Mutations at critical N-glycosylation sites reduce tyrosinase activity by altering folding and quality control

Tyrosinase is a copper-containing enzyme that regulates melanin biosynthesi s in mammals. Mutations at a single N-glycosylation sequon of tyrosinase ha ve been reported to be responsible for oculocutaneous albinism type IA in h umans, characterized by inactive tyrosinase and the total absence of pigmen tation. To probe the role that each N-glycosylation site plays in the synth esis of biologically active tyrosinase, we analyzed the calnexin mediated f olding of tyrosinase N-glycosylation mutants, We have determined that four of the six potential glycosylation sites, including that associated with al binism, are occupied. Analysis of the folding pathway and activity of 15 ty rosinase mutants lacking one our more of the occupied N-glycosylation sites shows that glycans at any two N-glycosylation sites are sufficient to inte ract with calnexin and give partial activity, but a specific pair of sites (Asn(86) and Asn(371)) is required for full activity. The mutants with less than two N-glycosylation sites do not interact with calnexin and show a co mplete absence of enzyme activity. Copper analysis of selected mutants sugg ests that the observed partial activity is due to two populations with diff erential copper content. By correlating the degree of folding with the acti vity of tyrosinase, we propose a local folding mechanism for tyrosinase tha t can explain the mechanism of inactivation of tyrosinase N-glycosylation m utants found in certain pigmentation disorders.