MOLECULAR-CLONING AND EXPRESSION OF CHICK-EMBRYO GAL-BETA-1,4GLCNAC-ALPHA-2,6-SIALYLTRANSFERASE - COMPARISON WITH THE MAMMALIAN ENZYME

DNA clones encoding beta-galactoside alpha 2,6-sialyltransferase have been isolated from chick embryonic cDNA libraries using sequence information obtained from the conserved amino acid sequence of the previously cloned enzymes. The cDNA sequence revealed an open reading frame coding for 413 amino acids, and the deduced amino acid sequence showed 57.6% identity with the sequence of rat liver Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase. The primary structure of this enzyme suggested a putative domain structure, similar to structures found in other glycosyltransferases, consisting of a short N-terminal cytoplasmic domain, a signal-membrane anchor domain, a proteolytically sensitive stem region and a large C-terminal active domain. The identity of this enzyme was confirmed by construction of a recombinant sialyltransferase in which the N-terminus part including the cytoplasmic tail, signal anchor domain and stem region was replaced with an immunoglobulin signal peptide sequence. The expression of this recombinant protein in COS-7 cells resulted in secretion of a catalytically active and soluble form of the enzyme into the medium. The expressed enzyme exhibited activity only towards the disaccharide moiety of Gal beta 1,4GlcNAc in glycoproteins.