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Volume 272, Number 40, Issue of October 3, 1997 pp. 24794-24799
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Expression Cloning of Rat cDNA Encoding UDP-galactose:G_D2 1,3-galactosyltransferase That Determines the Expression of G_D1b/G_M1/G_A1

(Received for publication, May 8, 1997, and in revised form, July 8, 1997)

Hiroshi Miyazaki ^§ , Satoshi Fukumoto , Masahiko Okada ^§ , Tomokazu Hasegawa , Keiko Furukawa ^** and Koichi Furukawa

From the  Department of Biochemistry II, Nagoya University School of Medicine, 65 Tsuramai, Nagoya 466, the ^§ Department of Oncology, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki 852, the  Department of Pediatric Dentistry, Nagasaki University School of Dentistry, 1-7-1 Sakamoto, Nagasaki 852, and the ^** Department of Biochemistry, Mie University School of Medicine, Edohashi, Tsu, Mie 514, Japan

Using an anti-G_D1b monoclonal antibody, expression cloning of a cDNA for the 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 were identical, as previously suggested based on enzymological analysis. In Northern blots of the 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.

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