Expression cloning of rat cDNA encoding UDP-galactose:G(D2) beta 1,3-galactosyltransferase that determines the expression of G(D1b)/G(M1)/G(A1)

Using an anti-G(D1b) monoclonal antibody, expression cloning of a cDNA for the beta 1,3-galactosyltransferase gene (EC 2.4.1.62) was performed, KF4C, mouse melanoma B16 transfected with polyoma T antigen gene, and G(M2)/G(D2) synthase cDNA was used as a recipient cell line for the cDNA library transfection. A cDNA clone of G(D3) synthase, pD3T-31 was co-transfected with a cDNA library prepared from rat brain RNA using the pcDNAI expression vector. The isolated cDNA clone pM1T-9 predicted a type II membrane protein with 4 amino acids of cytoplasmic domain, 21 amino acids of transmembrane region, and a large catalytic domain with 346 amino acids, Introduction of the cDNA clone into a mouse melanoma line B16 previously transfected with a G(M2)/G(D2) synthase gene resulted in the neo synthesis of G(M1). Co-transfection of the cell line with pM1T-9 and a G(D3) synthase cDNA resulted in the expression of G(D1b) as well as G(M1). Moreover, introduction of pM1T-9 into L cell (lacking G(M3) synthase), previously transfected with G(M2)/G(D2) synthase gene, resulted in the definite expression of asialo-G(M1). These results indicated that G(D1b)/G(M1)/G(A1) synthases mere identical, as previously suggested based on enzymological analysis. In Northern blots of the beta 1,3-galactosyltransferase gene with total RNA from various rat tissues, a 1.6-kilobase mRNA was strongly expressed in spleen, thymus, kidney, and testis, However, the expression level of the gene in the adult brain tissue was not especially high, On the other hand, this gene was expressed at high levels in the rat brain of embryonal day 12, and reached a peak at around birth, then fell to low level in the adult brain.