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 pathways, glycosylation genes, and mechanisms that regulate expression of a glycotype. Previous studies have shown that the gain-of-function Chinese hamster ovary (CHO) mutants LEC12, LEC29, and LEC30 express an N-ethylmaleimide-resistant alpha(1, 3)fucosyltransferase (alpha(1,3)Fuc-T) activity that is not detected in CHO cells and that generates the Lewis(X) but not the sialyl-Lewis(X) determinant. The three mutants differ, however, in lectin resistance 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-transcriptase (RT)-PCR strategies, we isolated coding portions of the CHO Fut4 and Fut9 genes. Each gene is present in a single copy in the CHO and mutant genomes. The Fut4 gene is expressed only in LEC30 cells, while all three mutants express the Fut9 gene. Interestingly, the fucosylation phenotypes of LEC12 and LEC29 cells do not correlate with the relative abundance of their Fut9 gene transcripts (LEC29 >> LEC12). Compared to LEC29 cells, LEC12 cells have an approximately 40-fold higher in vitro alpha(1,3)Fuc-T activity and bind the VIM-2 monoclonal antibody, whereas LEC29 cells do not bind VIM-2. Mixing experiments did not detect Fuc-TIX inhibitory activity in LEC29 cell extracts, and CHO cells expressing a transfected Fut9 gene behaved like LEC12 cells. Therefore, it seems that LEC29 cells may not translate their more abundant Fut9 gene transcripts efficiently or may not synthesize appropriate acceptors for internal alpha(1,3)fucosylation. Alternatively, LEC12 cells may possess, in addition to Fuc-TIX, a novel alpha(1,3)Fuc-T activity.
Patnaik
S K
SK
Department of Cell Biology, Albert Einstein College of Medicine, New York, New York, 10461, USA.
Zhang
A
A
Shi
S
S
Stanley
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015152:Blotting, Northern; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D000939:Epitopes / Q000276:immunology; D005647:Fucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D015870:Gene Expression; D006031:Glycosylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics; D010641:Phenotype; D010802:Phylogeny; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D014162:Transfection
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D000939:Epitopes; D012333:RNA, Messenger; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase