Glycosylation and secretion of human alpha-1-antitrypsin by yeast.
Human alpha-1-antitrypsin (alpha-AT) is a major serum protein with protease inhibitory activity. Three asparagine residues in alpha-AT are glycosylated with the mammalian 'complex' pattern of carbohydrate as the protein is secreted from cells in the liver. To examine the glycosylation and secretion of human alpha-AT by Saccharomyces cerevisiae, the yeast invertase secretion signal codons were substituted for the native secretion signal coding DNA of an alpha-AT cDNA, and the fusion gene was placed on an autonomously replicating yeast--Escherichia coli shuttle vector under control of the yeast triosephosphate isomerase (TPI) promoter. Yeast strains transformed with this plasmid produce human alpha-AT and secrete about one-fifth of it into the culture broth. Approximately 80% of the alpha-AT produced in yeast is not in the culture broth but is inside the cell within the secretory pathway. This internal alpha-AT is heterogeneous, consisting of molecules with core carbohydrate on either two or all three of the asparagine receptors. Human alpha-AT secreted into the culture broth contains, in addition to core carbohydrate, variable numbers of mannose outer chains, typical of secreted yeast proteins such as invertase. All carbohydrate is removed by endoglycosidase H treatment. Examination of alpha-AT secreted from an mnn9 mutant, which blocks addition of variable numbers of outer mannose chains, revealed a homogeneous alpha-AT product which, like alpha-AT isolated from human serum, bears carbohydrate on three asparagine residues per molecule.
Moir
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Department of Molecular Genetics, Collaborative Research, Inc., Bedford, MA 01730.
Dumais
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D001216:Asparagine / Q000378:metabolism; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D009154:Mutation; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*; D000515:alpha 1-Antitrypsin / Q000235:genetics / Q000378:metabolism*
D000515:alpha 1-Antitrypsin; D001216:Asparagine