Yutaka Sanai

Association of Src Family Tyrosine Kinase Lyn with Ganglioside GD3 in Rat Brain POSSIBLE REGULATION OF Lyn BY GLYCOSPHINGOLIPID IN CAVEOLAE-LIKE DOMAINS

Association of gangliosides with specific proteins in the central nervous system was examined by co-immunoprecipitation with anti-ganglioside antibody. Protein kinase activity was detected in precipitates with monoclonal antibody to ganglioside GD3 (R24) from membranal fraction of rat brain. Using in vitro kinase assay, several phosphorylated proteins of 40, 53, 56, and 80 kDa were isolated by gel electrophoresis. Of these proteins, the proteins of 53 and 56 kDa (p53/56) were identified as two isoforms of Src family tyrosine kinase Lyn, based on co-migration during gel electrophoresis, comparative peptide mapping, and sequential immunoprecipitation with anti-Lyn antibody. The identification was confirmed using a cDNA expression system in Chinese hamster ovary (CHO) cells, which express solely ganglioside GM3, the enzymatic substrate of GD3synthase. In co-transfection with GD3 synthase and Lyn expression plasmids, R24 immunoprecipitated Lyn and anti-Lyn antibody immunoprecipitated GD3. R24 treatment of rat primary cerebellar cultures induced Lyn activation and rapid tyrosine phosphorylation of several substrates including mitogen-activated protein kinases. Furthermore, sucrose density gradient analysis showed that Lyn of cerebellum and CHO transfectants were detected in a low density light-scattering band, i.e. the caveolae membrane fraction. R24 immunoprecipitated caveolin from Triton X-100 extract of CHO transfectants. These observations suggest that GD3 may regulate Lyn in a caveolae-like domain on brain cell membranes.

Involvement of Gangliosides in Glycosylphosphatidylinositol-anchored Neuronal Cell Adhesion Molecule TAG-1 Signaling in Lipid Rafts

The association of ganglioside GD3 with TAG-1, a glycosylphosphatidylinositol-anchored neuronal cell adhesion molecule, was examined by coimmunoprecipitation experiments. Previously, we have shown that the anti-ganglioside GD3 antibody (R24) immunoprecipitated the Src family kinase Lyn from the rat cerebellum, and R24 treatment of primary cerebellar cultures induced Lyn activation and rapid tyrosine phosphorylation of an 80-kDa protein (p80). We now report that R24 coimmunoprecipitates a 135-kDa protein (p135) from primary cerebellar cultures. Treatment with phosphatidylinositol-specific phospholipase C revealed that p135 was glycosylphosphatidylinositol-anchored to the membrane. It was identified as TAG-1 by sequential immunoprecipitation with an anti-TAG-1 antibody. Antibody-mediated cross-linking of TAG-1 induced Lyn activation and rapid tyrosine phosphorylation of p80. Selective inhibitor for Src family kinases reduced the tyrosine phosphorylation of p80. Sucrose density gradient analysis revealed that the TAG-1 and tyrosine-phosphorylated p80 in cerebellar cultures were present in the lipid raft fraction. These data show that TAG-1 transduces signals via Lyn to p80 in the lipid rafts of the cerebellum. Furthermore, degradation of cell-surface glycosphingolipids by endoglycoceramidase induced an alteration of TAG-1 distribution on an OptiPrep gradient and reduced the TAG-1-mediated Lyn activation and tyrosine phosphorylation of p80. These observations suggest that glycosphingolipids are involved in TAG-1-mediated signaling in lipid rafts.

Cloning and expression of a novel galactoside beta1, 3-glucuronyltransferase involved in the biosynthesis of HNK-1 epitope.

We isolated a cDNA encoding a novel glucuronyltransferase, designated GlcAT-D, involved in the biosynthesis of the HNK-1 carbohydrate epitope from rat embryo cDNA by the degenerate polymerase chain reaction method. The new cDNA sequence revealed an open reading frame coding for a protein of 324 amino acids with type II transmembrane protein topology. The amino acid sequence of GlcAT-D displayed 50.0% identity to rat GlcAT-P, which is involved in the biosynthesis of the HNK-1 epitope on glycoproteins. Expression of GlcAT-D in COS-7 cells resulted in the formation of the HNK-1 epitope on the cell surface. The enzyme expressed in COS-7 cells transferred a glucuronic acid (GlcA) not only to asialo-orosomucoid, a glycoprotein bearing terminal N-acetyllactosamine structure, but also to paragloboside (lacto-N-neotetraosylceramide), a precursor of the HNK-1 epitope on glycolipids. Furthermore, substrate specificity analysis using a soluble chimeric form of GlcAT-D revealed that GlcAT-D transfers a GlcA not only to Galβ1–4GlcNAcβ1–3Galβ1–4Glc-pyridylamine but also to Galβ1–3GlcNAcβ1–3Galβ1–4Glc-pyridylamine. Enzymatic hydrolysis and Smith degradation of the reaction product indicated that GlcAT-D transfers a GlcA through a β1,3-linkage to a terminal galactose. The GlcAT-D transcripts were detected in embryonic, postnatal, and adult rat brain. In situ hybridization analysis revealed that the expression pattern of GlcAT-D transcript in embryo is similar to that of GlcAT-P, but distinct expression of GlcAT-D was observed in the embryonic pallidum and retina. Regions that expressed GlcAT-D and/or GlcAT-P were always HNK-1-positive, indicating that both GlcATs are involved in the synthesis of the HNK-1 epitope in vivo.

Involvement of the acyl chain of ceramide in carbohydrate recognition by an anti-glycolipid monoclonal antibody: the case of an anti-melanoma antibody, M2590, to GM3-ganglioside

The effect of the chain length of the fatty acid residue of the ceramide moiety of ganglioside GM3 on the binding ability of monoclonal antibody M2590, which is specific for the carbohydrate structure of GM3-ganglioside, was examined by means of a direct binding assay on thin layer chromatography plates (TLC immunostaining) and a quantitative enzyme-linked immunosorbent assay (ELISA). Derivatives of GM3 with a long fatty acid chain reacted with the M2590 antibody, but those with a short fatty acid chain showed no reaction in either assay system. These results suggested that the acyl fatty acid moiety of the ganglioside played an important role in the formation or maintenance of the antigenic structure of the carbohydrate moiety of the ganglioside.

Gene transfer as a novel approach to the gene-controlled mechanism of the cellular expression of glycosphingolipids

Gene transfer is a useful technique to analyse the gene-controlled mechanism of cellular expression of glycosphingolipids. Using various oncogenes (DNA tumor virus oncogenes: adenovirus 12 E1 and its transcriptional subunits, myc and SV40 T antigens; RNA tumor retrovirus oncogens: fos, src and H-ras) for the transfer, it was demonstrated that these genes invariably caused remarkable and characteristic changes of the cellular expression of gangliosides in the same recipient rat embryonic cell line, 3Y1. As a consequence of the transfer, various ganglioside species appeared or disappeared, the extent of the changes varying according to the type of oncogenes used. Analysis of the enzymatic background of such changes was also carried out. Based on these results, the effects of oncogene transfer on glycosphingolipids were classified and discussed according to the mode of subcellular distribution of gene products.

Monoclonal antibody directed to a Hanganutziu-Deicher active ganglioside, GM2 (NeuGc)

Spleen cells from NZB mouse immunized with a membrane fraction of rabbit thymus tissue were fused with BALB/c 6-thioguanine-resistant myeloma cells, P3-X63-Ag8.653. One hybridoma clone (Y-2-HD-1) produced IgM immunoglobulin that bound to an N-glycolylneuraminic acid-containing GM2 ganglioside, GM2(NeuGc), which is known to be a Hanganutziu-Deicher antigen. The specificity of the Y-2-HD-1 monoclonal antibody was examined, using authentic glycosphingolipids structurally related to GM2(NeuGc), by means of an enzyme-linked immunosorbent assay and thin-layer chromatography/enzyme immunostaining, respectively. The monoclonal antibody was found to be highly specific to GM2(NeuGc) and the epitope was a non-reducing terminal GalNAc beta 1-4[NeuGc alpha 2-3]Gal structure. This monoclonal antibody (Y-2-HD-1) bound to native mouse erythrocytes, in which GM2(NeuGc) is a major ganglioside. These results indicate that GM2(NeuGc) is located on the surface of mouse erythrocytes.

Radioimmuno-thin-layer chromatographic detection of Forssman antigen in human carcinoma cell lines

Abstract Glycolipid antigen was examined by radioimmuno-thin-layer chromatography (RITLC), which is a combination of a thin-layer chromatography and radioimmunoassay. In this way Forssman antigen was studied in seven carcinoma cell lines. The usual Forssman antigen with a ceramide pentasaccharide structure was detected in cell lines of a gastric cancer and a breast cancer. In addition another glycolipid with slower mobility on thin-layer chromatography and with Forssman reactivity was found in cell lines of three gastric cancers and one lung cancer.

Analysis of cellular expression of gangliosides by gene transfection. I: GD3 expression in myc-transfected and transformed 3Y1 correlates with anchorage-independent growth activity

Transfection of c-myc DNA into rat fibroblastic 3Y1 cell line resulted in the neosynthesis of GD3 ganglioside, as has been previously shown to occur after transfection of 3Y1 cells with adeno El (Nakakuma, H., Sanai, Y., Shiroki, K., and Nagai, Y. (1984) J. Biochem. 96, 1471-1480); in both cases, the products are expressed intranuclearly. Moreover, a clear correlation was identified between levels of GD3 expression and colony-forming activity (in soft agar) of myc-transformed 3Y1 cells, implying that GD3 plays some specific role in myc-induced transformation of 3Y1 cell line.

Morphological and functional development of plasma membranes of mouse hepatocytes: An autoradiographic study of asialoglycoprotein uptake during the postnatal period

The morphological development of the postnatal mouse liver was studied with reference to the specialization of sinusoidal plasma membranes of hepatocytes. Numerous hemopoietic islands were distributed at random among hepatocytes at neonatal stages. The hemopoietic fronts of hepatocytes were flat and showed few structures indicating endocytotic and exocytotic activities. They gradually acquired structural characteristics of the sinusoidal region as the processes of the endothelial cells extended in the space between hemopoietic cells and hepatocytes. The degree of attenuation and fenestration of the sinusoidal endothelial cells increased as the postnatal day advanced. In the course of these changes, microvilli, pinocytotic pits, and vesicles of sinusoidal regions of hepatocytes increased as well. The functional development of sinusoidal regions of hepatocytes was investigated by the quantitative analysis of autoradiograms of [3H]asialoorosomucoid (ASOR). Silver grains were distributed only in the regions where the structural specializations of the sinusoidal region were observed. They increased as the maturation of sinusoidal structure advanced. These results suggest that the sinusoidal regions of hepatocytes specialize structurally and functionally, as regards ASOR-uptake capability, in correlation with the structural establishment of sinusoids.

Gangliosides and Related Compounds as Biological Response Modifiers

Several possibilities are discussed with regard to the biological potency of gangliosides and related compounds as biological response modifier. Evidence is presented that a ganglioside, GQ1b, but not other gangliosides examined exhibits a strong biological activity at a few nanomolar concentration for the promotion of neurite outgrowth as well as cell division in two cell lines of neuroblastoma.