Biosynthesis, Processing, and Secretion of M and Z Variant Human

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Val. 261, No. 21. Issue of July 25, pp. 9979-9989, 1986 Printed in U.S.A.

Biosynthesis, Processing, and Secretion of M and Z Variant Human al-Antitrypsin*

(Received for publication, January 24,1986)

Kathryn M. Verbanact and Edward C. Heath? From the Department of Biochemistry, The University of Iowa College of Medicine, Iowa City, Iowa 52242

The Z genetic variant of human al-antitrypsin (alAT) is associated with decreased serum alAT levels, hepatic inclusion bodies, and an increased risk of lung and liver disease. We studied the biosynthesis, processing, and secretion of normal and Z variant a1AT in cell-free translation systems, reconstituted in vitro processing systems, and in the Xenopus oocyte secretory system. Human liver mRNA was prepared from normal subjects (Pi”) and from individuals homozygous for alAT deficiency (PiZZ). Cell-free translation resulted in the synthesis of 49,000-Da preproteins with a 23-amino acid signal sequence. The genetic variants were synthesized at comparable levels and could be distinguished on the basis of charge. The majority of the amino acids in the ZZ signal peptide were identified and found to be the same as those comprising the MM signal sequence. These proteins were co-translationally processed with similar efficiency by dog pancreas microsomes, producing 52,000- Da glycoproteins which were completely translocated across the endoplasmic reticulum membrane. When the human liver RNA preparations were injected into Xen- opus oocytes, both of the alAT variants were synthesized intracellularly and alAT was detected in the medium of all oocytes injected with MM RNA. However, the Z variant accumulated within the microsomal vesicles of the cell and was undetectable or present at decreased levels in the medium. We conclude that the single amino acid substitution in .the Z variant of alAT does not affect its synthesis or co-translational processing but that it strongly affects its transport from the rough endoplasmic reticulum through the secretory pathway.

al-Antitrypsin (+AT’) is a glycoprotein that serves as a

* This work was supported by National Institutes of Health Grant AM19850. A preliminary report of this work has been presented (1). 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.

Submitted in partial fulfillment of the requirements for the Ph.D. degree. Present address: Biotechnology Laboratory, Central Re- search, Dow Chemical Co., Midland, MI 48674. To whom all corre- spondence should be addressed.

t Participated in the preparation of this paper but due to his untimely death he did not review the final manuscript.

The abbreviations used are: alAT, al-antitrypsin; IgG, immuno- globulin G; BSA, bovine serum albumin; poly(A), polyadenylated; PiM and PiZ, genetic variants of a,-proteinase inhibitor; Hepes, 4- (2-hydroxyethyl)-l-piperazineethanesulfonic acid; EGTA, [ethylene- bis(oxyethy1ene-nitri1o)ltetraacetic acid; Endo H, endoglycosidase H; SDS, sodium dodecyl sulfate, PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; ER, endoplasmic reticulum.

major proteinase inhibitor in mammalian blood (reviewed in Ref. 2). alAT has more recently been referred to as al- proteinase inhibitor, and its primary physiological target is leukocyte elastase. It is synthesized in the liver as a single polypeptide chain (Mr = 54,000) and contains three asparagine-linked carbohydrate side chains. The normal human serum alAT levels are 2 mg/ml, but these levels approximately double during the acute phase response to inflammation.

alAT production is determined by two autosomal codomi- nant alleles, and over 30 genetic variants of alAT have been identified which are functionally and immunologically identical (reviewed in Ref. 3). The proteinase inhibitor (Pi) variants are designated by letters which indicate their relative electrophoretic mobilities. The M variant is the “normal” most common form of alAT. The Z variant serum protein has been studied extensively and differs from the M variant by a single amino acid substitution (Glu - Lys). The carbohydrate compositions of the M and Z variant serum proteins are identical, and the proteins exhibit minimal differences in half- lives. Liver microsomal inclusion bodies containing alAT are found in all individuals carrying the Z allele, and homozygous PiZ individuals have 10-15% of the normal levels of circulat- ing alAT. ZZ alAT deficiency is clinically important because it is frequently associated with pulmonary emphysema and juvenile cirrhosis.

The accumulation of ZZ alAT in liver microsomes suggests that al-antitrypsin deficiency is a result of an alteration in the normal pathway of glycoprotein biosynthesis, processing, and secretion. This could occur at the level($) of protein synthesis, localization and stability, co-translational protein processing, and/or post-translational transport through intracellular secretory compartments. The present study was undertaken to investigate the mechanism of this defect and was made possible by the availability of human liver from individuals homozygous (PiZZ) for alAT deficiency. Cell-free translation experiments were carried out with RNA prepared from M and Z variant livers, and the ZZ signal sequence was partially determined by NH2-terminal sequence analysis. The in vitro processing of both alAT variants was examined in the presence of dog pancreas microsomes. Proteolytic enzymes and alkali treatment were used to probe the membrane segregation of the processed proteins. Finally, the in vivo synthesis and secretion of the a,AT variants were compared in microinjected Xenopus oocytes. These proteins were charac- terized by tunicamycin treatment and endoglycosidase H digestion.

EXPERIMENTAL PROCEDURES

Materials-Nontoasted hard red winter wheat germ was obtained as a gift from General Mills, Inc. Oligo(dT)-cellulose type 7 was purchased from Pharmacia P-L Biochemicals. [3H]Proline (114 Ci/ mmol), [‘Hlleucine (142 Ci/mmol), [’Hlalanine (57 Ci/mmol), [35S] methionine (1200 Ci/mmol), and [36S]cysteine (1000 Ci/mmol) were

9979

9980 M and Z Variant al-Antitrypsin Biosynthesis and Processing

obtained from Amersham Corp. Trypsin and micrococcal nuclease were supplied by Worthington. Placental ribonuclease inhibitor (RNasin) was purchased from Promega Biotec. Tunicamycin, protein A-Sepharose, chymotrypsin, and aprotinin were obtained from Sigma. ENHANCE and dog pancreas microsomes were purchased from New England Nuclear. Rabbit anti-human alAT IgG was obtained from Accurate, Atlantic Antibodies, and Boehringer Mannheim. Endogly- cosidase H was obtained from Dr. Frank Maley, Health Research, Inc. All other reagents were of the highest purity available commer- cially.

Tissue Source-One PiMZ and three PiZZ human liver samples were obtained from Dr. Patricia Eagon, the University of Pittsburgh, The MZ donor was a 4-year-old girl and the PiZZ donors were males of 5, 18, and 42 years of age. The six normal (PiM) donors were adults of both sexes. These samples were either obtained at autopsy (within 2-4 h of death) or during transplantation surgery at the University of Iowa or the University of Pittsburgh. Liver samples were cut into small pieces, frozen in liquid nitrogen, and stored at -70 'C. al-Antitrypsin phenotyping was conducted by serum analysis at Mayo Medical Laboratories.

Isolation of RNA and Rough Microsome-Human liver RNA was isolated by the method of Chirgwin et al. (4). The frozen tissue was placed directly in the guanidine thiocyanate solution before thawing. RNA for oocyte injections was extracted with phenol/chloroform (5) and subjected to oligo(dT)-cellulose chromatography according to the method of Stark (6) except that the 0.25 M NaCl wash was omitted. Microsomes enriched for rough endoplasmic reticulum were prepared as described (7).

Cell-free Protein Synthesis-RNA was translated in vitro in either a rabbit reticulocyte lysate system (8) or a wheat germ lysate system (9) in the presence of [35S]methionine for 1 h at 25 "C. Typical reticulocyte translation incubation mixtures contained 60 pCi of [%SI methionine, 25 pg of human liver RNA (or 1.2 A260 units rough microsomes), and 35 p1 of micrococcal nuclease-treated (10) lysate in a final volume of 50 pl.

Wheat germ embryos were separated from endosperm fragments by a selective organic solvent flotation procedure (111, and the S-23 fraction was prepared as described (9) with one modification. Only the first third to half of the turbid fractions eluting at the Sephadex G-25 void volume were pooled (12). Translation reaction mixtures typically contained 35 pCi of [%3]methionine, 20 mM Hepes (pH 7.5), 1.2 mM ATP, 100 p M GTP, 5.5 mM creatine phosphate, 200 pg of creatine phosphokinase/ml, 135 mM potassium acetate, 1.6 mM magnesium acetate, 80 p M spermine, 67 p M dithiothreitol, 33-200 p M amino acids minus methionine, 10 pg of RNA, 14 p1 of wheat germ extract in a final volume of 30 pl. The amount of wheat germ extract as well as the potassium and magnesium acetate concentrations were optimized for each preparation of wheat germ lysate.

Immunofogical and Electrophoretic Analyses-The immunoprecipitation method (13) involved partial denaturation of the antigen, incubation with IgG at both 25 and 37 'C, and adsorption of the immune complex by formalin-fixed Staphylococcus aureus (Cowan) cells or protein A-Sepharose. IgG which had been covalently coupled to Sepharose (14) was also used as a substitute for the two-step procedure, in which case the samples were constantly rotated during the incubations and alAT was dissociated in SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer without reductant.

Certain modifications of this procedure were used with the cellular and secreted products from oocytes. Thirty pl of the cellular homog- enate (from 1-2 oocytes) was diluted with 50 pl of 10 mM Tris (pH 7.4), 150 mM NaCl before subjection to the immunoprecipitation method. One mg/ml ovalbumin, 1 mg/ml goat IgG, and 1 mM unlabeled methionine were included in the dilution buffer to decrease nonspecific background. The immunoadsorbed proteins were dissociated from the IgG-Sepharose, rediluted, and submitted to the same immunoprecipitation protocol.

Media from oocyte incubations were thawed, made 1 mM in phenylmethylsulfonyl fluoride (PMSF), and centrifuged in a Microfuge for 5 min prior to immunoprecipitation of the supernatant. Nonspe- cific adsorption of labeled secreted human serum albumin (which comprised the majority of secreted products) was observed in the initial immunoprecipitations. Therefore, 20 pg of human serum albumin and 20 pg of goat IgG were added as carrier proteins prior to SDS solubilization, and 1 mg/ml ovalbumin, 1 mg/ml goat IgG, and 1 mM unlabeled methionine were included in the dilution buffer.

Antigen-antibody complexes were analyzed by SDS-PAGE (15) and autoradiography. Gradient (7-14%) slab gels were usually used. Molecular weight markers were phosphorylase a (93 kDa), BSA (68

kDa), ovalbumin (44 kDa), carbonic anhydrase (30 kDa), and soybean trypsin inhibitor (22 kDa). Immunoprecipitated samples were run under nonreducing conditions unless otherwise noted. This was done to remove interference by the heavy chain of IgG which exhibits a similar mobility to alAT under reducing conditions. Densitometric scans of autoradiograms were conducted on a Beckman DU-8 spec- trophotometer, using a gel scan module and integrating peak areas.

Equilibrium isoelectric focusing gels (0.5 X 125 mm) were prepared and electrophoresed by the method of O'Farrell (16). Gels were fixed in several changes of 12.5% trichloroacetic acid, stained with 0.02% Coomassie Blue G-250 in 3.5% perchloric acid, and destained with 10% acetic acid. Gels were dried at low heat between sheets of cellophane membrane and subjected to autoradiography.

Proteins were transferred from unfixed gels to nitrocellulose sheets using the Towbin buffer system (17). Lanes containing molecular weight standards were stained for protein in 0.2% Ponceau S in 3% trichloroacetic acid and 5% sulfosalicylic acid for 15 rnin (18). The remaining nitrocellulose was blocked for 1 h at room temperature or overnight at 4 "C in 3% BSA, 10% horse serum, 50 mM Tris/HCl (pH 7.4), and 200 mM NaCl. After rinsing in 50 mM Tris/HCl, pH 7.4, 200 mM NaCl (TBS) the nitrocellulose was incubated with 12 pg/ml rabbit anti-human IgG diluted in 25 ml of blocking solution for 4 h at room temperature on a rotational shaker. Nitrocellulose strips were extensively rinsed before incubation with affinity-purified 'Z51-labeled goat anti-rabbit IgG (1 pg/ml, 2 X lo6 cpmlpg in 25 ml of 3% BSA in TBS). After two consecutive 30-min incubations at room temperature and at 4 "C, the blots were washed in 0.2% SDS, 0.5% Triton X-100, 0.5% BSA, 0.01% sodium azide, 10 mM Tris (pH 7.4), and 0.9% NaCl. Alternatively, blots were treated with the following consecutive washes: 10 mM NaI in TBS, 0.05% Nonidet P-40 in the same buffer, blocking solution diluted 1:l in this buffer, and a final wash of NaI in saline. Filters were air dried, covered with plastic wrap, and subjected to autoradiography.

Repetitive Edman Degradation-For sequence determination of the precursor form of ZZ alAT, large-scale translations of human liver RNA were conducted using reticulocyte lysate that had been passed over a Sephadex G-25 column to remove endogenous amino acids (19, 20). Immunoprecipitated proteins were subjected to consecutive automated Edman degradation in a Beckman 890C sequencer using a modified 1M Quadrol program (21). The 2-anilino-5-thiazolinone amino acid derivatives were dried under nitrogen, dissolved in meth- anol, and their radioactivity was determined by liquid scintillation spectroscopy. Repetitive yields for carrier sperm whale apomyoglobin were determined by high pressure liquid chromatography on a 15-cm C18 column and ranged from 85-98%. Protein sequence analysis was performed by Glen Wilson, Protein Structure Facility, University of Iowa.

Post-translational Analysis of Protein for Segregation and Trans- location-In the first post-translational proteolysis experiment (Fig. 4), human liver RNA was translated for 75 min in the presence or absence of 2.5 Am units of dog pancreas microsomes. All samples were adjusted to 5 mM CaClz and trypsin (freshly prepared in 1 mM HC1) was added to appropriate samples at a final concentration of 100 pg/ml. One per cent Triton X-100 was also present during some digestions, as indicated in the figure legend. After incubation for 1 h on ice, proteolysis was terminated by the addition of 3000 Kallikrein units/ml of aprotinin and 10 mM EGTA. Samples were solubilized in 2% SDS and subjected to the usual immunoprecipitation procedure except that beef pancreas trypsin inhibitor was present during the antibody incubation (5 pg) and in the immunoprecipitation wash buffer (5 wg/ml).

In the second protease protection experiment (Fig. 5), human liver RNA was translated for 1 h in the presence of 2.5 A260 units of dog pancreas microsomes. Three mM tetracaine and 10 pg/ml cycloheximide were then added, and the samples were incubated for 10 min at 25 "C. All samples were made 1 mM in CaC12, and Triton X-100 was added to a final concentration of 1% to appropriate samples. Protease incubations were carried out in the presence of 100 Fg/ml trypsin and chymotrypsin in a final total volume of 50 pl for 45 min on ice. Aprotinin (120 Kallikrein units) was then added to inhibit further proteolysis. After a 5-min incubation at 4 "C, samples were made 2% in SDS and subjected to a slightly modified immunoprecipitation procedure. One mM PMSF, 500 pg/ml soybean trypsin inhibitor, and 10 kallikrein units/ml aprotinin were included in the dilution buffer

The procedure used for the alkali-induced release of microsomal

two published methods (22,23). MM and ZZ RNA were translated in contents and peripheral membrane proteins was a combination of

30-p1 wheat germ incubation mixtures, in the presence and absence

it4 and 2 Variant cq-Antitrypsin Biosynthesis and Processing 9981

of dog pancreas microsomes. After translation, the pH was adjusted to 11.5 and samples were incubated for 15 min at 4 "C. The pH of control samples was not altered. Samples were transferred to Beck- man Airfuge tubes containing 20 p1 of 2 M sucrose and 80 pl of 0.2 M sucrose and centrifuged a t 160,000 X g for 15 min. The sucrose solutions contained 130 mM potassium acetate and 1.5 mM magnesium acetate in either 20 mM Hepes/KOH (pH 7.5) or in 100 mM sodium carbonate (pH 11.5). After centrifugation, 50 pl of the supernatant solutions was removed, and microsomes (25 pl) were collected from the 0.2 M, 2.0 M sucrose interface and diluted with 25 pl of the appropriate buffer. Supernatant and microsomal fractions were adjusted to 1% SDS, boiled and diluted as usual, and neutralized before the addition of antibody.

Collection, Injection, and Culture of Oocytes-Stage VI oocytes (diameter 1.2-1.3 mm) were obtained from female Xenopus laeuis, manually defolliculated, and cultured in OR-2 medium (24). Oocytes were injected with 10-40 nl of poly(A) RNA (2 ng/nl) and incubated for 13-16 h. The oocytes that were viable after this recovery period (95%) were then injected with 20 nl of [35S]methionine (12.93 mCi/ ml) and transferred to 1.5-ml polypropylene tubes containing 200 pl of OR-2. Control oocytes were not injected with exogenous RNA, but were injected with [35S]methionine as described. After a 3-h labeling period, cells and media were separated, the cells were washed, and both fractions were quick-frozen in dry ice.

In subsequent experiments, oocytes were injected with 20 ng/20 nl poly(A) RNA. After recovery for 14-15 h, 100 pCi of [35S]methionine (1235 Ci/mmol) was added to 100 pl of OR-2 medium, and cells were cultured for an additional 24 h in microtiter plates, 8-10 oocytes/ well. After this time period, the media from the wells containing healthy oocytes was pooled and frozen. MM and ZZ RNA-injected oocytes (10 of each) were washed and frozen. Another group of oocytes (20 of each) were directly homogenized and subjected to subcellular fractionation in order to isolate the vesicular fraction. This procedure employed sucrose gradient microcentrifugation as described by Col- man and Morser (25). In studies with the glycosylation inhibitor tunicamycin, occytes were simultaneously injected with RNA and 1.6 pg of tunicamycin. After recovery for 3-4 h in OR-2 containing 2 pg/ ml tunicamycin, oocytes were transferred to microtiter plate wells containing 50 pCi/pl [35S]methionine in this same media and incubated for 18-19 h.

Extraction and Analysis of Oocytes-In the first set of experiments which used a 3-h labeling period, thawed oocytes were homogenized in 50 mM NaCl, 10 pg/ml cycloheximide, and 0.5 mM PMSF (100 pl/ oocyte). Samples were centrifuged in 1.5-ml polypropylene tubes in an HB4 rotor a t 5000 rpm for 8 min. The supernatant was analyzed by SDS-PAGE, immunoprecipitation, and by repeated precipitation with cold 10% trichloroacetic acid. Total [35S]methionine incorporation into oocytes was measured by determination of hot perchloric acid-insoluble radioactivity (26).

For analysis of samples generated in the second set of experiments, thawed oocytes were homogenized in 1% Nonidet P-40,l mM PMSF, 10 mM sodium phosphate (pH 7.6), 150 mM NaCl(20 pl/oocyte). The samples were centrifuged in a Microfuge for 10 min, and the supernatant was analyzed by SDS-PAGE, trichloroacetic acid precipitation, and immunoprecipitation. Total incorporation of label into protein was assayed by repeated precipitation by cold 10% trichloroacetic acid.

Endoglycosidose H-Immunoprecipitated cellular and secreted fractions from the long term labeling experiment were thawed and diluted 1: lO with 0.15 M sodium citrate, pH 5.3, containing 60 pg of BSA and 1 mM PMSF. Fourteen milliunits of endoglycosidase H were added to one of each of the duplicates, and samples were incubated for 5 h at 37 "C. Another 10 milliunits of enzyme were added, and the incubation was continued for 1 h a t which time samples were chilled on ice. Twenty pg of phosphorylase b was added as carrier protein, and cold trichloroacetic acid was added to a final concentration of 10%. After a 15-min incubation on ice, samples were centrifuged in a Microfuge and the protein pellets washed twice in cold ethanol. Pellets were dissolved in 60 pl of SDS-PAGE sample buffer containing reductant, and samples were boiled and analyzed by SDS-PAGE and fluorography.

RESULTS AND DISCUSSION

I n Vitro Synthesis of Pre-alAT-Our first objective was to investigate the in vitro synthesis and processing of M and Z variant alAT. We followed the same experimental protocol

that had been used in our previous study of rat alAT (7). RNA was isolated from normal (M) or ZZ variant human livers and translated in a cell-free rabbit reticulocyte system. The PiM and PiZ mRNA preparations translated with similar efficiencies, and the total RNA translation products from the different livers appeared to be very similar (Fig. 1, lunes 1 and 4). Primary a,AT translation products with molecular weights of 49,000 were immunoprecipitated by rabbit anti-human alAT IgG and were indistinguishable by SDS-PAGE (lanes 3 and 6). al-Antitrypsin constitutes ~ 1 % of the total protein synthesized from either mRNA source; therefore, PiM and PiZ livers contain comparable amounts of translatable alAT mRNA. This finding demonstrates that decreased serum levels of ZZ alAT are not a result of decreased ZZ alAT synthesis. As would be expected, the precursor proteins migrated faster than the mature glycosylated serum form of human alAT (M, = 54,000). The M and Z variants of pre-alAT could be distinguished on the basis of charge by isoelectric focusing (lunes 7-9). The Glu + Lys substitution that occurs in the Z variant protein caused it to migrate farther toward the cathode. Errington et ul. (27) reported the in vitro synthesis of a smaller 46-kDa alAT precursor protein. The authors proposed that the 46-kDa protein was a result of alAT cleavage by reticulocyte lysate thiol proteases. This protease activity may be responsible for the lower molecular weight proteins which were sometimes detected in our immunoprecipitations.

Although the amino acid substitution in mature ZZ alAT has been well established, the signal peptide sequence of ZZ pre-alAT has not been investigated. The amino-terminal amino acid sequence that comprises the signal peptide of secretory proteins is involved in the association of nascent

4 5 6 7 8 9 '" "' P@ pa +

a,AT-

44-

22-

crl

FIG. 1. In vitro synthesis of human alAT. RNA was isolated from the livers of MM or ZZ individuals and translated in the rabbit reticulocyte system in the presence of [35S]methionine. Products were analyzed by SDS-PAGE and autoradiography before and after immunoprecipitation with rabbit anti-human a1AT IgG. Lane 1 , total products from MM liver RNA; lane 2, preimmune IgG, lane 3, immunoprecipitated MM pre-alAT; lane 4, total products from ZZ liver RNA; lane 5, preimmune IgG; lane 6, immunoprecipitated ZZ pre-alAT. The notation alAT indicates the migration position of human serum a1AT (Mr = 54,000). Immunoprecipitated products were also analyzed by isoelectric focusing (pH 3.5-10.0) and autoradiography. Lune 7, immunoprecipitated MM pre-alAT; lane 8, immunoprecipitated ZZ pre-alAT, lane 9, immunoprecipitation products from combined MM and ZZ RNA translations. The positions of the anode and cathode are denoted + and -, respectively.

9982 M and 2 Variant cq-Antitrypsin Biosynthesis and Processing

polypeptide chains with the ER membrane and the subsequent translocation into the ER lumen. Partial amino-terminal sequence analysis of the ZZ a,AT primary translation product was undertaken in order to compare the sequence to that predicted for MM and SS pre-alAT by DNA sequencing (28,29). Consecutive automated Edman degradation was carried out with four immunoprecipitated preparations of ZZ pre- alAT, each radiolabeled with a different amino acid. The results are shown in Fig. 2.

The alignment of the ZZ alAT sequence information with the predicted amino-terminal sequence of the M and S var-

5

LEU

?? x W c t 3

?i ALA

CVS

SEQUENCER CYCLE

FIG. 2. Automated Edman degradation of ZZ pre-alAT. The primary in vitro translation products were synthesized in the rabbit reticulocyte system in the presence of [3H]proline, [3H)leucine, [3H] alanine, and [35S]cysteine prior to immunoprecipitation and sequencing. Numbered cycles indicate amino acid positions within pre-culAT.

iants is illustrated in Fig. 3. Twelve of the 24 amino acids of the signal sequence were identified, and these results indicate complete homology between the signal sequences of the M, S, and Z variants. Each preprotein contains a 24-residue signal sequence. Apparently, the initiator methionine residues are removed in the in uitro translation system. The structural features of the human alAT signal sequence is similar to other secretory proteins with respect to the abundance of uncharged hydrophobic amino acids and the presence of an alanyl residue at the cleavage site. Sequencing data thus suggests that a difference in the signal sequence is not responsible for decreased serum levels of ZZ alAT.

In Vitro Processing, Glycosylution, and Translocation-Dog pancreas microsomes contain the enzyme systems capable of signal peptide cleavage and co-translational transfer of core oligosaccharides to nascent polypeptides. Microsomes were included in a cell-free translation system with human liver mRNA to investigate if ZZ pre-alAT could undergo these initial co-translational processing events which are common to all secretory and membrane proteins. Immunoprecipita- tions of the translation mixtures indicated that both normal and ZZ pre-alAT were co-translationally processed to 52,000- Da proteins (Fig. 4, lanes 4 and 10; also see Figs. 5 and 6). The efficiency of polysome association with rough microsomes is approximately 50% under the in vitro conditions employed. The processed proteins are larger than the primary translation products, smaller than the serum forms of human aIAT, and bind to concanavalin A (data not shown). This is consistent with the microsome-catalyzed removal of the M and Z variant signal peptides and the addition of three core oligosaccharide side chains. The carbohydrate side chains are normally further modified in the Golgi apparatus prior to secretion.

Secretory proteins are completely translocated across the ER membrane into the lumen of the microsomal vesicle where they begin their transport through the Golgi apparatus and other secretory vesicles. Integral membrane proteins are only partially translocated to attain their specific transmembrane orientations. There is evidence that many membrane proteins contain a “stop” or “halt transfer” signal which signals a stop to the translocation process ((30) reviewed in Ref. 31). This signal is thought to consist of a 20-residue stretch of hydrophobic amino acids followed by a series of highly charged amino acids that are located near the carboxyl-terminal ends of a protein. The charged residues do not penetrate the phospholipid bilayer; thus, the remaining COOH-terminal portion of the protein remains outside the ER membrane in the cytoplasm. Since the Glu + Lys substitution in the ZZ a,AT variant results in a Lys342-Lys343 sequence near the COOH terminus of alAT, at least a portion of a stop signal could theoretically be generated and cause the nascent ZZ alAT to become lodged in the ER membrane. Several approaches were taken to determine if the ZZ protein is completely translocated across the ER membrane or integrated into the membrane.

Protection from protease digestion was used as one criterion for protein translocation and integration into microsomal vesicles. If ZZ alAT is processed like MM alAT and other secretory glycoproteins, the processed protein should be completely segregated within the ER lumen and thus protected from protease digestion. Proteolysis in the presence of deter- gents should abolish such protection. If the Glu + Lys substitution generates a stop signal, a 53 residue COOH-terminal portion of ZZ alAT would remain outside the ER, accessible to proteases even in the absence of detergent. Proteolytic removal of the COOH-terminal polypeptide would result in the immunoprecipitation of a smaller truncated form of aIAT.

M and Z Variant al-Antitrypsin Biosynthesis and Processing 9983 S i g n a l S e q u e n c e

-24 -20 -10 -I + I

Normal and SS Deduced Sequence Mat -Ro-~r -Ser -Val -Ser - l~y- l lbCerrLerAlbGly-LecrCysCysiw-Val -Ro-Val -Sar -Leu-Ala f rom the Genomic D N A Sequenc;

GkrAsp-ProGh-

22 o,AT Pr imary Translat ion Product +To- -Leu-Leu-Leu-Ala- -Leu-Cys-Cys-Leu- +To- -Leu-Ala +To-

FIG. 3. Comparison of NH2-terminal sequences of human alAT genetic variants. The sequence of MM and SS aIAT was deduced from the genomic DNA sequence (28, 29). The sequence of the ZZ alAT primary translation product was determined as described in the text. Residues are numbered from the amino terminus of the mature serum protein.

R M TRYPSIN TRITON

1

93-

68-

P T + + + P T + + + + + + + + + + + + + + + + + 2 3 4 5 6 7 8 9 1 0 1 1 12

1 2 3 4 5 6

93-

68 -

pre- apT- 44-

44 -

22-

29 -

FIG. 4. In vitro synthesis, processing, and segregation of MM and ZZ alAT by dog pancreas microsomes. RNA from M (lunes 1-6) or Z (lanes 7-12) variant human liver was translated in a wheat germ cell-free translation system in the presence or absence of dog pancreas microsomes. Immunoprecipitated alAT was analyzed by SDS-PAGE and autoradiography. The figure headings indicate specific sample treatments. RM, dog pancreas rough microsomes were included in the translation; PI", samples were incubated post-translationally with microsomes; trypsin, the translation was followed by incubation with 100 pg/ml trypsin for 1 h at 4 "C prior to SDS solubilization and immunoprecipitation; Triton, trypsin digestion was carried out in the presence of 1% Triton X-100. The arrowhead designates the migration of in uitro-glycosylated alAT. The immunoprecipitated samples were prepared for electrophoresis under nonreducing conditions.

The results of this experiment are shown in Fig. 4. As expected, pre-alAT (lanes 1 and 7) was susceptible to

trypsin digestion (lanes 2 and 8). The in vitro translocated forms of both normal and ZZ alAT (lanes 4 and 10) appeared to be completely segregated within microsomal vesicles since they were resistant to protease digestion (lanes 5 and 1 I). No shift in molecular weight, indicative of an exposed COOH- terminal polypeptide, was detected. When the integrity of the membrane was destroyed by detergent, the processed alAT species became susceptible to trypsin digestion (lanes 6 and 12). As expected, the pre-alAT that had not associated with the microsomes was proteolyzed under all of these conditions.

There are theoretically six trypsin-susceptible lysine residues in the alAT polypeptide sequence which are COOH-

FIG. 5. Protection of segregated MM and ZZ alAT from proteolysis by chymotrypsin and trypsin. The in vitro translations of MM (lanes 1-3) and ZZ (lanes 4-6) RNA in the presence of dog pancreas microsomes and subsequent analyses were carried out as described in Fig. 4 and in the text. Samples were immunoprecipitated either directly after translation (lanes 1 and 4 ) or after a subsequent incubation for 45 min on ice with a mixture of trypsin (100 pg/ml) and chymotrypsin (100 pg/ml) in the absence (lanes 2 and 5) or presence (lanes 3 and 6) of 1% Triton X-100. The arrowhead designates the migration of in vitro-glycosylated q A T . Immunopre- cipitated samples were prepared for electrophoresis under nonreducing conditions.

terminal to the Glu "-f Lys substitution. If all six sites were not cleaved, a slight decrease in molecular weight may not have been detected by SDS-PAGE. Therefore, the previous experiment was repeated using a mixture of trypsin and chymotrypsin, in order to more than double the number of protease-sensitive sites. The results of this experiment (Fig. 5) were consistent with those presented above, that is newly synthesized alAT is completely sequestered within microsomal vesicles. These results are consistent with those of Bathurst et al. (32) who reported the synthesis of a proteinase K-resistant core-glycosylated 59-kDa alAT from RNA translated in the presence of dog pancreas microsomes.

Although the protease protection experiments demonstrated that the entire alAT polypeptide enters the ER membrane, they did not exclude the possibility that a portion of the ZZ polypeptide may remain within the bilayer of the ER membrane. An alternative assay for membrane integration was used which is based on the observation that alkali treatment releases lumenal and peripheral membrane proteins in

9984 M and Z Variant al-Antitrypsin Biosynthesis and Processing

93-

68-

b

44-

1 2 3 4 5

93

6 8 30-

4 4 FIG. 6. Alkali-induced release of microsomal contents and

peripheral membrane proteins. RNA from M variant (lanes 1-3; 7 and 8) or Z variant (lanes 4-6; 9 and IO) human liver was translated in a wheat germ system in the presence or absence (lanes I and 4 ) of dog pancreas microsomes. As described in the text, samples were treated with alkali after translation and centrifuged through a sucrose gradient to separate the soluble alkali-released products (lanes 1-6) from those retained by the microsomes (lanes 7-10). Control samples (lanes 3, 6,8, and 10) were maintained at pH 7.5 and subjected to the same fractionation procedure. All samples were then immunoprecipitated and analyzed by SDS-PAGE and autoradiography. The arrowhead designates the mobility of in vitro-glycosylated aIAT.

soluble form (23), but integral membrane proteins are nonex- tractable by alkaline solutions (reviewed in Ref. 33). After translation in the presence or absence of dog pancreas microsomes, the pH was either adjusted to 11.5 with NaOH, or an equal volume of a pH 7.5 Hepes solution was added as a control. After a 15-min incubation a t 0 “C, the alkali-released materials were separated from the membrane-associated materials by centrifugation through a sucrose gradient. The immunoprecipitation products of each of these fractions are shown in Fig. 6. In both the MM and ZZ mRNA translations, all of the processed alAT was extracted by alkali treatment and detected in the supernatant fractions (lanes 2 and 5). alAT was not detected in the alkali-treated MM or ZZ microsomal fractions (lunes 7 and 9). Results from the control samples not subjected to alkali treatment indicated that the procedural manipulations per se did not release membrane- associated a,AT. Processed glycosylated alAT was not detected in the supernatant fractions of control samples (lunes 3 and 6) but was recovered in the microsomal fractions (lunes 8 and 10).

The protease protection experiments together with the alkali extraction study present strong evidence that, like other secretory proteins, both MM and ZZ alAT are completely translocated across the ER membrane.

alAT Association with Human Liver Microsomes-Rough microsomes consist of membrane-bound polysomes which contain nascent polypeptide chains inserted into the rough endoplasmic reticulum. Rough microsomes were prepared from normal and ZZ human livers by sucrose density gradient centrifugation. The ZZ liver tissue was fibrous and filled with connective tissue which made complete homogenization very difficult and microsomal yields lower than that achieved with normal healthy livers. Human rough microsomes were used to program the reticulocyte translation system, resulting in the in vitro completion of in vivo-initiated polypeptide chains (Fig. 7). Rough microsomes from normal (and MZ, data not shown) livers supported the translation of alAT (lane 3), whereas there was no immunodetectable alAT synthesized by membranes prepared from ZZ liver (lane 4). Care was taken

2 2

FIG. 7. alAT biosynthesis by human liver microsomes. Rough microsomes were isolated from M and Z human liver and translated in a rabbit reticulocyte system. The products were analyzed by SDS-PAGE and autoradiography, before and after immunoprecipitation. Lane 1, total MM rough microsome translation products; lane 2, total ZZ rough microsome translation products; lane 3, immunoprecipitated MM rough microsome translation products; lane 4, immunoprecipitated ZZ rough microsome translation products; lane 5, immunoprecipitated proteins from a mixture of M and Z rough microsome translation products, indicating quantitative recovery of MM a1AT.

to ensure quantitative immunoprecipitation of the in vitro- synthesized radiolabeled alAT (see lane 5 ) since endogenous unlabeled alAT had been detected in ZZ microsomes by Western analysis (Fig. 8). There appeared to be an absence of protein migrating at the position of alAT in the total ZZ rough microsome translation products, although the other liver proteins were synthesized at similar levels (compare lunes 1 and 2, Fig. 7). These results suggested an abnormal in situ association of ZZ alAT-synthesizing polysomes with ho- mologous microsomal membranes. As noted above, prolonged homogenization of the ZZ liver was necessary and may have dissociated polysomes from the ER membrane. However, it would be unlikely that the polysomes synthesizing alAT would be specifically dissociated if the alAT polysome-ER interaction was normal. Many attempts were made to strip these microsomal membranes of endogenous polysomes in order to use them in cell-free translations with ZZ and MM mRNA, but we were unable to recover processing activity in stripped human microsomes.

To examine the endogenous proteins associated with MM and ZZ liver microsomes in vivo, rough microsomal proteins (150 pg/lane) were subjected to SDS-PAGE, transferred to nitrocellulose, and incubated with rabbit antibodies against several human serum proteins. Following a second incubation with lZ5I-labeled goat anti-rabbit IgG, the nitrocellulose filters were washed, dried, and autoradiographed (Fig. 8). Rough

M and Z Variant cq-Antitrypsin Biosynthesis and Processing 9985

1

94 y m , EZz

- 2:

, ..

FIG. 8. Electrophoretic transfer and immunoblotting of human liver rough microsomal proteins. Rough microsomal proteins were isolated from M (lanes 1-5) and Z (lanes 6-10) livers, analyzed by SDS-PAGE (150 pgllane), transferred to nitrocellulose, and incubated with the following rabbit anti-human antibodies: lanes 2 and 6, nonimmune IgG lanes 3 and 7, anti-alAT IgG; lanes 4 and 8, anti-fibrinogen IgG; lanes 5 and 9, anti-albumin IgG. Following a second incubation with 1251-labeled goat anti-rabbit IgG, the filters were washed, dried, and autoradiographed. To examine total microsomal proteins (lanes 1 and 10) and molecular weight markers, nitrocellulose strips were stained with Ponceau S directly after transfer. The notation aIAT indicates the migration position of human serum qAT.

microsomes isolated from ZZ variant liver contained an immunoreactive alAT species (lane 7) which was not detected in normal liver membranes (lane 3) . These results are consistent with immunofluorescent studies (first reported in Ref. 34) which have located alAT-containing inclusion bodies in ZZ microsomes. It thus appears certain that ZZ alAT deficiency is not a result of rapid intracellular degradation of newly synthesized protein. alAT localization in ZZ liver rough microsomes also indicates that polysomes synthesizing alAT must have been membrane bound. The immunoblot also detected other serum proteins in both microsomal preparations. Fibrinogen appeared to be present a t elevated levels in the ZZ microsomes, perhaps as a result of the cirrhotic nature of the liver or perhaps as a result of the acute phase response to inflammation.

Biosynthesis and Secretion of alAT in Xenopus Oocytes- We wished to study alAT synthesis in intact cells in order to use approaches that cannot be applied to cell-free systems and to investigate alAT movement through the post-ER secretory pathway. In the mammalian liver cell, the core oligosaccharide side chain of a glycoprotein undergoes exten- sive processing as the protein is transported through several intracellular compartments. Glucose and mannose residues are removed in the rough endoplasmic reticulum and Golgi apparatus, and complex oligosaccharide side chains are com- pleted in the Golgi apparatus by the addition of the terminal sugars N-acetylglucosamine, galactose, fucose, and sialic acid.

Normal alAT synthesis and secretion was studied in three human hepatoma cell lines (HepG2, Hep3B and PLC/PRF/ 5).* However, the only cultured cells available from ZZ individuals were fibroblasts (GM2522) and lymphoblasts (GM3578), and no a,AT expression was detected in either of these cell lines? We thus decided to use the Xenopus oocyte as a surrogate system for studying alAT secretion after microinjection of mRNA. These studies were conducted at the Department of Biological Sciences a t Purdue University with the generous assistance of Drs. L. Smith and R. Gelfand.

* K. M. Verbanac and E. C. Heath, unpublished observations.

In the first set of experiments, defolliculated stage VI oocytes were injected with 40-80 ng of MM, MZ, or ZZ human liver poly(A) RNA. Control oocytes received no RNA injection. Oocytes were cultured for 15 h, injected with [35S] methionine, and labeled for 3 h. There was no difference in the total [35S]methionine incorporation into oocytes injected with MM or ZZ RNA, as assayed by hot perchloric acid- insoluble radioactivity. Neither was there a difference between oocytes injected with 20 and 80 ng of either RNA.

No qualitative differences in the total labeled cytosolic proteins of control or injected oocytes were detected by SDS- PAGE (data not shown). Therefore, the vast majority of the labeled cytosolic proteins appeared to be a result of the translation of endogenous Xenopus messenger RNA. A 54,000-Da protein that co-migrated with serum a,AT was immunoprecipitated from the oocyte extracts of both MM and ZZ RNA-injected cells (Fig. 9, lanes 1 and 2). No immunoreactive protein was observed in the control oocytes (lane 3). Only the MM alAT species could be detected in the culture medium (lanes 4-6). These results indicate that the information responsible for the aberrant secretion of ZZ alAT is inherent in the mRNA sequence. The possibility of ZZ liver- specific microsomal defects is thus excluded. The data is consistent with results recently reported by Foreman et al. (35) who also failed to detect ZZ alAT in the medium of microinjected oocytes.

Secreted MM alAT was only faintly detected; therefore, subsequent oocyte experiments were conducted under conditions designed to increase the levels of radiolabeled liver proteins in the medium. Oocytes were microinjected with RNA and after a 15-h recovery period were incubated for 24 h with [35S]methionine in the medium, as described under "Experimental Procedures." The immunoprecipitation of the posthomogenization supernatant of oocytes injected with MM RNA (lanes 1 3 ) or ZZ RNA (lanes 4-6) is presented in Fig. 10. The MM RNA-injected cells contained a predominant immunoreactive species with a molecular weight of approximately 54,000. This product was not detected with preimmune IgG (lane 1) and was markedly decreased by the presence of unlabeled serum alAT in the immunoprecipitation mixture

1 2 3 4 5 6

68-

a,AT-

44-

FIG. 9. The synthesis and secretion of alAT in 3-h labeled oocytes. Oocytes were microinjected with MM or ZZ liver poly(A) RNA followed 13-16 h later by an injection of [35S]methionine as described in the text. Control oocytes only received the [%]methionine injection. Oocyte extracts (lanes 1-3) and media (lanes 4-6) were analyzed after immunoprecipitation by SDS-PAGE and autoradiography. Products were immunoprecipitated from extracts of MM RNA-injected oocytes (lane I ) , ZZ RNA-injected oocytes (lane 2), and control oocytes (lane 3) . The incubation media of MM RNA-injected oocytes (lane 4) , ZZ RNA-injected oocytes (lane 5 ) , and control oocytes (lane 6) was also immunoprecipitated.

9986

93

68

44

M and Z Variant al-Antitrypsin Biosynthesis and Processing

1 2 3 4 5 6 7 8

FIG. 10. The synthesis and localization of mlAT in 24-h labeled oocytes. Oocytes were injected with RNA and incubated in the presence of [35S]methionine for 24 h. Some cells were immediately homogenized and fractionated on a sucrose gradient to isolate the vesicle fraction as described in the text. Others were frozen and later homogenized to prepare the oocyte cellular extract. All samples were immunoprecipitated, electrophoresed, and autoradiographed. Cellular extracts from MM RNA-injected oocytes were immunoprecipitated with nonimmune IgG (lane I ) , anti-alAT IgG (lane 2), or anti-alAT IgG in the presence of 20 pg of unlabeled alAT prepared from human serum (lane 3) . ZZ RNA-injected oocyte extracts were immunoprecipitated with nonimmune IgG (lane 4), anti-alAT IgG (lane 5), or anti- alAT IgG in the presence of unlabeled serum alAT (lane 6) . Lane 7, immunoprecipitated products of vesicles isolated from MM RNA- injected oocytes with anti-alAT IgG; lane 8, immunoprecipitated products of vesicles isolated from ZZ RNA-injected oocytes with anti- a1AT IgG.

(lune 3) . Analysis of ZZ-injected oocytes resulted in the immunoprecipitation of two very closely migrating species in this same molecular weight range (lune 5) . A larger protein with a molecular weight of about 58,000 was faintly detected in both MM and ZZ RNA-injected oocytes and was also apparently decreased in the competition experiments (lunes 3 and 6). It is clear from the autoradiogram that the oocytes injected with ZZ RNA contain more intracellular alAT than cells injected with MM RNA (compare Fig. 10, lunes 2 and 5) . Densitometric scans determined this difference to be approximately 7-fold. When 24-h labeled oocytes were subjected to subcellular fractionation, the same products that had been identified in the total cellular homogenates were localized to the vesicle fraction (lunes 7 and 8), indicating that newly synthesized alAT was associated with subcellular membranes. The intracellular accumulation of ZZ aIAT is very marked in the immunoprecipitations from vesicles; autoradiogram densitometric scans estimated that 10-fold more alAT was present in the vesicles of ZZ RNA-injected cells than in vesicles isolated from MM RNA-injected cells.

The secreted proteins were isolated from the incubation media of oocytes that were labeled for 24 h prior to subcellular fractionation. Albumin was the major protein secreted by the human liver RNA-injected oocytes (M, = 68,000) (Fig. 11, lunes 1 and 2). A protein migrating slower than serum alAT was immunoprecipitated from the media of both MM and ZZ RNA-injected oocytes (lunes 4 and 5). The proteins had a molecular weight of 57-58,000 and migrated identically under both reducing and nonreducing conditions (data not shown). As expected, both albumin and the alAT species were absent from the media of control oocytes (lanes 3 and 6). It is evident that not only was a greater amount of alAT secreted from MM RNA-injected cells (over twice as much as from ZZ RNA- injected oocytes) but also a greater proportion of the total

93 -

68- 1

44-

22-

FIG. 11. The secretion of alAT from 24-h labeled oocytes. The incubation media from oocytes subjected to subcellular fractionation (Fig. 10, lanes 7 and 8) were analyzed by immunoprecipitation, SDS-PAGE, and autoradiography. Lane 1, total secreted products from MM RNA-injected oocytes; lane 2, total secreted products from ZZ RNA-injected oocytes; lane 3, total secreted products from unin- jected control oocytes; lane 4, immunoprecipitated product from MM RNA-injected oocyte media; lane 5, immunoprecipitated product from ZZ RNA-injected oocyte media; lane 6, immunoprecipitated product from media of control oocytes.

MM alAT synthesized was secreted. The majority of the ZZ a,AT that was synthesized remained in the microsomal vesicles of the cell. Absolute rates of secretion cannot be obtained by measuring radioactivity levels because the specific activity of the radioactive methionine pool within the oocyte changes during the experiment (25).

As already described, the initial oocyte experiment (Fig. 9) employed mRNA preparations, frog oocytes, and radiolabeling conditions that differed from the subsequent experiments (Figs. 10-12). These were apparently responsible for two major differences in the results. In the experiment depicted in Fig. 9 (microinjection of [35S]methionine followed by a 3-h incubation) 54-kDa forms of alAT were synthesized and no secreted ZZ alAT was detected. Subsequent experiments (with cells that were cultured for 24 h in the presence of [35S] methionine) resulted in the detection of 58-kDa proteins in the medium of both MM and ZZ RNA-injected oocytes. All of the oocyte experiments were consistent on the most important point; microinjected oocytes secrete less ZZ alAT than MM alAT. This is true both with respect to the absolute amounts of alAT secreted and with respect to the percentage of alAT secreted of the total synthesized.

There is no known physiological secretory function for the frog oocyte which normally secretes few endogenous proteins. However, several co-translational (signal peptide cleavage and core glycosylation) and post-translational (NH2-terminal acetylation and protein phosphorylation) modification events are known to occur normally within the microinjected oocyte. Terminally glycosylated membrane proteins synthesized from injected RNA have been localized in active forms on the cell surface (36). There are several potential explanations for the secretion of a larger form of alAT by frog oocytes than in the human liver. Among these are the possibilities that 1) the signal peptide was not cleaved; 2) an additional N- or 0- linked carbohydrate side chain was transferred (co- or post-

M and Z Variant al-Antitrypsin Biosynthesis and Processing 9987

1 2 3 4 5 6 7 8 9 1 0 1 1 12

93 - 68-

“UZ.

,i.

, . -58 - 54

-45 44-

FIG. 12. Glycosylation of alAT by microinjected oocytes; analysis by treatment with tunicamycin and endo-b-N-ace- tylglucosaminidase H. Oocytes were injected with either MM liver RNA (lanes 1 and 2) or with MM RNA simultaneously with 1.6 pg of tunicamycin (lanes 3 and 4). Cells were incubated for 3-4 h in the presence or absence of 2 pg/ml tunicamycin. Oocytes were then transferred to media containing [35S]methionine in the presence or absence of 2 pg/ml tunicamycin for 18 h. Intracellular products from oocyte extracts were immunoprecipitated with anti-alAT IgG (lanes 1 and 3) or anti-alAT IgG in the presence of 10 pg of unlabeled serum alAT (lanes 2 and 4) . Secreted proteins from untreated (lane 5 ) or tunicamycin-treated oocytes (lane 6 ) were immunoprecipitated from the incubation media with anti-alAT IgG. Immunoprecipitated products were analyzed by electrophoresis and autoradiography. The nature of alAT glycosylation in oocytes was also investigated by the enzymatic digestion of immunoprecipitated MM and ZZ proteins from the 24-h labeling experiment presented in Figs. 10 and 11. Immunoreactive cellular (lanes 7 and 8) and secreted (lanes 9 and 10) proteins from oocytes injected with MM RNA were incubated for 6 h a t 37 “C in the presence (lanes 8 and 10) or absence (lanes 7 and 9) of endoglycosidase H as described in the text. Immunoreactive secreted proteins from ZZ RNA-injected oocytes were also incubated in the presence (lane 12) or absence ( l u n e 11) of endo H. All samples were analyzed by electrophoresis and autoradiography.

translationally) to the alAT polypeptide chain; and 3) the post-translational trimming of the core oligosaccharide and addition of terminal sugars occurred very differently in the oocyte. Several of these questions were investigated in the following experiment with the use of tunicamycin, which prevents the assembly of the core oligosaccharide and is thus an inhibitor of N-linked carbohydrate side chain biosynthesis.

The effect of the glycosylation inhibitor tunicamycin on alAT synthesis and processing in MM liver mRNA-injected oocytes was studied during a 24-h labeling period. Tunica- mycin treatment resulted in the intracellular appearance of a 45,000-Da protein and a decrease in the amount of 54-kDa protein detected (Fig. 12, lanes 1 and 3). The specificity of the immunoprecipitation ( i e . the relatedness of the labeled proteins to alAT) was confirmed by the observation that the presence of human serum alAT in the immunoprecipitation mixture reduced the detection of both the glycosylated (54 kDa) and unglycosylated (45 kDa) proteins (lunes 2 and 4). The primary translation product pre-alAT was earlier determined to have a M , of 49,000 and a 24-amino acid signal sequence (see Figs. 1 and 3). Therefore, the size of the unglycosylated protein synthesized in the presence of tunicamycin (M, = 45,000) indicated that proteolytic removal of the signal peptide was indeed occurring in the oocyte and that the 54- kDa glycoprotein found within the cell does not contain 0- linked carbohydrate side chains.

Two major proteins (58 and 45 kDa) were secreted from tunicamycin-treated oocytes (lane 6). Thus, inhibition of alAT glycosylation did not prevent alAT secretion. These results have also been observed with tunicamycin-treated rat hepatocytes (7). The inhibition of glycosylation was apparently incomplete; the 58-kDa glycoprotein that was secreted from untreated cells (lane 5 ) was detected at lower levels in the medium of tunicamycin-treated oocytes (lune 6). Since no 58-kDa protein was detected intracellularly (lune 3), this glycoprotein may have been secreted preferentially or a t a

i

faster rate than unglycosylated alAT. The nature of the oligosaccharide side chains attached in

the oocyte was investigated by subjecting the immunoprecipitated proteins to digestion by endoglycosidase H. Endo H catalyzes the cleavage of high mannose type, asparagine- linked oligosaccharide side chains whereas complex oligosaccharide side chains are resistant to endo H digestion. The susceptibility of immunoprecipitated cellular alAT to endo H indicated that the 54-kDa species possessed high mannose or core oligosaccharide side chains (lanes 7 and 8). The 45-kDa product from endo H digestion was very similar in size to the product from tunicamycin treatment (compare lanes 3,6, and 8) confirming that proteolytic removal of the signal sequence was taking place and that all of the carbohydrate side chains of intracellular alAT were asparagine linked, as is true for the liver-synthesized protein. The 58-kDa secretory protein that was immunoprecipitated from the media of MM and ZZ RNA-injected oocytes was resistant to endo H digestion (lanes 9-12) and, therefore, contained complex asparagine-linked carbohydrate side chains.

It is still uncertain whether the increased size of the secretory a,AT isolated from the oocyte medium is a result of the post-translational addition of a fourth N-linked or 0-linked carbohydrate side chain. Since we demonstrated that all of the alAT detected intracellularly contains only asparagine- linked carbohydrate, the addition of an 0-linked side chain would have to occur immediately before alAT secretion. There is a fourth potential site for the attachment of an N-linked side chain at Asn3’’ of alAT. However, the acceptor tripeptide sequence at this site would be Asn-Pro-Thr, and a proline residue in the central tripeptide position is extremely rare. It is perhaps more likely that three core oligosaccharides undergo a different type of processing that results in a heavier carbohydrate complement. For example, instead of producing alAT that contains one tri- and two biantennary carbohydrate side chains (as occurs in the liver), oocyte processing of mlAT may result in tri- and/or tetra-antennary side chains. Such atypical highly branched carbohydrate chains have been observed in alAT isolated from a human hepatoma cell line (37).

We have eliminated many of the possible mechanisms for decreased ZZ alAT secretion. We found comparable amounts of translatable alAT RNA in MM, MZ, and ZZ livers; thus, ZZ alAT appears to be synthesized at normal levels. The M and Z variant signal sequences appear to be identical with respect to the position and number of amino acids, and they are co-translationally processed with similar efficiency by dog pancreas microsomes. The detection of accumulated aIAT in ZZ rough microsomes demonstrated that alAT is synthesized on membrane-bound polysomes and that newly synthesized ZZ alAT is not rapidly degraded. Both genetic variants were localized in microsomal vesicles in microinjected oocytes, confirming the observation that ZZ alAT enters the ER membrane. Protease digestion and alkali release experiments determined that alAT was completely translocated into the ER lumen and thus disproved the possibility that alAT became lodged in the ER membrane. A ZZ liver-specific defect in the secretory apparatus was ruled out by the observation that decreased secretion of ZZ alAT also occurs in the microinjected oocyte.

In summary, our experiments have detected no differences in the synthesis and stability of the M and Z variant precursor proteins, the nature and proteolytic removal of the signal peptides, or in the glycosylation and translocation of the nascent polypeptide chains. However, a marked accumulation of alAT was observed in ZZ liver-derived rough microsomes

9988 M and Z Variant cul-Antitrypsin Biosynthesis and Processing

and in microsomal vesicles of oocytes microinjected with ZZ RNA. In addition, alAT was undetectable or present at decreased levels in the medium of oocytes injected with ZZ mRNA. Therefore, it is the transport of ZZ alAT from the lumen of the rough endoplasmic reticulum through the secretory pathway to the outside of the cell that is severely affected.

There is evidence from the study of genetic variants and mutants in other secretory systems that the synthesis and cleavage of a signal sequence is not sufficient for subsequent secretion of the protein. Genetic analysis in Escherichia coli has revealed a number of genes that specify components required for protein localization (reviewed in Ref. 38). Distinct stages in protein transport have been defined though the characterization of temperature-sensitive yeast mutants that accumulate secretory proteins intracellularly (reviewed in Ref. 39). Double mutant studies have shown that as many as 21 gene products are required for post-translational transport through the yeast secretory pathway for arrival in vacuoles (lysosomes), at cell surfaces, or outside the cell.

A distinct gene product that directs p-glucuronidase localization in liver and kidney cells has been studied in some detail (reviewed in Ref. 40). A single gene codes for p-glucuronidase which is both lysosomal and microsomal. The localization of the enzyme in the ER is controlled by the Eg gene locus which codes for egasyn, an ER-bound glycoprotein that noncovalently attaches to the enzyme subunits. There is much evidence that egasyn is required to anchor and/or stabilize the enzyme in the ER membrane and serve as a type of retention signal. A mutation in the Eg locus results in a nearly complete loss of microsomal enzyme. Levels of egasyn and microsomal @-glucuronidase are strongly correlated among cell types and during tissue development. Since only -10% of total liver egasyn is bound to @-glucuronidase, egasyn may serve as an integral membrane “receptor” to regulate the membrane binding and localization of a variety of proteins.

The mannose 6-phosphate receptor is a well studied Golgi protein that is responsible for guiding lysosomal enzymes to their proper intracellular destination (reviewed in Ref. 41). In fibroblasts, the phosphorylated carbohydrate of lysosomal enzymes serves as a recognition marker and is essential for proper intracellular localization. Two human storage syn- dromes (mucolipidosis I1 and 111) are caused by the absence of phosphotransferase activity; many unphosphorylated lysosomal enzymes are secreted instead of being delivered to lysosomes.

In these two examples, the interaction between proteins is a determinative event governing protein transport. An alteration in the primary structure of a protein can have a profound influence on its interactions with other molecules. Although segregation of lysosomal proteins relies on a carbohydrate structure, specific features of the polypeptide must create an appropriate substrate that is recognized by the phosphotransferase. Carbohydrate does not serve as a direct signal for the localization of secretory or membrane glycoproteins. As we demonstrated for alAT, the presence of carbohydrate is not required for protein secretion. However, glycosylation has been shown to affect the conformation of at least certain proteins. The role of carbohydrate in the synthesis and membrane insertion of vesicular stomatitis virus envelope G protein has been investigated, and these studies indicate that the maintenance of a given conformation may be necessary for transit through the secretory pathway (42).

Alterations in the primary structure of a protein can result in decreased transport to the cell surface. In an example most analogous to alAT, a X-producing MOPC 315.37 nonsecreted mutant was found to differ from the secreted wild-type protein

by a single amino acid (Arg + Gly) (43). The phenotype was cell type independent since it was observed in RNA-microinjected Xenopus oocytes as well as in the plasmacytoma cell line (44). The authors proposed that a selective interaction process was involved in the transfer of proteins from the lumen of the ER to the exterior of the cell and that the proper tertiary structure of a protein may be essential for secretion.

The amino acid substitution in the Z variant of &,AT may alter alAT recognition by protein receptors located within the secretory organelles of the cell. The mutation may adversely affect protein-protein interactions that would be necessary for proper transport of a secretory protein or it may promote inappropriate associations. Altered recognition could be a result of the amino acid difference per se, that is if amino acid 394 is located at or near a transport protein’s binding site, the change in charge may affect requisite ionic interactions. On the other hand, the Z variant protein may have a different folding pattern such that certain domain(s) are poorly recognized by transport proteins.

The crystal structure has recently been reported for a,AT that has been proteolytically cleaved at the reactive site peptide Met358-Ser359 after complex formation with elastase (45). These residues are located in strands at opposite ends of the crystallized protein. Since these residues are covalently linked in intact alAT, peptide bond cleavage must be accom- panied by a major structural rearrangement. The authors suggest that movement of the central (4A) strand of the p sheet A occurs, such that it is absent from the @ sheet in the intact inhibitor and present in the proteolytically modified protein. G ~ u ~ ~ ~ is located in the P-bend between strands 4A and 5A and forms a salt bridge with LysZgo of strand 6A. The authors suggest that this salt bridge has an effect on the rate of a,AT folding. Replacement of G ~ u ~ ~ ~ by a lysine in the Z variant prevents the formation of this important salt bridge.

If this model is accurate and the intact inhibitor consists of an imperfect (and less stable) p sheet A that lacks strand 4A, the folding of the M and Z variants may be very different. Slow rates of folding may cause lengthy exposure of the hydrophobic segments of ZZ alAT and result in polypeptide aggregation and/or precipitation. Perhaps the simplest expla- nation for the reduced secretion of a,AT is that the Glu + Lys substitution results in a protein that has limited solubility in the microenvironment of the secretory pathway membranes. Structure crystallographic data of the Z variant of alAT will be essential to evaluate the effect of the amino acid substitution on the protein’s conformation.

Acknowledgments-I wish to thank Dr. Patricia Eagon, University of Pittsburgh, for kindly providing the samples of PiZZ human liver; Dr. Earl Rose, Dr. James Kisthard, and Dr. David Stump, University of Iowa, for the normal liver samples obtained by autopsy or as a result of transplantation surgery; Glen Wilson, University of Iowa Protein Structure Facility, for conducting the protein-sequencing work; and Cay Wieland for typing the manuscript.

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