β1-4N-acetylgalactosaminyltransferase can synthesize both asialoglycosphingolipid GM2 and glycosphingolipid GM2in 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 beta 1-4N-acetylgalactosaminyltransferase (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-GM2 [Taki, T., Hirabayashi, Y., Ishiwata, Y., Matsumoto, M., and Kojima, K. (1979) Biochim. Biophys. Acta 572, 113-120]. The cDNA, pGNA56, was isolated by screening AH7974F cDNA library in lambda gt10 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 GM2 and GD2 from GM3 and GD3 respectively, but also asialo-GM2 from CDH. It also acted on GSL substrates, including GM1b, sialylparagloboside and GD1 alpha. 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-GM2 and GM2 on the plasma membrane. Therefore, we concluded that the formation of asialo-GM2, GM2 and GD2 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 alpha-pathways such as asialo-GM1 and GD1 alpha 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-GM2, GM2 and GD2 in vivo.

Purification and characterization of a soluble recombinant human ST6Gal I functionally expressed in Escherichia coli

“…Previous studies demonstrated that PA-sugar chains are good substrates for glycosyltransferases [22,30,31]. In Figure 4, under our HPLC conditions, PA-001 was commonly eluted around at 7 min post injection.…”

“…(I) HPLC-based assay: The assay for PAoligosaccharide as an acceptor was carried out as described previously [30,31]. Sialylated products were resolved on an HPLC system equipped with an ion-exchange column, PALPAK Type N (6.4 mm × 25 cm) (Takara Bio, Inc.).…”

β1,4-N-Acetylgalactosaminyltransferase (GM2 Synthase) Is Released from Golgi Membranes as a Neuraminidase-sensitive, Disulfide-bonded Dimer by a Cathepsin D-like Protease

“…Thus, cathepsin D or another unknown cathepsin D-like protease appeared to mediate the release of GalNAcT/myc from transfected CHO cells. Interestingly, the apparent cathepsin D cleavage site that we have identified in human GalNAcT, LL2YASTR (Table II), is also present in the rat homolog (18) and only slightly modified (LL2YSSTR) in the murine homolog. 4 The soluble form of GalNAcT released into the culture medium appeared as an intermolecular disulfide-bonded species of a size consistent with that of a homodimer (Fig.…”

Cloning and Transcriptional Regulation of Genes Responsible for Synthesis of Gangliosides

“…The GalNac T gene is highly expressed in tumor cell lines such as neuroblastoma and melanoma cell lines [83,84], and in mouse brain at the late stage of development [18,59]. The 5′-flanking fragment of the human GalNac T gene was the first cloned promoter for a ganglioside synthase gene [85].…”

“…The cDNA for GalNAc T has been cloned from human [58], rat [59], and mouse [60,61]. The gene consists of 11 exons.…”