Abstract
UDP-glucuronosyltransferases (UGTs) are glycoproteins in endoplasmic reticulum membranes. UGT2B7 is an important UGT isoenzyme expressed in human liver and glucuronidates various endogenous and exogenous substances. Although this enzyme has three potential N-glycosylation sites (asparagine at positions 67, 68 and 315), no information is available on the actual glycosylated sites and the effects of N-glycosylation on its enzymatic functions. We thus constructed HEK293 cells expressing wild-type UGT2B7 and five mutants (N67Q, N68Q, N315Q, N68Q/N315Q and N67Q/N68Q/N315Q) in which an asparagine at one or more potential N-glycosylation sites was substituted with a glutamine. An immunoblot analysis of whole cell lysate (S9) fractions with or without treatment with an endoglycosidase revealed that UGT2B7 was N-glycosylated at Asn-68 and Asn-315 but not Asn-67. Kinetic analysis employing the S9 fractions as enzyme sources and zidovudine (AZT) and morphine as typical substrates demonstrated that the abolition of N-glycosylation decreased the affinity for AZT but increased that for morphine without affecting reaction velocities, while it decreased the affinity for UDPGA as a cofactor regardless of the substrate used. These results suggest that N-glycosylation differentially affects the glucuronidation of AZT and morphine by human UGT2B7.
