Glycosylation and secretion of human alpha-1-antitrypsin by yeast

Abstract

Human α-1-antitrypsin (α-AT) is a major serum protein with protease inhibitory activity. Three asparagine residues in a-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 α-AT by Saccharomyces cerevisiae, the yeast invertase secretion signal codons were substituted for the native secretion signal coding DNA of an α-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 α-AT and secrete about one-fifth of it into the culture broth. Approximately 80% of the α-AT produced in yeast is not in the culture broth but is inside the cell within the secretory pathway. This internal α-AT is heterogeneous, consisting of molecules with core carbohydrate on either two or all three of the asparagine receptors. Human α-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 a-AT secreted from an mnn9 mutant, which blocks addition of variable numbers of outer mannose chains, revealed a homogeneous α-AT product which, like α-AT isolated from human serum, bears carbohydrate on three asparagine residues per molecule.