MOLECULAR-CLONING AND EXPRESSION OF GAL-BETA-1,3GALNAC-ALPHA-2,3-SIALYTRANSFERASE FROM MOUSE-BRAIN

DNA clones encoding beta-galactoside alpha2,3-sialyltransferase have b een isolated from mouse brain cDNA libraries using sequence informatio n obtained from the conserved amino acid sequence of the previously cl oned enzymes. The cDNA sequence revealed an open reading frame coding for 337 amino acids, and the deduced amino acid sequence showed 80% id entity with that of porcine submaxillary gland Galbeta1,3GalNAc alpha2 ,3-sialyltransferase. The primary structure of this enzyme suggested a putative domain structure, like that in other glycosyltransferases, c onsisting of a short NH2-terminal cytoplasmic domain, a signal-membran e anchor domain, a proteolytically sensitive stem region, and a large COOH-terminal active domain. The identity of this enzyme was confirmed by construction of a recombinant sialyltransferase in which the NH2-t erminal part including the cytoplasmic tail, signal-anchor domain and stem region was replaced with an immuno-globulin signal sequence. The expression of this recombinant in COS-7 cells resulted in secretion of a catalytically active and soluble form of the enzyme into the medium . This enzyme exhibited the transferase activity toward only the disac charide moiety of Galbeta1,3GalNAc of glycoproteins and glycolipids, n o significant activity being detected for the other substrates tested.