The Effects of N-Glycosylation on the Glucuronidation of Zidovudine and Morphine by UGT2B7 Expressed in HEK293 Cells

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.

Modulation of the UGT2B7 Enzyme Activity by C-Terminally Truncated Proteins Derived from Alternative Splicing

“…We also report the presence of i1, i2, and i4 in the Golgi, a feature that is compatible with scarce reports of weak UGT activity in this organelle (von Bahr et al, 1972;Bossuyt and Blanckaert, 2001). However, it is also plausible that these three proteins undergo chemical modifications in the Golgi; indeed, UGT2B7 is glycosylated (Nagaoka et al, 2012), a chemical modification mainly initiated and/or terminated in the Golgi (Stanley, 2011). Western blotting of centrifugation fractions and comparison of UGT2B7 signals with subcellular compartment-specific markers further substantiate that i1, i2, and i4 are all residents of the ER and Golgi, whereas i2 and i4 can also be found in the cytoplasm, in which they may have roles distinct from that of i1 inhibition.…”

In vitro assessment of the glucose-lowering effects of berberrubine-9-O-β-D-glucuronide, an active metabolite of berberrubine

“…Many drugs, such as metoclopramide, chloramphenicol, p-aminobenzoic acid, and zidovudine, can be metabolized into their respective glucuronides [13][14][15][16] . Fortunately, morphine-6-glucuronied possesses similar pharmacological activity as morphine and has been successfully developed as a frontline analgesic drug.…”

Human UDP-Glucuronosyltransferase Expression in Insect Cells: Ratio of Active to Inactive Recombinant Proteins and the Effects of a C-Terminal His-Tag on Glucuronidation Kinetics

“…The nearly identical results and conclusions of the two studies indicate that the similarity between active recombinant UGTs that were expressed in HEK293 cells and His-tagged UGTs from baculovirus-infected insect cells is very high. It may be added here that in contrast to the results we (unpublished data) and Nakajima et al (2010) got with UGT1A9, a very recent study found that N-glycosylation affects the activity of UGT2B7 that was expressed in HEK293 cells (Nagaoka et al, 2012). The reason for this difference is currently unclear.…”