Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Exogenous gangliosides affect the angiogenic activity of fibroblast growth factor-2 (FGF-2), but their mechanism of action has not been elucidated. Here, a possible direct interaction of sialo-glycolipids with FGF-2 has been investigated. Size exclusion chromatography demonstrates that native, but not heat-denatured, 125I-FGF-2 binds to micelles formed by gangliosides GT1b, GD1b, or GM1. Also, gangliosides protect native FGF-2 from trypsin digestion at micromolar concentrations, the order of relative potency being GT1b > GD1b > GM1 = GM2 = sulfatide > GM3 = galactosyl-ceramide, whereas asialo-GM1, neuraminic acid, and N-acetylneuramin-lactose were ineffective. Scatchard plot analysis of the binding data of fluorochrome-labeled GM1 to immobilized FGF-2 indicates that FGF-2/GM1 interaction occurs with a Kd equal to 6 microM. This interaction is inhibited by the sialic acid-binding peptide mastoparan and by the synthetic fragments FGF-2(112-129) and, to a lesser extent, FGF-2(130-155), whereas peptides FGF-2(10-33), FGF-2(39-59), FGF-2(86-96), and the basic peptide HIV-1 Tat(41-60) were ineffective. These data identify the COOH terminus of FGF-2 as a putative ganglioside-binding region. Exogenous gangliosides inhibit the binding of 125I-FGF-2 to high-affinity tyrosine-kinase FGF-receptors (FGFRs) of endothelial GM 7373 cells at micromolar concentrations. The order of relative potency was GT1b > GD1b > GM1 > sulfatide a = sialo-GM1. Accordingly, GT1b,GD1b, GM1, and GM2, but not GM3 and asialo-GM1, prevent the binding of 125I-FGF-2 to a soluble, recombinant form of extracellular FGFR-1. Conversely, the soluble receptor and free heparin inhibit the interaction of fluorochrome-labeled GM1 to immobilized FGF-2. In agreement with their FGFR antagonist activity, free gangliosides inhibit the mitogenic activity exerted by FGF-2 on endothelial cells in the same range of concentrations. Also in this case, GT1b was the most effective among the gangliosides tested while asialo-GM1, neuraminic acid, N-acetylneuramin-lactose, galactosyl-ceramide, and sulfatide were ineffective. In conclusion, the data demonstrate the capacity of exogenous gangliosides to interact with FGF-2. This interaction involves the COOH terminus of the FGF-2 molecule and depends on the structure of the oligosaccharide chain and on the presence of sialic acid residue(s) in the ganglioside molecule. Exogenous gangliosides act as FGF-2 antagonists when added to endothelial cell cultures. Since gangliosides are extensively shed by tumor cells and reach elevated levels in the serum of tumor-bearing patients, our data suggest that exogenous gangliosides may affect endothelial cell function by a direct interaction with FGF-2, thus modulating tumor neovascularization.
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PMID:Interaction of fibroblast growth factor-2 (FGF-2) with free gangliosides: biochemical characterization and biological consequences in endothelial cell cultures. 995 Jun 79

Gangliosides are physiological components of the outer cell membrane. In the present study, the role of ganglioside expression during differentiation of human mast cells was evaluated. After 11 days of culture in medium known to induce mast cell differentiation, 70% of peripheral blood mononuclear cells (PBMC) showed positive staining for the high affinity IgE receptor and tryptase on immunocytochemistry and an associated 20-fold increase of ganglioside GM3 expression. Furthermore, exogenous addition of GM3 during cultivation of PBMC in medium containing low levels of growth factors induced an increase of mast cell specific tryptase. The association of ganglioside expression with mast cell differentiation was confirmed by experiments with the human mast cell line HMC-1. FcepsilonRI-positive cultured cells enriched with immunobeads exhibited a 3-fold higher expression of GM3, compared to FcepsilonRI negative HMC-1 cells. Furthermore, measurable amounts of the gangliosides GM2, GM1 and GD1a were found only in the FcepsilonRI positive cells. A corresponding transient increase of mRNA for GalNAcT, the key enzyme in the production of these latter gangliosides, could be detected preceding the expression of these gangliosides and the FcepsilonRI by RT-PCR. Taken together, these data point to a functional role of gangliosides in the differentiation of human mast cells.
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PMID:Alterations in ganglioside expression during the differentiation of human mast cells. 1053 64

Exogenous glycosphingolipids, especially gangliosides, are used to study transport and metabolism of their endogenous counterparts as well as their role in cell adhesion, cell recognition and signal transduction. Unlike monodispersed solutes, in aqueous media ganglioside molecules aggregate into micelles (or bilayer structures) with a very low critical micellar concentration. Upon addition to cells in culture, exogenous gangliosides bind to the cell surface in three operationally defined modes: loosely associated micelles removable by serum; tightly attached micelles removable by proteases such as trypsin; and ganglioside molecules inserted into the outer leaflet of the plasma membrane. As shown by a biotin-labeled derivative of the ganglioside GM1 these inserted molecules are endocytosed and transported to intralysosomal membranes for catabolism. The benefit from using (partially) nondegradable as well as semi-truncated glycosphingolipids in transport studies is discussed.
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PMID:Uptake and metabolism of exogenous glycosphingolipids by cultured cells. 1129 82

Precipitate of Campylobacter jejuni cytotonic enterotoxin(CE) performed in an 80% saturated solution of ammonium sulfateit indicated that there were some little molecular proteins except the 68 kD main band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), whereas the eluate from GM1 ganglioside affinity column chromatography exhibited only one 68 kD band on SDS-PAGE. The results suggest that CE mainly be consisted of 68 kD protein. The toxin is heat-labile, pH dependent and resistant to trypsin, It could be completely inactivated by heating at either 56 degrees C and 60 degrees C for 30 min or 100 degrees C for 15 min. The activity was maximum at pH 6.0 and was completely inactivate at pH 3.0 and pH 9.0, and rapidly reduced after storage over 3 d at 4 degrees C. The anti-LT serum could completely inhibited the activity of CE.
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PMID:[Study on the physicochemical properties of Campylobacter jejuni enterotoxin]. 1254 83

To investigate the effect of the ceramide moiety of GM1 ganglioside on its association with detergent resistant membrane domains (DRMs) in human leukemia HL-60 cells, [(3)H] labeled GM1 molecular species (GM1s) with ceramides consisting of C18 sphingosine acetylated or acylated with C(8), C(12), C(14), C(16), C(18), C(22), C(24), C(18:1), C(22:1), or C(24:1) fatty acids (FAs), or C20 sphingosine acetylated or acylated with C(8) or C(18) FA were prepared and added to culture media. GM1s uptake by HL-60 cells was affected by the structure of their ceramides. Resistance to removal with trypsin and the stoichiometry of [(125)I] cholera toxin (CT) binding indicated that the added GM1s were incorporated into the membranes of the cells used for the isolation of DRMs in a manner resembling endogenous gangliosides. The ceramide moieties of the GM1s determined their occurrence in DRMs and the dependence of their recovery in this membrane fraction on the amount of Triton X-100 (TX) used for extraction as well as on cholesterol depletion. The GM1s with sphingosine acylated with C(14), C(16), C(18) C(22), or C(24) FAs were similarly abundant in DRMs. GM1s acylated with C(18:1), C(22:1), or C(24:1) were less abundant than those acylated with saturated FA of the same length. GM1s acetylated or acylated with C(8) FA were detected in DRMs in the lowest proportion. Depletion of 73% of cell cholesterol with methyl-beta-cyclodextrin significantly affected the recovery in DRMs of GM1s acetylated or acylated with C(8) or unsaturated FAs but not of GM1 acylated with C(18), C(22), or C(24) FAs. After cross-linking with CT B subunit, all GM1s were recovered in DRMs in a similarly high proportion irrespective of their ceramide structure or cholesterol depletion. DRMs prepared with low TX concentration at the TX/cell protein ratio of 0.3:1 were separated by multistep sucrose density gradient centrifugation into two fractions. The GM1s with sphingosine acetylated or acylated with C(18) or C(18:1) FAs occurred in these fractions in different proportions.
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PMID:Structure of the ceramide moiety of GM1 ganglioside determines its occurrence in different detergent-resistant membrane domains in HL-60 cells. 1276 45

Lipid rafts and the formation of an immunological synapse are crucial for T-cell activation. Binding of cholera toxin B subunit (CTB) to ganglioside GM1 is a marker to identify lipid rafts. Primary human T cells were isolated from healthy donors and were stimulated with superantigen staphylococcus enterotoxin B (SEB) and stained with cholera toxin B-fluorescein isothiocyanate (CTB-FITC). An optimized staining procedure is required to stain lipid rafts exclusively on the cell surface. Unstimulated T cells show a few CTB binding spots on the cell surface. The size and number of CTB-binding lipid rafts are strongly upregulated during T-cell activation in SEB-stimulated CD4(+) T cells. However, our data show that the specificity of CTB for GM1 ganglioside is limited, because the binding capacity is partly resistant to inhibition of ganglioside synthesis and sensitive to trypsin digestion. Our results indicate that the binding of FITC-labeled CTB can be divided into at least three different categories: a specific binding of CTB to ganglioside GM1, a nonspecific binding of CTB probably to glycosylated surface proteins and a nonspecific binding of FITC to the cell surface.
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PMID:Cholera toxin binds to lipid rafts but has a limited specificity for ganglioside GM1. 1732 93

Alexa Fluor 350 hydrazide (AF) was coupled to the aldehyde group at C-6 of terminal galactose of oxidized GM1 gangliosides containing different fatty acid residues (GM1s). The AF-GM1 hydrazones obtained were reduced with NaBH(4) or [3H]NaBH(4) and purified by high-performance thin layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC). Final yields of AF-GM1s exceeded 30%, purity was better than 97%, and radiochemical purity of 3H-labeled AF-GM1s was more than 94.5%. Structures of AF-GM1s were confirmed by electrospray ionization-mass spectrometry (ESI-MS). When added to HL-60 cell culture media, more than 81.6 or 78.9% of the AF-[3H]GM1s were taken up by cells in a bovine serum albumin- or trypsin-resistant manner, respectively. Approximately 70% of the AF-[3H]GM1s were recovered in HL-60 total plasma membrane fraction.
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PMID:Preparation of Alexa Fluor 350-conjugated nonradioactive or 3H-labeled GM1 ganglioside derivatives with different ceramides. 1898 10

Epididymosomes are small membranous vesicles secreted by epithelial cells within the luminal compartment of the epididymis. In bovine, many proteins are associated with epididymosomes, and some of them, such as the glycosylphosphatidylinositol (GPI)-anchored protein P25b, macrophage migration inhibitory factor (MIF), and aldose reductase (AKR1B1), are transferred to spermatozoa during the epididymal maturation process. P25b is associated with detergent-resistant membrane (DRM) domains of epididymal spermatozoa, whereas MIF and AKR1B1 are cytosolic proteins associated with detergent-soluble fractions. In this study, we tested the hypothesis that DRM domains are also present in the epididymosomes and that P25b DRM-associated proteins in these vesicles are transferred to the DRMs of spermatozoa. The presence of DRMs in epididymosomes was confirmed by their insolubility in cold Triton X-100 and their low buoyant density in sucrose gradient. Furthermore, DRMs isolated from epididymosomes are characterized by the exclusive presence of ganglioside GM1 and by high levels of cholesterol and sphingomyelin. Biochemical analysis indicated that P25b is linked to DRM in epididymosomes, whereas MIF and AKR1B1 are completely excluded from these membrane domains. Proteolytic treatment of epididymosomes and immunoblotting studies showed that P25b is affected by trypsin or pronase proteolysis. In contrast, MIF and AKR1B1 are not degraded by proteases, suggesting that they are localized within epididymosomes. Interaction studies between epididymosomes and epididymal spermatozoa demonstrated that P25b is transferred from the DRM of epididymosomes to the DRM of the caput epididymal spermatozoa as a GPI-anchored protein. Together, these data suggest that specific localization and compartmentalization of proteins in the epididymosomes coordinate the association of epididymal proteins with the different functional structures of spermatozoa.
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PMID:Compartmentalization of proteins in epididymosomes coordinates the association of epididymal proteins with the different functional structures of bovine spermatozoa. 1916 73

An immunohistochemical method utilizing anti-ganglioside GM1 antiserum combined with the peroxidase-antiperoxidase technique was applied to a mixed cell population in primary cultures of newborn rat brain. Ganglioside GM1 was demonstrated to be present in neurons and oligodendroglia, but was absent in astroglia. This demonstration was confirmed using a newly developed biotinylated choleragen-avidin-peroxidase procedure. Primary cultures from newborn rat brain cells that had been subjected to a single treatment with trypsin (first passage) and then cultured for 14 days were predominately (95%) composed of astrocytes that stained positively for glial fibrillary acidic protein but were negative for GM1 ganglioside. This preparation contained only 0.34 nmol ganglioside NeuNAc per mg protein compared to 23.9 nmol gangliosidic NeuNAc/mg protein for a five day culture of newborn rat brain mixed cell culture that had not been subjected to passage. Prolongation of culture time from 5 to 21 days in the latter preparation reduced the ganglioside NeuNAc content to 4.9 nmol gangliosidic NeuNAc/mg protein as the proportion of astrocytes in the culture increased. Ganglioside GM1 could not be detected by TLC analysis of the lipid extract obtained from the "pure" astrocyte culture, although small amounts of GM3 and some polysialogangliosides were detected. About half of the label incorporated upon 24 h incubation of astrocytes in the presence of N-[ (3)H]acetylmannosammine appeared in ganglioside GM3. It is concluded that astrocytes in mixed cell primary cultures from newborn rat brain, as well as astrocytes in astroglial preparations derived from such cultures, do not contain ganglioside GM1.
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PMID:Absence of ganglioside GM1 in astroglial cells from newborn rat brain. 2048 24


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