The Polymorphic Variant P24T of UDP-Glucuronosyltransferase 1A4 and Its Unusual Consequences.
The P24T polymorphic variant of the human UDP-glucuronosyltransferase 1A4 (UGT1A4*2, 70C>A) occurs within the signal peptide, five amino acids upstream of the cleavage site and the start of the mature protein. Bioinformatic analysis of the variant suggested that the signal peptide of part of the translated protein is cleaved two residues upstream of the regular site, whereas the rest is cleaved as usual. To test this, recombinant UGT1A4-P24T, with a C-terminal His-tag, was expressed in sf9 insect cells and affinity-purified for N-terminal protein sequencing. The results were in agreement with the in silico prediction. About half of the mutant protein was cleaved at the regular site, between S28 and G29, whereas the other half was cleaved two amino acids upstream, between A26 and E27. The glucuronidation of two substrates, dexmedetomidine and trifluoperazine, was assayed using membrane-enriched UGT1A4-P24T and wild-type UGT1A4. The variant exhibited much lower glucuronidation rates, but kinetic analyses revealed large differences between them only in the Vmax values. The Km values for both substrates were not affected by the mutation and its consequences. This might suggest that the unusual signal peptide cleavage in UGT1A4-P24T somehow disturbs protein folding. Moreover, it raises the possibility that the effect of UGT1A4-P24T on the glucuronidation rate in mammalian expression systems would be mild since they contain more effective post-translation protein control systems in the endoplasmic reticulum. In summary, our results reveal the effect of a polymorphic mutation on the signal sequence cleavage and thereby also the mature UGT.
Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland.
Johanna
J
Troberg
Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland Moshe.Finel@helsinki.fi.
Moshe
M
Finel
D000818:Animals; D014644:Genetic Variation / Q000235:genetics*; D014453:Glucuronosyltransferase / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D011110:Polymorphism, Genetic / Q000235:genetics*; D014268:Trifluoperazine / Q000378:metabolism / Q000494:pharmacology
C019787:bilirubin glucuronoside glucuronosyltransferase; D014268:Trifluoperazine; D014453:Glucuronosyltransferase