N-Glycosylation and Residue 96 Are Involved in the Functional Properties of UDP-Glucuronosyltransferase Enzymes^,

Abstract

The recent cloning of several human and monkey UDP-glucuronosyltransferase (UGT) 2B proteins has allowed the characterization of these steroid metabolic enzymes. However, relatively little is known about the structure-function relationship, and the potential post-translational modifications of these proteins. The mammalian UGT2B proteins contain at least one consensus asparagine-linked glycosylation site NX(S/T). Endoglycosidase H digestion of the human and monkey UGT2B proteins demonstrates that only UGT2B7, UGT2B15, UGT2B17, and UGT2B20 are glycosylated. Although UGT2B15 and UGT2B20 contain three and four potential glycosylation sites, respectively, site-directed mutagenesis revealed that both proteins are glycosylated at the same first site. In both proteins, abolishing glycosylation decreased glucuronidation activity; however, the K(m) values and the substrate specificities were not affected. Despite the similarities between UGT2B15 and UGT2B20, UGT2B20 is largely more labile than UGT2B15. Treating HK293 cells stably expressing UGT2B20 with cycloheximide for 2 h decreased the enzyme activity by more than 50%, whereas the activity of UGT2B15 remained unchanged after 24 h. The UGT2B20 protein is unique in having an isoleucine at position 96 instead of an arginine as found in all the other UGT2B enzymes. Changing the isoleucine in UGT2B20 to an arginine stabilized enzyme activity, while the reciprocal mutation in UGT2B15 R96I produced a more labile enzyme. Secondary structure predictions of UGT2B proteins revealed a putative alpha-helix in this region in all the human and monkey proteins. This alpha-helix is shortest in UGT2B20; however, the helix is lengthened in UGT2B20 I96R. Thus, it is apparent that the length of the putative alpha-helix between residues 84 and 100 is a determining factor in the stability of UGT2B enzyme activity. This study reveals the extent and importance of protein glycosylation on UGT2B enzyme activity and that the effect of residue 96 on UGT2B enzyme stability is correlated to the length of a putative alpha-helix.

N-Glycosylation plays a role in protein folding of human UGT1A9

“…Thus, it is conceivable that human UGT1As are commonly N-glycosylated at the sites corresponding to N292 and N344 of UGT1A9, which are encoded from common exons 2 and 5, although the extent of glycosylation and/or size of the glycan are different. The mutation of these asparagine residues significantly decreased the Vmax values, but not the Km values (Table 2), consistent with a previous study of human UGT2B15 and monkey UGT2B20 [8]. To investigate whether the decreased velocity was due to the abolished glycosylation, not by the change in primary structure, we further studied the enzyme activity of unglycosylated and deglycosylated UGT1A9 proteins.…”

“…However, it should be pointed out that some UGT2B isoforms are not glycosylated. Barbier et al [8] reported that human UGT2B4, monkey UGT2B18 and UGT2B23 are not N-glycosylated in spite of the presence of the potential N-glycosylation sites. Additionally, there is no potential N-glycosylation site for rat UGT2B3 [9] and UGT2B6 [10].…”

“…Barbier et al [8] have reported that human UGT2B15 and monkey UGT2B20, in which the glycosylation was abolished by the mutation of asparagine residues, showed decreased velocity of glucuronidation, but no changes in the Km values and substrate specificities. Additionally, it has been demonstrated that some other UGT isoforms including human UGT2B4, monkey UGT2B23, and rat UGT2B3 are not glycosylated [8,11].…”

Key Amino Acid Residues Responsible for the Differences in Substrate Specificity of Human UDP-Glucuronosyltransferase (UGT)1A9 and UGT1A8

“…Such higher activities in the HEK293 system than the Supersomes were also observed for the glucuronidations of 5-(4Ј-hydroxyphenyl)-5-phenylhydantoin (Nakajima et al, 2007) and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (Malfatti and Felton, 2004;Dellinger et al, 2007). The differences in the membrane environment or lipid components and/or the differences in the post-translational modulation of UGT such as glycosylation or phosphorylation between the host cells (Barbier et al, 2000;Basu et al, 2005) may be involved in the inconsistency. In the present study using Supersomes, the V max values of UGT1A8 and UGT1A10 were considerably lower than those of UGT1A7 and UGT1A9 for 4-MU and p-NP O-glucuronidations.…”

Gender-Related Differences in Mycophenolate Mofetil-Induced Gastrointestinal Toxicity in Rats

“…This discrepancy may be explained by the fact that the relationship between amount of mRNA and "signal" in the branched chain assay (method used by Shelby et al, 2003) is not necessarily constant, due to the sequences of the oligonucleotide probes and their specific characteristics. Alternatively, these inconsistencies may be explained by post-translational changes in UGT activity resulting from phosphorylation or glycosylation or by inconsistent relationships between mRNA and protein expression when different proteins are compared (Barbier et al, 2000;Basu et al, 2003).…”