Structural studies of alpha-N-acetylgalactosaminidase: effect of glycosylation on the level of expression, secretion efficiency, and enzyme activity.

alpha-N-Acetylgalactosaminidase (alphaNAGAL, EC 3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal alpha-linked N-acetylgalactosamine from oligosaccharide chains. After cloning of its cDNA, the recombinant alphaNAGAL (ralphaNAGAL) 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, ralphaNAGAL 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 ralphaNAGAL 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 Gln residue. The results indicate that ralphaNAGAL 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 ralphaNAGAL 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.