STRUCTURAL STUDIES OF ALPHA-N-ACETYLGALACTOSAMINIDASE - EFFECT OF GLYCOSYLATION ON THE LEVEL OF EXPRESSION, SECRETION EFFICIENCY, AND ENZYME-ACTIVITY

alpha-N-Acetylgalactosaminidase (alpha NAGAL, EC 3.2.1.49) is an exogl ycosidase specific for the hydrolysis of terminal alpha-linked N-acety lgalactosamine from oligosaccharide chains. After cloning of its cDNA, the recombinant alpha NAGAL (r alpha NAGAL) was produced in Pichia pa storis, a methylotrophic yeast strain, The enzyme was hyperglycosylate d by the host cells, resulting in a protein with a molecular mass of a pproximately 50 kDa, which was 7 kDa larger than that of its native co unterpart. When deglycosylated with endoglycosidase H under nondenatur ing conditions, r alpha NAGAL remained fully active, suggesting that t he glycosylation is not required for enzyme activity, Data derived fro m mass spectrometry indicated that all three putative N-glycosylation sites [Asn residues at positions 161 (N1), 185 (N2), and 369 (N3)] in the enzyme were glycosylated, and a high-mannose structure, which was possibly phosphorylated, was attached to the sites N1 and N2. In order to examine the effect of individual N-linked oligosaccharide chains o n the expression of r alpha NAGAL in P. pastoris, we mutated each of t he N-glycosylation sites, as well as all three sites in the same prote in molecule, by substituting the Asn with a Gin residue. The results i ndicate that r alpha NAGAL mutations in any of the three glycosylation sites, N2 being the most profound, impaired the expression level, alt ered subcellular distribution, and decreased the efficiency of secreti on. Our data suggest that the N-glycosylation of r alpha NAGAL express ed in P. pastoris may be important in protein folding and resistance t o protease degradation during protein synthesis, although it is appare ntly not required for enzyme activity. (C) 1998 Academic Press.