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Abstract
Protection of lung elastin fibers from proteolytic destruction is compromised by inefficient secretion of incompletely folded allelic variants of human alpha1-antitrypsin from hepatocytes. Pulse-chase radiolabeling with [35S]methionine and sucrose gradient sedimentation and coimmunoprecipitation techniques were employed to investigate quality control of human alpha1-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 alpha-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.
Highlights
Physical association with one or more molecular chaperones and are retained in the ER prior to intracellular disposal
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
Since hepatocytes are the major site for biosynthesis and secretion of AAT [28, 46], stably transfected mouse hepatoma cells were used to study conformation-based quality control of human AAT secretion
Summary
Chemicals and Reagents—Protein G-agarose, castanospermine, and 1-deoxymannojirimycin were purchased from Calbiochem. For metabolic radiolabeling of proteins, confluent cell monolayers were washed with phosphate-buffered saline and incubated for the specified period in methionine-free Dulbecco’s modified Eagle’s medium containing [35S]methionine (150 – 300 Ci). Immunocomplexes were washed by agitation at 4 °C with cell lysis buffer containing 0.5 M NaCl. For detection of newly synthesized null(Hong Kong) bound to calnexin, double immunoprecipitations were performed as described previously [6]. H1A/N13 cells were incubated for 5 h with medium containing [35S]methionine, and the soluble cell lysate was subjected to sucrose gradient sedimentation prior to immunoprecipitation of human AAT from gradient fractions. C, proteins in a gel identical to that in B were electrophoretically transferred to nitrocellulose and incubated with a 1:1000 dilution of antiserum against canine calnexin to detect immunoreactivity by ECL Western blotting. Immunoreactivity was detected by enhanced chemiluminescent Western blotting (ECL, Amersham Corp.) according to the manufacturer’s instructions, except that SuperSignal reagent (Pierce) was used as the detection reagent
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