Ligand-of-Numb protein X is an endocytic scaffold for junctional adhesion molecule 4.

Junctional adhesion molecule 4 (JAM4) is a cell adhesion molecule that interacts with a tight junction protein, membrane-associated guanylate kinase inverted 1 (MAGI-1). Our previous studies suggest that JAM4 is implicated in the regulation of paracellular permeability and the signalings of hepatocyte growth factor. In this study, we performed yeast two-hybrid screening to search for an unidentified JAM4-binding protein and obtained one isoform of Ligand-of-Numb protein X1 (LNX1), LNXp70, that is an interactor of Numb. Ligand-of-Numb protein X1 is expressed in kidney glomeruli and intestinal epithelial cells, where JAM4 is also detected. Immunoprecipitation from kidney lysates supports the in vivo interaction of proteins. Biochemical studies reveal that JAM4 directly binds the second PDZ domain of LNX1 through its carboxyl terminus. Junctional adhesion molecule 4, LNX1 and Numb form a tripartite complex in vitro and are partially colocalized in heterologous cells. Ligand-of-Numb protein X1 facilitates endocytosis of JAM4 and is involved in transforming growth factor beta -induced redistribution of JAM4 in mammary epithelial cells. Experiments using dominant-negative constructs and RNA interference insure that Numb is necessary for the LNX1-mediated endocytosis of JAM4. All these findings indicate that LNX1 provides an endocytic scaffold for JAM4 that is implicated in the reorganization of cell junctions.

Human hepatoma gangliosides: occurrence of a novel I-type glycolipid with NeuAc alpha 2-6Gal structure.

Gangliosides with NeuAc alpha 2-6Gal structure have been studied in human hepatocellular carcinoma. The gangliosides were purified to homogeneity by a DEAE-Sephadex A-25 column chromatography and by repeated silica beads column chromatography. Three gangliosides containing NeuAc alpha 2-6Gal structure were isolated and were structurally characterized by using monoclonal antibodies, proton nuclear magnetic resonance, fast atom bombardment mass spectrometry, methylation analysis by gas chromatography-mass spectrometry, and exoglycosidase treatments. The first compound was identified as NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer. The structures of 2 other components were concluded to be as follows: [formula: see text] The first compound is a ganglioside that is characteristic of human meconium. The second compound has the same structure as a ganglioside recently found by us (Taki, T., Rokukawa, C., Kasama, T., Kon, K., Ando, S., Abe, T., and Handa, S., J. Biol. Chem., 267: 11811-11817, 1992) in meconium. The third compound is a novel type of ganglioside having blood group I-type structure as the core sequence. In addition to these gangliosides, 5 others were detected, and all except for GM3 were glycolipids with neolacto-series core structure. These results suggest that enzymes for the synthesis of neolacto type and NeuAc alpha 2-6Gal structure of glycolipids are activated in hepatoma.

Had-1, a uridine 5'-diphosphogalactose transport-defective mutant of mouse mammary tumor cell FM3A: composition of glycolipids, cell growth inhibition by lactosylceramide, and loss of tumorigenicity.

Glycolipid compositions of mouse mammary tumor cell FM3A and its Newcastle disease virus-resistant mutant cell, Had-1, which was also characterized as a defective mutant of UDP-galactose transport to Golgi apparatus, have been studied. The major neutral glycolipid in FM3A was Gal beta 1-4Glc beta 1-1Cer (LacCer) (95%) and the rest was Glc beta 1-1Cer. The concentration of neutral glycolipids in Had-1 was only about one-fifth of that in FM3A. GlcB1-1Cer in Had-1 accounted for 79% of neutral glycolipids and the rest was LacCer, the content of which was decreased to 4% of that in FM3A. Ganglioside patterns of the two cell lines were similar, although gangliosides with N-glycolylneuraminic acid were increased in Had-1 cells compared with that in FM3A cells. The presence of NeuAc alpha 2-3-Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer, NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-2Cer, GM3, and GD3 was demonstrated by thin-layer chromatography immunostaining. 125I-Labeled Newcastle disease virus bound only poorly to gangliosides extracted from either FM3A or Had-1 cells on a high performance thin-layer chromatography plate. The effects of glycolipids on the growth of the two cell lines were also studied. Had-1 cells were more sensitive to glycolipids added exogenously than FM3A cells. Addition of GM3 had a stimulative effect on cell growth of Had-1. LacCer, Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 4-1Cer, and Glc beta 1-1Cer inhibited the growth of Had-1 cells. LacCer was the most potent inhibitor. LacCer immobilized on the culture plate also inhibited the growth of Had-1 cells. The inhibitory effect was recovered completely overcome by transferring the cells to LacCer-free medium. Had-1 cells were not tumorigenic in C3H/He mice, and furthermore the tumorigenic activity of FM3A cells was suppressed by the prior administration of Had-1 cells.

Glycolipid composition of a mutant cell line of mouse FM3A cells, and the effect of exogenous glycolipids on cell growth.

Had-1 isolated from mouse mammary tumour FM3A cells as a non-permissive cell line to Newcastle disease virus infection is deficient in NDV receptors, and galactosylation of the complex type sugar chains of the glycoproteins is extensively reduced compared to FM3A cells. It is also deficient in UDP-galactose transport into Golgi vesicles. The major neutral glycolipids in FM3A is Lac-Cer, whereas, in Had-1 cell, Glc-Cer is the major glycolipid and the concentration of neutral glycolipids is one-tenth as low as that in FM3A. GM3, GD3 and sialyl i- and I-type lactosaminylceramide are the gangliosides present in both FM3A and Had-1, although their presence in both cells is only in traces. Had-1 contains relatively high N-glycolyl-neuraminic acid. Among the several glycolipids tested, Lac-Cer, Gg-4-Cer and Glc-Cer showed inhibitory effect on proliferation of Had-1 cells, but did not show any appreciable effect on that of FM3A cells. Lac-Cer had the most potent inhibitory effect and this inhibitory effect was completely reversible. While mice injected with 5 x 10(6) cells of FM3A died in one month, those injected of Had-1 cells at the same dose survived for more than 6 months. Thus glycolipids on the cell surface play an essential role during cell growth both in vivo and in vitro.

Direct analysis of glycolipids on thin-layer plates by matrix-assisted secondary ion mass spectrometry: application for glycolipid storage disorders.

The lipids accumulated in organs of patients with Gaucher's, Tay-Sachs, and Fabry's disease were identified by means of the combination of thin-layer chromatography and matrix-assisted secondary ion mass spectrometry. The total lipid extract of each lipidosis tissue was chromatographed on a TLC plate and then analyzed directly by mass spectrometry without elution of the sample from the TLC plate. The amount of material needed to obtain an adequate spectrum is in the order of a few micrograms of lipids per band for both positive and negative ion detection. By scanning the plates, mass spectral and chromatographic information can be obtained simultaneously, which was shown to be useful for the qualitative identification of the components on the plates.

Glycolipids of mouse erythroleukemia cells (Friend cells) and their alteration during differentiation.

Neutral and acidic glycosphingolipids of Friend cells were characterized in 1) undifferentiated Friend cells (745A), 2) differentiated Friend cells induced with dimethyl-sulfoxide, and 3) solid tumors grown in mice after subcutaneous implantation of Friend cells. The structures of the isolated glycosphingolipids were determined by means of compositional analysis, methylation analysis and enzyme treatment. Gangliosides GD1a and N-acetylgalactosaminyl-GD1a, followed by GM1a and GM2, were the main gangliosides in undifferentiated Friend cells. GD1a and N-acetylgalactosaminyl-GD1a accounted for 45 and 25% of the total gangliosides, respectively. On differentiation, ganglioside GM2 decreased significantly, from 10% to a trace amount. In solid tumors, GD1a was the major ganglioside, whereas in contrast to the situation in the cultured cells, N-acetylgalactosaminyl-GD1a was almost completely absent, and ganglioside GM1b, but not GM1a, was detected. In addition, ganglioside GD1 alpha was detected in the solid tumors. Galactosylceramide, glucosylceramide, and lactosylceramide were the main neutral components in both types of cells, while globotetraosylceramide (globoside), IV3-N-acetyl-galactosaminyl globotetraosylceramide (Forssman glycolipid) and gangliotetraosylceramide (GA1) were major in solid tumors grown in vivo.