No abstract

Two membrane subfractions, one enriched in GM3 ganglioside and the other containing caveolin, were separated from low density detergent-insoluble membrane fraction prepared by sucrose density gradient centrifugation of postnuclear fraction of mouse melanoma B16 cells. The GM3-enriched subfraction, separated by anti-GM3 monoclonal antibody DH2, contained sphingomyelin, cholesterol, c-Src, and Rho A but not caveolin. In contrast, the caveolin-containing subfraction, separated by anti-caveolin antibody, contained neither GM3, c-Src, nor Rho A but did contain glucosylceramide, Ras, a very small quantity of sphingomyelin, and a very large quantity of cholesterol. The GM3/c-Src-enriched membrane subfraction was characterized by (i) maintenance of GM3-dependent adhesion and (ii) susceptibility to being activated for signal transduction through GM3.32 P-Phosphorylation of c-Src (M r 60,000) together with two other components (M r 45,000 and 29,000) was enhanced in the fraction bound to dishes coated with asialo-GM2 (Gg3) or with anti-GM3 monoclonal antibody DH2, detected by incubation with [␥-32 P]ATP at 37°C for 5 min. GM3-dependent adhesion of B16 cells to Gg3-coated dishes and associated signaling were not reduced or abolished in the presence of either filipin or nystatin, which are cholesterol-binding reagents known to abolish caveolae structure and function. B16 melanoma cells incubated with filipin (0.16 -0.3 g/ml) or with nystatin (25 g/ml) for 30 min showed depletion of cholesterol in detergentinsoluble membrane fraction but were still capable of binding to Gg3-coated plate and capable of the associated signaling. Thus, the GM3-enriched subfraction, involved in cell adhesion and capable of sending signals through GM3, represents a membrane domain distinguishable from caveolin-containing subfraction or caveolae. This microdomain is hereby termed the "glycosphingolipid signaling domain" or "glycosignaling domain".

We studied chemical level and glycosylation status of haptoglobin in sera of patients with prostate cancer, as compared to benign prostate disease and normal subjects, with the following results. (i) Haptoglobin level was enhanced significantly in sera of prostate cancer. (ii) Sialylated bi-antennary glycans were the dominant structures in haptoglobins from all 3 sources, regardless of different site of N-linked glycan. The N-linked glycans at N184 were exclusively bi-antennary, and showed no difference between prostate cancer vs. benign prostate disease. (iii) Tri-antennary, N-linked, fucosylated glycans, carrying at least 1 sialyl-Lewis x/a antenna, were predominantly located on N207 or N211 within the amino acid 203-215 sequence of the b-chain of prostate cancer, and were minimal in benign prostate disease. Fucosylated glycans were not observed in normal subjects. A minor tri-antennary N-linked glycan was observed at N241 of the b-chain in prostate cancer, which was absent in benign prostate disease. (iv) None of these N-linked structures showed the expected presence of disialylated antennae with GalNAcb4(NeuAca3)Galb3(NeuAca6)GlcNAcbGal, or its analogue, despite cross-reactivity of prostate cancer haptoglobin with monoclonal antibody RM2. (v) Minor levels of O-glycosylation were identified in prostate cancer haptoglobin for the first time. Monoand disialyl core Type 1 O-linked structures were identified after reductive b-elimination followed by methylation and mass spectrometric analysis. No evidence was found for the presence of specific RM2 or other tumor-associated glycosyl epitopes linked to this Oglycan core. In summary, levels of haptoglobin are enhanced in sera of prostate cancer patients, and the N-glycans attached to a defined peptide region of its b-chain are characterized by enhanced branching as well as antenna fucosylation. ' 2007 Wiley-Liss, Inc.

In the study of apoptosis initiated by various signals including ligands binding to cell membrane receptors such as Fas and TNFRI, the sphingomyelin pathway and its resulting metabolites, the sphingolipids, have been suggested to be involved in the signaling pathway. In earlier studies we presented data which indicated that sphingosine (Sph) itself was increased during apoptosis induced by phorbol myristate acetate (PMA) in HL60 cells and tumor necrosis factor (TNF) in neutrophils, and when added exogenously was able to induce apoptosis. We report here that Sph and its methylated derivative N,N,-dimethylsphingosine (DMS) are able to induce apoptosis in cancer cells of both hematopoietic and carcinoma origin. In human leukemic cell lines CMK-7, HL60 and U937, treatment with 20 microM Sph for 6 hr caused apoptosis in up to 90% of cells. Human colonic carcinoma cells HT29, HRT18, MKN74 and COLO205 were shown to be more susceptible to apoptosis upon addition of DMS (>50%) than of Sph (<50%), yet were weakly or not sensitive to N,N,N-trimethylsphingosine (TMS). Under the same conditions, in the presence of serum, neither Sph-1-phosphate nor ceramide analogues C2-, C6- or C8-ceramide were able to induce apoptosis in any cell lines. However, in the absence of serum, ceramide analogues induced apoptosis in leukemia cell lines after 18 hr, yet much less so than Sph or DMS. Furthermore, apoptosis induced by Sph or DMS could not be inhibited by the ceramide synthase inhibitor fumonisin B1. Apoptosis was not induced by sphingolipids in primary culture cells, such as HUVEC or rat mesangial cells, but was apparent in transformed rat mesangial cells. Additionally, apoptosis induced by Sph, DMS or C2Cer was inhibited by protease inhibitors. Our data further support the evidence that the catabolic pathway of sphingomyelin involving Sph and other metabolites is an integral part of the apoptosis pathway.

The extent and the specificity of the initial cell attachment induced by various proteins coated on plastic surfaces have been studied with the following results: (a) Cell adhesion on the surfaces coated with sialidase and ,6-galactosidase was as strong as on concanavalin A and Limulus lectin-coated surfaces and the reactions were strongly inhibited by glycosidase inhibitors or by competitive substrates . The adhesion on sialidase was inhibited by 2-deoxy-2,3-dehydro-N-acetylneuram in ic acid and by polysialoganglioside (GT1b) at low concentration (0 .05-0.1 mM) . The cell adhesion on a-gal actosidase coat was inhibited by 1,4-D-galactonolactone and a-methylgalactoside but not by a-methylgalactoside . Thus, the initiation of cell adhesion on glycosidase surfaces could be mediated through the interactions of the specific binding sites of the enzyme surface with the cell surface substrates under physiological conditions . (b) Cell adhesion on various lectins could be blocked by various competing monosaccharides at the concentrations similar to the inhibitory concentrations for binding of lectins from solution to the cells. (c) Cell adhesion on fibronectin surfaces as well as on gelatincoated surfaces was equally inhibited by GT 1b at relatively high concentrations (0 .25-0.5 mM) . Lower concentrations of GT 1b (0 .05-0.1 mM) inhibited the cell adhesion on surfaces of Limulus lectin and sialidase . It is suggested that the cell adhesion mediated by fibronectin is based on yet unknown interactions in contrast to a specific cell adhesion through glycosidases and lectins.The complex carbohydrates at the cell surface have been implicated to play an essential role in determining the specificity and the reactivity of cell to cell or cell to substratum (e .g., basement membrane) interaction in multicellular system and in tissue . A remarkable change of the carbohydrate structure at the cell surface, associated with oncogenic transformation (24, 65) and differentiation (16,25,47), and the presence of lectins at the animal cell membranes (reviewed in references 1 and 59) have supported this concept. However, the biochemical mechanism of cell-cell interaction and adhesion is far from being clear, and the topic has received much discussion in current studies, particularly in relation to the function of fibronectin, which promotes cell adhesion and spreading (23, 27, 68; reviewed in references 11, 22, and 67 Cell adhesion has been studied on lectins coated on nylon and plastic surfaces (22,29,56), on fibronectin and gelatin coated on plastic plates (11,13,22,23,27,67,68), and on galactose-gel particles (66). These assay systems are sensitive and can be used as a good model to study the mechanism of cell-to-cell or cell-to-substratum adhesion . This paper describes the intensity and the specificity of cell adhesion on two classes of carbohydrate-binding proteins (lectins and glycosidases) as compared with the adhesion on fibronectin-coated surfaces .

No abstract

N‐glycosylation status of purified β‐haptoglobin from sera of 17 patients, and from sera of 14 healthy volunteer subjects, was compared by blotting with various lectins and antibodies. Patients in this study were diagnosed as having colon cancer through histological examination of each tumor tissue by biopsy. Blotting index of serum β‐haptoglobin with Aleuria aurantia lectin (AAL) was clearly higher for cancer patients than for healthy subjects. No such distinction was observed for blotting with three other lectins and two monoclonal antibodies. To determine tumor‐associated reactivity of AAL binding as compared to inflammatory processes in colonic tissues, β‐haptoglobin separated from sera of 5 patients with Crohn's disease (CD), and 4 patients with ulcerative colitis (UC), was studied. All these cases, except one case of UC, showed AAL index lower than that in cancer cases, similarly to healthy subjects. The higher AAL binding of β‐haptoglobin in colon cancer patients than in healthy subjects appeared to be due to α‐L‐fucosyl residue, since it was eliminated by bovine kidney α‐fucosidase treatment. N‐linked glycans of serum haptoglobin from colon cancer patients vs. healthy subjects were released by N‐glycanase, fluorescence‐labeled, and subjected to normal‐phase high performance liquid chromatography (NP‐HPLC). Glycan structures were determined based on glucose unit (GU) values and their changes upon sequential treatment with various exoglycosidases. Glycosyl sequences and their branching status of glycans from 14 cases of serum β‐haptoglobin were characterized. The identified glycans were sialylated or nonsialylated, bi‐antennary or tri‐antennary structures, with or without terminal fucosylation.

G2 was identified by1 H NMR and mass spectrometry as having a structure similar to Structure I but without the GalNAc␤134 substitution and showed strong reactivity with mAb FH9 reported previously to be specific for disialosyl lacto-series type 1 (disialosyl Lc 4 ) having vicinal ␣233 and ␣236 sialosyl residues, an antigen associated with human colonic cancer. Clinicopathological studies indicate that expression of these disialoganglioside antigens in RCC tissue is correlated with the metastatic potential of RCC.