Expression cloning of a GM3-specific alpha-2,8-sialyltransferase (GD3 synthase).
A cDNA encoding a GM3-specific alpha-2,8-sialyltransferase (GD3 synthase) was obtained from an expression cDNA library of human melanoma cell line WM266-4 by enrichment of Namalwa KJM-1 cells highly expressing GD3 using an anti-GD3 antibody and a fluorescence-activated cell sorter. Selection of B-cell line Namalwa cells expressing transfected cDNAs in the presence of anti-GD3 monoclonal antibody KM641 gave a cDNA (pAMo-GD3) encoding a protein with a type II transmembrane topology as found for mammalian glycosyltransferases. The following evidence confirms that the cDNA encodes an alpha-2,8-sialyltransferase, which specifically converts GM3 to GD3. (i) Transfection of pAMo-GD3 into Namalwa KJM-1 cells leads to the appearance of GD3 and a GD3 synthase activity. (ii) Northern blot analysis revealed a correlation between the expression of this gene and GD3 in several cell lines. (iii) The putative COOH-terminal active domain of this cloned enzyme fused with protein A has been purified with IgG-Sepharose beads and has been shown to possess GD3-synthesizing activity, excluding the possibility that the cloned cDNA encodes a transacting factor inducing a GD3 synthase. The deduced primary sequence also contains the "sialyl motif" conserved among all the sialyltransferases cloned to date. The polymerase chain reaction analysis reveals that this gene is located on chromosome 12.
Sasaki
K
K
Tokyo Research Laboratories, Kyowa Hakko Kogyo, Co., Ltd., Japan.
Kurata
K
K
Kojima
N
N
Kurosawa
N
N
Ohta
S
S
Hanai
N
N
Tsuji
S
S
Nishi
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D005434:Flow Cytometry; D005679:G(M3) Ganglioside / Q000378:metabolism*; D005732:Gangliosides / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D008545:Melanoma / Q000201:enzymology; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012799:Sialyltransferases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; D005679:G(M3) Ganglioside; D005732:Gangliosides; D012799:Sialyltransferases; C043662:alpha-N-acetylneuraminate alpha-2,8-sialyltransferase