Serum al-Antitrypsin Deficiency Associated with the Common S-type

THE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 264, No. 18, Issue of June 25, pp. 10477-10466,1989 Printed in U.S.A.

Serum al-Antitrypsin Deficiency Associated with the Common S-type ( G ~ u ~ ‘ ~ I) Val) Mutation Results from Intracellular Degradation of al-Antitrypsin Prior to Secretion*

(Received for publication, January 9, 1989)

David T. CurielS, Anna Chytil, Michael Courtney, and Ronald G. Crystal From the Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 and Transgene, I 1 rue De Molsheim, Strasbourg, France

The S-type a1-antitrypsin (alAT) deficiency allele differs from the normal M1(ValaI3) allele by a single amino acid substitution ( G ~ u ~ ~ ~ + Val). To evaluate the molecular pathophysiology responsible for the reduced serum levels of alAT associated with the S-type allele, alAT gene expression was examined in blood monocytes, cells which normally produce alAT, as well as murine fibroblasts modified by retroviral gene transfer to express the S-type and normal “type human a1AT genes. Northern analysis and S1 protection analysis demonstrated that monocytes of M and S homozygotes both express 1.8-kilobase a1AT mRNA transcripts in comparable levels and similar in structure. Pulse-chase labeling studies demonstrated that both M and S monocytes synthesized and secreted a 52-kDa protein, but the S monocytes secreted significantly less. The cellular lysates of both M and S monocytes contained a newly synthesized 50-kDa precursor form of alAT, but the S monocytes contained reduced amounts. Pulse-chase labeling in the presence of tunicamycin, an inhibitor of core oligosaccharide addition, demonstrated that S monocytes exhibited a selective inhibition of secretion of 45-kDa nonglycosylated alAT not observed in M monocytes. Consistent with these observations, murine fibroblasts modified by retroviral gene transfer to contain an integrated human S-type alAT cDNA demonstrated reduced secretion of alAT compared with fibroblasts containing an integrated human M-type a1 AT cDNA and also reproduced the abnormality of alAT biosynthesis observed with S-type monocytes. Furthermore, in the presence of leupeptin, an inhibitor of cellular proteinases, the S - type modified fibroblasts demonstrated a selective aug- mentation of human alAT secretion not observed for the “type. Together, these observations are consistent with the concept that the single A + T mutation of the S-type alAT gene results in reduced cellular secretion of alAT because the newly synthesized S-type alAT protein is degraded intracellularly prior to secretion.

a1-Antitrypsin (alAT),’ a 52-kDa serum glycoprotein pro-

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

$To whom correspondence and reprint requests should be ad- dressed Pulmonary Branch, Bldg. 10, Rm. 6D03, National Institutes of Health, Bethesda, MD 20892.

The abbreviations used are: alAT, al-antitrypsin; SDS, sodium dodecyl sulfate; kb, kilobase(s).

duced by hepatocytes and mononuclear phagocytes, serves as the major inhibitor of neutrophil elastase in the lower respiratory tract (I, 2). In its secreted form, a l A T is composed of 394 amino acids and three carbohydrate side chains of the N - linked asparaginyl type (3-5). Its principal function is to inactivate neutrophil elastase, a powerful serine protease ca- pable of degrading the major components of the connective tissue matrix (5, 6). When serum levels of a l A T are below 35% of normal, as occurs in the hereditary disorder a1AT deficiency, there are insufficient amounts to protect the lower respiratory tract from its burden of neutrophil elastase, plac- ing the affected individual at high risk for the development of pulmonary emphysema (1, 2, 7, 8).

a l A T is coded for by a highly pleomorphic, single copy gene of 12.2 kb on chromosome 14 at q31-32.3 (2, 9). The a lAT phenotype is determined by codominant expression of the two parental alleles (10-12). In USA Caucasians, the most common normal a l A T variants are those of the “family (com- bined allelic frequency, >go%) and the most common deficiency variants are the S-type (frequency 0.02-0.04) and the Z-type (frequency 0.01-0.02) (1, 2, 3, 10, 12, 13-17).

Unlike the Z variant, in which the mechanism responsible for the deficiency state is understood to result from intracellular aggregation of the aberrant molecule causing reduced a l A T secretion (4, 18, 19), little is known as to why the S allele is associated with reduced serum levels of a1AT. It is known, however, that the coding exons of S-type a l A T gene differ from the common normal Ml(Val2l3)-type a1AT gene by a single base (M1(Va1213) GAA + S GTA) causing the amino acid substitution G ~ u ~ ~ ~ Z V a l (20-22). This knowledge, together with the observations that the S-type protein has a normal serum half-life (23) and an association rate constant for neutrophil elastase that is close to normal (24), has led to the assumption that the GIuzfi4 to Val mutation causes a deficiency in a l A T biosynthesis, resulting in deficient secretion of a l A T by a l A T synthesizing cells (4, 18).

To evaluate this concept, we have examined a l A T mRNA transcripts and a1AT synthesis in mononuclear phagocytes recovered from individuals homozygous for the S-type a1AT gene. Then, using retroviral gene transfer to establish permanent murine fibroblast lines expressing the S-type and normal M1(Va1213)-type human a l A T genes, we have reproduced the abnormalities observed in the alAT synthesizing cells of the S homozygotes. Together, the observations are consistent with the concept that the single A T mutation in the S-type a1AT gene causes a serum a l A T deficiency because the newly synthesized a l A T is degraded in a l A T synthesizing cells prior to secretion.

MATERIALS AND METHODS

Source of al-Antitrypsin Synthesizing Cells-Blood monocytes, cells that synthesize and secrete alAT (25, 26), were obtained from

10477

10478 S-type a1 -Antitrypsin Deficiency normals ("type homozygotes), S-type homozygotes, and, as controls, 2-type homozygotes. The determination of the M, S, and Z states was made by a combination of measurement of serum alAT levels determined by radial immunodiffusion (Calbiochem), a lAT typing by isoelectric focusing of serum, and family studies (1, 2, 10-12). The normal individuals (n = 7) all were of phenotype MM (i.e. various combinations of the normal alleles M1(Va1'13), Ml(Ala2l3), M2, and M3) (12) and had normal a lAT serum levels (295 * 35 mg/dl)." The homozygous S-type alAT-deficient group included five individuals with alAT serum levels of 142 +_ 40 mg/dl. The homozygous Z-type alAT deficient group included two individuals with alAT serum levels of 34 & 6 mg/dl. The monocytes were isolated utilizing adher- ence purification of mononuclear cells obtained by monocytapheresis as described previously (26).

RNA Analysis of Monocytes-alAT mRNA transcripts of the S- type and M-type monocytes were evaluated by Northern analysis, S1 nuclease protection analysis, and quantitative dot blot analysis. Total cellular RNA was prepared from purified blood monocytes by guani- dine hydrochloride extraction and CsCl centrifugation (26).

Northern analysis was carried out with agarose gel electrophoresis under denaturing conditions, transfer to nitrocellulose filters, and hybridization utilizing a 3ZP-labeled full length human alAT cDNA plasmid probe (pPBO1). Exposure of the autoradiograms was for 48 h at -70 "C. S1 nuclease analysis was carried out using total cellular RNA hybridized under denaturing conditions for 3 h at 56 "C to a 1.4-kb single strand anti-sense a lAT cDNA probe complementary to the protein coding regions of the a1AT gene. Digestion with S1 nuclease (200 units/ml; Pharmacia LKB Biotechnology Inc.) was for 30 min at 37 "C. The resulting protected fragments were electrophoresed under alkaline conditions, transferred to nitrocellulose filters, and detected by hybridization to a 32P-labeled full length alAT cDNA. The resulting autoradiograms were exposed for 48 h at -70 "C.

Quantification of steady state a lAT mRNA levels was performed using dot blot hybridization (26). Total cellular RNA was denatured and applied to nitrocellulose filters with a minifold apparatus (Schleicher & Schuell) in serial dilutions of 10-1.25 pg. The filters were hybridized as described above and the amount of specific hybridization determined by densitometric analysis of the autoradiograms with results expressed as densitometric units of a lAT mRNA/ pg total RNA.

Synthesis and Secretion ofalAT by Monocytes-The synthesis and secretion of alAT by the monocytes was evaluated using [35S]methionine as a label and anti-human a1AT antibody (Boehringer Mann- heim) immunoprecipitation of the supernatants and cell lysates for the presence of 35S-labeled a1AT as described previously (26).

Modification of Oligosaccharide Side Chain Addition and Process- ing-Modification of steps in oligosaccharide side chain addition and processing of the alAT synthesized by the monocytes was accomplished by adding tunicamycin, an inhibitor of oligosaccharide side chain addition (27-33) (10 pg/ml, Boehringer Mannheim), or swainsonine, an inhibitor of the distal oligosaccharide processing enzyme Golgi mannosidase I1 (27,32,34,35) (5 pg/ml, Boehringer Mannheim) during the 12-h period prior to the [35S]methionine labeling and during the pulse labeling and chase periods. Immunoprecipitation, sodium dodecyl sulfate (SDS)-gel electrophoresis, and fluorography were as described previously above.

Retroviral Gene Transfer of Human M-type and S-type alAT cDNAs to Murine Fibroblasts-Murine fibroblasts were modified to contain the human alAT cDNAs of M-type or S-type utilizing a retroviral vector as described previously (33). Briefly, the N2 vector (36) was modified to contain M-type or S-type alAT cDNAs (pN2- alAT-M and pN2-alAT-S, respectively) by standard procedures. Sequence analysis confirmed that pN2-alAT-S differed from pN2- alAT-M exclusively by the single nucleotide mutation corresponding to the S mutation. Integration of pN2-alAT-M and pN2-alAT-S into the genome of $2 cells (37), a helper virus-free packaging cell line, was by calcium phosphate transfection (38). Supernatants of individual $2 clones transfected with pN2-alAT-M or pN2-alAT-S (clones referred to as $2/alAT-M and $2/alAT-S, respectively) were titered for infectious retrovirus and used to generate polyclonal populations of infected NIH-3T3 cells. In the context that the retroviral vector randomly integrates single copies of the provirus, and with the knowledge that individual clones with the integrated alAT cDNA produce a1AT at different levels (39), polyclonal populations (rather than individual cell populations) were used to ensure that the NIH-

'All data are presented as the mean k S.E., and all statistical comparisons were made using the two-tailed Student's t test.

3T3 fibroblasts with integrated M- and S-type a1AT cDNAs would represent average populations of these cells.

Analysis of Modified Murine Fibroblasts Containing M-type and S- type alAT cDNAs-Confirmation of integration of the full length human alAT cDNAs of M-type and S-type into the genome of mouse fibroblasts modified by the retroviral transfer was carried out by Southern analysis using a 32P-labeled a lAT cDNA probe (33, 40). Human alAT mRNA transcripts were identified in the NIH-3T3/ alAT-M and NIH-3T3/alAT-S cells by Northern analysis (33). Quantification of total cellular RNA for steady state human alAT mRNA levels was as described above for M- and S-type monocytes.

The secretion of human alAT by NIH-3T3/alAT-M and NIH- 3T3/alAT-S cells was evaluated by [35S]methionine pulse-chase labeling followed by immunoprecipitation of secreted labeled alAT as described previously (33). The effect of modification of oligosaccharide side chain addition and processing on secretion of human alAT by the cells was accomplished by incubating the cells for 6 h with tunicamycin (10 &g/ml) or swainsonine (5 pg/ml) prior to pulse labeling and during pulse labeling and chase periods.

Modification of Intracellular Proteolysis by Inhibition of Cellular Proteinases-The effect of inhibition of intracellular proteinases on the secretion of human alAT by NIH-3TB/alAT-M and NIH-3T3/ a1AT-S cells was examined utilizing leupeptin (Boehringer Mann- heim), a broad spectrum inhibitor of cellular proteinases (41-47). The cells were plated as before with incubation in the presence of leupeptin (0.5 mM) for 24 h prior to analysis and during pulse labeling and chase period. The dose of leupeptin was based on previous studies in fibroblast cell systems (46). Pulse labeling, immunoprecipitation, gel electrophoresis, and fluorography were as described above.

RESULTS

Evaluation of the Form of S-type alAT mRNA Transcripts in Monocytes-The A -+ T substitution of the S mutation not only causes an amino acid change (G1uZa + Val) but as noted by Schindler et al. (48), also creates a region within exon 111 of the S-type alAT gene with a high degree of homology to the consensus 5' donor splice site, thus creating a potential aberrant donor splice site (Fig. lA). In this regard, it is conceivable that the pathogenesis of the serum deficiency of alAT associated with the S mutation results from abnormal d A T mRNA processing, causing a truncated form of a1AT mRNA, and a resultant abnormal protein. Three lines of evidence, however, suggest that this putative splice donor site is not recognized. First, Northern blot analysis of RNA of M- type and S-type monocytes demonstrated alAT mRNA transcripts of identical size (1.8 kb) in both cell types (Fig. 1B). Second, evaluation of the exon I11 region of the alAT mRNA transcripts utilizing S1 protection analysis with a 1.4-kb single strand anti-sense alAT cDNA probe demonstrated that this region of the alAT mRNA transcripts of S-type monocytes was identical to that of normal "type monocytes (Fig. IC). In contrast, if the potential aberrant 5' donor splice site in exon I11 had been utilized, fragments of 0.9 and 0.5 kb would have been observed (see Fig. lA). Third, comparison of the amounts of alAT mRNA in S-type and "type alAT monocytes demonstrated equal levels of alAT transcripts ( p > 0.3) (Fig. 2).

Secretion of alAT by S-type Monocytes-Evaluation of the form of alAT synthesized and secreted by S-type monocytes demonstrated a 52-kDa protein, identical to that of normal "type monocytes (Fig. 3A). In this regard, pulse labeling for 1 h in the presence of [35S]methionine followed by a 2-h chase period revealed a 52-kDa mature form of a1AT that was specifically immunoprecipitated by anti-a1AT antibody from the supernatants of S-type monocytes, similar to that of the normal "type monocytes and, as an additional control, Z- type monocytes. However, assessment of the relative amount of newly synthesized alAT secreted by the S-type monocytes showed that it was less than that secreted by normal "type monocytes. Following a 1-h pulse with [35S]methionine, the

S-type a1 -Antitrypsin Deficiency 10479

A.

8. Northern

MM SS

IA !E IC 11 111 IV v H 100 bp

1-1 Normal Splice

1 I Probe pPBO1

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4 4 4 0.9 kb 0.5 kb

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FIG. 1. Evaluation of the form of a l A T mRNA transcripts present in monocytes of individuals homozygous for M- and S-type a lAT. A, schematic of the a lAT gene showing the S-type mutation in exon 111. Included are exons I,-V of the a lAT gene. The nomenclature used for the numbering of the exons (1) is directed toward preserving the established nomenclature (22) for the coding exons (11-V) but to include the recent observations (71) of additional noncoding exons (IA, IB) 5' to the original exon I (now IC) (22). The single nucleotide substitution (A + T) in exon I11 responsible for the S-type mutation (G1uZa +Val) is indicated. As pointed out (48), this results in a sequence (cross-hatched area) with homology to the 5' consensus donor splice site. Theoretically, this sequence could be recognized as a splice donor site. If this occurs, instead of the normal 1.8-kb a l A T mRNA observed with "type alAT, the S-type mutation would result in a premature splice, with the consequence of a truncated a1AT mRNA with the 60 base pairs ( b p ) of exon I11 3' to the S mutation site lost. An arrow indicates the site of the potential aberrant splice region in exon 111 created by the S mutation. Shown in the lower portion of the panel are the possible results of S1 nuclease protection analysis of the S- and M-type mRNAs utilizing a single strand anti-sense a1AT cDNA probe (pPBO1) complementary to the coding regions of the a lAT gene. The normally spliced a1AT mRNA would yield a 1.4-kb protected fragment. However, if utilized the aberrant splice site would generate fragments of 0.9 and 0.5 kb resulting from nuclease digestion of the nonprotected region of the probe (depicted as a gap) corresponding to the region of exon I11 which would be deleted by aberrant splicing (cross-hatched region in exon 111). Arrows indicate sites of potential nuclease digestion. B, comparison of M- and S-type a1AT mRNA transcripts by Northern analysis. Following electrophoresis and transfer, total cellular RNA (20 pg/lane) was hybridized with a 32P-labeled a1AT cDNA probe. Lane 1, MM, "type a1AT. Lane 2, SS, S-type alAT. The position of the normal 1.8-kb a l A T mRNA transcripts are indicated. C, comparison of the a lAT transcripts of M- and S-type monocytes by S1 nuclease protection. Total cellular RNA (20 pg) was hybridized to a 1.4-kb single strand anti-sense a1 AT cDNA and digested with S1 nuclease. The resulting RNA/DNA heteroduplex protected fragments were electrophoresed under alkaline conditions, transferred to nitrocellulose filters, and hybridized with a full length 32P-labeled a1AT cDNA probe. Lane 3, "type a1AT. Lane 4, S-type alAT. The S- type mRNA yielded a normal 1.4-kb protected fragment indicating that all detectable a l A T transcripts utilize only the normal slice sites, i.e. the putative "consensus-like" sequence in exon I11 of the S-type mRNA is not used.

S-type monocytes secreted 35S-labeled a l A T in a time-dependent fashion with the labeled a l A T detected in the culture supernatant within 30 min and the amount of labeled a1AT increasing until 2 h, thereafter leveling off, similar to the pattern observed with "type monocytes (not shown). Quan- titatively, however, there was a significant difference in the amounts of newly synthesized a l A T secreted by the S- and "type cells, with S-type monocytes secreting 42 f 10% of the amount of a1AT as "type cells (p < 0.01; Fig. 3B). In contrast, while Z-type monocytes had a similar time-dependent pattern of appearance of 35S-labeled a l A T in the super-

natant, the amount secreted by Z-type cells was markedly less (12 f 2% of "type, p < 0.01). These observations are consistent with the knowledge that the serum levels of Z homozygotes are approximately 10-15% of normal, whereas that of S-homozygotes is 50-60% of normal (1, 2, 15).

Intracellular Newly Synthesized alAT in S-type Mono- cytes-Analysis of the cellular lysates of 35S-labeled blood monocytes of homozygous a1AT S-type individuals demonstrated that a 50-kDa intracellular form of a1AT could be specifically immunoprecipitated in a fashion similar to that observed from normal "type and deficient Z-type cells (Fig.

10480 S-type a1 -Antitrypsin Deficiency

0.6

0.5

0.4

0.2

0.:

0.1

MM

ss

T "

MM ss FIG. 2. Comparison of the levels of a lAT mRNA transcripts

in M- and S-type monocytes (MM and SS, respectively). Total cellular RNA was evaluated for a1AT mRNA transcripts using dot blot analysis with a 3ZP-labeled a1AT cDNA probe. Resulting autoradiograms were quantified by densitometry. Shown is the amount of a l A T mRNA expressed as units/pg total RNA. Inset, example of the a l A T dot blot analysis.

4A). In addition, a minor fraction of mature 52-kDa a1AT was immunoprecipitated from the cellular lysates as well as a nonspecific 43-kDa band not blocked by an excess of unlabeled alAT. The 50-kDa major intracellular species of a1AT corresponds to an immature precursor form of a1AT localized to the rough endoplasmic reticulum and characterized by the presence of high mannose-type oligosaccharide side chains (32,49) as confirmed by sensitivity to digestion with endogly- cosidase H (data not shown). However, despite the fact that the S-, M-, and Z-type cells all contained a form of newly synthesized a lAT tha t was qualitatively similar, quantitation of the amount of newly synthesized intracellular a1AT was markedly different among the different a1AT types. In this regard, the amount of newly synthesized intracellular a l A T isolated from S-type monocytes was reduced compared to M- type monocytes ( p < 0.01). This is in marked contrast to Z- type monocytes, in which newly synthesized a1AT accumu- lated, resulting in more intracellular a l A T compared to normal "type monocytes ( p < 0.01). Thus, while the reduced secretion of a l A T of Z-type cells occurs in conjunction with an intracellular accumulation of a l A T compared to normal, the pattern associated with the S-type cells is very different; the reduced secretion of a l A T of S-type monocytes is associated with reduced amounts of newly synthesized intracellular alAT.

Effect of Modification of Oligosaccharide Side Chain Proc- essing on Secretion of alAT by S-type Monocytes-The finding of reduced amounts of the intracellular high mannose form of a l A T associated with S-type monocytes compared to normal "type monocytes suggests that the abnormality in the biosynthesis of the S-type alAT occurs at, or prior to, the level of the rough endoplasmic reticulum. To further evaluate this concept, inhibitors of oligosaccharide side chain addition (27, 28) and processing (27, 34) were utilized. These agents act at defined steps in the carbohydrate side chain biosyn- thetic and processing pathways and thus may exaggerate an abnormality of S-type a1AT biosynthesis through ablation of

A.

MU

kDa 52- m

1

ss n

mm

2 3

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ss zz FIG. 3. Secretion of newly synthesized a lAT by monocytes

from individuals homozygous for the M-, S-, and Z-types (MM, SS, and 22, respectively) of alAT. A, fluorograms of SDS- acrylamide gel analysis of "'S-labeled proteins secreted by monocytes (1-h pulse with ['"S]methionine and 2-h chase with label-free media) and immunoprecipitated with an anti-alAT antibody. Lane 1, monocytes of an individual with a1AT M-type. Lane 2, a1AT S-type. Lane 3, a l A T Z-type. The position of normal purified plasma a1AT is indicated. B, quantification of the relative amount of a l A T secreted by monocytes. Shown for M-, S-, and Z-type monocytes is the amount of 35S-labeled a1AT secreted by lo6 cells following a 1-h pulse with [35S]methionine and a 2-h chase with label-free media. All immunoprecipitations were carried out on samples containing lo6 dpm of total trichloroacetic acid (TCA)-precipitable 3'S-labeled protein. Quantification was carried out by densitometric analysis of fluorograms as in A.

stabilizing effects oligosaccharide side chains contribute to a1AT structure prior to secretion.

In the presence of swainsonine, an inhibitor of the oligosaccharide processing enzyme Golgi mannoside 11, monocytes of normal "type individuals secreted a1AT molecules with an apparent molecular mass of 50 kDa (Fig. 5A, lanes 1 and 2). This size corresponds to a species of a l A T shown in previous studies of a1AT biosynthesis to possess "hybrid" type oligosaccharide side chains (32, 35). Quantitatively, despite the difference in the form of the oligosaccharide side chains, the amount of a1AT secreted by the "type monocytes in the presence of swainsonine was similar to that of the untreated cells (Fig. 5B; p > 0.2). When normal "type cells were incubated in the presence of tunicamycin, an inhibitor that prevents core oligosaccharide addition in the rough endoplasmic reticulum (27, 28), the newly synthesized secreted a l A T had a molecular mass of 45 kDa, the known molecular mass of nonglycosylated a l A T (32, 33, 49) (Fig. 5A, lane 3) . Quantitatively, despite the fact that the a1AT secreted in the presence of tunicamycin has no carbohydrate side chains, the normal cells still secrete a lAT a t a level about 68 + 3% that of untreated normal cells (Fig. 5B), consistent with the observation in previous studies that the presence of oligosaccharide side chains is not normally a prerequisite for the secretion of a l A T (30-33).

S-type a1 -Antitrypsin Deficiency

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FIG. 4. Evaluation of intracellular alAT newly synthesized by M-, S - , and Z-type (I", SS, and 22, respectively) blood monocytes. A, SDS-acrylamide gel electrophoresis and fluorographic analysis of lysates of blood monocytes of individuals homozygous for a l A T types M, S, and Z incubated in the presence of ["S]methionine (1-h pulse with ["S]methionine, no chase). The [3sS]methionine- labeled proteins in the lysates were immunoprecipitated with an anti- alAT antibody prior to analysis. Blocking experiments were accomplished by immunoprecipitating in the presence of excess unlabeled alAT. Lane 1, a l A T M-type. Lane 2, identical to lane 1 but with immunoprecipitation carried out with excess unlabeled a1AT. Lune 3, a l A T S-type. Lune 4, a l A T Z-type. The positions of the 52-kDa mature form and 50-kDa intracellular form of a l A T are indicated. A nonspecific band not blocked by excess unlabeled a l A T is seen a t 43 kDa. B, quantification of the relative amount of intracellular newly synthesized alAT. Following a 1-h pulse with ["SIalAT (no chase), the cell lysates were immunoprecipitated with an anti-a1AT antibody and analyzed as described in the legend to Fig. 3. All immunoprecipitations were carried out on samples containing lo6 dpm total trichloroacetic acid (TCA)-precipitable 3sS-labeled protein.

With the S-type cells, prevention of trimming of the oligosaccharide side chains with swainsonine had comparable effects on the form (Fig. 5A, lanes 4 and 5 ) and amount (Fig. 5B; p > 0.2) of a l A T secreted as that observed in "type cells. In contrast, prevention of core glycosylation with tunicamycin had dramatically different effects. In this regard, incubation of the S-type monocytes with tunicamycin resulted in the absence of detectable a l A T in the supernatants (Fig. 5A, lane 6; Fig. 5B). In addition, 45-kDa nonglycosylated a l A T was also not detectable within the cellular lysates of the S-type monocytes treated with tunicamycin (data not shown). Although it is conceivable that this finding could be explained by loss of antigenicity of the nonglycosylafed S- type alAT, the use of a polyclonal anti-alAT antibody for immunoprecipitation argues against this. Since oligosaccharide side chains contribute to the stability of protein structure, these observations suggest that the S-type a1AT protein has a higher degree of intrinsic instability than the corresponding normal "type a l A T protein, a phenomenon which is exaggerated by inhibition of oligosaccharide side chain addition. In addition, the differential effects of tunicamycin and swain-

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FIG. 5. Effect of modification of proximal and distal oligosaccharide side chain processing on the secretion of a l A T by homozygous M- (MM) and S-type (SS) monocytes. All cultures utilized a 1-h pulse with [R"S]methionine followed by a 2-h chase with unlabeled media. A, SDS-acrylamide gel electrophoresis and fluorographic analysis of a l A T immunoprecipitated from supernatants of monocytes cultured with ["S]methionine in the presence of swainsonine (5 pg/ml), a blocker of distal oligosaccharide side chain processing, or tunicamycin (10 pglml), a blocker of core oligosaccharide side chain addition. Lane I , control (Untreated) monocytes, a l A T "type. Lane 2, similar to lane 1, but with the monocytes cultured with swainsonine. Lane 3, similar to lane I , but cultured in the presence of tunicamycin. Lane 4, control (Untreated) monocytes, S- type. Lune 5, similar to lane 4, but cultured in the presence of swainsonine. Lune 6, similar to lane 4, but cultured in the presence of tunicamycin. Indicated are the 52-kDa mature form of alAT, the 50-kDa form of a l A T containing hybrid oligosaccharide side chains, and the 45-kDa nonglycosylated form of a1AT. B, quantification of the relative amounts of a l A T secreted by monocytes cultured with swainsonine and tunicamycin compared to that of control monocytes. Quantification was performed by densitometric analysis of fluorograms as in A. All immunoprecipitations were carried out with samples containing lo6 dpm total trichloroacetic acid-precipitable 3sS- labeled protein. Shown is the relative amount a l A T secreted by M- and S-type monocytes cultured with swainsonine or tunicamycin expressed as a percentage of that secreted by control homozygous M- and S-type monocytes, respectively. *, S-type monocytes cultured with tunicamycin did not secrete any detectable 3sS-labeled a1AT.

sonine on the biosynthesis of the S-type alAT suggest that it is the presence of oligosaccharide side chains which are obli- gated to effect stabilization of the S-type protein, and that the final structure of the side chains does not appear to be a

10482 S-type al-Antitrypsin Deficiency

critical component of this effect. A. Integration and Expression of the Human alAT cDNAs of

M-type and S-type into the Genome of Murine Fibroblasts- NIH-3T3 NIH9T3 Permanent integration of the human alAT cDNAs of M-type and S-type into murine fibroblasts was accomplished by retroviral gene transfer (see Fig. 6 for details concerning the constructs used). The polyclonal populations of modified mouse fibroblasts contained the a1AT cDNA and expressed human a l A T RNA transcripts. Southern blot analysis of NIH-3T3 cells infected with the M-type or S-type human a1AT cDNAs (NIH-3T3/alAT-M and NIH-3T3/alAT-S, respectively) cut with the restriction endonucleases Hind111 and XhoI and evaluated utilizing a 32P-labeled human alAT cDNA probe demonstrated the presence of the intact, integrated a l A T cDNA (Fig. 7A, lanes 1 and 2) . Furthermore, Northern analysis of poly(A)+-selected RNA isolated from the two cell populations demonstrated that both contained three a1AT mRNA transcripts containing sequences complementary to the human alAT cDNA, similar to that observed in clonal cell populations of NIH-3T3 cells modified with the "type a l A T cDNA (33) (Fig. 7B, lanes 3 and 4) . In this regard, the 5.8-kb transcript represents a genomic transcript originating from the 5' long terminal repeat, the 4.8-kb transcript is the likely product of a spliced genomic transcript generated from a cryptic splice acceptor signal between the 5' long terminal repeat and the neoR gene, and the 2.4-kb transcript is the size predicted to be generated from the transcripts driven by the SV40 promoter (33). Importantly, the presence of the S mutation sequence did not appear to alter RNA processing in the a1AT S-type cDNA containing cells, since the same alAT mRNA species were observed in S-type cells as in the "type cells. Furthermore, dot blot analysis of total cellular RNA isolated from the two cell populations demonstrated that the amount of a l A T mRNA transcripts expressed in the NIH-3T3IalAT-M and NIH-3T3/alAT-S cells was

a1 AT-M a1 AT4

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FIG. 6. DNA plasmid map of the pN2-alAT-M and pN2- alAT-S retroviral vectors containing full length human a l - antitrypsin cDNAs of "type and S-type, respectively. The plasmids were constructed from the plasmid pN2, a retroviral vector

1 2

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NlH-3T3/alAT-M r) @ 0 NIH-3T3/alAT-S clb 0 '

T

based on the plasmid pBR322 together with a-5' long terminal repeat (LTR) containing a viral packaging signal, the bacterial neomycin FIG. 7. Evaluation of murine fibroblasts modified by retro- resistance gene (NE@), and a 3' long terminal repeat (36). The M- viral gene transfer of "type and S-type alAT cDNAs. A , type and S-type OlAT cDNAs differ by a single nucleotide substitu- identification of human a l A T cDNAs of "type and S-tYPe inte- tion (A + T) causing the replacement of glutamic acid by valine a t grated into the genome of mouse fibroblasts. Shown are data from amino acid position 264 of the coded protein. bp, base pairs. polyclonal populations of NIH-3T3 cells infected with the G2-alAT-


NIH-3T3klAT-M

+ + Untreated Swalnsonlna Tunicamycin

+ + Unlreated Swainsonins Tunicamycin

koa 52-

kDa - 50- 0

koa

45-

NiH-3T3kl AT-S

1 2 3 4 5 6

FIG. 8. Effect of modification of proximal and distal oligosaccharide side-chain processing on the secretion of human a lAT by mouse fibroblasts containing the integrated a lAT "type or S-type cDNAs. NIH-3T3 cells containing the a l A T cDNAs were pulsed with ["S]methionine for 1 h followed by a 2-h chase with unlabeled media. Shown are SDS-acrylamide gel electrophoresis and fluorographic analysis of human a1AT immunoprecipitated from supernatants of the modified mouse fibroblasts cultured with ["S]methionine in the presence of swainsonine (5 pg/ml) or tunicamycin (10 pglml). Lane I , control (Untreated) NIH-3T3IalAT- M cells. Lane 2, similar to lane 1, but with cells cultured with swainsonine. Lane 3, similar to lane 1, but cultured in the presence of tunicamycin. Lane 4, control (Untreated) NIH-3T3/alAT-S cells. Lane 5, similar to lane 4, but cultured in the presence of swainsonine. Lane 6, similar to lane 4, but cultured in the presence of tunicamycin. Indicated are the 52-kDa mature secreted form of human alAT, the 50-kDa secreted form of a l A T containing hybrid oligosaccharide side chains and the 45-kDa nonglycosylated form of alAT.

comparable (Fig. 7C, p > 0.2). Thus, at the mRNA level, murine fibroblasts containing the a lAT cDNAs of "type and S-type expressed the M and S a l A T cDNAs in a fashion comparable to that of M and S blood monocytes.

Synthesis and Secretion of Human alAT by Murine Fibro- blasts Modified to Contain the Human alAT cDNAs of M - type and S-type-Quantification of the relative amount of human a lAT secreted by murine fibroblasts containing integrated human M- or S-type a1AT cDNAs demonstrated that the S-type cells secreted relatively less alAT. Polyclonal populations of NIH-3T3/alAT-M and NIH-3T3-alAT-S fibroblasts pulsed with [35S]methionine for 1 h secreted in increasing amounts of 3sS-labeled 52-kDa human a1AT (Fig. 8, lanes 1 and 4 ) in a time-dependent fashion until plateauing at 2 h (not shown). Immunoprecipitation of supernatants of

M and $2/alAT-S generated retroviruses (NIH-3T3/alAT-M and NIH-3T3/alAT-S, respectively). DNA extracted from the two cell populations was digested with the restriction enzymes Hind111 and XhoI. The DNA (10 pg/lane) was analyzed by Southern blots hybridized with a 32P-labeled human a l A T cDNA probe. Lane I , NIH-3T3/ alAT-M; lane 2, NIH-3T3/alAT-S. The 1.4-kb a1AT cDNA insert is present in both the NIH-3T3/alAT-M and NIH-3TB/alAT-S cells. B, identification of a1AT mRNA transcripts in mouse fibroblasts containing the integrated human n lAT M-type or S-type cDNAs. Shown are data from NIH-3T3 cells infected with the $2/ a1AT-M and $2/alAT-S generated retroviruses. Poly(A)'-selected RNA (2 pg) from the cell lines was evaluated by RNA blot-analysis utilizing a J2P-labeled human a l A T cDNA probe. Lane 3, NIH-3T3/ alAT-M, lane 4, NIH-3T3/alAT-S. Both the NIH-3T3/alAT-M and the NIH-3T3IalAT-S cells contain a l A T mRNA transcripts of comparable size. C, comparison of the levels of a l A T mRNA transcripts in mouse fibroblasts containing the integrated human a l A T M-type or S-type cDNAs. Total cellular RNA was evaluated for a l A T mRNA transcripts using dot blot analysis with a 32P-labeled a1AT cDNA probe. Resulting autoradiograms were quantified by densitometry. Shown is the amount of a l A T mRNA expressed as unitslpg total RNA. Inset, example of a l A T dot blot analysis.

12

10

8

6

4

2

NIH-3T3 NIH-3T3 alAT-M alAT-S

+ + + + Swalnsonlne Tunlcamycln Swainsonine Tunicamycin

LNIH-3T3/alAT-MJ LNIH-3T3hlAT-SJ

FIG. 9. Replication of the a lAT S-type defect in murine fibroblasts modified to contain the a lAT "type or S-type cDNAs. A, quantification of the relative amount of human a l A T secreted by murine fibroblasts containing the integrated a l A T M- type or S-type cDNAs. Polyclonal populations of NIH-3T3 cells containing the a1AT M-type or S-type cDNAs were pulsed with [35S] methionine for 1 h followed by a 2-h chase with unlabeled media. SDS-acrylamide gel electrophoresis, fluorography, and densitometric quantification of the 35S-labeled human a l A T immunoprecipitated from supernatants of the modified mouse fibroblasts were as in Fig. 8. All immunoprecipitations were carried out on samples containing IO6 dpm total trichloroacetic acid (TCA)-precipitable 3sSS-labeled protein. B, quantification of the relative amount of human a1AT secreted by modified murine fibroblasts cultured with swainsonine and tunicamycin compared to that of control cells. Quantification was performed by densitometric analysis of fluorograms as in Fig. 8. All immunoprecipitations were carried out with samples containing lo6 dpm total precipitable "S-labeled protein. Shown is the relative amount of human a l A T secreted by NIH-3T3IalAT-M and NIH- 3T3/alAT-S cells cultured with swainsonine or tunicamycin expressed as a percentage of that secreted by control NIH-3T3/alAT- M and NIH-3T3/alAT-S cells, respectively.

modified mouse fibroblasts of both cell types after a 1-h "pulse" with [3sS]methionine and a 2-h chase with unlabeled media followed by SDS-acrylamide gel electrophoresis, fluorography, and densitometric quantification of the labeled


180 r

I NIH-3T3 NIH-3T3 alAT-M alAT-S

+ + Leupeptin Leupeptin

FIG. 10. Effect of inhibition of cellular proteinases on secretion of human a1 AT by murine fibroblasts modified to contain the a l A T "type or S-type cDNAs. Polyclonal populations of NIH-3T3 cells containing the LvlAT M-type or S-type cDNAs (NIH- 3T3/alAT-M and NIH-3T3/alAT-S, respectively) were treated with leupeptin (0.5 mM), an inhibitor of cellular proteinases, for 24 h prior to pulse labeling with [36S]methionine as before. All immunoprecipitations were carried out on samples containing IO6 dpm total precipitable 36S-labeled protein. Quantification was performed by densitometric analysis of fluorograms. Shown is the relative amount of human alAT secreted by NIH-3T3/alAT-M and NIH-3TS/alAT-S cells treated with the antiproteinase expressed as a percentage of that secreted by untreated control NIH-3T3IalAT-M and NIH-3T3/ alAT-S cells, respectively.

secreted human alAT demonstrated that NIH-3T3/alAT-S cells secreted 45 f 5% the amount of human alAT compared to the NIH-3T3/alAT-M cells (Fig. 9A, p < 0.01).

Importantly, not only did the murine fibroblasts containing the a1AT S-type cDNA secrete less human alAT, but the S- type a1AT cDNA containing fibroblasts exhibited a similar abnormality in alAT biosynthesis as observed in S-type monocytes. In this regard, blockade of distal oligosaccharide side chain processing with swainsonine resulted in the secretion of a 50-kDa species of a1AT for both of the cell populations (Fig. 8, lanes 2 and 5 ) in amounts comparable to untreated controls (Fig. 9B). In contrast, blockade of oligosaccharide side chain addition with tunicamycin resulted in the secretion of a 45-kDa nonglycosylated human alAT by both cell populations (Fig. 8, lanes 3 and 6). However, the relative reduction in alAT secretion by treated cells compared to untreated controls was dramatically greater for the S-type than for the "type cDNA containing cells (Fig. 9B), i.e. as observed in the monocytes, the nonglycosylated S-type alAT protein appears to be more dependent than the M-type alAT on the presence of oligosaccharide side chains for secretion.

Effect of Inhibition of Cellular Proteinases on Secretion of Human alAT by Modified Murine Fibroblasts-Inhibition of cellular proteinases in murine fibroblasts modified to synthesize and secrete human alAT demonstrated a selective aug- mentation in secretion of alAT by cells containing the S- type alAT cDNA not observed in the cells containing the M- type cDNA. When NIH-3T3/alAT-M and NIH-3T3/alAT- S cells were treated with leupeptin, followed by pulse-chase labeling with [35S]methionine and evaluation for the amount of labeled, human a1AT secreted by the cells, NIH-3T3/ alAT-M secreted 102 & 10% the amount of human alAT as untreated M-type control cells. In marked contrast, NIH- 3T3/alAT-S cells treated in a similar manner secreted 155 f 12% the amount of alAT as the untreated S-type control (Fig. 10; p < 0.01). Thus, it appears that intracellular proteolysis contributes to the abnormality in biosynthesis associ-

ated with the S-type mutation and, furthermore, that this process can be modified by inhibition of cellular proteinases.

DISCUSSION

Together, the data in the present study suggest that the pathogenesis of reduced serum levels of alAT associated with the a1AT S allele results from exaggerated intracellular degradation of newly synthesized alAT resulting in reduced secretion of a1AT. This conclusion is based on several lines of evidence, including the following. 1) Utilizing peripheral blood monocytes as an in vitro model system of alAT gene expression, evaluation of a1AT mRNA transcripts demonstrated that the form and steady state levels of the a1AT mRNA associated with the S-type allele are identical to that of the normal "type allele. 2) Monocytes of S homozygotes secrete 42 k 10% of the amount of alAT as do normal monocytes, comparable to the level of serum alAT deficiency observed in the S homozygote. 3) Inhibition of addition of oligosaccharide side chains to the newly synthesized S-type alAT molecule results in markedly reduced secretion of nonglycosylated alAT compared to that of similarly treated M- type monocytes. 4) Retroviral gene transfer to modify murine fibroblasts to contain the "type or S-type human alAT cDNAs demonstrated that the resulting S-type murine fibroblasts contained alAT mRNA transcripts of similar form and level as the normal "type fibroblasts, but the S-type fibroblasts secreted 45 + 5% less human alAT; i.e. the S-type modified murine fibroblasts behaved identically to the S-type monocytes, strongly suggesting that the abnormal biosynthesis of the S-type a1AT is a consequence of the single nucleotide substitution (A + T) generating the G1uzU + Val amino acid substitution and is not dependent on the cell synthesizing the protein. 5) The exaggerated decreased secretion of S-type a1AT in the presence of a core glycosylation inhibitor could be reproduced in the S-type murine fibroblasts. 6) The alAT secretory defect associated with the S-type mutation could be significantly prevented by treating the alAT synthesizing cells with an inhibitor of cellular proteinases, suggesting a role of intracellular proteolysis in the reduced secretion of a1AT by S-type cells.

This scenario for the pathogenesis of the deficient levels of a1AT associated with the S-type gene is markedly different from that conceptualized to occur in association with the Z- type allele. The Z mutation results from a G I u ~ ~ ' + Lys substitution that results in exaggerated intracellular accumulation of newly synthesized alAT, not exaggerated proteolysis as apparently occurs in association with the S mutation. Although the mechanisms responsible for the intracellular aggregation of alAT associated with the Z mutation are not completely understood, it likely results from the combination of a disruption of a critical intramolecular salt bridge together with a charge alteration at residue 342, culminating in im- paired assumption of the correct three-dimensional conformation during synthesis and intracellular aggregation at the level of the rough endoplasmic reticulum (19, 50). This aggregation can be visualized histologically within hepatocytes of individuals homozygous for Z-type alAT, and analysis of this intracellular alAT has demonstrated alAT molecules with immature high mannose-type oligosaccharide side chains (51).

Unlike individuals with the Z mutation, histologic exami- nation of liver of individuals homozygous for S-type mutation shows no intracellular aggregation of a1AT (52). In addition, studies comparing the serum half-life of the S-type alAT protein to normal "type alAT protein have demonstrated a nearly identical rate of disappearance of the two molecules (23). Together, these clinical observations are consistent with


the concept derived in the present study that the observed deficient level of serum alAT seen in association with the S homozygous state results from exaggerated intracellular degradation of the newly synthesized alAT.

One possible explanation of the apparent reduced synthesis of alAT of S-type compared to "type monocytes is that there is reduced expression of the a1AT gene at the level of mRNA processing. In this regard, analysis of the sequence of the S-type alAT gene has lead to the hypothesis that the S mutation generates an area within the 3"terminal region of exon I11 that has a high degree of homology to the consensus 5' splice donor sequence (48). If recognized as a splicing site during mRNA processing, a truncated alAT mRNA would be generated which, if stable, would result in an altered transla- tion reading frame and premature termination of an abnormal protein. Alternatively, an abnormal spliced mRNA might be unstable, resulting in reduced levels of alAT mRNA transcripts in a1AT producing cells. However, the demonstration of a correctly processed alAT mRNA product of the S-type allele as well as normal levels of steady state alAT mRNA associated with the S homozygous state excludes the possibil- ity that the potential aberrant splice site is recognized or, if it is recognized, excludes its pathophysiologic relevance.

Oligosaccharide side chains serve an important function in conferring protein stabilization during biosynthesis of many secretory and membrane glycoproteins (53, 54). For example, in the absence of their normal complement of oligosaccharide side chains, fibronectin (55), acetylcholine receptor (47), human chorionic gonadotropin receptor (56), and the lympho- cyte cell surface protein CD4 (57) are all degraded within the cell. Interestingly, the finding of secretion of near-normal amounts of "type alAT in the presence of blockade of oligosaccharide addition suggests that oligosaccharide side chains are not obligatory for the structural stabilization or secretion of the normal form of a1AT (30-33). In marked contrast, however, the ability of the newly synthesized S-type alAT to remain intact and be secreted by the cell was criti- cally dependent upon the presence of oligosaccharide side chains. One explanation of this finding is that the S-type protein is less stable than the "type protein, a disparity which is exaggerated through loss of the stabilizing effect of oligosaccharide side chains, i.e. the S-type protein is more dependent on the conformational stabilization afforded by the oligosaccharide side chains than the normal "type alAT. Consistent with this concept, crystallographic analysis has shown that the S mutation ( G ~ u ' ~ ~ -+ Val) results in the loss of an intramolecular salt bridge with Lys3s7 (50). Since this interaction is likely critical to the tertiary structure of the protein, i t is reasonable to conclude that the S-type mutation would have profound consequences on the conformational stability of the alAT molecule. In this regard, abnormal proteins with perturbations of tertiary conformation have been shown to be more susceptible to intracellular proteolysis in studies with incorporation of amino acid analogues (46,58) and in naturally occurring mutations (59, 60). Therefore, the demonstration of a selective increase in secretion of S-type a l A T from synthesizing cells treated with a broad spectrum inhibitor of cellular proteinases suggests that intracellular proteolysis contributes to the abnormal pattern of biosynthesis of a1AT associated with the S-type allele and that this proteolysis likely occurs because the S-type alAT molecule is conformationally unstable. Importantly, the fact the abnormality of biosynthesis is reproduced in murine fibroblasts modified with the S-type a1AT cDNA strongly argues that the S-type mutation renders the protein intrinsically less stable within the cell, independent of the synthesizing cell.

Multiple mechanisms of intracellular protein degradation have been characterized (61-64). In this context, both lysosomal and non-lysosomal pathways have been implicated in the degradation of abnormal proteins within the cell (46, 62, 65-67). The partial reversal of the S-type defect by treatment with a broad spectrum inhibitor of cellular proteinases con- firms that intracellular degradation of the protein is partici- patory in the aberrant biosynthesis of alAT associated with the Glu264 + Val mutation. The proteinase inhibitor utilized in these studies, leupeptin, has been demonstrated to inhibit lysosomal proteinases such as the thiol proteinases cathepsin B and cathepsin L (42,43) as well as various serine proteinases (68). In addition, this agent has inhibitory activity against the calcium-dependent proteinases, calpains, which comprise a component of the non-lysosomal pathway of intracellular degradation (44, 45,69). It is thus not possible, based on this limited analysis, to characterize the specific intracellular pathways of protein degradation involved in the proteolysis of the S-type alAT. In addition, the fact that the rescue of the S-type alAT from proteolysis was only partial suggests that additional degradative pathways not inhibited by leupeptin, such as the ubiquitin pathway (67, 70), may be contribu- tory.

Acknowledgments-We would like to thank Dr. Susan Leitman and the nurses of the National Institutes of Health Clinical Center Blood Bank for performing cytapheresis of the study patients and Dr. Robert F. Ritchie, Foundation for Blood Research, Scarbor- ough, ME, for referral of several of the study patients. We would also like to thank L. Sichert for excellent editorial assistance.

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