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Abstract
Hepatocyte growth factor (HGF) is a heparin-binding pleiotropic factor that acts on a variety of epithelial cells. The interaction of human HGF with glycolipids was studied by overlaying them with 125I-HGF on thin layer chromatograms and by a solid-phase assay using lipids adsorbed on microtiter plates. Among various glycolipids tested, HGF was found to bind to sulfoglycolipids, including galactosylceramide sulfate (SM4), lactosylceramide sulfate (SM3), and gangliotriaosylceramide bis-sulfate. In contrast, HGF failed to bind to gangliosides or neutral glycolipids. HGF binding to SM4 was strongly inhibited by dextran sulfate, heparin, and fucoidan, whereas neither keratan sulfate nor hyaluronic acid had any inhibitory activity. When glycolipids from a renal cancer cell line, SMKT-R3, which overexpresses sulfoglycolipids, were developed on a thin layer chromatogram, SM4 and SM3 were the only glycolipids that bound HGF. We further examined the effect of the incorporation of glycolipids into SMKT-R3 cells on HGF binding to the cells. The incorporation of SM4 into the cells enhanced HGF binding to SMKT-R3 cells, while that of galactosylceramide, a precursor of SM4, had no effect. These observations indicated that SM4 exogenously incorporated into the cell membranes could react with HGF and suggested that endogenous sulfoglycolipids on SMKT-R3 cells might function as reservoirs for HGF.
Highlights
Hepatocyte growth factor(HGF)is a heparin-binding cifically to several proteins, including laminin
Binding of HGF to Sulfoglycolipids-To examine the ability of glycolipids on renal cancer cells to serve as receptors for HGF, binding to glycolipids was investigated in two ways [19]. 6
HGF bound to SM4 and SM3 extracted from a renal cancer cell line, SMKTR3, which expresses SM4, SM3, and SM2 [3],but not to other acidic or neutral glycolipids from the cells (Fig. 1B )
Summary
R3, which overexpresses sulfoglycolipids, were devel- Materials-NalzsI (17 Ci’mg) waspurchased from New England oped on athin layer chromatogram,SM4 and SM3 were Nuclear. Human recombinant HGF was purifiedfrom culture medium the only glycolipids that boundHGF.We further examined the effect of the incorporation of glycolipids into SMKT-R3cells on HGF binding to the cells. SMKT-R3 cells, while that of galactosylceramide, a pre- coidan, dextran sulfate, keratan sulfate, and hyaluronic acid were obcursor ofSM4, had no effect. In human renal cell carcinoma tissue, sulfoglycolipid conalkaline hydrolysis t.o destroy ester lipids and fractionated into neutral andacidic lipid fractions by DEAE-Sephadex A-25 column chromatography. Assay of ‘Z,51-HGFBinding to Glycolipids-Binding of lZsII-HGF to glycolipids on HPTLC plates wasperformed as previously described for. Solid-phase radioassayosf HGF binding to glycolipids adsorbed on 96-well microtiter plates (Falcon 3912).
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