Molecular and functional characterisation of microsomal UDP-glucuronic acid uptake by members of the nucleotide sugar transporter (NST) family

Tsutomu Kobayashi , Judith E Sleeman , Michael Wh Coughtrie (1) , Brian Burchell

1 Division of Pathology & Neuroscience

Transport of the co-substrate UDP-glucuronic acid (UDPGA) into the lumen of the endoplasmic reticulum is an essential step in glucuronidation reactions due to the intraluminal location of the catalytic site of the enzyme UDP-glucuronosyltransferase (UGT). We have characterised the function of several nucleotide sugar transporters (NSTs) and UGTs as potential carriers of UDPGA for glucuronidation reactions. UDP-N-acetylglucosamine (UDPGlcNAc)-dependent UDPGA uptake was found both in rat liver microsomes and in microsomes prepared from the rat hepatoma cell line H4IIE. The latency of UGT activity in microsomes derived from rat liver and V79 cells expressing UGT1A6 correlated well with mannose-6-phosphatase latency, confirming the UGT in the recombinant cells retained a physiology similar to rat liver microsomes. Four cDNAs coding for nucleotide sugar transporters (NSTs) were obtained; two were previously reported (UGTrel1, UGTrel7) and two newly identified in this work (huYEA4, huYEA4S). Localization of NSTs within the human genome sequence revealed that huYEA4S is an alternatively spliced form of huYEA4. All the cloned NSTs were stably expressed in Chinese hamster fibroblast (V79) cells, and were able to transport UDPGA after preloading of isolated microsomal vesicles with UDPGlcNAc. The highest uptake was seen with UGTrel7, which displayed a V max} about 1 % of rat liver microsomes. Treatment of H4IIE cells with {beta}-naphthoflavone induced UGT protein expression but did not affect the rate of UDPGA uptake. Furthermore, microsomes from UGT1-deficient Gunn rat liver showed UDPGA uptake similar to those from control rats. These data show that NSTs can act as UDPGA transporters for glucuronidation reactions, and indicate that UGTs of the 1A family do not function as UDPGA carriers in microsomes. The cell line H4IIE is a useful model for the study of UDPGA transporters for glucuronidation reactions.

Mots clés: Life Sciences

Tsutomu Kobayashi, Judith E Sleeman, Michael Wh Coughtrie, Brian Burchell. Molecular and functional characterisation of microsomal UDP-glucuronic acid uptake by members of the nucleotide sugar transporter (NST) family. Biochemical Journal, 2006, 400 (2), pp.281-289. ⟨10.1042/BJ20060429⟩. ⟨hal-00478560⟩