Three glucosamine-containing sphingoglycolipids were isolated from human lung tissue of a blood group-A subject. They were hexaglycosyl, pentaglycosyl, and tetraglycosyl ceramides. The hexaglycosyl ceramide exhibited blood group-A antigenicity, and the following chemical structure was proposed on the basis of sequential glycosidase treatment and methylation analysis. (Formula: see text) The pentaglycosyl ceramide for human lung was a blood group H(O)-active glycolipid having a carbohydrate composition of Fuc : GlcNAc : Gal : Glc (1 : 1 : 2 : 1). The tetraglycosyl ceramide is probably paragloboside (lactoneotetraosyl ceramide).
Background. It is important to develop new immunosuppressive agents without clinical drawbacks. In this article, we reveal the possibility of a chemically synthetic sulfonolipid that acts as a novel immunosuppressive drug. Methods. We evaluated the immunosuppressive effect of 3-O-(6-deoxy-6-sulfono-β-D-glucopyranosyl)-1,2-di-O-acylglycerol (β-SQDG) that contains a saturated C18 fatty acid, which is designated as β-SQDG(18:0) by mixed lymphocyte reaction (MLR) and rat allogeneic skin graft. Then, we investigated the mechanism of immunosuppressive effect of β-SQDG(18:0). Results. β-SQDG(18:0) inhibited human MLR in a dose-dependent manner without overt cytotoxic effect and prolonged rat skin allograft rejection in vivo. β-SQDG(18:0) did not inhibit the direct activation of responder T. This reagent could not affect the expression of either major histocompatibility antigen complex (MHC) class I or class II molecules on the cell surface of the stimulator cells, antigen-presenting cells. In contrast, β-SQDG(18:0) was demonstrated to inhibit the binding among allogeneic lymphocytes. However, the expression of known cell surface accessory and adhesion molecules, such as CD4, CD28, leukocyte function-associated antigen 1, intercellular adhesion molecule 1, and CTLA-4, was not affected by β-SQDG(18:0) treatment. Conclusions. β-SQDG(18:0) might be a new class of the immunosuppressive reagent, and the inhibition of responder T-lymphocyte activation in MLR by β-SQDG(18:0) may be responsible for certain three-dimensional structures of this reagent or its quinovose binding to sulfonic acid.
Arylsulfatase A was purified from human lung to apparent homogeneity as determined by electrophoresis in the presence of sodium dodecyl sulfate. The enzyme from normal lung as well as that from lung adenocarcinoma showed considerable microheterogeneity when examined by isoelectric focussing, with an isoelectric point (pI) ranging from 5.1 to 4.6. The tumor enzyme was more heterogeneous and contained more acidic components than the normal lung enzyme. The cause of the charge heterogeneity was examined by treatment with exogenous hydrolases. Upon treatment with sialidase, phosphatase or endo-β- N -acetylglucosaminidase H (endoglycosidase H), the acidic enzyme forms shifted to an alkaline region on isoelectric focussing gels. Combined treatment of the arylsulfatase A with endoglycosidase H and sialidase resulted in complete loss of the most acidic components to give the less acidic components with pI 5.1, 5.0, and 4.9. These results strongly suggest that the charge heterogeneity of arylsulfatase A is due not only to sialylation but also to phosphorylation at the carbohydrate moiety of the enzyme, and the extent of substitution by acidic groups is markedly increased in the tumor enzyme.
Accumulation of sulfolipids associated with elevated levels of glycolipid sulfotransferase activities has previously been demonstrated in renal cell carcinoma cells. To investigate the role of protein kinase C in the synthesis of sulfolipids, the effects of 12-O-tetradecanoylphorbol-13-acetate and protein kinase C inhibitors on glycolipid sulfotransferase activity levels were examined in a human renal cell carcinoma cell line, SMKT-R3. Continuous treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate caused a dose- and time-dependent reduction of the sulfotransferase activity levels. Similarly, protein kinase C inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride and staurosporine, reduced the enzyme activities in a dose-dependent manner. These observations suggest that the glycolipid sulfotransferase activity levels are regulated by protein kinase C in SMKT-R3 cells. Furthermore, long-term 12-O-tetradecanoylphorbol-13-acetate treatment resulted in a reduction of sulfolipid synthesis and a decrease of the expression of sulfolipids on the cell surface. Taken together, it is suggested that protein kinase C is involved in the synthesis of sulfolipids through the regulation of the glycolipid sulfotransferase activity levels in renal cell carcinoma cells.
beta-Glucuronidase purified from human placenta and chronic myelogenous leukemic cells was composed of three components of 18, 64 and 80 kDa, though the relative contents of the components were different between the sources. Analysis of their N-terminal amino-acid sequences showed that the 18-kDa and 64-kDa components were derived from the 80-kDa component by cleavage between Val159 and Gly160. Furthermore, the enzyme was found to be glycosylated at Asn173 and Asn420 with high mannose-type oligosaccharides, based on the electrophoretic mobility of the components as well as the endopeptidic peptides before and after endoglycosidase treatment. The enzyme purified from leukemic cells was poorly phosphorylated by N-acetylglucosamine 1-phosphotransferase as compared to the placental enzyme.
Biosynthesis of sulfoglycolipid is markedly increased in a human renal cancer cell line, SMKT-R3. We investigated the sulfotransferase catalyzing the transfer of sulfate from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to gangliotriaosylceramide (Gg3Cer) in SMKT-R3 cells. On thin-layer chromatography, the reaction product comigrated with Gg3Cer III3-sulfate (SM2b), not with Gg3Cer II3-sulfate (SM2a). To examine which monosaccharide of Gg3Cer was sulfated, the product was treated with β-hexosaminidase A. Unlike authentic SM2a, of which the non-reducing terminal N-acetylgalactosamine was cleaved off, the product was not hydrolyzed. These results suggest that sulfate was transferred to the non-reducing terminal N-acetylgalactosamine. Gg3Cer sulfotransferase activity was independent of divalent cations but stimulated by Fe2+, and the optimal pH was approximately 6.5. The apparent Km values were 102 μM for PAPS and 348 μM for Gg3Cer. Gg3Cer II3, III3-bis-sulfate (SB2) and a sulfotransferase activity synthesizing SB2 from SM2a were also detected in SMKT-R3 cells
Gangliosides are glycosphingolipids found on the cell surface. They act as recognition molecules or signal modulators and regulate cell proliferation and differentiation. N-glycolylneuraminic acid (NeuGc)-containing gangliosides have been detected in some neoplasms in humans, although they are usually absent in normal human tissues. Our aim was to evaluate the presence of NeuGc-containing gangliosides including GM3 (NeuGc) and assess their relationship with the prognosis of non-small-cell lung cancer (NSCLC). NeuGc-containing ganglioside expression in NSCLC tissues was analyzed immunohistochemically using the mouse monoclonal antibody GMR8, which is specific for gangliosides with NeuGc alpha 2,3Gal-terminal structures. On the basis of NeuGc-containing ganglioside expression, we performed survival analysis. We also investigated the differences in the effects of GM3 (N-acetylneuraminic acid [NeuAc]) and GM3 (NeuGc) on inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase in A431 cells. As a result, the presence of NeuGc-containing gangliosides was evident in 86 of 93 (93.5%) NSCLC samples. The NSCLC patients with high NeuGc-containing ganglioside expression had a low overall survival rate and a significantly low progression-free survival rate. In the in vitro study, the inhibitory effect of GM3 on EGFR tyrosine kinase in A431 cells after exposure to GM3 (NeuGc) was lower than that after exposure to GM3 (NeuAc). In conclusion, NeuGc-containing gangliosides including GM3 (NeuGc) are widely expressed in NSCLC, and NeuGc-containing ganglioside expression is associated with patient survival. The difference in the effects of GM3 (NeuGc) and GM3 (NeuAc) on the inhibition of EGFR tyrosine kinase might contribute to improvement in the prognosis of NSCLC patients.
