MOLECULAR-CLONING AND EXPRESSION OF GALNAC ALPHA-2,6-SIALYLTRANSFERASE

cDNA clones encoding GalNAc alpha2,6-sialyltransferase (EC 2.4.99.3) h ave been isolated from chick embryo cDNA libraries using sequence info rmation obtained from the conserved amino acid sequence of the previou sly cloned enzymes. The cDNA sequence included an open reading frame c oding for 566 amino acids, and the deduced amino acid sequence showed 12% identity with that of Galbeta1,4GlcNAc alpha2,6-sialyltransferase from chick embryo. The primary structure of this enzyme suggested a pu tative domain structure, like that in other glycosyltransferases, cons isting of a short NH2-terminal cytoplasmic domain, a signal-membrane a nchor domain, a proteolytically sensitive stem region, and a large COO H-terminal active domain. The identity of this enzyme was confirmed by the construction of a recombinant sialyltransferase in which the NH2- terminal part (232 amino acid residues) was replaced with the immunogl obulin signal sequence. The expression of this recombinant in COS-7 ce lls resulted in secretion of a catalytically active and soluble form o f the enzyme into the medium. The expressed enzyme exhibited activity toward only asialomucin and (asialo)fetuin, no significant activity be ing detected toward the other glycoprotein and glycolipid substrates t ested. C-14-Sialylated glycols obtained from asialomucin re-sialylated with this enzyme were identical to NeuAcalpha2,6-GalNAc-ol and GlcNAc beta1,3(NeuAca2,6) GalNAc-ol. Synthetic GalNAc-SerNAc also served as a n acceptor for alpha2,6-sialylation. These results clearly showed that the expressed enzyme is GalNAc alpha2,6-sialyltransferase.