Isolation and characterization of murine alpha(1 going to 3)fucosyltransferase genes.

Abstract

Murine $\alpha(1\to3$)fucosyltransferase genes provide the means to understand the function of $\alpha(1\to3$)fucosylated glycoconjugates during development and in the adult animal. Towards this end, we have isolated two murine genes which encode enzymes that participate in the synthesis of $\alpha(1\to3$)fucosylated oligosaccharides. Sequence comparisons, gene expression data, kinetic analyses, and chromosomal localization experiments indicate that one of these sequences is the murine homologue of the human Fuc-TIV gene. The murine Fuc-TIV gene is most highly expressed in bone marrow and the gastrointestinal tract, and, in transfected cells, encodes an enzyme which determines expression of cell surface Lewis$\rm\sp{X}$ but not sialyl Lewis$\rm\sp{X}$ epitopes. The second gene, referred to as Fuc-TVII, represents a novel sequence which differs from previously described $\alpha(1\to3$)fucosyltransferase sequences in genomic organization. The Fuc-TVII gene contains at least three exons which may be alternatively spliced to produce three different transcripts. The Fuc-TVII enzyme also has unique substrate specificities, catalyzing the construction of sialyl Lewis$\rm\sp{X}$ but not Lewis$\rm\sp{X}$ epitopes. In addition, Fuc-TVII expression is restricted primarily to myeloid tissues and cell lines, suggesting that Fuc-TVII enzyme constructs the ligands for E- and P-selectin. The Fuc-TVII gene has been localized to the proximal portion of mouse chromosome 2, which is syntenic with human chromosomes 9 and 10. Molecular data indicates that there is a human Fuc-TVII homologue. Anti-Fuc-TIV and anti-Fuc-TVII polyclonal antisera have been generated in rabbits using recombinant peptides synthesized in Escherichia coli. These antibodies were used to localize the Fuc-TIV and Fuc-TVII enzymes to the Golgi apparatus at the light microscopic level and may be used in future experiments to determine the subcellular localization of the proteins at the electron microscopic level. The anti-fucosyltransferase antibodies may also be used to investigate the biosynthesis of the Fuc-TIV and Fuc-TVII enzymes and define their expression patterns. We have begun isolating additional $\alpha(1\to3$)fucosyltransferase sequences in a variety of species using degenerate oligonucleotides and the polymerase chain reaction. We have isolated a human sequence from a HL60 cDNA library that has sequence similarity to the murine Fuc-TVII sequence. We have also isolated two distinct chicken genomic DNAs that exhibit sequence similarity to the human Fuc-TIII cDNA.