Activation of beta1,3-N-acetylglucosaminyltransferase-2 (beta3Gn-T2) by beta3Gn-T8. Possible involvement of beta3Gn-T8 in increasing poly-N-acetyllactosamine chains in differentiated HL-60 cells.
Enzymatic activities of some glycosyltransferases are markedly increased via complex formation with other transferases or cofactor proteins. We previously showed that beta1,3-N-acetylglucosaminyltransferase-2 (beta3Gn-T2) and beta3Gn-T8 can form a heterodimer in vitro and that the complex exhibits much higher enzymatic activity than either enzyme alone (Seko, A., and Yamashita, K. (2005) Glycobiology 15, 943-951). Here we examined this activation and the biological significance of complex formation in differentiated HL-60 cells. beta3Gn-T2 and -T8 were co-immunoprecipitated from the lysates of both-transfected COS-7 cells, indicating their association in vivo. We prepared inactive mutants of both enzymes by destroying the DXD motifs. The mixture of mutated beta3Gn-T2 and intact beta3Gn-T8 did not exhibit any activation, whereas the mixture of intact beta3Gn-T2 and mutated beta3Gn-T8 had increased activity, indicating the activation of beta3Gn-T2 via complex formation. Next, we compared expression levels of beta3Gn-T1-T8 in HL-60 cells and DMSO-treated differentiated HL-60 cells, which produce larger poly-N-acetyllactosamine chains. The expression level of beta3Gn-T8 in the differentiated cells was 2.6-fold higher than in the untreated cells. Overexpression of beta3Gn-T8, but not beta3Gn-T2, induced an increase in poly-N-acetyllactosamine chains in HL-60 cells. These results raise a possibility that up-regulation of beta3Gn-T8 in differentiated HL-60 cells increases poly-N-acetyllactosamine chains by activating intrinsic beta3Gn-T2.
Seko
Akira
A
Innovative Research Initiatives, Tokyo Institute of Technology, Yokohama 226-8503, Japan.
Yamashita
Katsuko
K
D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002522:Chlorocebus aethiops; D003451:Cryoprotective Agents / Q000494:pharmacology; D019281:Dimerization; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004789:Enzyme Activation / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000235:genetics; D018922:HL-60 Cells; D006801:Humans; D051379:Mice; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D041681:NIH 3T3 Cells; D011134:Polysaccharides / Q000096:biosynthesis* / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
D003451:Cryoprotective Agents; D011134:Polysaccharides; C037199:poly-N-acetyllactosamine; C496333:B3GNT8 protein, human; D017351:N-Acetylglucosaminyltransferases; C428132:B3GNT2 protein, human; D004121:Dimethyl Sulfoxide