T. Pillot et al., GLUCURONIDATION OF HYODEOXYCHOLIC ACID IN HUMAN LIVER - EVIDENCE FOR A SELECTIVE ROLE OF UDP-GLUCURONOSYLTRANSFERASE-2B4, The Journal of biological chemistry, 268(34), 1993, pp. 25636-25642
Monospecific polyclonal antibodies were raised against a variable amin
o-terminal domain (amino acids 14-150) of a human liver form of UDP-gl
ucuronosyltransferase conjugating bile acids, UGT2B4 (Jackson, M. R.,
McCarthy, L. R., Harding, D., Wilson, S., Coughtrie, M. W., and Burche
ll, B. (1987) Biochem. J. 242, 581-588), expressed as a fusion protein
in Escherichia coli. The antibodies were able to recognize the protei
n, stably expressed in a genetically engineered eukaryotic V79 cell li
ne, against which they were directed. The specificity of these antibod
ies allowed their use for analyzing the substrate specificity of this
isoform in human liver, as well as for determining its contribution to
the total hepatic and extra-hepatic glucuronidation of hyodeoxycholic
acid. Western blot analysis of microsomal proteins demonstrated the p
resence of UGT2B4 exclusively in human liver and not in human kidney.
In human liver microsomes, the antibodies were able to inhibit and pre
cipitate up to 90% of the total hyodeoxycholic acid 6-O-glucuronidatio
n activity, but had no effect on activities toward several other subst
rates, such as phenols, bilirubin, or other bile acids, especially hyo
cholic acid and the steroids 4-hydroxyestrone and estriol. Moreover, W
estern blot analysis and immunoinhibition studies of human liver micro
somes from healthy patients and from patients presenting liver disease
s revealed a good correlation between the glucuronidation rate of hyod
eoxycholic acid and the UGT2B4 expression level. The absence of immuno
inhibition of hyodeoxycholic acid conjugation with UDP sugars other th
an UDP-glucuronic acid suggests the involvement of different enzymatic
systems in the glucosidation and xylosylation of hyodeoxycholic acid.
Altogether, the results provided strong evidence for the specific and
predominant involvement of UGT2B4 in the 6-O-glucuronidation of this
bile acid via a UDP-glucuronic acid-dependent mechanism.