alpha(1,3)fucosyltransferases expressed by the gain-of-function Chinese hamster ovary glycosylation mutants LEC12, LEC29, and LEC30

Gain-of-function glycosylation mutants provide access to glycosylation path ways, glycosylation genes, and mechanisms that regulate expression of a gly cotype. Previous studies have shown that the gain-of-function Chinese hamst er ovary (CHO) mutants LEC12, LEC29, and LEC30 express an N-ethylmaleimide- resistant alpha(1,3)fucosyltransferase (alpha(1,3)Fuc-T) activity that is n ot detected in CHO cells and that generates the Lewis(X) but not the sialyl -Lewis(X) determinant. The three mutants differ, however, in lectin resista nce properties, expression of fucosylated antigens, and in vitro alpha(1,3) Fuc-T activities. In this paper we show that each mutant expresses Fuc-TIX, but only LEC30 cells express Fuc-TIV. Using genomic PCR and reverse-transc riptase (RT)-PGR strategies, we isolated coding portions of the CHO Fut4 an d Fut9 genes. Each gene is present in a single copy in the CHO and mutant g enomes. The Fut4 gene is expressed only in LEC30 cells, while all three mut ants express the Fut9 gene. Interestingly, the fucosylation phenotypes of L EC12 and LEC29 cells do not correlate with the relative abundance of their Fut9 gene transcripts (LEC29 >> LEC12). Compared to LEC29 cells, LEC12 cell s have an similar to 40-fold higher in vitro alpha(1,3)Fuc-T activity and b ind the VIM-2 monoclonal antibody, whereas LEC29 cells do not bind VIM-2. M ixing experiments did not detect Fuc-TIX inhibitory activity in LEC29 cell extracts, and CHO cells expressing a transfected Fut9 gene behaved like LEC 12 cells. Therefore, it seems that LEC29 cells may not translate their more abundant Fut9 gene transcripts efficiently or may not synthesize appropria te acceptors for internal alpha(1,3)-fucosylation. Alternatively, LEC12 cel ls may possess, in addition to Fuc-TIX, a novel alpha(1,3)Fuc-T activity. ( C) 2000 Academic Press.