Expression cloning of a novel alpha 1,3-fucosyltransferase that is involved in biosynthesis of the sialyl Lewis x carbohydrate determinants in leukocytes.
The sialyl Lewis x (NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)Glc-NAc) determinants serve as ligands in the selectin-mediated adhesion of leukocytes to activated endothelium or platelets. In our efforts to identify glycosyltransferases involved in the biosynthesis of those ligands, we achieved expression cloning of a novel human alpha 1,3-fucosyltransferase termed Fuc-TVII from a THP-1 cDNA library by enrichment of the Namalwa cells highly expressing that determinant with a fluorescence-activated cell sorter. Expression of the COOH-terminal catalytic domain of Fuc-TVII showed an alpha 1,3-fucosyltransferase activity for a type II oligosaccharide with a terminal alpha 2,3-linked sialic acid among various acceptors, consistent with that in vivo acceptor specificity. Alignment of the primary sequences of five alpha 1,3-fucosyltransferases and assignment of the chromosomal location of Fuc-TVII gene, together with that acceptor specificity, indicate that Fuc-TVII consists of a unique class of the alpha 1,3-fucosyltransferase family. Determination of the expression levels of these alpha 1,3-fucosyltransferases in various cells revealed that both Fuc-TVII and a myeloid fucosyltransferase Fuc-TIV were significantly expressed in myeloid lineage cells. Fuc-TVII-transfected Namalwa cells exhibited significant binding to E-selectin in contrast to little binding of the Fuc-TIV-transfected cells. These results suggest that Fuc-TVII may participate in the biosynthesis of the selectin ligands.
Sasaki
K
K
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Japan.
Kurata
K
K
Funayama
K
K
Nagata
M
M
Watanabe
E
E
Ohta
S
S
Hanai
N
N
Nishi
T
T
D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002051:Burkitt Lymphoma; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis; D005647:Fucosyltransferases / Q000096:biosynthesis / Q000097:blood* / Q000302:isolation & purification; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology*; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000097:blood / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D000080506:Sialyl Lewis X Antigen; D012799:Sialyltransferases / Q000096:biosynthesis / Q000097:blood; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D018076:DNA, Complementary; D009844:Oligosaccharides; D011994:Recombinant Proteins; D000080506:Sialyl Lewis X Antigen; D004247:DNA; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; D012799:Sialyltransferases