Expression and Functional Domains of Rabbit Liver UDP-glucuronosyltransferase 2B16 and 2B13
Qing Li, Xiaojing Lou, Marie-Anne Peyronneau, Petra Obermayer Straub, Robert H. Tukey
Journal of Biological Chemistry
Southern blot analysis has demonstrated that the 5 portion of the rabbit liver dexamethasone-inducible UDP-glucuronosyltransferase (UGT) 2B13 RNA is related in sequence to a family of UGT genes (Tukey, R. H., Pendurthi, U. R., Nguyen, N. T., Green, M. D., and Tephly, T. R. (1993) J. Biol. Chem. 268, 15260 -15266). To identify these additional gene transcripts, rabbit liver cDNA libraries were screened with a 5 conserved 330base pair UGT2B13 cDNA fragment, resulting in the isolation and
... ization of several rabbit liver UGT cDNAs. One such clone, called pGT11, encodes a putative glycoprotein that is 78% similar to rabbit UGT2B13. The new UGT has been designated UGT2B16. The UGT2B16 gene is expressed as a single 4200-base RNA transcript that is regulated only in adult rabbits. The predicted NH 2 -terminal 25 amino acids of UGT2B16 are identical to that of rabbit liver UGT2B13, with the remainder of the protein being 77% similar to UGT2B13. Expressed UGT2B16 protein in COS-1 cells was active toward 4-hydroxybiphenyl, similar to that of UGT2B13. However, UGT2B16 efficiently conjugated 4-hydroxyestrone and 4-tert-butylphenol, substrates that are not efficiently catalyzed by UGT2B13. To further characterize the structural domains of UGT2B16 and UGT2B13, a series of chimeric cDNAs were constructed that contained portions of both UGT2B16 and UGT2B13. Chimeric 2B16 300 2B13 531 , which contained the amino-terminal UGT2B16 amino acids 1-300 followed by amino acids 301-531 of UGT2B13, as well as chimeric 2B16 358 2B13 531 and 2B16 434 2B13 531 proteins, catalyzed the glucuronidation of 4-hydroxyestrone, indicating that the carboxyl terminus of UGT2B13 could substitute for those same regions on UGT2B16. However, the replacement of the carboxyl end of UGT2B13 with 2B16 300 -531 or 2B16 434 -531 dramatically impaired the catalytic function of the chimeric proteins. These results indicate that the carboxyl end of UGT2B13 plays an important role in the functional and possible conformational state of the protein.