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KMID : 0617319980070010150
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Journal of Pharmacetical Sceiences Ewha Womans University
1998 Volume.7 No. 1 p.150 ~ p.155
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Cloning and Expression of Human Liver UDP-Glucuronosyltransferase cDNA, UDPGTh2
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Dong, Misook
Owens, Ida S./Sheen, Yhun Yhong
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Abstract
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The human liver cDNA clone UDPGTh2, encoding a liver UDP-glucuronosyltransferas (UDPGT) was isolated from a λ gt 11 cDNA library by hybridization to mouse transferase cDNA clone, UDPGTm1. UDPGTh2 encoded a 529 amino acid protein with an amino terminu membrane-insertion signal peptide and a carboxyl terminus membrane-spanning region. There were three potential asparagine-linked glycosylation sites at residues 67, 68, and 315. In order to obtain UDPGTh2 protein encoded from cloned human liver UDP-glucuronosyltransferas cDNA, the clone was inserted into the pSVL vector (pUDPGTh2) and expressed in COS cells, The presence of a transferase with Mr-52,000 in transfected cells cultured in the presence of [^35S]methionine was shown by immunocomplexed products with goat antimouse transferase lgG and protein A-Sepharose and analysis by sodium dodecyl sulfate-polycrylamide gel electrophoresis and autoradiography. The expressed UDPGT was a glycoprotein as indicated by electrophoresis mobility shift in Mr~3,000-4,000 when expressed in the presence of tunicamycin. The extent of glycosylation was difficult to assess, although one could assume that glycosyl structures incorporated at the level of endoplasmic reticulum were always the core oligosaccharides. Thus, it is likely that at least two moieties inserted can account for the shift of Mr~3,000-4,000. This study demonstrates the cDNA and deduced amino acid sequence of human liver UDP-glucuronosyltransferase cDNA, UDPGTh2.
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