Expression cloning of a novel Gal beta (1-3/1-4) GlcNAc alpha 2,3-sialyltransferase using lectin resistance selection.
This report describes the isolation of a cDNA encoding a novel human Gal beta (1-3/1-4)GlcNac alpha 2,3-sialyl-transferase involved in the biosynthesis of the sialyl Lewis x determinant (NeuAc alpha 2-3 Gal beta 1-4(Fuc alpha 1-3)GlcNAc). A cDNA library of the human melanoma cell line WM266-4 was constructed in an Epstein-Barr virus-based cloning vector. Selection of the B-cell line Namalwa expressing transfected cDNAs in the presence of the cytotoxic lectin Ricinus communis agglutinin 120 gave a cDNA encoding a protein with type II transmembrane topology, as found for mammalian glycosyltransferases. The use of this lectin, which is specific to galactose residues (especially the Gal beta 1-4GlcNAc structure), originates from our prediction that the modification of the Gal beta 1-4GlcNAc structure (a backbone of the sialyl Lewis x structure) by glycosyltransferases may increase the levels of resistance to this lectin. Comparison of this cDNA sequence with those of three other cloned sialyltransferases revealed two conserved regions shared by all four enzymes. Expression of the COOH-terminal catalytic domain of this protein showed alpha 2,3-sialyltransferase activity with substrate specificity different from that of CMP-N-acetylneuraminate:N-acetyllactosaminide alpha-2,3-sialyltransferase (Gal-beta 1-3(4)GlcNAc alpha 2,3-sialyltransferase, EC 2.4.99.6). Furthermore, expression of this cDNA in Namalwa cells increased the level of sialyl Lewis x antigens. The cloning approach based on lectin resistance may be useful for the isolation of cDNAs encoding other mammalian glycosyltransferases.
Sasaki
K
K
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Japan.
Watanabe
E
E
Kawashima
K
K
Sekine
S
S
Dohi
T
T
Oshima
M
M
Hanai
N
N
Nishi
T
T
Hasegawa
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004351:Drug Resistance; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D016256:Lewis X Antigen / Q000096:biosynthesis; D008545:Melanoma; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051381:Rats; D015183:Restriction Mapping; D012276:Ricin / Q000633:toxicity*; D017386:Sequence Homology, Amino Acid; D012799:Sialyltransferases / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D017931:DNA Primers; D018076:DNA, Complementary; D016256:Lewis X Antigen; D012276:Ricin; D012799:Sialyltransferases; C029156:N-acetyllactosaminide alpha-2,3-sialyltransferase; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase