Expression cloning of a G_M3-specific α-2,8-Sialyltransferase (G_D3 Synthase)

A cDNA encoding a G_M3-specific α-2,8-sialyltransferase (G_D3 synthase) was obtained from an expression cDNA library of human melanoma cell line WM266-4 by enrichment of Namalwa KJM-1 cells highly expressing G_D3 using an anti-G_D3 antibody and a fluorescence-activated cell sorter. Selection of B-cell line Namalwa cells expressing transfected cDNAs in the presence of anti-G_D3 monoclonal antibody KM641 gave a cDNA (pAMo-GD3) encoding a protein with a type II transmembrane topology as found for mammalian glycosyltransferases. The following evidence confirms that the cDNA encodes an α-2,8-sialyltransferase, which specifically converts G_M3 to G_D3. (i) Transfection of pAMo-GD3 into Namalwa KJM-1 cells leads to the appearance of G_D3 and a G_D3 synthase activity, (ii) Northern blot analysis revealed a correlation between the expression of this gene and G_D3 in several cell lines, (iii) The putative COOH-terminal active domain of this cloned enzyme fused with protein A has been purified with IgG-Sepharose beads and has been shown to possess G_D3-synthesizing activity, excluding the possibility that the cloned cDNA encodes a transacting factor inducing a G_D3 synthase. The deduced primary sequence also contains the "sialyl motif" conserved among all the sialyltransferases cloned to date. The polymerase chain reaction analysis reveals that this gene is located on chromosome 12.