Identification and characterization of novel alpha 1--3 fucosyltransferases

Abstract

alpha1-3 fucosyltransferases (alpha1-3 Fuc-Ts) catalyze the transfer of fucose from GDP-fucose to glycans on lipids and proteins. This leads to the creation of glycan epitopes, such as the LewisX (Le X), VIM-2 and sialyl LeX structures, that are known to be important for biological processes such as those involved in embryo compaction, cancer metastasis and leukocyte trafficking. Two alpha1-3 Fuc-T proteins in mouse and five in humans had been characterized when this project was initiated. Identification and characterization of additional novel alpha1-3 Fuc-Ts can elucidate new mechanisms of fucosylation and further our understanding of the importance of fucosylation in biological processes. Two approaches that relied on the phylogenetically conserved homology of alpha1-3 Fuc-T protein sequences were thus taken to identify novel alpha1-3 Fuc-T genes.;In the first approach, potentially novel alpha1-3 Fuc-T genes of LEC12, LEC29 and LEC30 CHO mutant cell-lines, shown previously to express alpha1-3 Fuc-T activities, were sought using degenerate primers specific for conserved regions of known mammalian alpha1-3 Fuc-T proteins. All three lines were found to express the hamster ortholog of the Fut9 gene, while LEC30 was found to express the hamster Fut4 gene ortholog as well.;It was noticed that LEC12 cells have 10-20 fold lower Fut9 mRNA but ∼40-fold higher Fut9 protein level than LEC29 cells. This suggested the existence of a post-transcriptional regulatory mechanism controlling Fut9 activity that differed in the two cell lines. Full length Fut9 cDNA was sequenced from LEC12 and LEC29. The two sequences were found to be identical except for the first half of the 5' untranslated region (5' UTR). LEC29 cells transfected with full-length (ORF and both UTRs) LEC29 Fut9 cDNA had less alpha1-3 Fuc-T activity and Fut9 protein than those transfected with full-length LEC12 Fut9 cDNA. This and data from other experiments suggests that LEC29 5' UTR may be inhibitory to translation of the Fut9 gene ORF. However, in luciferase reporter studies LEC29 5' UTR, compared to LEC12 5' UTR, was not inhibitory to translation. This indicates that the coding or 3' UTR regions of Fut9 mRNA might play a role in the mediation of translation by the 5' UTR.;While LEC29 cells have slightly less LeX containing glycans than LEC12 cells, VIM-2 containing glycan structures are almost absent on LEC29 but not LEC12 cells. It was found that a recombinant form of E-selectin could bind to LEC12 but not LEC29 cells. LEC29 cells, on the other hand, bound three different galectin proteins better than LEC12 (Abstract shortened by UMI.).