EXPRESSION CLONING OF CDNA-ENCODING A HUMAN BETA-1,3-N-ACETYLGLUCOSAMINYLTRANSFERASE THAT IS ESSENTIAL FOR POLY-N-ACETYLLACTOSAMINE SYNTHESIS

The structure and biosynthesis of poly-N-acetyllactosamine display a d ramatic change during development and oncogenesis. Poly-N-acetyllactos amines are also modified by various carbohydrate residues, forming fun ctional oligosaccharides such as sialyl Le(x), Herein we describe the isolation and functional expression of a cDNA encoding beta-1,3-N-acet ylglucosaminyltransferase (iGnT), an enzyme that is essential for the formation of poly-N-acetyllactosamine. For this expression cloning, Bu rkitt lymphoma Namalwa KJM-1 cells were transfected with cDNA librarie s derived from human melanoma and colon carcinoma cells, Transfected N amalwa cells overexpressing the i antigen were continuously selected b y fluorescence-activated cell sorting because introduced plasmids cont aining Epstein-Barr virus replication origin can be continuously ampli fied as episomes. Sibling selection of plasmids recovered after the th ird consecutive sorting resulted in a cDNA clone that directs the incr eased expression of i antigen on the cell surface, The deduced amino a cid sequence indicates that this protein has a type II membrane protei n topology found in almost all mammalian glycosyltransferases cloned t o date, iGnT, however, differs in having the longest transmembrane dom ain among glycosyltransferases cloned so far. The iGnT transcript is h ighly expressed in fetal brain and kidney and adult brain but expresse d ubiquitously in various adult tissues, The expression of the presume d catalytic domain as a fusion protein with the IgG binding domain of protein A enabled us to demonstrate that the cDNA encodes iGnT, the en zyme responsible for the formation of GlcNAc beta 1 --> 3Gal beta 1 -- > 4GlcNAc --> R structure and poly-N-acetyllactosamine extension.