Cloning of a novel alpha 2,3-sialyltransferase that sialylates glycoprotein and glycolipid carbohydrate groups.
Sialyltransferases are a family of 10-12 enzymes that catalyze the transfer of sialic acid to carbohydrate groups of glycoproteins and glycolipids. Three sialyltransferase cDNAs have been cloned, revealing a highly conserved sialylmotif in the catalytic domain of these enzymes. Using a polymerase chain reaction-based approach, we cloned a 150-base pair fragment of a new sialymotif from human placenta mRNA, which was then used as a probe to clone the complete coding sequence of the corresponding gene from a cDNA library. Like the other members of the sialyltransferase gene family cloned to date, the new cDNA coded for a protein predicted to have an NH2-terminal signal-anchor sequence and had the sialylmotif located in the center of the molecule. Comparison with the three other cloned sialyltransferases revealed extensive sequence homology that was not recognized earlier. Expression of a soluble recombinant form of the protein in COS-1 cells produced an active sialyltransferase, which used oligosaccharide, glycoprotein, and glycolipid acceptor substrates with terminal galactose in the Gal beta 1,3GalNAc and Gal beta 1, 4GlcNAc sequences but not the Gal beta 1,3GlcNAc sequence. The sialylated products were sensitive to digestion with the Newcastle disease virus sialidase, which is specific for sialic acid-galactose linkages in the alpha 2,3 linkage. The results suggest that this new member of the sialyltransferase gene family is the enzyme previously described as a glycolipid sialyltransferase activity (SAT-3), which forms the terminal sequences NeuAc alpha-2,3Gal beta 1,3GalNAc-R and NeuAc alpha 2,3Gal beta 1, 4GlcNAc-R.
Kitagawa
H
H
Cytel Corporation, San Diego, California 92121.
Paulson
J C
JC
D000328:Adult; D000367:Age Factors; D001483:Base Sequence; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression; D006017:Glycolipids / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007231:Infant, Newborn; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012799:Sialyltransferases / Q000235:genetics*; D013379:Substrate Specificity
D017931:DNA Primers; D006017:Glycolipids; D006023:Glycoproteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012799:Sialyltransferases