused in combination with the polymerase chain reaction to generate a 740-bp DNA fragment encompassing the entirety of exon II of the alATgene plus flanking regions. Arrows below the schematic of the exon II region indicate AvaIl recognition sites in the normal Ml(Ala213) gene.Above the map are indicated the sequences of the normal Ml(Ala213) and Mmi,,.ner spngs alAT genes from residues 66 to 69. Underlinednucleotides in the Ml(Ala213) sequence indicate the recognition sequence for Avall. The Mmieral springs mutation [Ml(Ala213) Gly-67(GQC)M,iner sprng, Glu-67 (GAG)] results in the loss of an Avall site in this region. Below the exon II map are indicated the fragmentlengths (in base pairs) expected by this analysis. For the Ml(Ala213) gene, fragments of 120, 220, and 400 bp are expected; for Mmineral springs'

fragments of 120 and 620 bp should be generated. (B) Identification of the Minineral spnings gene in genomic DNA by using the AvaIIpolymorphism. After amplification of exon II, the DNA was digested with Avall and subjected to agarose gel electrophoresis. Arrows indicatesizes of DNA fragments. Lanes: 1, MlMl control [Ml refers to the Ml(Ala213) allele]; 2, M1Mmineral springs' family member I2; 3,Mmineral springsMmineral spings index case (II1). Detection of the novel 620-bp fragment indicates the presence of the alAT Mminernc springs gene.

the alAT Mmineral springs gene causing the alAT deficiencystate is likely to manifest at a posttranslational level of alATgene expression.

Abnormalities of alAT biosynthesis associated with thealAT M. . sP1, gene. The Mmjneraj sprigs cells secretedless alAT than did cells from an Ml(Val' ) homozygotecontrol (Fig. 9); i.e., despite the normal levels of alATmRNA transcript, the Mminera7 springs mutation was associ-ated with reduced secretion of alAT from cxlAT-synthe-sizing cells. To demonstrate that this abnormality was aconsequence of the Mmineral springs mutation per se and tocharacterize the abnormal intracellular events in alAT bio-synthesis associated with the alAT Mmninera springs gene,NIH 3T3 murine fibroblasts were modified by retroviral gene

transfer to contain human atlAT cDNAs of Ml or

Mminera springs type (Fig. 1OA). Polyconal cell populationscontaining the Ml or Mniner, springs human alAT cDNAsand expressing comparable levels of human alAT mRNAtranscripts (not shown; P > 0.5) were labeled with[35S]methionine and evaluated for intracellular and secretedhuman alAT. Cells containing the Mminera springs otlATcDNA secreted a 52-kDa human alAT protein but in loweramounts than did cells containing the normal Ml alATcDNA (Fig. lOB, lanes 1 and 2); i.e., the aLlATMmineral springs mutation was etiologic of the aLlAT-secretorydefect associated with this gene, independent of the synthe-sizing cell.To exclude differential uptake or turnover of the mutant

A.

5' 3'exon II5(+)

M1 (Ala213)M,nerai sDrings

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52 CURIEL ET AL.

z

KD

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index Case * *a)C-)

0

z

Kt-.

VI'auz_S

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L-

K

2 -

2 Qi

Genomic DNA blotted (kig)FIG. 6. Determination of aslAT gene copy number of the index

case. Genomic DNA of the index case (0) and M1M2 control (0)was digested with PstI and denatured, and equivalent amounts wereblotted on Zeta-Probe membranes in serial dilutions (2.5, 1.25, and0.625 ,ug); the blots were hybridized by using a 32P-labeled ailATexon II probe, and the resulting autoradiograms were quantified bydensitometry. Ordinate is densitometric units of alAT DNA de-tected; abscissa is micrograms of DNA blotted. Inset shows exam-ples of the analysis.

protein as the basis for the observed secretory defect,35S-labeled Mminera s and Ml(Va1213) aclAT producedby the respective modified murine fibroblasts was exposed tounmodified NIH 3T3 cells for 24 h, and the remaining, intactalAT was evaluated by immunoprecipitation, SDS-acryl-amide gel electrophoresis, fluorography, and densitometricquantification (37). By this analysis, the amount of oalATremaining after exposure to unmodified murine fibroblastsdid not differ from a comparable starting amount of unex-posed alAT for both the Mminerad springs and Ml(Val2l3)alAT (not shown; P > 0.5). Thus, the reduced secretion ofhuman alAT by the murine fibroblasts containing theMminerW sps alAT cDNA compared with secretion bycells containing the normal Ml(Val2l3)aAT cDNA was notthe result of differential uptake or turnover.

Analysis of the cell lysates directly after the labelingperiod demonstrated a 50-kDa intracellular alAT, a speciescorresponding to the immature rough endoplasmic reticu-lum-associated protein with high mannose-type oligosaccha-ride side chains (34). Whereas both the Ml andMminer-l springs alAT cDNA-containing cells exhibited simi-lar intracellular species, the Mmineral sprngs cDNA-containingcells were associated with less intracellular alAT than werethe normal Ml cDNA-containing cells (Fig. lOB, lanes 3 and4). Importantly, after a 2-h chase period, there was very littledetectable intracellular alAT in the normal Ml cDNA-containing cells, i.e., the protein having been converted tothe mature form and secreted (lane 5). Likewise, theMminera spnngs cDNA-containing cells had no detectable in-tracellular alAT after the 2-h chase period (Fig. lOB, lane 6),excluding intracellular accumulation of the Mmineral springsprotein as the basis for the observed alAT secretion defect.Thus, the Mmineral springs mutation was associated with re-duced amounts of alAT secretion from synthesizing cellsand reduced amounts of intracellular clAT despite normal

5

4

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2

ml (Val ?l:5.) * q q oM **

i.

Ml (Val2-3) M pnraig,sFIG. 7. Determination of alAT mRNA transcript level associ-

ated with the alAT Mminerd springs gene. Cytoplasmic RNA of bloodmonocytes of an Ml(VaE13) homozygote control and theMmineral springs homozygote index case was analyzed by cytoplasmicblot analysis with a 32P-labeled alAT cDNA probe, and the resultingautoradiograms were quantified by densitometry. Shown is theamount of alAT mRNA transcripts expressed as densitometricunits of alAT mRNA per 106 cells. Inset shows example of thecytoplasmic blot analysis.

levels of alAT mRNA transcripts and normal translation ofthese transcripts. This pattern of abnormal posttranslationalalAT biosynthesis is thus distinct from that associated withthe alAT Z allele, in which abnormal intracellular accumu-lation of the mutant protein is etiologic of the reduced alATsecretion (22, 47, 54).

kDa

47 --4 4~4U

2

FIG. 8. Evaluation of the translational efficiency of alATMinineral springs mRNA transcripts. Synthetic alAT mRNA tran-scripts of Ml(Val213) and Mmineral springs type were derived by in vitrotranscription from corresponding alAT cDNAs. Comparableamounts (1.0 ,ug) of these transcripts were used to direct translationin a rabbit reticulocyte lysate system with [35S]methionine as thelabel, followed by SDS-acrylamide gel electrophoresis, fluorog-raphy, and densitometric quantification. Lanes: 1, Ml(Val213); 2,Mn,ineral springs, The size of the 47-kDa primary translation product isindicated.

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al-ANTITRYPSIN Mi,mjnra sprngs ALLELE 53

A.

kDa

Leu56 GIy5 Thr6a.CTG GGG ACC.

IlM nlAT CDNA

_Z n _ -Mm,nera, s'r-gs a1AT cDNA

CTG GAG ACC .

Leu66 GIu67 Thrf

1 2

FIG. 9. Secretion of alAT by monocytes of the Mmineraj springshomozygote compared with the normal level. Blood monocytes ofan Ml(Val213) homozygote control and an Mminera springs homozy-gote index case were labeled with [35S]methionine for 1 h, followedby a 2-h chase period with unlabeled medium. Secreted, labeledalAT in the supernatant was evaluated by immunoprecipitationwith an anti-alAT antibody, SDS-acrylamide gel electrophoresis,and fluorography. Lanes: 1, Ml(Val2l3) homozygote; 2,Mmineral springs homozygote. The size of the 52-kDa secreted form ofalAT is indicated.

DISCUSSION

At the level of the lower respiratory tract, the insufficientlevels of oalAT associated with different mutations causingalAT deficiency are similar; all permit unimpeded proteo-lytic destruction by NE, with the clinical consequences ofpulmonary emphysema. However, the various examples ofalAT deficiency in which the mutation is known all resultfrom abnormalities of coding exons of the alAT gene. Theresulting molecular abnormalities associated with these mu-tations are diverse, with mutations including those thatcause absent alAT mRNA transcripts, intracellular degra-dation of newly synthesized alAT protein, and intracellularaccumulation of the newly synthesized alAT (13-15, 22, 28,43, 47, 51, 52, 54; M. Holmes, D. Curiel, M. Brantly, and R.G. Crystal, Am. Rev. Respir. Dis., in press), all culminatingin reduced or absent secretion of alAT from synthesizingcells. This study identifies and characterizes the alATMmineral sprngs gene, an alAT allele associated with alATdeficiency, alAT dysfunction, and the development of em-physema resulting from a single-nucleotide-substitution mu-tation in exon II of the alAT gene that causes a change in theencoded protein (Gly-67 [GaG]--Glu-67 [GAG]).

Analysis of alAT gene expression associated with thealAT Mnineral spnngs allele demonstrated reduced alAT se-cretion from synthesizing cells due to abnormal posttransla-tional alAT biosynthesis. In this regard, alAT-synthesizingcells of the Mminer. springs homozygote index case revealedlevels of steady-state cytoplasmic alAT mRNA transcriptscomparable to those of normal Ml(Val213) cells, and thesetranscripts exhibited a normal capacity to direct the transla-tion of alAT. Cells expressing the Mmineral springs gene,however, exhibited less alAT secretion than did normalM1(Val213) cells; in addition, heterologous cells in which the

minera sprngs alAT cDNA was inserted demonstrated lesssecretion of alAT than did cells containing the normalM1(Val213) alAT cDNA, confirming that the alATMmineir springs mutation was etiologic of the associatedalAT-secretory defect.

Studies of other alAT mutations have demonstrated twogeneral aberrant posttranslational patterns of biosynthesis

5r

B.

kDa

52 ._ _50 -_0-

1 2 3 4 5 6

FIG. 10. Analysis of expression of the alAT Mmineru springs genein murine fibroblasts modified by retroviral gene transfer to containhuman alAT Mm,ner sprngs cDNA. (A) DNA plasmid map of the N2retroviral vector used to transfer human alAT cDNAs of Ml orMgn,nral springs type to murine fibroblasts. The vector contains 5' and3' long terminal repeats (LTR) plus the simian virus 40 (SV40) earlypromoter and the alAT cDNA inserted into the XhoI site of theneomycin resistance gene (NEOR). The Ml and Mmineral springs alATcDNAs differed by a single-nucleotide mutation (Ml Gly-67[GQUGH+Mmiiner, springs Glu-67 [GAG]). (B) Synthesis and secretionof human alAT by murine fibroblasts modified to contain humanalAT cDNAs of Ml or Mmineral springs type. Polyclonal populationsof NIH 3T3 cells modified by retroviral gene transfer to containhuman alAT cDNAs of Ml or Mnine,..j spri type and expressingcomparable levels of human alAT mRNA transcripts were labeledwith [35S]methionine for 1 h, followed by a 2-h chase with unlabeledmedium. Cell lysates were evaluated after 0- and 2-h chase periods,and cell supernatants were evaluated after a 2-h chase for thepresence of labeled human alAT, using immunoprecipitation withan anti-human alAT antibody, SDS-acrylamide gel electrophoresis,and fluorography. Lanes: 1 and 2, analysis after a 2-h chase forextracellular secreted alAT in supernatants from cells containingthe normal alAT Ml cDNA (lane 1) and the alAT Mmnjneraj springscDNA (lane 2); 3 and 4, analysis after a 0-h chase for intracellularalAT in lysates from Ml-containing (lane 3) and MminerW springs-containing (lane 4) cells; 5 and 6, analysis after a 2-h chase of lysatesfrom Ml-containing (lane 5) and Mmineral springs-containing (lane 6)cells. Positions of the 52-kDa secreted form and 50-kDa intracellularform of alAT are indicated. A lower-molecular-size non-alAT,nonspecific band was observed in all cell lysates.

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54 CURIEL ET AL.

resulting in reduced alAT secretion from alAT-synthesizingcells: intracellular accumulation and intracellular degrada-tion of the newly synthesized protein. Intracellular accumu-lation is the basis of the alAT deficiency associated with twoalAT genes, Z and Mmalton, For the Z-type mutation (Glu-342 [-jAG]-*Lys-342 [AAG]), the substitution is a positivelycharged residue for a negatively charged one in a 13-sheetregion of the molecule (35, 42). This substitution is thoughtto result in the loss of a normal salt bridge between Glu-342and Lys-290, although the secondary structural features ofalAT assumed in this hypothesis are based on crystallo-graphic analysis of a modified inhibitor (35). Furthermore,there is increasing evidence that there are also local chargeeffects of the Glu-342--+Lys mutation around residue 342 thatresult in accumulation of the Z-type alAT molecule in therough endoplasmic reticulum (5, 23, 37, 53). The Mmaitonmutation is a triple-nucleotide-deletion mutation causing theloss of a single amino acid residue (Phe-52 [TTC]--delete)(15). Like the Z-type mutation, this alteration occurs in a1-sheet region of the molecule, but it is not apparent whythis results in intracellular accumulation of the newly syn-thesized alAT molecule. Interestingly, the liver diseaseassociated with alAT deficiency has been restricted to thesetwo alleles, suggesting that it is not the alAT-deficiencystate which elicits the hepatic injury.One alAT mutation, the S allele, is clearly associated with

alAT deficiency and increased intracellular degradation (14,36). Analysis of the S-type mutation (Glu-264 [GAA]-*Val-264 [GIA]) demonstrates that it is in an a-helical region ofthe molecule and, like the Z mutation, has been hypothe-sized to interrupt an important intramolecular salt bridge(Glu-264--Lys-387) (56). The net effect of this change is analAT protein that is less stable during biosynthesis, with theconsequence of intracellular degradation of the newly syn-thesized protein, resulting in reduced secretion of aLlAT.For the alAT Mmineral springs allele, the single-nucleotide

mutation results in the substitution of a bulky negativelycharged amino acid for a small, neutral residue (Gly-67[G!jG}-*Glu-67 [GAG]) in an a-helical region (helix B) of themolecule. Given that glycine residues are small and are oftenfound in positions of protein molecules where there arespace limitations (20, 56), the interposition of glutamic acid,an amino acid with a larger side chain, for glycine in theMminerai spnngs protein could effect a net change in overallprotein conformation. The potential consequence of such aconformational change is structural destabilization of theprotein, rendering it susceptible to intracellular proteolysis.

In addition to having an alAT deficiency, the alATMmineral springs allele is also dysfunctional. In this regard,whereas the Mnineral springs homozygote index case exhibitedmarked pulmonary emphysema consistent with serum alATdeficiency, his levels varied, with the lowest alAT levelsbelow the threshold thought necessary to protect the lungbut with levels also above this range, suggesting adequatelower respiratory tract protection at least part of the time.This dichotomy of disease risk suggested an additional basisof impaired lower respiratory tract antielastase defenses.Evaluation of the Mminer,l springs alAT as an inhibitor of NEdemonstrated markedly lower than normal function. Thus,despite serum levels of alAT in the protective range, thefunctional impairment of the Mmineral spnngs protein rendersthe index case effectively alAT deficient. Thus, like thealAT Z allele (44), the Mminerai springs allele predisposes tothe development of emphysema on the basis of both serumalAT deficiency and alAT dysfunction.

Interestingly, the common alAT alleles associated with

alAT deficiency and emphysema are confined almost en-tirely to Caucasians of European descent (6, 12, 21, 32, 49).In this regard, the Mmineral springs mutation may have oc-curred spontaneously in a population different from that ofthe common Z- or S-deficiency alleles (6, 11, 12, 21, 32). Inthis context, the Mmineral springs allele is identical to thecommon Ml(Ala213) allele except for the single-nucleotidesubstitution in the codon for residue 67. In an evolutionarysense, Ml(Ala213) is the oldest alAT, closest of all knownalAT alleles to the chimpanzee counterpart (6). However,assuming that the Mmineral springs allele had the same evolu-tionary pressures as did the common Z-deficiency allele, therarity of Mmineral springs suggests that it is a relatively newalAT mutation. However, the fact that it does exist and thatit occurred in a black kindred suggests that the diagnosticconsideration ofalAT deficiency should not be dismissed denovo as a cause of emphysema in blacks.

ACKNOWLEDGMENTS

We thank M. Brantly, M. Holmes, and H. Takahashi for help withthis study and L. Sichert for excellent editorial assistance.

LITERATURE CITED

1. Armentano, D., S.-F. Yu, P. W. Kantoff, T. von Ruden, W. F.Anderson, and E. Gilboa. 1987. Effect of internal viral sequenceson the utility of retroviral vectors. J. Virol. 61:1647-1650.

2. Bates, P. F., and R. A. Swift. 1983. Double cos site vectors:simplified cosmid cloning. Gene 26:137-146.

3. Bathurst, I. C., J. Stenflo, D. M. Errington, and R. W. Carrell.1983. Translation and processing of normal (PiMM) and abnor-mal (PiZZ) human al-antitrypsin. FEBS Lett. 153:270-274.

4. Beatty, K., J. Bieth, and J. Travis. 1980. Kinetics of associationof serine proteinases with native and oxidized a-1-proteinaseinhibitor and a-1-antichymotrypsin. J. Biol. Chem. 255:3931-3934.

5. Brantly, M., M. Courtney, and R. G. Crystal. 1988. Repair of thesecretion defect in the Z form of al-antitrypsin by addition of asecond mutation. Science 242:1700-1702.

6. Brantly, M., T. Nukiwa, and R. G. Crystal. 1988. Molecularbasis of alpha-1-antitrypsin deficiency. Am. J. Med. 84:13-31.

7. Carrell, R. W. 1986. ac-Antitrypsin: molecular pathology, leu-kocytes, and tissue damage. J. Clin. Invest. 78:1427-1431.

8. Carrell, R. W., J.-O. Jeppsson, C.-B. Laurell, S. 0. Brennan, M.C. Owen, L. Vaughan, and D. R. Boswell. 1982. Structure andvariation of human ac-antitrypsin. Nature (London) 298:329-334.

9. Constans, J., M. Viau, and C. GouailHard. 1980. piM4: anadditional PiM subtype. Hum. Genet. 55:119-121.

10. Cox, D. W., A. M. Johnson, and M. K. Fagerhol. 1980. Reportof nomenclature meeting for ac-antitrypsin. Hum. Genet. 535:429-433.

11. Cox, D. W., S. L. C. Woo, and T. Mansfield. 1985. DNArestriction fragments associated with al-antitrypsin indicate asingle origin for deficiency allele PI Z. Nature (London) 316:79-81.

12. Crystal, R. G., M. L. Brantly, R. C. Hubbard, D. T. Curiel, D.J. States, and M. D. Holmes. 1989. The alpha1-antitrypsin geneand its mutations. Chest 95:196-208.

13. Curiel, D., M. Brantly, E. Curiel, L. Stier, and R. G. Crystal.1989. al-antitrypsin deficiency caused by the al-antitrypsinNuflM.,taW5 gene. J. Clin. Invest. 83:1144-1152.

14. Curiel, D. T., A. Chytil, M. Courtney, and R. G. Crystal. 1989.Serum ac-antitrypsin deficiency associated with the commonS-type (Glu2M4+Val) mutation results from intracellular degra-dation of a1-antitrypsin prior to secretion. J. Biol. Chem.264:10477-10486.

15. Curiel, D. T., M. D. Holmes, H. Okayama, M. L. Brantly, C.Vogelmeier, W. D. Travis, L. Stier, W. H. Perks, and R. G.Crystal. 1989. Molecular basis of the liver and lung disease

MOL. CELL. BIOL.

on February 4, 2018 by guest

http://mcb.asm

.org/D

ownloaded from

Vtl-ANTITRYPSIN Mmipnera springs ALLELE 55

associated with the al-antitrypsin deficiency allele Mmalton. J.Biol. Chem. 264:13938-13945.

16. Dalgleish, R., B. C. Trapneil, R. G. Crystal, and P. Tolstoshev.1982. Copy number of a human type I a2 collagen gene. J. Biol.Chem. 257:13816-13822.

17. Dykes, D. D., S. A. Mifler, and H. F. Polesky. 1984. Distributionof oil-antitrypsin variants in a US white population. Hum.Hered. 34:308-310.

18. Elbein, A. D. 1987. Inhibitors of the biosynthesis and processingof N-linked oligosaccharide chains. Annu. Rev. Biochem. 56:497-534.

19. Errington, D. M., I. C. Bathurst, E. D. Janus, and R. W. Carrell.1982. In vitro synthesis of M and Z forms of human al-antitrypsin. FEBS Lett. 148:83-86.

20. Esser, V., and D. W. Russell. 1988. Alterations in the cysteine-rich and cysteine-poor regions of the protein block intracellulartransport. J. Biol. Chem. 263:13276-13281.

21. Fagerhol, M. K., and D. W. Cox. 1981. The pi polymorphism:genetic, biochemical, and clinical aspects of human al-anti-trypsin. Adv. Hum. Genet. 11:1-62.

22. Foreman, R. C., J. D. Judah, and A. Colman. 1984. Xenopusoocytes can synthesis but do not secrete the Z variant of humanal-antitrypsin. FEBS Lett. 168:84-88.

23. Forman, R. C. 1987. Disruption of the Lys-290-Glu-342 saltbridge in human alpha-1-antitrypsin does not prevent its synthe-sis and secretion. FEBS Lett. 216:79-82.

24. Frants, R. R., and A. W. Eriksson. 1976. a1-Antitrypsin: com-mon subtypes of Pi M. Hum. Hered. 26:435-440.

25. Gadek, J. E., and R. G. Crystal. 1982. al-Antitrypsin deficiency,p. 1450-1467. In J. L. Goldstein and M. S. Brown (ed.),Metabolic basis of inherited disease. McGraw-Hill Book Co.,New York.

26. Gadek, J. E., G. A. Fells, R. L. Zimmerman, S. I. Rennard, andR. G. Crystal. 1981. Antielastases of the human alveolar struc-tures. J. Clin. Invest. 68:889-898.

27. Garver, R. I., A. Chytil, S. Karlsson, G. A. Fells, M. L. Brantly,M. Courtney, P. W. Kantoff, A. W. Nienhuis, W. F. Anderson,and R. G. Crystal. 1987. Production of glycosylated physiolog-ically "normal" human al-antitrypsin by mouse fibroblastsmodified by insertion of a human al-antitrypsin cDNA using aretroviral vector. Proc. Natl. Acad. Sci. USA 84:1050-1054.

28. Garver, R. I., J.-F. Mornex, T. Nukiwa, M. Brandy, M. Court-ney, J.-P. LeCocq, and R. G. Crystal. 1986. Alpha1-antitrypsindeficiency and emphysema caused by homozygous inheritanceof non-expressing alpha1-antitrypsin genes. N. Engl. J. Med.314:762-766.

29. Hirschberg, C. B., and M. D. Snider. 1987. Topography ofglycosylation in the rough endoplasmic reticulum and golgiapparatus. Annu. Rev. Biochem. 56:63-87.

30. Janoff, A. 1985. Elastases and emphysema: current assessmentof the protease-antiprotease hypothesis. Am. Rev. Respir. Dis.132:417-433.

31. Krieg, P. A., and D. A. Melton. 1984. Functional messengerRNAs are produced by SP6 in vitro transcription of clonedcDNAs. Nucleic Acids Res. 12:7057-7071.

32. Kueppers, F. 1978. Inherited differences in alpha-l-antitrypsin,p. 23-74. In S. D. Litwin (ed.), Genetic determinants of pulmo-nary disease. Marcel Dekker, Inc., New York.

33. Kueppers, F., and M. J. Christopherson. 1978. Alpha1-anti-trypsin: further genetic heterogeneity revealed by isoelectricfocusing. Am. J. Hum. Genet. 30:359-365.

34. Lodish, H. F., and N. Kong. 1984. Glucose removal fromN-linked oligosaccharides is required for efficient maturation ofcertain secretory glycoproteins from the rough endoplasmicreticulum to the golgi complex. J. Cell Biol. 98:1720-1729.

35. Loebermann, H., R. Tokuoka, J. Deisenhofer, and R. Huber.1984. Human al-proteinase inhibitor. J. Mol. Biol. 177:531-556.

36. Long, G. L., T. Chandra, S. L. C. Woo, E. W. Davie, and K.Kurachi. 1984. Complete sequence of the cDNA for humanal-antitrypsin and the gene for the S variant. Biochemistry23:4828-4837.

37. McCracken, A. A., K. B. Kruse, and J. L. Brown. 1989.Molecular basis for defective secretion of the Z variant of

human alpha-1-proteinase inhibitor: secretion of variants havingaltered potential for salt bridge formation between amino acids290 and 342. Mol. Cell Biol. 9:1406-1414.

38. Melton, D. A., P. A. Krieg, M. R. Rebagliati, T. Maniatis, K.Zinn, and M. R. Green. 1984. Efficient in vitro synthesis ofbiologically active RNA and RNA hybridization probes forplasmids containing a bacteriophage SP6 promoter. NucleicAcids Res. 12:7035-7056.

39. Mornex, J.-F., A. Chytil-Weir, Y. Martinet, M. Courtney, J.-P.LeCocq, and R. G. Crystal. 1986. Expression of the alpha-1-antitrypsin gene in mononuclear phagocytes of normal andalpha-l-antitrypsin-deficient individuals. J. Clin. Invest. 77:1952-1961.

40. Nukiwa, T., M. Brantly, F. Ogushi, G. Fells, K. Satoh, L. Stier,M. Courtney, and R. G. Crystal. 1987. Characterization of theMl(Ala213) type of al-antitrypsin, a newly recognized, common"normal" al-antitrypsin haplotype. Biochemistry 26:5259-5267.

41. Nukiwa, T., M. L. Brantly, F. Ogushi, G. A. Fells, and R. G.Crystal. 1988. Characterization of the gene and protein of thecommon al-antitrypsin normal M2 allele. Am. J. Hum. Genet.43:322-330.

42. Nukiwa, T., K. Satoh, M. L. Brantly, F. Ogushi, G. A. Fells, M.Courtney, and R. G. Crystal. 1986. Identification of a secondmutation in the protein-coding sequence of the Z type alpha1-antitrypsin gene. J. Biol. Chem. 261:15989-15994.

43. Nukiwa, T., H. Takahashi, M. Brantly, M. Courtney, and R. G.Crystal. 1987. al-Antitrypsin NUIIG.ile FaUls' a nonexpressingal-antitrypsin gene associated with a frameshift to stop mutationin a coding exon. J. Biol. Chem. 262:11999-12004.

44. Ogushi, F., G. A. Fells, R. C. Hubbard, S. D. Straus, and R. G.Crystal. 1987. Z-type al-antitrypsin is less competent thanMl-type al-antitrypsin as an inhibitor of neutrophil elastase. J.Clin. Invest. 80:1366-1374.

45. Perlino, E., R. Cortese, and G. Ciliberto. 1987. The humanal-antitrypsin gene is transcribed from two different promotersin macrophages and hepatocytes. EMBO J. 6:2767-2771.

46. Perlmutter, D. H., F. S. Cole, P. Kilbridge, T. H. Rossing, andH. R. Colten. 1985. Expression of the al-proteinase inhibitorgene in human monocytes and macrophages. Proc. Natl. Acad.Sci. USA 82:795-799.

47. Perlmutter, D. H., R. M. Kay, F. S. Cole, T. H. Rossing, D. VanThiel, and H. R. Colten. 1985. The cellular defect in al-proteinase inhibitor (a,-PI) deficiency is expressed in humanmonocytes and in xenopus oocytes injected with human livermRNA. Proc. Natl. Acad. Sci. USA 82:6918-6921.

48. Pfeffer, S. R., and J. E. Rothman. 1987. Biosynthetic proteintransport and sorting by the endoplasmic reticulum and golgi.Annu. Rev. Biochem. 56:829-852.

49. Pierce, J. A., B. Eradio, and T. A. Dew. 1975. Antitrypsinphenotypes in St. Louis. J. Am. Med. Assoc. 231:609-612.

50. Rabin, M., M. Watson, V. Kidd, S. L. C. Woo, W. R. Breg, andF. H. Ruddle. 1986. Regional location of al-antichymotrypsinand al-antitrypsin genes on human chromosome 14. SomaticCell Mol. Genet. 12:209-214.

51. Satoh, K., T. Nukiwa, M. Brandy, R. I. Garver, Jr., M. Hofker,M. Courtney, and R. G. Crystal. 1988. Emphysema associatedwith complete absence of al-antitrypsin in serum and thehomozygous inheritance of stop codon in an al-antitrypsin-coding exon. Am. J. Hum. Genet. 42:077-083.

52. Sifers, R. N., S. Brashears-Macatee, V. J. Kidd, H. Muensch,and S. L. C. Woo. 1988. A frameshift mutation results in atruncated al-antitrypsin that is retained within the rough endo-plasmic reticulum. J. Biol. Chem. 263:7330-7335.

53. Sifers, R. N., C. P. Hardick, and S. L. C. Woo. 1989. Disruptionof the 290-342 salt bridge is not responsible for the secretorydefect of the PiZ al-antitrypsin variant. J. Biol. Chem. 264:2997-3001.

54. Verbanac, K. M., and E. C. Heath. 1986. Biosynthesis, proc-essing, and secretion of M and Z variant human a1-antitrypsin.J. Biol. Chem. 261:9979-9989.

55. Vieira, J., and J. Messing. 1982. The pUC plasmids, andM13mp7-derived system for insertion mutagenesis and sequenc-

VOL. 10, 1990

on February 4, 2018 by guest

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MOL. CELL. BIOL.

ing with synthetic universal primer. Gene 19:259-268.56. Vogel, B. E., R. R. Minor, M. Freund, and D. J. Prockop. 1987.

A point mutation in a type I procollagen gene converts glycine748 of the al chain to cysteine and destabilizes the triple helix ina lethal variant of osteogenesis inperfecta. J. Biol. Chem.262:14737-14744.

57. Wewers, M. D., M. A. Casolaro, S. E. Sellers, S. C. Swayze, K.M. McPhaul, J. T. Witters, and R. G. Crystal. 1987. Replace-ment therapy for alpha1-antitrypsin deficiency associated withemphysema. N. Engl. J. Med. 316:1055-1062.

58. White, B. A., and F. C. Bancroft. 1982. Cytoplasmic dothybridization. J. Biol. Chem. 257:8569-8572.

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