A study of the effects of altering the sites for N-glycosylation in alpha-1-proteinase inhibitor variants M and S.
alpha-1-Proteinase inhibitor (A1Pi) is a monomeric secreted protein glycosylated at asparagines 46, 83, and 247. For this study cDNAs for M (normal) and S (Glu264-->Val) variants of A1Pi were altered by site-directed mutagenesis to produce the combinations of single, double, and triple mutants that can be generated by changing the codons normally specifying these Asn residues to encode Gln. The fates of the mutant proteins were followed in transiently transfected COS-1 cells. All variants with altered glycosylation sites are secreted at reduced rates, are partially degraded, accumulate intracellularly, and some form Nonidet P-40-insoluble aggregates. The carbohydrate attached at Asn83 seems to be of particular importance to the export of both A1PiM and A1PiS from the endoplasmic reticulum. All mutations affecting glycosylation of A1PiS notably reduce secretion, cause formation of insoluble aggregates, and influence degradation of the altered proteins. The variant of A1PiS missing all three glycosylation sites is poorly secreted, is incompletely degraded, and accumulates in unusual perinuclear vesicles. These studies show that N-linked oligosaccharides in A1Pi are vital to its efficient export from the endoplasmic reticulum and that the consequences of changing the normal pattern of glycosylation vary depending upon the sites altered and the variant of A1Pi bearing these alterations.
Samandari
T
T
Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver 80262.
Brown
J L
JL
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017830:Octoxynol; D010957:Plasmids; D011092:Polyethylene Glycols; D012995:Solubility; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology; D000515:alpha 1-Antitrypsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000515:alpha 1-Antitrypsin; D014415:Tunicamycin; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D006596:Hexosaminidases