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Abstract
We and others have previously described the isolation of three human alpha (1,3)fucosyltransferase genes which form the basis of a nascent glycosyltransferase gene family. We now report the molecular cloning and expression of a fourth homologous human alpha (1,3)fucosyltransferase gene. When transfected into mammalian cells, this fucosyltransferase gene is capable of directing expression of the Lewis x (Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc), sialyl Lewis x (NeuNAc alpha 2-->3Gal beta 1-->4 [Fuc alpha 1-->3]GlcNAc), and difucosyl sialyl Lewis x (NeuNAc alpha 2-->3Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc beta 1-->3 Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc) epitopes. The enzyme shares 85% amino acid sequence identity with Fuc-TIII and 89% identity with Fuc-TV but differs substantially in its acceptor substrate requirements. Polymerase chain reaction analyses demonstrate that the gene is syntenic to Fuc-TIII and Fuc-TV on chromosome 19. Southern blot analyses of human genomic DNA demonstrate that these four alpha (1,3)fucosyltransferase genes account for all DNA sequences that cross-hybridize at low stringency with the Fuc-TIII catalytic domain. Using similar methods, a catalytic domain probe from Fuc-TIV identifies a new class of DNA fragments which do not cross-hybridize with the chromosome 19 fucosyltransferase probes. These results extend the molecular definition of a family of human alpha (1,3)fucosyltransferase genes and provide tools for examining fucosyltransferase gene expression.
Highlights
From the $Departmentof Pediatrics, Division of Pediatric Hematology/Omology,the §Howard Hughes Medical Institute, and the TDepartment of Pathlogy, Universityof Michigan Medical School, Ann Arbor, Michigan 48109-0650
We report themolecular cloning and A subset of a(l,3)-fucosylated cell surface carbohydrate expression of a fourth homologous human a(l,3)fuco- molecules serve as oligosaccharide ligands for two members syltransferase gene
The carbohydrate recognition domain of each of these selectins mediates cell adhesion through calcium-dependent interactions with surface-localized a(2,3)-sialylated, a(l,3)-fucosylated lactosaminoglycans represented by the sialyl Lewis x1 determinant and related monofucosylated or difucosylated moieties [23,24,25,26,27,28,29,30]. a(2,3)-Sialylated a(l,4)-fucosylatedglycans function as stantially in its acceptor substrate requirements
Summary
Assays indicated that the Dowex column procedure removes more Molecular Cloning of a Human GenomicDNA Segment than 95% of the radiolabeled GDP-fucose in the assay, yet does not Homologous to, but Distinct from, the Human Fw-TIII, -IV, retain detectable amounts of the sialylated product. Minal halves of these three protein(sFig. 2 A ) .Four asparagine In summaryt the cross-hybridizing open reading frame has residues that represent potential N-glycosylation sitesare the potential toencode a type I1transmembrane protein with found within the Fuc-TVI predicted protein (Fig. 1).Two of marked amino acid sequence similarity to two other cloned these, located at amino acid residues 153and 184,correspond human fucosyltransferases. This relationship is most salient t o N-linked glycosylation sitespresent a t precisely corre- in a region corresponding to the catalytic domain of these sponding positions in Fuc-TI11 and Fuc-TV (Fig. 2 A ). F~c-Ttll v v f 361 88 fected into the COS-1 cell line, a mammalian host that does not normally express a(1,3)fucosyltransferase activity [44]
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