THE ROLE OF GLYCOSYLATION AND PHOSPHORYLATION IN THE EXPRESSION OF ACTIVE HUMAN BETA-GLUCURONIDASE

Phosphorylation of mannose residues on N-linked oligosaccharide side c hains of lysosomal enzymes targets them to lysosomes. We used site-dir ected mutagenesis to observe the effect of eliminating selective glyco sylation sites from human beta-glucuronidase on enzyme sorting. Expres sion studies allowed us to determine which of four potential sites wer e glycosylated, preferentially phosphorylated, and required for cataly tic activity. All four sites of the human enzyme were glycosylated, wh ereas in the mouse and rat enzymes, only three of four sites are used. Sites 2 and 3 were preferentially phosphorylated. Elimination of site s 2 and 3 in combination markedly decreased sorting to lysosomes and i ncreased enzyme secretion. Each of the four glycosylation sites could be eliminated individually without drastic reduction in catalytic acti vity. Activity was progressively lost as combinations of two, three, a nd four sites were eliminated. Wild-type enzyme produced in the presen ce of tunicamycin was also inactive, indicating that glycosylation is required for realization of enzyme activity. However, active enzyme co uld be deglycosylated with only minimal loss of activity. Mutant enzym e completely lacking glycosylation did not form tetramers. Partial res toration of tetramerization was achieved by the co-expression of norma l rat enzyme, provided that the normal rat enzyme supplied at least tw o subunits to the tetramer.