Molecular cloning and expression of GalNAc alpha 2,6-sialyltransferase.

N Kurosawa,T Hamamoto,Y.C Lee,T Nakaoka,N Kojima,S Tsuji

doi:10.1016/s0021-9258(17)42272-1

Abstract

cDNA clones encoding GalNAc alpha 2,6-sialyltransferase (EC 2.4.99.3) have been isolated from chick embryo cDNA libraries using sequence information obtained from the conserved amino acid sequence of the previously cloned enzymes. The cDNA sequence included an open reading frame coding for 566 amino acids, and the deduced amino acid sequence showed 12% identity with that of Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase from chick embryo. The primary structure of this enzyme suggested a putative domain structure, like that in other glycosyltransferases, consisting of a short NH2-terminal cytoplasmic domain, a signal-membrane anchor domain, a proteolytically sensitive stem region, and a large COOH-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 immunoglobulin 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. The expressed enzyme exhibited activity toward only asialomucin and (asialo)fetuin, no significant activity being detected toward the other glycoprotein and glycolipid substrates tested. 14C-Sialylated glycols obtained from asialomucin re-sialylated with this enzyme were identical to NeuAc alpha 2,6-GalNAc-ol and GlcNAc beta 1,3(NeuAc alpha 2,6) GalNAc-ol. Synthetic GalNAc-SerNAc also served as an acceptor for alpha 2,6-sialylation. These results clearly showed that the expressed enzyme is GalNAc alpha 2,6-sialyltransferase.

Highlights

From Glyco ~ o ~ e c u~~iaorl o Fg r~ontier Research Program, The ~ nstio~f uPh~ysical and e ~ ~ ~Recseaarclh ~ R ~ ~ N ~, Wako, Saitama 351-02,Japan cDNA clones encoding GalNAc &,&sialyltransferase cellular processes
For understanding the expressed enzyme exhibited activity toward only asiamloe-aning of the diversity and the regulatory mechanism for mucin and (asialolfetuin,no significant activity being sialylation of glycoconjugates, it is necessary to obtainknowldetected toward the other glycoprotein and glycolipid edge on the enzymesthemselves and the gene structure of substrates tested. 14C-Sialylated glycols obtained from sia~yltransferasesS. o far, five s~alyltransferases havbeeen puasialomucin re-sialylated withis enzyme wereidenti- rified, and theyexhibit strict specificity for acceptorsubstrates cal to NeuAccuZ,6-GalNAcc-o1and GicNAcfll,3(NeuAca2,6) (18-21). cDNAs encoding Galpl,4GlcNAc a2,6-sialyltransfer
Cell surface sialic acid and a few small stretches (291, tentatively named the ”sialyloccurs in a variety of structures, which change in a regulated motif“ by Livingstone and Paulson (30).Protein motifs are ofmannerduring development, di~erentiation, and oncogenic ten used to identify other members of the same gene family transformation (8,9).Very little is known about the regulation of cell surface sialyl oligosaccharides, which are involved in Theabbreviationsusedare:Gal&4GlcNAc-a6ST,Galfil,4GlcNAc a2,6-sialyltransferase(EC 2.4.99.1); GalNAc-a6ST, GalNAc a2,6-sialyl

Summary

Introduction

From Glyco ~ o ~ e c u~~iaorl o Fg r~ontier Research Program, The ~ nstio~f uPh~ysical and e ~ ~ ~Recseaarclh ~ R ~ ~ N ~ , Wako, Saitama 351-02,Japan cDNA clones encoding GalNAc &,&sialyltransferase cellular processes. This gave a fusion protein containing the putative active domain of the enzyme and the immunoglobulin signal peptide sequence.

Results

Conclusion