Structural studies of α-N-acetylgalactosaminidase: Effect of glycosylation on the level of expression, secretion efficiency, and enzyme activity

Abstract

α-N-Acetylgalactosaminidase (αNAGAL, EC 3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal α-linked N-acetylgalactosamine from oligosaccharide chains. After cloning of its cDNA, the recombinant αNAGAL (rαNAGAL) was produced in Pichia pastoris, a methylotrophic yeast strain. The enzyme was hyperglycosylated by the host cells, resulting in a protein with a molecular mass of approximately 50 kDa, which was 7 kDa larger than that of its native counterpart. When deglycosylated with endoglycosidase H under nondenaturing conditions, rαNAGAL remained fully active, suggesting that the glycosylation is not required for enzyme activity. Data derived from 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 on the expression of rαNAGAL in P. pastoris, we mutated each of the N-glycosylation sites, as well as all three sites in the same protein molecule, by substituting the Asn with a Gin residue. The results indicate that rαNAGAL mutations in any of the three glycosylation sites, N2 being the most profound, impaired the expression level, altered subcellular distribution, and decreased the efficiency of secretion. Our data suggest that the N-glycosylation of rαNAGAL expressed in P. pastoris may be important in protein folding and resistance to protease degradation during protein synthesis, although it is apparently not required for enzyme activity.