A rare UGT2B7 variant creates a novel N-glycosylation site at codon 121 with impaired enzyme activity

UDP-glucuronosyltransferase (UGT) superfamily are glycoproteins resident of the endoplasmic reticulum membranes that undergo post-translational modifications (PTM). UGT2B7 is of particular interest because of its action on a wide variety of drugs. Most studies currently survey common variants and are only examining a small fraction of the genetic diversity. However, rare variants (frequency <1%) might have significant effect as they are predicted to greatly outnumber common variants in the human genome. Here, we discovered a rare single nucleotide UGT2B7 variant of potential pharmacogenetic relevance that encodes a nonconservative amino acid substitution at codon 121. This low-frequency variation, found in two individuals of a population of 305 healthy volunteers, leads to the translation of an asparagine (Asn) instead of an aspartic acid (Asp) (UGT2B7 p.D121N). This amino acid change was predicted to create a putative N-glycosylation motif NX(S/T) subsequently validated upon endoglycosidase H treatment of microsomal fractions and inhibition of N-glycosylation of endogenously produced UGT2B7 with tunicamycin from HEK293 cells. The presence of an additional N-linked glycan on the UGT2B7 enzyme, likely affecting proper protein folding, resulted in a significant decrease, respectively by 49 and 40%, in the formation of zidovudine and mycophenolic acid glucuronides. A systematic survey of the dbSNP database uncovered 32 rare and naturally occurring missense variations predicted to create or disrupt N-glycosylation sequence motifs in the other UGT2B enzymes. Collectively, these variants have the potential to increase the proportion of variance explained in the UGT pathway due to changes in PTM such as N-linked glycosylation with consequences on drug metabolism

Un nouveau variant rare entrainant la modification post-traductionnelle de l'enzyme UGT2B7 et affectant son activité

La superfamille des UDP-glucuronosyltransférases (UGT) est constituée de glycoprotéines résidant au réticulum endoplasmique et sujettes aux modifications post-traductionnelles (PTM, post-translational modifications). L’enzyme UGT2B7 est d’un intérêt particulier vu son action sur une grande variété de médicaments. La plupart des études actuelles n’ont pour sujet que les variants communs de cette enzyme et n’examinent donc qu’une fraction de la diversité génétique de celle-ci. En effet, les variants rares (fréquence allélique en deçà de 1%) peuvent potentiellement avoir un effet considérable puisqu’ils sont prédits comme étant bien plus nombreux que les variants communs au sein du génome humain. La présente étude fait état de la découverte d’un variant rare d’UGT2B7 possédant un intérêt pharmacogénétique potentiel et encodant une substitution d’acide aminé au codon 121. Cette variation peu fréquente, retrouvée chez deux individus au sein d’une population de 305 sujets sains, mène à la traduction d’une asparagine (Asn) plutôt qu’un acide aspartique (Asp) au codon 121 (UGT2B7 p.D121N). Cette substitution est prédite comme créant un motif de N-glycosylation NX(S/T) subséquemment validé par traitement à l’endoglycosidase de fractions microsomales issues de surexpressions dans des cellules HEK293 et par inhibition à la tunicamycine de la N-glycosylation d’UGT2B7 produites de façon endogène dans des HEK293. De plus, la présence d’un oligosaccharide additionnel sur l’enzyme UGT2B7, affectant potentiellement son repliement, résulte en la diminution, respectivement par 49 et 40%, de la formation de glucuronides à partir de la zidovudine et de l’acide mycophénolique. Une analyse de la base de données dbSNP a permis la découverte de 32 variants rares pouvant potentiellement créer ou abolir des motifs de N-glycosylation au sein d’enzymes UGT2B. Ensemble, ces variants ont le potentiel d’augmenter la proportion de la variance de la voie des UGT qui est expliquée par des modifications post-traductionnelles telles la N-glycosylation, affectant ainsi le métabolisme des médicaments.The UDP-glucuronosyltransferase (UGT) superfamily consists of glycoproteins resident of the endoplasmic reticulum membranes that undergo post-translational modifications (PTM). UGT2B7 is of particular interest because of its action on a wide variety of drugs. Most studies currently survey common variants and are only examining a small fraction of the genetic diversity. However, rare variants (frequency &lt; 1%) might have significant effect as they are predicted to greatly outnumber common variants in the human genome. Here, we discovered a rare single nucleotide UGT2B7 variant of potential pharmacogenetic relevance that encodes a nonconservative amino acid substitution at codon 121. This low-frequency variation, found in two individuals of a population of 305 healthy volunteers, leads to the translation of an asparagine (Asn) instead of an aspartic acid (Asp) (UGT2B7 p.D121N). This amino acid change was predicted to create a putative N-glycosylation motif NX(S/T) subsequently validated upon endoglycosidase H treatment of microsomal fractions and inhibition of N-glycosylation of endogenously produced UGT2B7 with tunicamycin from HEK293 cells. The presence of an additional N-linked glycan on the UGT2B7 enzyme, likely affecting proper protein folding, resulted in a significant decrease, respectively by 49 and 40%, in the formation of zidovudine and mycophenolic acid glucuronides. A systematic survey of the dbSNP database uncovered 32 rare and naturally occurring missense variations predicted to create or disrupt N-glycosylation sequence motifs in the other UGT2B enzymes. Collectively, these variants have the potential to increase the proportion of variance explained in the UGT pathway due to changes in PTM such as N-linked glycosylation with consequences on drug metabolism

Endogenous Protein Interactome of Human UDP-Glucuronosyltransferases Exposed by Untargeted Proteomics

The conjugative metabolism mediated by UDP-glucuronosyltransferase enzymes (UGTs) significantly influences the bioavailability and biological responses of endogenous molecule substrates and xenobiotics including drugs. UGTs participate in the regulation of cellular homeostasis by limiting stress induced by toxic molecules, and by controlling hormonal signaling networks. Glucuronidation is highly regulated at genomic, transcriptional, post-transcriptional and post-translational levels. However, the UGT protein interaction network, which is likely to influence glucuronidation, has received little attention. We investigated the endogenous protein interactome of human UGT1A enzymes in main drug metabolizing non-malignant tissues where UGT expression is most prevalent, using an unbiased proteomics approach. Mass spectrometry analysis of affinity-purified UGT1A enzymes and associated protein complexes in liver, kidney and intestine tissues revealed an intricate interactome linking UGT1A enzymes to multiple metabolic pathways. Several proteins of pharmacological importance such as transferases (including UGT2 enzymes), transporters and dehydrogenases were identified, upholding a potential coordinated cellular response to small lipophilic molecules and drugs. Furthermore, a significant cluster of functionally related enzymes involved in fatty acid β-oxidation, as well as in the glycolysis and glycogenolysis pathways were enriched in UGT1A enzymes complexes. Several partnerships were confirmed by co-immunoprecipitations and co-localization by confocal microscopy. An enhanced accumulation of lipid droplets in a kidney cell model overexpressing the UGT1A9 enzyme supported the presence of a functional interplay. Our work provides unprecedented evidence for a functional interaction between glucuronidation and bioenergetic metabolism

Divergent Expression and Metabolic Functions of Human Glucuronosyltransferases through Alternative Splicing

Maintenance of cellular homeostasis and xenobiotic detoxification is mediated by 19 human UDP-glucuronosyltransferase enzymes (UGTs) encoded by ten genes that comprise the glucuronidation pathway. Deep RNA sequencing of major metabolic organs exposes a substantial expansion of the UGT transcriptome by alternative splicing, with variants representing 20% to 60% of canonical transcript expression. Nearly a fifth of expressed variants comprise in-frame sequences that may create distinct structural and functional features. Follow-up cell-based assays reveal biological functions for these alternative UGT proteins. Some isoforms were found to inhibit or induce inactivation of drugs and steroids in addition to perturbing global cell metabolism (energy, amino acids, nucleotides), cell adhesion, and proliferation. This work highlights the biological relevance of alternative UGT expression, which we propose increases protein diversity through the evolution of metabolic regulators from specific enzymes

Deciphering multivalent glycocluster–lectin interactions through AFM characterization of the self-assembled nanostructures

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