Cloning of a novel alpha 2,3-sialyltransferase that sialylates glycoprotein and glycolipid carbohydrate groups.

H Kitagawa,J.C Paulson

doi:10.1016/s0021-9258(17)42271-x

Abstract

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.

Highlights

From Cytel Corporation and the Demrtments o f Chemistry andMolecular Biology, Scripps Research Institute, Sun Diego, Calijbrnia 92121
Three sialyl- The sialyltransferase family consistsof 10-12 glycosyltranstransferasecDNAs have been cloned, revealing a highfleyrases grouped by their common function of transferring sialic conserved sialylmotif in the catalytic domain of these acid from CMP-NeuAc toterminal positions on thesugar enzymes
Expressionof a solublere- parison of the cloned sialyltransferases has revealed that all combinant formof the protein inCOS-1cells produced have a short NHz-tenninal cytoplasmic tail, a signal-anchor an active sialyltransferasew, hich used oligosaccharide, domain, and an extended stem region followedby a large glycoprotein, and glycolipid acceptor substrates with COOH-terminal catalytic domain characteristic of all cloned terminalgalactose in the Ga1Bly3GalNAcandGalpl, glycosyltransferases [12]

Summary

RESULTS

Ase (Stratagene), and the products were digested with BarnHI and Hind111 and subcloned into these sites of Bluescript SK (Stratagene). The 50 clones were sequenced using a T7 primer (Stratagene), and sialylmotif is dispersed throughout the sialyltransferase cataclones of them contained the new sia~ylmotifragment, SMZ. Assuming that the first of the three is used, it codes fora protein of 332 amino acids and four N-linked glycosylationsites (Fig. 2) It should be noted, ,that the first ATGcodon has a poor sequence context for translation initiation [22, 23]. A Kyte-Doolittle hydropathy analysis [24] revealed one potential membrane-spanning region consisting of 18 hydrophobic residues, located 7 residues from the amino terminus (Fig. 2) This structural featuresuggests that theSTZ protein, like the other glycosyltransferases which have been cloned, has a type I1 membrane topology anchored by a noncleavable amino-terminal signal-anchor domain [12].

L e u Ala Met L e u Ala Leu ValLeu Val Val Met V a l Trp Tyr Ser Long-P-A

IIIIIIII II Ill IIllI II I I I I IIII I I

IIII IIII IIIIIIIIIIIIIII I II I IIIII Ill I

Findings

DISCUSSION