Molecular cloning and characterization of a human beta-Gal-3'-sulfotransferase that acts on both type 1 and type 2 (Gal beta 1-3/1-4GlcNAc-R) oligosaccharides.
A novel sulfotransferase gene (designated GP3ST) was identified on human chromosome 2q37.3 based on its similarity to the cerebroside 3'-sulfotransferase (CST) cDNA (Honke, K., Tsuda, M., Hirahara, Y., Ishii, A., Makita, A., and Wada, Y. (1997) J. Biol. Chem. 272, 4864-4868). A full-length cDNA was obtained by reverse transcription-polymerase chain reaction and 5'- and 3'-rapid amplification of cDNA ends analyses of human colon mRNA. The isolated cDNA clone predicts that the protein is a type II transmembrane protein composed of 398 amino acid residues. The amino acid sequence indicates 33% identity to the human CST sequence. A recombinant protein that is expressed in COS-1 cells showed no CST activity, but did show sulfotransferase activities toward oligosaccharides containing nonreducing beta-galactosides such as N-acetyllactosamine, lactose, lacto-N-tetraose (Lc4), lacto-N-neotetraose (nLc4), and Gal beta 1-3GalNAc alpha-benzyl (O-glycan core 1 oligosaccharide). To characterize the cloned sulfotransferase, a sulfotransferase assay method was developed that uses pyridylaminated (PA) Lc4 and nLc4 as enzyme substrates. The enzyme product using PA-Lc4 as an acceptor was identified as HSO(3)-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1- 4Glc-PA by two-dimensional (1)H NMR. Kinetics studies suggested that GP3ST is able to act on both type 1 (Gal beta 1-3GlcNAc-R) and type 2 (Gal beta 1-4GlcNAc-R) chains with a similar efficiency. In situ hybridization demonstrated that the GP3ST gene is expressed in epithelial cells lining the lower to middle layer of the crypts in colonic mucosa, hepatocytes surrounding the central vein of the liver, extravillous cytotrophoblasts in the basal plate and septum of the placenta, renal tubules of the kidney, and neuronal cells of the cerebral cortex. The results of this study indicate the existence of a novel beta-Gal-3'-sulfotransferase gene family.
Honke
K
K
Department of Biochemistry, Osaka University Medical School, Suita, Osaka 565-0871, Japan. khonke@biochem.med.osaka-u.ac.jp
Tsuda
M
M
Koyota
S
S
Wada
Y
Y
Iida-Tanaka
N
N
Ishizuka
I
I
Nakayama
J
J
Taniguchi
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002240:Carbohydrate Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017403:In Situ Hybridization; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000145:classification / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D008670:Metals; D009844:Oligosaccharides; D012333:RNA, Messenger; D015238:Sulfotransferases; C020556:galactosylceramide sulfotransferase