EXPRESSION CLONING OF A G(M3)-SPECIFIC ALPHA-2,8-SIALYLTRANSFERASE (G(D3) SYNTHASE)

A cDNA encoding a G(M3)-specific alpha-2,8-sialyltransferase (G(D3) sy nthase) was obtained from an expression cDNA library of human melanoma cell line WM266-4 by enrichment of Namalwa KJM-1 cells highly express ing G(D3) using an anti-G(D3) antibody and a fluorescence-activated ce ll sorter. Selection of B-cell line Namalwa cells expressing transfect ed cDNAs in the presence of anti-G(D3) monoclonal antibody KM641 gave a cDNA (pAMo-GD3) encoding a protein with a type II transmembrane topo logy as found for mammalian glycosyltransferases. The following eviden ce confirms that the cDNA encodes an alpha-2,8-sialyltransferase, whic h specifically converts G(D3) to G(D3). (i) Transfection of pAMo-GD3 i nto Namalwa KJM-1 cells leads to the appearance of G(D3) and a G(D3) s ynthase activity. (ii) Northern blot analysis revealed a correlation b etween the expression of this gene and G(D3) in several cell lines. (i ii) The putative COOH-terminal active domain of this cloned enzyme fus ed with protein A has been purified with IgG-Sepharose beads and has b een shown to possess G(D3)-synthesizing activity, excluding the possib ility that the cloned cDNA encodes a transacting factor inducing a G(D 3) synthase. The deduced primary sequence also contains the ''sialyl m otif'' conserved among all the sialyltransferases cloned to date. The polymerase chain reaction analysis reveals that this gene is located o n chromosome 12.