RESUMO
Phosphomannan polysaccharides and fucoidan, a polymer of fucose 4-sulfate, have been demonstrated to inhibit adhesion of lymphocytes to tissue sections that contain high endothelial venules (Stoolman, L. M., T. S. Tenforde, and S. D. Rosen, 1984, J. Cell Biol., 99:1535-1540). We have investigated the potential cell surface carbohydrate receptors involved by quantitating adhesion of rat cervical lymph node lymphocytes to purified polysaccharides immobilized on otherwise inert polyacrylamide gels. One-sixth of the lymphocytes adhered specifically to surfaces derivatized with PPME (a phosphomannan polysaccharide prepared from Hansenula holstii yeast), whereas up to half of the cells adhered to surfaces derivatized with fucoidan. Several lines of evidence demonstrated that two distinct receptors were involved. Adhesion to PPME-derivatized gels was labile at 37 degrees C (decreasing to background levels within 120 min) whereas adhesion to fucoidan-derivatized gels was stable. Soluble PPME and other phosphomannans blocked adhesion only to PPME-derivatized gels; fucoidan and a structurally related fucan blocked adhesion to fucoidan-derivatized gels. Other highly charged anionic polysaccharides, such as heparin, did not block adhesion to either polysaccharide-derivatized gel. Adhesion to PPME-derivatized gels was dependent on divalent cations, whereas that to fucoidan-derivatized gels was not. The PPME-adherent lymphocytes were shown to be a subpopulation of the fucoidan-adhesive lymphocytes which contained both saccharide receptors. These data reveal that at least two distinct carbohydrate receptors can be found on peripheral lymphocytes.
Assuntos
Linfócitos/citologia , Receptores de Superfície Celular/análise , Animais , Anticorpos Monoclonais , Cátions Bivalentes , Adesão Celular , Cromatografia de Afinidade/métodos , Técnicas In Vitro , Linfonodos/citologia , Linfócitos/classificação , Masculino , Polissacarídeos , Ratos , Ratos Endogâmicos , Relação Estrutura-Atividade , TemperaturaRESUMO
The selectins are a family of three calcium-dependent lectins that mediate adhesive interactions between leukocytes and the endothelium during normal and abnormal inflammatory episodes. Previous work has implicated the carbohydrate sialyl Lewis(x) (sLe(x); sialic acid alpha 2-3 galactose beta 1-4 [Fucose alpha 1-3] N-acetyl glucosamine) as a component of the ligand recognized by E- and P-selectin. In the case of P-selectin, other components of the cell surface, including 2'6-linked sialic acid and sulfatide (galactose-4-sulfate ceramide), have also been proposed for adhesion mediated by this selectin. We have recently defined a region of the E-selectin lectin domain that appears to be directly involved with carbohydrate recognition and cell adhesion (Erbe, D. V., B. A. Wolitzky, L. G. Presta, C. R. Norton, R. J. Ramos, D. K. Burns, R. M. Rumberger, B. N. N. Rao, C. Foxall, B. K. Brandley, and L. A. Lasky. 1992. J. Cell Biol. 119:215-227). Here we describe a similar analysis of the P-selectin lectin domain which demonstrates that a homologous region of this glycoprotein's lectin motif is involved with carbohydrate recognition and cell binding. In addition, we present evidence that is inconsistent with a biological role for either 2'6-linked sialic acid or sulfatide in P-selectin-mediated adhesion. These results suggest that a common region of the E- and P-selectin lectin domains appears to mediate carbohydrate recognition and cell adhesion.
Assuntos
Moléculas de Adesão Celular/fisiologia , Adesão Celular , Glicolipídeos/metabolismo , Glicoproteínas da Membrana de Plaquetas/fisiologia , Sequência de Aminoácidos , Sítios de Ligação , Moléculas de Adesão Celular/química , Selectina E , Citometria de Fluxo , Humanos , Técnicas In Vitro , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Selectina-P , Glicoproteínas da Membrana de Plaquetas/química , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
The selectins (lectin-EGF-complement binding-cell adhesion molecules [LEC-CAMs]) are a family of mammalian receptors implicated in the initial interactions between leukocytes and vascular endothelia, leading to lymphocyte homing, platelet binding, and neutrophil extravasation. The three known selectins, L-selectin (leukocyte adhesion molecule-1 [LECAM-1]), E-selectin (endothelial-leukocyte adhesion molecule-1 [ELAM-1]), and P-selectin (GMP-140) share structural features that include a calcium-dependent lectin domain. The sialyl Lewis(x) carbohydrate epitope has been reported as a ligand for both E- and P-selectins. Although L-selectin has been demonstrated to bind to carbohydrates, structural features of potential mammalian carbohydrate ligand(s) have not been well defined. Using an ELISA developed with a sialyl Lewis(x)-containing glycolipid and an E-selectin-IgG chimera, we have demonstrated the direct binding of the L-selectin-IgG chimera to sialyl Lewis(x). This recognition was calcium dependent, and could be blocked by Mel-14 antibody but not by other antibodies. Recognition was confirmed by the ability of cells expressing the native L-selectin to adhere to immobilized sialyl Lewis(x). These data suggest that the sialyl Lewis(x) oligosaccharide may form the basis of a recognition domain common to all three selectins.
Assuntos
Moléculas de Adesão Celular/metabolismo , Gangliosídeos/metabolismo , Antígenos CD15/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Animais , Sequência de Carboidratos , Adesão Celular , Células Cultivadas , Selectina E , Epitopos , Gangliosídeos/química , Técnicas In Vitro , Selectina L , Ligantes , Camundongos , Dados de Sequência Molecular , Selectina-P , Proteínas Recombinantes de Fusão/metabolismoRESUMO
E-selectin elicits cell adhesion by binding to the cell surface carbohydrate, sialyl Lewis X (sLe(x)). We evaluated the effects of mutations in the E-selectin lectin domain on the binding of a panel of anti-E-selectin mAbs and on the recognition of immobilized sLe(x) glycolipid. Functional residues were then superimposed onto a three-dimensional model of the E-selectin lectin domain. This analysis demonstrated that the epitopes recognized by blocking mAbs map to a patch near the antiparallel beta sheet derived from the NH2 and COOH termini of the lectin domain and two adjacent loops. Mutations that affect sLe(x) binding map to this same region. These results thus define a small region of the E-selectin lectin domain that is critical for carbohydrate recognition.
Assuntos
Moléculas de Adesão Celular/metabolismo , Adesão Celular/fisiologia , Antígenos CD15/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Sítios de Ligação/fisiologia , Moléculas de Adesão Celular/química , Análise Mutacional de DNA , Selectina E , Imunofluorescência , Humanos , Antígenos CD15/química , Glicoproteínas de Membrana/química , Dados de Sequência Molecular , Conformação Proteica , Coelhos , Receptores Imunológicos/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Complex carbohydrates coat the surfaces of cells and have the potential to carry the information necessary for cell-cell recognition. Sugar-specific receptors (lectins) are also present on cells, and can interact with sugars on apposing cells. This may result in the adhesion of the two cells via carbohydrates and specific cell-surface receptors. Such carbohydrate-directed cell adhesion appears to be important in many intercellular activities including infection by bacteria and viruses, communication among cells of lower eukaryotes, specific binding of sperm to egg; and recirculation of lymphocytes, among others. New approaches involving synthesis of chemically defined cell-surface analogs, in conjunction with inhibition experiments, are beginning to reveal the mechanics of a potential carbohydrate "language" involved in intercellular interactions.
Assuntos
Carboidratos/fisiologia , Lectinas/fisiologia , Receptores de Superfície Celular/fisiologia , Adesividade , Animais , Antígenos de Superfície/fisiologia , Fenômenos Fisiológicos Bacterianos , Adesão Celular , Agregação Celular , Comunicação Celular , Endotélio/fisiologia , Feminino , Fertilização , Glicolipídeos/fisiologia , Glicoproteínas/fisiologia , Fígado/citologia , Linfócitos/fisiologia , Macrófagos/citologia , Masculino , Neurônios/fisiologia , Ácidos Siálicos/fisiologia , Interações Espermatozoide-Óvulo , Fenômenos Fisiológicos ViraisAssuntos
Carboidratos , Adesão Celular , Fenômenos Fisiológicos Celulares , Células Eucarióticas/fisiologia , Resinas Acrílicas , Adsorção , Animais , Divisão Celular , Células Cultivadas , Cromatografia em Camada Fina/instrumentação , Cromatografia em Camada Fina/métodos , Meios de Cultura , Células Eucarióticas/citologia , Géis , Glicoesfingolipídeos , Indicadores e ReagentesRESUMO
Carbohydrates are ubiquitous constituents of cell surfaces, and possess many characteristics that make them ideal candidates for recognition molecules. In many systems where cell adhesion plays a critical role, carbohydrate binding proteins have been shown to bind to cell surface carbohydrates and participate in cell-cell interactions. Such systems include fertilization, development, pathogen-host recognition and inflammation. In particular the recent discovery of the LEC-CAMs and their importance in leukocyte biology has refocused attention on lectin-mediated cell adhesion. The LEC-CAMs offer good targets for the development of therapeutics based on carbohydrate structures.
Assuntos
Carboidratos/fisiologia , Moléculas de Adesão Celular/fisiologia , Adesão Celular/fisiologia , Lectinas/metabolismo , Animais , Aderência Bacteriana/fisiologia , Sequência de Carboidratos , Carboidratos/uso terapêutico , Fertilização , Humanos , Dados de Sequência Molecular , Vírus/metabolismoRESUMO
The movement of cells up an adhesive substratum gradient has been proposed as a mechanism for directing cell migration during development and metastasis. Critical evaluation of this hypothesis (haptotaxis) benefits from the use of quantifiable, stable substratum gradients of biologically relevant adhesion molecules. We report covalent derivatization of polyacrylamide surfaces with quantifiable gradients of a nonapeptide containing the adhesive Arg-Gly-Asp sequence. Cell migration was studied by seeding derivatized surfaces evenly with B16F10 murine melanoma cells. Within 8 hr, cells on gradients redistributed markedly; higher cell densities were found at gel positions having higher immobilized peptide densities. In contrast, cells seeded on control gels with uniform concentrations of adhesive peptide did not redistribute. Redistribution occurred on gradients in both serum-free and serum-containing media. Experiments with uniform density peptide-derivatized gels demonstrated that redistribution on gradients was not due to preferential initial cell attachment or preferential growth on the higher density of immobilized peptide, but must have been due to cell translocation. Cells on exponential gradients of immobilized peptide migrated to a position on the gel surface corresponding to the highest immobilized peptide density, while cells on linear gradients of the same peptide migrated to a position of intermediate peptide density. These data suggest that the B16F10 cells respond to proportional changes in immobilized peptide density rather than to absolute changes, implying a sensing mechanism which utilizes adaptation. These results demonstrate that (1) a gradient of a small adhesive peptide is sufficient to generate redistribution of cell populations and (2) controlled quantifiable substratum gradients can be produced and used to probe the underlying cellular mechanisms of this behavior.
Assuntos
Antígenos de Superfície/fisiologia , Adesão Celular , Melanoma Experimental/patologia , Animais , Moléculas de Adesão Celular , Divisão Celular , Movimento Celular , Géis , Técnicas In Vitro , Camundongos , Peptídeos/metabolismo , Células Tumorais CultivadasRESUMO
A synthetic nonapeptide (Tyr-Ala-Val-Thr-Gly-Arg-Gly-Asp-Ser), which includes the adhesive Arg-Gly-Asp (RGD) sequence, was covalently immobilized on chemically well-defined polyacrylamide gel surfaces utilizing N-succinimidyl active esters. The amount of peptide immobilized varied linearly with the concentration added to the gels. Immobilization was approximately 80% efficient (based on peptide added), resulting in up to 17.5 nmol peptide/cm2 gel surface. Balb/c 3T3 mouse fibroblast cells adhered readily to peptide-derivatized surfaces, even in the absence of serum. Furthermore, surfaces derivatized with 2 nmol peptide/cm2 gel supported long-term fibroblast growth at a rate and to an extent comparable to that on tissue culture plastic. Surfaces derivatized with a control nonapeptide having no RGD sequence were nonsupportive of cell attachment or growth. The immobilization technology used to derivatize the gel surfaces with adhesive nonapeptide can be modified to allow coderivatization with proteins, glycoproteins, glycosides, or other amine-containing compounds to test their effects on long-term cell behaviors.
Assuntos
Adesão Celular , Divisão Celular , Oligopeptídeos/fisiologia , Resinas Acrílicas , Animais , Fibroblastos/citologia , Fibroblastos/fisiologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Cell-cell adhesion is a multi-step process which may be initiated by binding of cell surface carbohydrates to complementary carbohydrate receptors on apposing cell surfaces. We have modeled such interactions using polyacrylamide gels covalently derivatized with glycosides, to which intact cells specifically adhere; chicken hepatocytes adhere to gels derivatized with N-acetylglucosamine (GlcNAc). Initially adhesion is blocked (or reversed) by soluble GlcNAc, but becomes sugar-resistant rapidly at 37 degrees C, perhaps due to cellular modification of the carbohydrate-derivatized surface (Guarnaccia, S. P., Kuhlenschmidt, M. S., Slife, C. W., and Schnaar, R. L. (1982) J. Biol. Chem. 257, 14293-14299). We report here that, subsequent to recognition and adhesion, intact chicken hepatocytes transfer phosphate covalently to GlcNAc-derivatized gels. Metabolically radiolabeled cells (32Pi) were incubated on polyacrylamide gels derivatized with various aminohexyl glycosides. Noncovalently bound material was then removed from the gels by extensive washing in detergents and salt solutions. Subsequent radiochemical analysis revealed that phosphate was transferred selectively to GlcNAc-derivatized gels (up to 20-fold more than to glucose-, galactose-, or mannose-derivatized gels). Soluble GlcNAc (but not other sugars) or low temperature inhibited phosphate transfer. The phosphorylation was mediated by intact cells; cell lysate was itself incapable of specific phosphate transfer and attenuated specific transfer when added to intact cells. When GlcNAc was immobilized using a cleavable (disulfide-containing) linker arm the transferred phosphate radiolabel could be solubilized by disulfide reduction and recovered for further analysis. The released phosphorylated product migrated as a single low molecular weight species upon gel permeation chromatography, paper electrophoresis, and cellulose thin layer chromatography. Acid hydrolysis of the phosphorylated product generated a compound with the mobility of GlcNAc-6-P in five different separation systems. Treatment with alkaline phosphatase converted the radiolabel to a compound with the properties of inorganic phosphate. These data indicate that; subsequent to carbohydrate recognition and adhesion, intact hepatocytes generate phosphomonoesters of recognized carbohydrates outside of their plasma membranes.
Assuntos
Acetilglucosamina/metabolismo , Metabolismo dos Carboidratos , Glucosamina/análogos & derivados , Fígado/metabolismo , Resinas Acrílicas , Fosfatase Alcalina/metabolismo , Animais , Adesão Celular , Comunicação Celular , Galinhas , Cromatografia em Gel , Cromatografia em Camada Fina , Ditiotreitol/farmacologia , Eletroforese em Papel , Géis , Hidrólise , Cinética , Fígado/citologia , Fosfatos/metabolismo , FosforilaçãoRESUMO
Ligands and proteins were covalently but reversibly immobilized on polyacrylamide gels using novel acrylic monomers whose syntheses are reported here. These reagents have an acrylyl group at one end for copolymerization into gels, an N-succinimidyl ester at the other allowing rapid immobilization of molecules having an available primary amino group, and a cleavable disulfide bond in the middle. Two immobilization methods were developed using these reagents. In the first method, a ligand with a primary amino group was treated with the immobilization reagent in anhydrous ethanol and the resulting amide derivative was purified and copolymerized with acrylamide and bisacrylamide resulting in the desired reversible immobilization. In the second method, the immobilization reagents (at densities up to 50 mumol/ml) were directly copolymerized with acrylamide and bisacrylamide to form activated gels of the desired shape and porosity. Proteins or other ligands in aqueous buffers were then added to the activated gels resulting in their covalent immobilization. Ligands or proteins immobilized using the methods reported here remained stably bound even when gels were subjected to boiling in detergents or high-ionic-strength buffers. Immobilized ligands were readily released (greater than 97%) from gels by treatment with quantitative amounts of aqueous dithiothreitol (DTT) under mild conditions. Immobilized proteins were also released (up to 87%) from the gels by DTT treatment. Small ligands (e.g., aminohexyl glycosides), active enzymes, and glycoproteins were immobilized, and then recovered, using these reagents.
Assuntos
Proteínas/análise , Fosfatase Alcalina/análise , Ditiotreitol , Eletroforese em Gel de Poliacrilamida/métodos , Enzimas Imobilizadas/análise , Glicoproteínas/análise , Indicadores e Reagentes/síntese química , Cinética , L-Lactato Desidrogenase/análise , LigantesRESUMO
Important cellular characteristics, including selective adhesion, growth rate, motility, and differentiation, are controlled, in part, by signals received at the cell surface. The molecular mechanisms for the cell surface control of these cell behaviors are largely unknown. In order to probe the role of specific extracellular molecules in controlling cell function, we report the development of synthetic surfaces which generally support the long-term growth of cells yet can be readily derivatized with a wide variety of molecules of biological interest. Polyacrylamide gels containing a gradient of active ester groups were prepared and then the esters were displaced with ligands to generate a gradient of carboxylic acid, tertiary amine, or hydroxyl groups. When untransformed mouse fibroblasts (BALB/3T3) were seeded on the various surfaces, they attached and grew only on those derivatized with carboxylic acids or hydroxyl groups within narrow concentration ranges. Cell growth rate, density, and morphology on polyacrylamide gels containing the optimal concentration of carboxylic acid groups (approximately 30 mumol/ml) were comparable to those on tissue culture plastic, whereas growth on hydroxyl group-derivatized gels was less extensive. In contrast, short-term (90-min) adhesion to hydroxyl group-derivatized gels was greater than that to carboxylic acid-derivatized gels. Both short-term adhesion and long-term growth required serum. Growth-supportive polyacrylamide gels were readily derivatized with molecules of biological interest. The techniques reported here are applicable to other types of cell in culture since the nature and concentration of substratum functional groups can be easily varied and tested for support of long-term cell growth.
Assuntos
Resinas Acrílicas , Adesão Celular , Animais , Divisão Celular , Fenômenos Químicos , Química , Fibroblastos/citologia , Géis , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Polyacrylamide surfaces covalently derivatized with quantifiable gradients of glycosides superimposed on a uniform adhesive background of coimmobilized Arg-Gly-Asp-containing adhesion peptide were synthesized. Substrate-directed cell redistribution (haptotaxis) was measured by seeding derivatized surfaces uniformly with B16F10 murine melanoma cells. After 4-32 hr, cells on gradients of N-acetylglucosamine (GlcNAc) redistributed markedly; higher cell densities were found at gel positions having a higher immobilized GlcNAc density. In contrast, cells seeded on otherwise identical gels having a uniform concentration of immobilized GlcNAc, or on gels having gradients of glucose or galactose, did not redistribute. Soluble inhibitors containing nonreducing terminal GlcNAc (but not those with terminal GalNAc or Gal) blocked redistribution on immobilized GlcNAc gradients. Redistribution was not affected by the presence or absence of serum in the medium. An affinity-purified antibody against beta-1,4-galactosyltransferase, a GlcNAc-binding protein reported to be expressed on B16F10 cell surfaces, attenuated GlcNAc-directed redistribution. When cells were seeded on surfaces derivatized with various uniform densities of immobilized GlcNAc coimmobilized with an invariant density of immobilized Arg-Gly-Asp-peptide, neither cell attachment nor proliferation rate were enhanced on the gels having a higher GlcNAc density. These data indicate that the redistribution on immobilized GlcNAc gradients was due to cell motility. Although gels derivatized with Arg-Gly-Asp-peptide alone supported strong B16F10 cell adhesion, surfaces derivatized with uniform high concentrations of GlcNAc did not. We conclude that cell recognition of substratum gradients that support, at best, weak adhesion (GlcNAc) on an otherwise uniform strongly adhesive background (Arg-Gly-Asp-peptide) may be sufficient to direct cell migration.
Assuntos
Acetilglucosamina/farmacologia , Resinas Acrílicas , Movimento Celular/efeitos dos fármacos , Glucosamina/análogos & derivados , Animais , Adesão Celular , Divisão Celular/efeitos dos fármacos , Técnicas In Vitro , Melanoma/patologia , CamundongosRESUMO
To further characterize the molecular mechanisms that govern carcinoma cell adhesion to stimulated endothelium, we studied the adhesion of a human colon carcinoma cell line, KM12-L4, to an E-selectin-IgG1 chimera and interleukin (IL)-1 beta-stimulated human umbilical vein endothelial cells (HUVEC) under in vitro fluid flow conditions. Between 0.6 and 1.8 dynes/cm2, KM12-L4 cells attach to and roll on IL-1 beta-stimulated HUVEC. The adhesion is E-selectin dependent and diminished upon pretreatment of the KM12-L4 cells with neuraminidase (neuraminidase sensitive). Between 0.7 and 1.8 dynes/cm2, surfaces coated with an E-selectin-IgG1 chimera support attachment and rolling of KM12-L4 cells. The adhesion to the E-selectin-IgG1 chimera is blocked by an antibody to the lectin domain of E-selectin and is neuraminidase sensitive. Rolling KM12-L4 cells exhibit variable velocity motion over both IL-1 beta-stimulated HUVEC and E-selectin-IgG1 chimera-coated surfaces. Our results provide the first direct evidence that sialylated moieties are involved in the adhesion of carcinoma cells to IL-1 beta-stimulated endothelium under flow conditions; E-selectin-IgG1 chimeras can support cell attachment and rolling under defined flow conditions; the topology of the endothelium is not the sole cause of variable velocity motion observed in cell rolling systems.
Assuntos
Adesão Celular/fisiologia , Neoplasias do Colo/fisiopatologia , Endotélio Vascular/fisiologia , Adesão Celular/efeitos dos fármacos , Selectina E/fisiologia , Citometria de Fluxo , Humanos , Imunoglobulina G/farmacologia , Imunoglobulina G/fisiologia , Interleucina-1/farmacologia , Interleucina-1/fisiologia , Neuraminidase/metabolismo , Neuraminidase/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Reologia , Propriedades de Superfície , Células Tumorais Cultivadas/fisiologia , Veias Umbilicais/fisiologiaRESUMO
Early in inflammation, adhesion occurs between leukocytes and endothelium when selectins bind to sialyl Lewis X (sLex) and related oligosaccharides. We tested novel compounds that mimic sLex for their ability to inhibit selectin-mediated adhesion of human eosinophils and neutrophils in vitro. Neutrophils and eosinophils were isolated by density gradient centrifugation, and eosinophils were further purified by immunomagnetic negative selection. Adhesion to unstimulated or interleukin-1beta-stimulated (5 ng/ml, 4-6 h) umbilical vein endothelial monolayers was tested under static or rotating conditions, where adhesion is primarily E- or L-selectin dependent, respectively. P-selectin-dependent adhesion was tested on immobilized platelets treated with or without phorbol myristate acetate (10(-7) M, 10 min). Stimulus-induced adhesion was always at least 4-fold higher than without stimulus, and selectin dependence was confirmed with specific blocking monoclonal antibodies. E-selectin-dependent adhesion of eosinophils and neutrophils was inhibited by compound GM2296 (the concentration producing 50% inhibition of adhesion [IC50] approximately 0.5-1 mM). E-selectin-dependent adhesion of neutrophils, but not eosinophils, was also inhibited by another compound, sLex with a lipid tail (30 +/- 6% inhibition at 3 mM), whereas compound GM1292 slightly inhibited adhesion of both (23 +/- 5 and 20 +/- 6% inhibition, respectively, at 1 mM). L-selectin-dependent adhesion was more effectively inhibited by GM2296 (IC50 approximately 0.2-0.5 mM), although P-selectin-dependent adhesion was also inhibited (IC50 approximately 1 mM). Inhibition was reversible without affecting viability, and no effect was seen with these compounds in assays testing neutrophil adhesion to immobilized intercellular adhesion molecule-1. Thus, compound GM2296, a carbon-fucosylated derivative of glycyrrhetinic acid, inhibits E-, L-, and P-selectin-dependent eosinophil and neutrophil adhesion. The ability of these and perhaps other related glycomimetic compounds to interfere with the function of more than one type of selectin makes them desirable candidates as anti-inflammatory agents.
Assuntos
Adesão Celular/efeitos dos fármacos , Eosinófilos/metabolismo , Neutrófilos/metabolismo , Oligossacarídeos/farmacologia , Selectinas/farmacologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Sequência de Carboidratos , Ácido Glicirrízico/química , Ácido Glicirrízico/farmacologia , Humanos , Inflamação/tratamento farmacológico , Dados de Sequência Molecular , Estrutura Molecular , Oligossacarídeos/química , Antígeno Sialil Lewis XRESUMO
The acute inflammatory response requires that circulating leukocytes bind to and penetrate the vascular wall to access the site of injury. Several receptors have been implicated in this interaction, including a family of putative carbohydrate-binding proteins. We report here the identification of an endogenous carbohydrate ligand for one of these receptors, endothelial-leukocyte adhesion molecule 1 (ELAM-1). Radiolabeled COS cells transfected with a plasmid containing the cDNA for ELAM-1 were used as probes to screen glycolipids extracted from human leukocytes. COS cells transfected with this plasmid adhered to a subset of sialylated glycolipids resolved on TLC plates or adsorbed on polyvinyl chloride microtiter wells. Adhesion to these glycolipids required calcium but was not inhibited by heparin, chondroitin sulfate, keratan sulfate, or yeast phosphomannan. Monosaccharide composition, linkage analysis, and fast atom bombardment mass spectrometry of the glycolipids indicate that the ligands for ELAM-1 are terminally sialylated lactosylceramides with a variable number of N-acetyllactosamine repeats and at least one fucosylated N-acetylglucosamine residue.
Assuntos
Moléculas de Adesão Celular/metabolismo , Adesão Celular , Animais , Sequência de Bases , Configuração de Carboidratos , Sequência de Carboidratos , Carboidratos , Moléculas de Adesão Celular/genética , Linhagem Celular , Clonagem Molecular , Selectina E , Glicolipídeos/metabolismo , Humanos , Ligantes , Espectrometria de Massas , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Neutrófilos/fisiologia , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , TransfecçãoRESUMO
The selectins are a family of carbohydrate-binding proteins that have been implicated in the initial interaction between leukocytes and the vascular endothelium. The three members of this family will bind to the sialyl-Lewisx epitope [Sia alpha 2-3 Gal beta 1-4 (Fuc alpha 1-3) GlcNAc] and related oligosaccharides. In this report, we examine the molecular details of that recognition using synthesized carbohydrates with specific modifications on the sialyl-Lewisx epitope. E- and L-Selectin require hydroxyl groups at the 2, 3 and 4 positions of the fucose residue. P-Selectin, however, requires only the 3-position hydroxyl group, while tolerating removal of the oxygen at positions 2 or 4 of fucose residue. Modifications of the glycerol side chain or the N-acetyl group of the sialic acid have little effect on the binding of any of the selectins. All three selectins bind efficiently to an oligosaccharide with a sulphate replacement for the sialic acid [sulpho-Lewisx, or SO4-3Gal beta 1-4 (Fuc alpha 1-3) Glc-ceramide]. For E-Selectin, binding to sulpho-Lewisx appears to be equivalent to binding to sialyl-Lewisx, while for L- and P-Selectin binding to the sulphated structure shows characteristics distinct from sialyl-Lewisx recognition. Taken together, these data indicate that, while all three selectins can recognize sialyl-Lewisx, E-, L- and P-Selectin each display distinct carbohydrate ligand preferences.
Assuntos
Metabolismo dos Carboidratos , Moléculas de Adesão Celular/metabolismo , Fucose/metabolismo , Ácidos Siálicos/metabolismo , Sulfatos/metabolismo , Sequência de Carboidratos , Carboidratos/química , Moléculas de Adesão Celular/química , Células Cultivadas , Selectina E , Fucose/química , Humanos , Selectina L , Antígenos CD15/química , Antígenos CD15/metabolismo , Ligantes , Dados de Sequência Molecular , Ácido N-Acetilneuramínico , Selectina-P , Glicoproteínas da Membrana de Plaquetas/química , Glicoproteínas da Membrana de Plaquetas/metabolismo , Ácidos Siálicos/química , Relação Estrutura-Atividade , Sulfatos/químicaRESUMO
A receptor uniquely found on the surface of rat Kupffer cells was shown previously to bind oligosaccharides terminating in galactose, N-acetylgalactosamine, and fucose. To analyze further the binding specificity of the receptor, receptor-mediated adhesion of transfected COS cells to immobilized glycolipids of known structure was measured. The glycolipid Gb4Cer (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1Cer) was the best ligand. Gb5Cer (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1Cer) and LacCer (Gal beta 1-4Glc beta 1Cer) bound more weakly (five times less than Gb4Cer) and Gb3Cer (Gal alpha 1-4Gal beta 1-4Glc beta 1Cer), and g3Cer(GalNAc beta 1-4Gal beta 1-4Glc beta 1Cer) bound even more weakly (60 times less than Gb4Cer). Gangliosides did not support adhesion of transfected cells. The adhesion of COS cells transfected with plasmids encoding variants of the receptor was also examined. In each variant, either tryptophan 498 or 523, which are conserved in most C-type lectins, was replaced by one of several amino acids. Variants that retained binding activity had the same specificity as the normal receptor. Differences between variants were noted, however, in maximal levels of adhesion and these differences correlated with altered expression of the receptor variants in COS cells.
Assuntos
Células de Kupffer/metabolismo , Receptores Mitogênicos/metabolismo , Acetilgalactosamina/metabolismo , Animais , Sequência de Carboidratos , Adesão Celular , Linhagem Celular , Cromatografia em Camada Fina , Fucose/metabolismo , Galactose/metabolismo , Dados de Sequência Molecular , Plasmídeos , Ratos , Especificidade por Substrato , TransfecçãoRESUMO
The selectins, a family of adhesion receptors involved in leukocyte extravasation, recognize sialyl Lewis X (sLe(x); NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc) and related oligosaccharides. We used conformational energy computations, high field NMR, and structure-function studies to define distance parameters of critical functional groups of sLe(x). This sLe(x) pharmacophore was used to search a three-dimensional data base of chemical structures. Compounds that had a similar spatial relationship of functional groups were tested as inhibitors of selectin binding. Glycyrrhizin, a triterpene glycoside, was identified and found to block selectin binding to sLe(x) in vitro. We substituted different sugars for the glucuronic acids of glycyrrhizin and found the L-fucose derivative to be the most active in vitro and in vivo. A C-fucoside derivative, synthesized on a linker designed for stability and to more closely approximate the original sLe(x) pharmacophore, resulted in an easily synthesized, effective selectin blocker with anti-inflammatory activity.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Moléculas de Adesão Celular/efeitos dos fármacos , Ácido Glicirretínico/análogos & derivados , Oligossacarídeos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Sequência de Carboidratos , Selectina E , Ácido Glicirretínico/farmacologia , Ácido Glicirrízico , Selectina L , Camundongos , Dados de Sequência Molecular , Oligossacarídeos/química , Selectina-P , Glicoproteínas da Membrana de Plaquetas/efeitos dos fármacos , Receptores Imunológicos/efeitos dos fármacos , Antígeno Sialil Lewis XRESUMO
The interaction of basic FGF (bFGF) with heparin, heparan sulfate and related sugars can potentiate or antagonize bFGF activity, depending on the size of the saccharide used. Oligosaccharides based on heparin structures, as small as six sugar residues, have been demonstrated to bind to bFGF and block its activity, while larger structures (> 10 sugar residues) tend to potentiate bFGF. In this study we have synthesized a series of compounds designed to test the requirements of size and sulfation for binding of oligosaccharides to bFGF. These oligosaccharides are not derived from heparin, but rather, are linear chains of glucose linked alpha 1-4 (malto-oligosaccharides) that have been chemically sulfated. In addition to bFGF binding, these compounds were tested for their ability to block basic functions of endothelial cells that are known to be mediated, at least in part, by bFGF. We report that the ability of sulfated malto-oligosaccharides to block binding of bFGF to heparan sulfate was dependent on the size (at least a tetrasaccharide is required), and the degree of sulfation. The activity profile in the bFGF ELISA closely correlated with the ability of these compounds to block REEC or HMVEC tube formation on Matrigel. There was a similar relationship of size and sulfation to the ability of the sulfated malto-oligosaccharides to inhibit endothelial cell growth for most human and rat EC types tested. The single exception was REEC cell growth. One isolate of these cells was stimulated by sulfated malto-oligosaccharides rather than inhibited by them, while a second isolate was neither stimulated nor inhibited. This stimulation showed no correlation with inhibition of bFGF binding in the ELISA assay, suggesting that growth of this cell type was probably not dependent on bFGF. Compounds derived from this series of sulfated, malto-oligosaccharides have the potential to function as bFGF antagonists, are relatively easy to produce, and possess relatively low anticoagulant properties.