Portal de Periódicos da CAPES

Intracellular Disposal of Incompletely Folded Human α1-Antitrypsin Involves Release from Calnexin and Post-translational Trimming of Asparagine-linked Oligosaccharides

1997; Elsevier BV; Volume: 272; Issue: 12 Linguagem: Inglês

10.1074/jbc.272.12.7946

1083-351X

Yan Liu, Priya Choudhury, Christopher M. Cabral, Richard N. Sifers,

Vector-Borne Animal Diseases

Protection of lung elastin fibers from proteolytic destruction is compromised by inefficient secretion of incompletely folded allelic variants of human α1-antitrypsin from hepatocytes. Pulse-chase radiolabeling with [35S]methionine and sucrose gradient sedimentation and coimmunoprecipitation techniques were employed to investigate quality control of human α1-antitrypsin secretion from stably transfected mouse hepatoma cells. The secretion-incompetent variant null(Hong Kong) (Sifers, R. N., Brashears-Macatee, S., Kidd, V. J., Muensch, H., and Woo, S. L. C. (1988) J. Biol. Chem. 263, 7330-7335) cannot fold into a functional conformation and was quantitatively associated with the molecular chaperone calnexin following biosynthesis. Assembly with calnexin required cotranslational trimming of glucose from asparagine-linked oligosaccharides. Intracellular disposal of pulse-radiolabeled molecules coincided with their release from calnexin. Released monomers and intracellular disposal were nonexistent in cells chased with cycloheximide, an inhibitor of protein synthesis. Post-translational trimming of asparagine-linked oligosaccharides and intracellular disposal were abrogated by 1-deoxymannojirimycin, an inhibitor of α-mannosidase activity, without affecting the monomer population. The data are consistent with a recently proposed quality control model (Hammond, C., Braakman, I., and Helenius, A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 913-917) in which intracellular disposal requires dissociation from calnexin and post-translational trimming of mannose from asparagine-linked oligosaccharides. Protection of lung elastin fibers from proteolytic destruction is compromised by inefficient secretion of incompletely folded allelic variants of human α1-antitrypsin from hepatocytes. Pulse-chase radiolabeling with [35S]methionine and sucrose gradient sedimentation and coimmunoprecipitation techniques were employed to investigate quality control of human α1-antitrypsin secretion from stably transfected mouse hepatoma cells. The secretion-incompetent variant null(Hong Kong) (Sifers, R. N., Brashears-Macatee, S., Kidd, V. J., Muensch, H., and Woo, S. L. C. (1988) J. Biol. Chem. 263, 7330-7335) cannot fold into a functional conformation and was quantitatively associated with the molecular chaperone calnexin following biosynthesis. Assembly with calnexin required cotranslational trimming of glucose from asparagine-linked oligosaccharides. Intracellular disposal of pulse-radiolabeled molecules coincided with their release from calnexin. Released monomers and intracellular disposal were nonexistent in cells chased with cycloheximide, an inhibitor of protein synthesis. Post-translational trimming of asparagine-linked oligosaccharides and intracellular disposal were abrogated by 1-deoxymannojirimycin, an inhibitor of α-mannosidase activity, without affecting the monomer population. The data are consistent with a recently proposed quality control model (Hammond, C., Braakman, I., and Helenius, A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 913-917) in which intracellular disposal requires dissociation from calnexin and post-translational trimming of mannose from asparagine-linked oligosaccharides. INTRODUCTIONConformational maturation of nascent polypeptides is facilitated by transient physical interaction with molecular chaperones. The general consensus is that rounds of binding prevent misfolding and subsequent entrance of partially folded intermediates into nonproductive folding pathways (1Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3573) Google Scholar). In eukaryotic cells, nascent polypeptides destined to traverse compartments of the secretory pathway are translocated into the lumen of the endoplasmic reticulum (ER) 1The abbreviations used are: ERendoplasmic reticulumAATα1-antitrypsinPAGEpolyacrylamide gel electrophoresis. during biosynthesis (2Walter P. Gilmore R. Blobel G. Cell. 1984; 38: 5-8Abstract Full Text PDF PubMed Scopus (392) Google Scholar). Incompletely folded proteins often exhibit a persistent physical association with one or more molecular chaperones and are retained in the ER prior to intracellular disposal (for a review, see 3Klausner R.D. Sitia R. Cell. 1990; 62: 611-614Abstract Full Text PDF PubMed Scopus (455) Google Scholar). This mechanism has been termed "quality control" (4Hurtley S.M. Helenius A. Annu. Rev. Cell Biol. 1989; 5: 277-307Crossref PubMed Scopus (774) Google Scholar) and apparently functions to ensure transport of only correctly folded proteins beyond the ER.Calnexin (also designated p88 or IP90), a calcium-binding molecular chaperone of the ER membrane (5Wada I. Rindress D. Cameron P.H. Ou W.-J. Doherty II, J.J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Abstract Full Text PDF PubMed Google Scholar), forms a transient noncovalent association with several newly synthesized proteins in the ER (6Ou W.-J. Cameron P.H. Thomas D.Y. Bergeron J.M. Nature. 1993; 364: 771-776Crossref PubMed Scopus (483) Google Scholar, 7Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (708) Google Scholar, 8Wada I. Ou W.-J. Liu M.-C. Scheele G. J. Biol. Chem. 1994; 269: 7464-7472Abstract Full Text PDF PubMed Google Scholar, 9Pind S. Riordan J.R. Williams D.B. J. Biol. Chem. 1994; 269: 12784-12788Abstract Full Text PDF PubMed Google Scholar, 10Jackson M.R. Cohen-Doyle M.F. Peterson P.A. Williams D.B. Science. 1994; 263: 384-387Crossref PubMed Scopus (220) Google Scholar, 11Hammond C. Helenius A. Science. 1994; 266: 456-458Crossref PubMed Scopus (273) Google Scholar, 12Tector M. Salter R.D. J. Biol. Chem. 1995; 270: 19638-19642Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 13Kim P.S. Arvan P. J. Cell Biol. 1995; 128: 29-38Crossref PubMed Scopus (168) Google Scholar, 14van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 9660-9665Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). An important feature of calnexin is that it exhibits an affinity for monoglucosylated oligosaccharides (15Ware F.E. Vassilakos A. Peterson P.A. Jackson M.R. Lehrman M.A. Williams D.B. J. Biol. Chem. 1995; 270: 4697-4704Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar), which are intermediates formed during cotranslational trimming by ER α-glucosidases (16Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3750) Google Scholar). Importantly, it can also be generated by post-translational reglucosylation of glycans, an event catalyzed by the ER resident protein UDP-glucose:glycoprotein glucosyltransferase (17Trombetta S. Bosch M. Parodi A.J. Biochemistry. 1989; 28: 8108-8116Crossref PubMed Scopus (134) Google Scholar, 18Sousa M.C. Ferrero-Garcia M.A. Parodi A.J. Biochemistry. 1992; 31: 97-105Crossref PubMed Scopus (267) Google Scholar, 19Trombetta S.E Ganan S. Parodi A.J. Glycobiology. 1991; 1: 155-161Crossref PubMed Scopus (44) Google Scholar). Interaction with calnexin can be prevented by drugs that either inhibit asparagine-linked glycosylation or arrest cotranslational trimming of attached glucose residues (6Ou W.-J. Cameron P.H. Thomas D.Y. Bergeron J.M. Nature. 1993; 364: 771-776Crossref PubMed Scopus (483) Google Scholar, 7Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (708) Google Scholar, 20Moore S.E.H. Spiro R.G. J. Biol. Chem. 1993; 268: 3809-3812Abstract Full Text PDF PubMed Google Scholar, 21Kearse K.P. Williams D.B. Singer A. EMBO J. 1994; 13: 3678-3686Crossref PubMed Scopus (110) Google Scholar, 22Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (486) Google Scholar) and is virtually nonexistent in mutant cell lines deficient in ER α-glucosidase I or II (23Ora A. Helenius A. J. Biol. Chem. 1995; 270: 26060-26062Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar).Protein folding and quality control machinery may participate in the molecular pathogenesis of plasma α1-antitrypsin (AAT) deficiency (24Sifers R.N. Finegold M.J. Woo S.L.C. Am. J. Respir. Cell Mol. Biol. 1989; 1: 341-345Crossref PubMed Scopus (26) Google Scholar, 25Sifers R.N. Finegold M.J. Woo S.L.C. Arias I.M. Boyer J.L. Fausto N. Jakoby W.B. Schachter D.A. Shafritz D.A. The Liver: Biology and Pathobiology. 3rd Ed. Raven Press, Ltd., New York1994: 1357-1365Google Scholar). Human AAT is a monomeric glycoprotein of 394 amino acids (26Carrell R.W. Boswell D.R. Barrett A.J. Salvesen G. Proteinase Inhibitors. Elsevier Science Publishers B. V., Amsterdam1986: 403-420Google Scholar, 27Long G.L. Chandra T. Woo S.L.C. Davie W.W. Kurache K. Biochemistry. 1984; 23: 4828-4837Crossref PubMed Scopus (289) Google Scholar) and is secreted from liver hepatocytes (28Peters Jr., T. Glaumann H. Peters Jr., T. Redman C. Plasma Protein Secretion by the Liver. Academic Press, New York1983: 1-5Google Scholar). It is a member of the serine proteinase inhibitor superfamily (29Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar) and protects lung elastin fibers from proteolytic destruction by inhibiting the activity of elastase released from activated neutrophils (30Travis J. Salvesen G.S. Annu. Rev. Biochem. 1983; 52: 655-709Crossref PubMed Scopus (1473) Google Scholar). Several allelic variants of the inhibitor exist (31Brantly M. Nukiwa Y. Crystal R.G. Am. J. Med. 1988; 84: 13-31Abstract Full Text PDF PubMed Scopus (396) Google Scholar), and many exhibit a distinct mutation predicted to preclude conformational maturation of the encoded polypeptide following biosynthesis (32Stein P.E. Carrell R.W. Nat. Struct. Biol. 1995; 2: 96-113Crossref PubMed Scopus (390) Google Scholar). Defective intracellular transport of the aberrantly folded protein through compartments of the secretory pathway can diminish circulating levels of the inhibitor (25Sifers R.N. Finegold M.J. Woo S.L.C. Arias I.M. Boyer J.L. Fausto N. Jakoby W.B. Schachter D.A. Shafritz D.A. The Liver: Biology and Pathobiology. 3rd Ed. Raven Press, Ltd., New York1994: 1357-1365Google Scholar). Proteolytic destruction of lung elastin is associated with severe plasma AAT deficiency and is implicated in the pathogenesis of chronic obstructive lung disease (33Beith J. Front. Matrix Biol. 1978; 6: 1-4Google Scholar).Intracellular retention of most "null" AAT variants results from mutations that cause premature truncation of the polypeptide at its carboxyl terminus (34Fabretti G. Sergi C. Consales G. Faa G. Brisigotti M. Romeo G. Callea F. Liver. 1992; 12: 296-301Crossref PubMed Scopus (17) Google Scholar), a phenomenon predicted to prevent formation of specific secondary structural features (32Stein P.E. Carrell R.W. Nat. Struct. Biol. 1995; 2: 96-113Crossref PubMed Scopus (390) Google Scholar, 35Loebermann H. Tobuoka R. Deisenhofer J. Huber R. J. Mol. Biol. 1984; 177: 531-556Crossref PubMed Scopus (607) Google Scholar). In this study, conformation-based quality control of human AAT secretion was investigated in mouse hepatoma cells stably expressing the nonfunctional allelic variant QO Hong Kong (null(Hong Kong)), which is incapable of folding into the appropriate native structure. Null(Hong Kong) exhibits a Ca2+-sensitive physical interaction with the molecular chaperone calnexin during intracellular retention (36Le A. Steiner J.L. Ferrell G.A Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar) and requires release from the molecular chaperone as well as post-translational trimming of its asparagine-linked oligosaccharides for normal disposal. Predicted roles for each of these events in the quality control of human AAT secretion are discussed.DISCUSSIONSince hepatocytes are the major site for biosynthesis and secretion of AAT (28Peters Jr., T. Glaumann H. Peters Jr., T. Redman C. Plasma Protein Secretion by the Liver. Academic Press, New York1983: 1-5Google Scholar, 46Sifers R.N. Carlson J.A. Clift S.M. DeMayo F.J. Bullock D.W. Woo S.L.C. Nucleic Acids Res. 1987; 15: 1459-1475Crossref PubMed Scopus (73) Google Scholar), stably transfected mouse hepatoma cells were used to study conformation-based quality control of human AAT secretion. Variant null(Hong Kong) was employed for this analysis because truncation of amino acids at its carboxyl terminus prevents formation of all three large β-sheet structures common to members of the serine proteinase inhibitor superfamily (32Stein P.E. Carrell R.W. Nat. Struct. Biol. 1995; 2: 96-113Crossref PubMed Scopus (390) Google Scholar, 35Loebermann H. Tobuoka R. Deisenhofer J. Huber R. J. Mol. Biol. 1984; 177: 531-556Crossref PubMed Scopus (607) Google Scholar). The correctly folded variant PI M1(Val-213) is rapidly secreted from these cells, whereas null(Hong Kong) is subjected to intracellular retention and disposal (37Sifers R.N. Brashears-Macatee S. Kidd V.J. Muensch H. Woo S.L.C. J. Biol. Chem. 1988; 263: 7330-7335Abstract Full Text PDF PubMed Google Scholar, 40Le A. Graham K.S. Sifers R.N. J. Biol. Chem. 1990; 265: 14001-14007Abstract Full Text PDF PubMed Google Scholar). In this study, cosedimentation and coimmunoprecipitation analyses indicated that newly synthesized molecules sedimented at 6.8 S, which coincided with their quantitative association with the molecular chaperone calnexin. This indicates that assembly of the complex occurred cotranslationally or immediately following biosynthesis.Interaction with calnexin was prevented by inhibiting asparagine-linked glycosylation with tunicamycin or by arresting cotranslational trimming of attached glucose residues with castanospermine. This indicates that like many other glycoproteins (11Hammond C. Helenius A. Science. 1994; 266: 456-458Crossref PubMed Scopus (273) Google Scholar, 47Helenius A. Mol. Biol. Cell. 1994; 5: 253-265Crossref PubMed Scopus (558) Google Scholar), oligosaccharides may somehow function to facilitate assembly between null(Hong Kong) and calnexin. Intracellular disposal of nascent null(Hong Kong) was accelerated in cells preincubated with either tunicamycin or castanospermine, suggesting that disposal occurs in the absence of the bound molecular chaperone. This idea was consistent with appearance of the 4.5 S species at a time point that coincided with the onset of intracellular disposal. The absence of associated calnexin and sedimentation at 4.5 S suggests that this population of molecules represents the released monomer (36Le A. Steiner J.L. Ferrell G.A Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar, 39Le A. Ferrell G.A. Dishon D.S. Le Q.-Q.A. Sifers R.N. J. Biol. Chem. 1992; 267: 1072-1080Abstract Full Text PDF PubMed Google Scholar). No changes in the monomer population were observed when cell lysis was performed under less stringent conditions (data not shown), suggesting that the 4.5 S population does not merely reflect weakening of the null(Hong Kong)-calnexin interaction during retention, but represents unassociated molecules. It should be pointed out that in an earlier report (36Le A. Steiner J.L. Ferrell G.A Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar), an error in our fractionation method, overlap between the 4.5 S and 6.8 S species, and incomplete dissociation of complexes in response to incubation with EGTA led to the incorrect conclusion that an additional sedimenting species was detected under steady-state conditions. It is now apparent that the most abundant forms of null(Hong Kong) are the 4.5 S and 6.8 S species. However, we cannot rule out the possibility that additional, more transient species exist.Intracellular disposal of null(Hong Kong) was arrested during incubation of pulse-radiolabeled cells with cycloheximide, and this coincided with complete absence of the 4.5 S monomer population. Cycloheximide had no demonstrable effect on the intracellular stability of null(Hong Kong) in cells preincubated with either tunicamycin or castanospermine, which supported the idea that dissociation from calnexin required protein synthesis. Although our experiments have not addressed the mechanism by which dissociation was inhibited, one possibility is that a short-lived protein is required for normal disruption of the null(Hong Kong)-calnexin complex. Cycloheximide treatment had the added effect of preventing post-translational trimming of oligosaccharides attached to null(Hong Kong). At present, we do not know whether this reflects inaccessibility of glycans for trimming while null(Hong Kong) is bound to calnexin. However, previous experiments have suggested that the null(Hong Kong)-calnexin interaction is stabilized by a peptide-peptide interaction (36Le A. Steiner J.L. Ferrell G.A Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar). Furthermore, incubation with several combinations of monosaccharides has failed to dissociate the complex after coprecipitation, 2R. N. Sifers, unpublished data. suggesting that the glycan moiety is not involved in stabilizing the interaction after assembly. These data are in agreement with a two-step binding model proposed by Tector and Salter (12Tector M. Salter R.D. J. Biol. Chem. 1995; 270: 19638-19642Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) in which the function of the monoglucosylated oligosaccharide is to initiate assembly of glycoproteins with calnexin. However, we cannot disregard the possibility that a lectin-like interaction does exist, but is disrupted during immunoprecipitation.Intracellular disposal of null(Hong Kong) was also arrested in cells treated with 1-deoxymannojirimycin, which also prevented demonstrable post-translational trimming of asparagine-linked oligosaccharides. Originally, only one α-mannosidase activity had been assigned to the ER (16Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3750) Google Scholar). However, in recent years, several α-mannosidases have been identified in the ER, many of which are inhibited by 1-deoxymannojirimycin (42Rizzolo L.J. Kornfeld R. J. Biol. Chem. 1988; 263: 9520-9525Abstract Full Text PDF PubMed Google Scholar, 43Elbein A.D. FASEB J. 1991; 5: 3055-3063Crossref PubMed Scopus (345) Google Scholar, 44Bause E. Breuer W. Schweden J. Roeser R. Geyer R. Eur. J. Biochem. 1992; 208: 451-457Crossref PubMed Scopus (58) Google Scholar, 45Bonay P. Roth J. Hughes C. Eur. J. Biochem. 1992; 205: 399-407Crossref PubMed Scopus (22) Google Scholar). Unlike cycloheximide treatment, in which inhibition of disposal coincided with a complete absence of the 4.5 S peak, inhibition with 1-deoxymannojirimycin did not alter the monomer population. Taken together, data from this study suggest that variant null(Hong Kong) is subjected to a quality control pathway identical to that recently proposed by Hammond and Helenius (11Hammond C. Helenius A. Science. 1994; 266: 456-458Crossref PubMed Scopus (273) Google Scholar). Their model proposes that persistent association between unfolded glycoproteins and calnexin reflects a continuous cycle of binding in which assembly of the complex is facilitated by reglucosylation of asparagine-linked oligosaccharides by the ER resident enzyme UDP-glucose:glycoprotein glucosyltransferase (17Trombetta S. Bosch M. Parodi A.J. Biochemistry. 1989; 28: 8108-8116Crossref PubMed Scopus (134) Google Scholar, 18Sousa M.C. Ferrero-Garcia M.A. Parodi A.J. Biochemistry. 1992; 31: 97-105Crossref PubMed Scopus (267) Google Scholar, 19Trombetta S.E Ganan S. Parodi A.J. Glycobiology. 1991; 1: 155-161Crossref PubMed Scopus (44) Google Scholar, 48Ganan S. Cazzulo J.J. Parodi A.J. Biochemistry. 1991; 30: 3098-3104Crossref PubMed Scopus (52) Google Scholar). Importantly, the model predicts that permanent dissociation from the binding cycle will occur when hydrolysis of mannose residues generates an oligosaccharide unable to participate as a glucose acceptor, thereby leading to disposal of the unfolded glycoprotein. Consistent with this model, inhibition of oligosaccharide trimming by 1-deoxymannojirimycin abrogated intracellular disposal of variant null(Hong Kong) without affecting the percent population of 4.5 S monomers present in the cell. This latter finding would be expected if the protein is degraded only when it can no longer participate in the cycle of binding to calnexin, which would result after extensive post-translational trimming of glycans by α-mannosidase. Further dissection of this quality control pathway will be the subject of future investigations and will enhance our current understanding of how incompletely folded human AAT variants are retained and degraded in hepatocytes. INTRODUCTIONConformational maturation of nascent polypeptides is facilitated by transient physical interaction with molecular chaperones. The general consensus is that rounds of binding prevent misfolding and subsequent entrance of partially folded intermediates into nonproductive folding pathways (1Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3573) Google Scholar). In eukaryotic cells, nascent polypeptides destined to traverse compartments of the secretory pathway are translocated into the lumen of the endoplasmic reticulum (ER) 1The abbreviations used are: ERendoplasmic reticulumAATα1-antitrypsinPAGEpolyacrylamide gel electrophoresis. during biosynthesis (2Walter P. Gilmore R. Blobel G. Cell. 1984; 38: 5-8Abstract Full Text PDF PubMed Scopus (392) Google Scholar). Incompletely folded proteins often exhibit a persistent physical association with one or more molecular chaperones and are retained in the ER prior to intracellular disposal (for a review, see 3Klausner R.D. Sitia R. Cell. 1990; 62: 611-614Abstract Full Text PDF PubMed Scopus (455) Google Scholar). This mechanism has been termed "quality control" (4Hurtley S.M. Helenius A. Annu. Rev. Cell Biol. 1989; 5: 277-307Crossref PubMed Scopus (774) Google Scholar) and apparently functions to ensure transport of only correctly folded proteins beyond the ER.Calnexin (also designated p88 or IP90), a calcium-binding molecular chaperone of the ER membrane (5Wada I. Rindress D. Cameron P.H. Ou W.-J. Doherty II, J.J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Abstract Full Text PDF PubMed Google Scholar), forms a transient noncovalent association with several newly synthesized proteins in the ER (6Ou W.-J. Cameron P.H. Thomas D.Y. Bergeron J.M. Nature. 1993; 364: 771-776Crossref PubMed Scopus (483) Google Scholar, 7Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (708) Google Scholar, 8Wada I. Ou W.-J. Liu M.-C. Scheele G. J. Biol. Chem. 1994; 269: 7464-7472Abstract Full Text PDF PubMed Google Scholar, 9Pind S. Riordan J.R. Williams D.B. J. Biol. Chem. 1994; 269: 12784-12788Abstract Full Text PDF PubMed Google Scholar, 10Jackson M.R. Cohen-Doyle M.F. Peterson P.A. Williams D.B. Science. 1994; 263: 384-387Crossref PubMed Scopus (220) Google Scholar, 11Hammond C. Helenius A. Science. 1994; 266: 456-458Crossref PubMed Scopus (273) Google Scholar, 12Tector M. Salter R.D. J. Biol. Chem. 1995; 270: 19638-19642Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 13Kim P.S. Arvan P. J. Cell Biol. 1995; 128: 29-38Crossref PubMed Scopus (168) Google Scholar, 14van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 9660-9665Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). An important feature of calnexin is that it exhibits an affinity for monoglucosylated oligosaccharides (15Ware F.E. Vassilakos A. Peterson P.A. Jackson M.R. Lehrman M.A. Williams D.B. J. Biol. Chem. 1995; 270: 4697-4704Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar), which are intermediates formed during cotranslational trimming by ER α-glucosidases (16Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3750) Google Scholar). Importantly, it can also be generated by post-translational reglucosylation of glycans, an event catalyzed by the ER resident protein UDP-glucose:glycoprotein glucosyltransferase (17Trombetta S. Bosch M. Parodi A.J. Biochemistry. 1989; 28: 8108-8116Crossref PubMed Scopus (134) Google Scholar, 18Sousa M.C. Ferrero-Garcia M.A. Parodi A.J. Biochemistry. 1992; 31: 97-105Crossref PubMed Scopus (267) Google Scholar, 19Trombetta S.E Ganan S. Parodi A.J. Glycobiology. 1991; 1: 155-161Crossref PubMed Scopus (44) Google Scholar). Interaction with calnexin can be prevented by drugs that either inhibit asparagine-linked glycosylation or arrest cotranslational trimming of attached glucose residues (6Ou W.-J. Cameron P.H. Thomas D.Y. Bergeron J.M. Nature. 1993; 364: 771-776Crossref PubMed Scopus (483) Google Scholar, 7Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (708) Google Scholar, 20Moore S.E.H. Spiro R.G. J. Biol. Chem. 1993; 268: 3809-3812Abstract Full Text PDF PubMed Google Scholar, 21Kearse K.P. Williams D.B. Singer A. EMBO J. 1994; 13: 3678-3686Crossref PubMed Scopus (110) Google Scholar, 22Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (486) Google Scholar) and is virtually nonexistent in mutant cell lines deficient in ER α-glucosidase I or II (23Ora A. Helenius A. J. Biol. Chem. 1995; 270: 26060-26062Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar).Protein folding and quality control machinery may participate in the molecular pathogenesis of plasma α1-antitrypsin (AAT) deficiency (24Sifers R.N. Finegold M.J. Woo S.L.C. Am. J. Respir. Cell Mol. Biol. 1989; 1: 341-345Crossref PubMed Scopus (26) Google Scholar, 25Sifers R.N. Finegold M.J. Woo S.L.C. Arias I.M. Boyer J.L. Fausto N. Jakoby W.B. Schachter D.A. Shafritz D.A. The Liver: Biology and Pathobiology. 3rd Ed. Raven Press, Ltd., New York1994: 1357-1365Google Scholar). Human AAT is a monomeric glycoprotein of 394 amino acids (26Carrell R.W. Boswell D.R. Barrett A.J. Salvesen G. Proteinase Inhibitors. Elsevier Science Publishers B. V., Amsterdam1986: 403-420Google Scholar, 27Long G.L. Chandra T. Woo S.L.C. Davie W.W. Kurache K. Biochemistry. 1984; 23: 4828-4837Crossref PubMed Scopus (289) Google Scholar) and is secreted from liver hepatocytes (28Peters Jr., T. Glaumann H. Peters Jr., T. Redman C. Plasma Protein Secretion by the Liver. Academic Press, New York1983: 1-5Google Scholar). It is a member of the serine proteinase inhibitor superfamily (29Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar) and protects lung elastin fibers from proteolytic destruction by inhibiting the activity of elastase released from activated neutrophils (30Travis J. Salvesen G.S. Annu. Rev. Biochem. 1983; 52: 655-709Crossref PubMed Scopus (1473) Google Scholar). Several allelic variants of the inhibitor exist (31Brantly M. Nukiwa Y. Crystal R.G. Am. J. Med. 1988; 84: 13-31Abstract Full Text PDF PubMed Scopus (396) Google Scholar), and many exhibit a distinct mutation predicted to preclude conformational maturation of the encoded polypeptide following biosynthesis (32Stein P.E. Carrell R.W. Nat. Struct. Biol. 1995; 2: 96-113Crossref PubMed Scopus (390) Google Scholar). Defective intracellular transport of the aberrantly folded protein through compartments of the secretory pathway can diminish circulating levels of the inhibitor (25Sifers R.N. Finegold M.J. Woo S.L.C. Arias I.M. Boyer J.L. Fausto N. Jakoby W.B. Schachter D.A. Shafritz D.A. The Liver: Biology and Pathobiology. 3rd Ed. Raven Press, Ltd., New York1994: 1357-1365Google Scholar). Proteolytic destruction of lung elastin is associated with severe plasma AAT deficiency and is implicated in the pathogenesis of chronic obstructive lung disease (33Beith J. Front. Matrix Biol. 1978; 6: 1-4Google Scholar).Intracellular retention of most "null" AAT variants results from mutations that cause premature truncation of the polypeptide at its carboxyl terminus (34Fabretti G. Sergi C. Consales G. Faa G. Brisigotti M. Romeo G. Callea F. Liver. 1992; 12: 296-301Crossref PubMed Scopus (17) Google Scholar), a phenomenon predicted to prevent formation of specific secondary structural features (32Stein P.E. Carrell R.W. Nat. Struct. Biol. 1995; 2: 96-113Crossref PubMed Scopus (390) Google Scholar, 35Loebermann H. Tobuoka R. Deisenhofer J. Huber R. J. Mol. Biol. 1984; 177: 531-556Crossref PubMed Scopus (607) Google Scholar). In this study, conformation-based quality control of human AAT secretion was investigated in mouse hepatoma cells stably expressing the nonfunctional allelic variant QO Hong Kong (null(Hong Kong)), which is incapable of folding into the appropriate native structure. Null(Hong Kong) exhibits a Ca2+-sensitive physical interaction with the molecular chaperone calnexin during intracellular retention (36Le A. Steiner J.L. Ferrell G.A Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar) and requires release from the molecular chaperone as well as post-translational trimming of its asparagine-linked oligosaccharides for normal disposal. Predicted roles for each of these events in the quality control of human AAT secretion are discussed.