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Expression cloning of a GM3-specific alpha-2,8-sialyltransferase (GD3 synthase).

1994; Elsevier BV; Volume: 269; Issue: 22 Linguagem: Inglês

10.1016/s0021-9258(17)40773-3

1083-351X

Keisuke Sasaki, Keigo Kurata, Nobuhiko Kojima, Nobuyuki Kurosawa, Shigenori Ohta, Nobuo Hanai, Shingo Tsuji, Tatsunari Nishi,

Carbohydrate Chemistry and Synthesis

A cDNA encoding a G,-specific a-2,B-sialyltransferase (G,, synthase) was obtained from an expression cDNA library of human melanoma cell line WM266-4 by enrichment of Namalwa K J " 1 cells highly expressing G,, using an anti-G,, antibody and a fluorescence-activated cell sorter.Selection of B-cell line Namalwa cells expressing transfected cDNh in the presence of anti-G,, monoclonal antibody K M 6 4 1 gave a cDNA (pAMo-GD3) encoding a protein with a type I1 transmembrane topology as found for mammalian glycosyltransferases.The following evidence confirms that the cDNA encodes an a-2,8-sialyltransferase, which specifically converts G,, to GD3. (i) Transfection of pAMo-GD3 into Namalwa K J " 1 cells leads to the appearance of G,, and a G,, synthase activity.(ii) Northern blot analysis revealed a correlation between the expression of this gene and G,, 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,,-synthesizing activity, excluding the possibility that the cloned cDNAencodes a transacting factor inducing aG,, 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.Gangliosides comprise a structurally diverse subset of sialylated glycosphingolipids that are expressed predominantly on the outer leaflet of animal cell surface membranes (1, 2).The biological roles of gangliosides have become increasingly appreciated, particularly in regard to intercellular adhesion (3-71, immune modulation (8, 91, growth control, and receptor function (10-16).Among gangliosides, NeuAca2,8NeuAca2,-3Galpl,4Glc-Cer (G,,)' has attracted attention in various