Molecular cloning and expression of chick embryo Galβ1,4GlcNAcα2,6‐sialyltransferase

Open Access
Publisher Website
Google Scholar
Abstract
DNA clones encoding β‐galactoside α2,6‐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 Galβ1,4GlcNAcα2,6‐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 Galβ1,4GlcNAc in glycoproteins.