Intracellular disposal of incompletely folded human alpha1-antitrypsin involves release from calnexin and post-translational trimming of asparagine-linked oligosaccharides.
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.
Liu
Y
Y
Department of Pathology, Section of Molecular Pathobiology, Baylor College of Medicine, Houston, Texas 77030, USA.
Choudhury
P
P
Cabral
C M
CM
Sifers
R N
RN
D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D005947:Glucose / Q000378:metabolism; D006801:Humans; D051379:Mice; D009844:Oligosaccharides / Q000378:metabolism*; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D014407:Tumor Cells, Cultured; D000515:alpha 1-Antitrypsin / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D009844:Oligosaccharides; D000515:alpha 1-Antitrypsin; D037281:Calnexin; D017485:1-Deoxynojirimycin; D001216:Asparagine; D005947:Glucose