β1-4N-acetylgalactosaminyltransferase can synthesize both asialo-glycosphingolipid G(M2) and glycosphingolipid G(M2) in vitro-and in vivo: Isolation and characterization of a β1-4N-acetylgalactosaminyltransferase cDNA clone from rat ascites hepatoma cell line AH7974F

Abstract

We have cloned a cDNA encoding β1-4N-acetylgalactosaminyl-transferase (EC 2.4.1.92) (GalNAc-T) from rat ascites hepatoma of the free-cell type AH7974F. The cell line only expressed asialo-series glycosphingolipids (GSLs) including asialo-G(M2). The cDNA, pGNA56, was isolated by screening AH7974F cDNA library in λgt10 with a probe. The probe was obtained from AH7974F cDNA by PCR using primers with the nucleotide sequence of the human GalNAc-T cDNA. The amino acid sequence deduced from the nucleotide sequence of pGNA56 exhibited 88% similarity to the human GalNAc-T sequence. The enzyme was a typical type II membrane protein, which consisted of a short N-terminal residue, a transmembrane region, and a long C-terminal residue, including the catalytic domain. The substrate specificity of rat GalNAc-T was determined using homogenates from cells into which the cDNA clone was transfected. The enzyme catalysed not only the formation of G(M2) and G(D2) from G(M3) and G(D3) respectively, but also asialo-G(M2) from CDH. It also acted on GSL substrates, including G(M1b), sialylparagloboside and G(D1α). On the other hand, the enzyme did not transfer GalNAc to soluble substrates such as glycoproteins and oligosaccharide. The GSL compositional and immunocytochemical analyses of stable transfectants obtained by transfection of the cDNA showed simultaneous expression of asialo-G(M2) and G(M2) on the plasma membrane. Therefore, we concluded that the formation of asialo-G(M2), G(M2) and G(D2) was catalysed by the single GalNAc-T. Northern-blot hybridization showed that the GalNAc-T mRNA was strongly expressed in rat brain, testis, and spleen. The gene was also expressed in rat normal liver to a lesser extent. We found the GSLs in asialo- and α-pathways such as asialo-G(M1) and GD(D1α) in the rat tissues by using a sensitive t.l.c.-immunostaining method. These observations also supported our conclusion that the single GalNAc-T synthesizes asialo-G(M2), G(M2) and G(D2) in vivo.