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E Concord

Publications and source records attributed to E Concord.

9 recordsLinked to original sources
PubMed2000

Self-assembly of the vascular endothelial cadherin ectodomain in a Ca2+-dependent hexameric structure.

Vascular endothelial cadherin (VE-cadherin) is a transmembrane protein essential for endothelial cell monolayer integrity (Gulino, D., Delachanal, E., Concord, E., Genoux, Y., Morand, B., Valiron, M. O., Sulpice, E., Scaife, R., Alemany, M., and Vernet, T. (1998) J. Biol. Chem. 273, 29786-29793). This molecule belongs to the cadherin family of cell-cell adhesion receptors, for which molecular details of homotypic interactions are still lacking. In this study, a recombinant fragment encompassing the four N-terminal modules of VE-cadherin (VE-EC1-4) was shown to associate, in solution, as a stable Ca(2+)-dependent oligomeric structure. Cross-linking experiments combined with mass spectrometry demonstrated that this oligomer is a hexamer. Gel filtration chromatography experiments and analytical ultracentrifugation analyses revealed the existence of an equilibrium between the hexameric and monomeric species of VE-EC1-4. The concentration at which 50% of VE-EC1-4 is in its hexameric form was estimated as 1 microm. The dimensions of the hexamer, measured by cryoelectron microscopy to be 233 +/- 10 x 77 +/- 7 A, are comparable to the thickness of adherens endothelial cell-cell junctions. Altogether, the results allow us to propose a novel homotypic interaction model for the class II VE-cadherin, in which six molecules of cadherin form a hexamer.

Animals↗
PubMed1998

Alteration of endothelial cell monolayer integrity triggers resynthesis of vascular endothelium cadherin.

Although cadherins appear to be necessary for proper cell-cell contacts, the physiological role of VE-cadherin (vascular endothelium cadherin) in adult tissue has not been clearly determined. To shed some light on this question, we have disturbed the adhesive function of VE-cadherin in human endothelial cell culture using a polyclonal anti-VE-cadherin antibody. This antibody disrupts confluent endothelial cell monolayers in vitro and transiently generates numerous gaps at cell-cell junctions. The formation of these gaps correlates with a reversible increase in the monolayer permeability. We present evidence that destruction of the homotypic interactions between the extracellular domains of VE-cadherin induces a rapid resynthesis of VE-cadherin, leading to restoration of endothelial cell-cell contacts. The expression of new molecules of VE-cadherin correlates with a modest but significant increase in VE-cadherin mRNA synthesis. Altogether, these results establish a critical role for VE-cadherin in the maintenance and restoration of endothelium integrity.

Amino Acid Sequence↗
PubMed1996

Sequence 274-368 in the beta 3-subunit of the integrin alpha IIb beta 3 provides a ligand recognition and binding domain for the gamma-chain of fibrinogen that is independent of platelet activation.

Several bacterial-expressed recombinant fragments encompassing the extracellular part of the beta 3 subunit of the integrin alpha IIb beta 3 were shown to recognize and bind soluble and immobilized forms of fibrinogen. Two of them, designated as rIII-11 (beta 3 274-368) and rIII-13 (beta 3 274-403), did not contain the established RGD-ligand binding sequence. In fact, they interacted, in a Ca(2+)-independent manner, with the C-terminal part of the fibrinogen gamma chain. Both beta 3 fragments blocked the participation of fibrinogen in the induction of platelet aggregation induced by adenosine diphosphate. Fragment rIII-13 was recognized by the anti-beta 3 monoclonal antibody B2A. This antibody, which possesses an epitope exposed on both resting and activated platelets, inhibited fibrinogen binding as well as platelet adhesion and aggregation. In conclusion, the results demonstrated that the 274-368 sequence of the beta 3 subunit of integrin alpha IIb beta 3 constitutes a fibrinogen ligand binding domain, distinct from the RGD-binding site, that is required for both platelet adhesion and aggregation.

Adenosine Diphosphate↗
PubMed1995

Expression and purification of a soluble functional form of the platelet alpha IIb beta 3 integrin.

Platelet glycoproteins alpha IIb and beta 3 are membrane proteins that associate to form a Ca(2+)-dependent heterodimer which constitutes an inducible member of the integrin family at the surface of the cell. To produce a soluble form of this complex, alpha IIb and beta 3 were both deleted of their transmembrane and cytoplasmic domains and were expressed in COS cells. Production of the truncated subunits and their mode of assembly were examined by immunoprecipitation experiments and compared to those of wild-type alpha IIb beta 3. Synthesis and processing of the truncated heterodimer proceeded via a pathway similar to that observed for the wild-type alpha IIb beta 3 in COS cells or in human megakaryocytes. The truncated beta 3 subunit associated with the Pro-truncated form of the alpha IIb subunit. This precursor form was not secreted. After proteolytic cleavage of the Pro-truncated alpha IIb, the mature heterodimer was secreted into the culture supernatant. To quantify the molar ratio of the various secreted soluble forms, an immunocapture assay was designed. All secreted tr-alpha IIb subunits associated with tr-beta 3. In contrast, tr-beta 3 was produced and secreted in excess as the free form. Immunoreactivity of the wild-type and soluble truncated complexes was identical since all the monoclonal antibodies used reacted with surface-located epitopes on both complexes. This indicated that the soluble truncated heterodimer adopted a native conformation. To purify this soluble heterodimer, tr-alpha IIb beta 3-containing culture supernatant was adsorbed on an RGDW-affinity column and eluted with a solution of the free peptide RGDW. In the RGD-eluted material, the amount of each subunit was stoichiometric, suggesting that the complex was not disrupted during purification. The capacity of the wild-type and truncated RGD-eluted complexes to interact with soluble fibrinogen was compared using a solid-phase immunocapture assay. tr-alpha IIb beta 3 and platelet alpha IIb beta 3 exhibited similar fibrinogen-binding capacity. For both complexes, these interactions were mediated by RGD and gamma fibrinogen signals.

Amino Acid Sequence↗
PubMed1992

Ca(2+)-binding properties of the platelet glycoprotein IIb ligand-interacting domain.

Glycoprotein (GP) IIb is the alpha subunit of platelet integrin GPIIb-IIIa. Analysis of the primary structure of this subunit has indicated the presence of four stretches of amino acid residues that are highly conserved among various integrin alpha subunits and that have been suggested to be putative calcium-binding sites. To verify the Ca(2+)-binding capacity of these conserved domains and their implication in integrin adhesive functions, a fragment corresponding to the amino acid sequence of GPIIb from positions 171 to 464 was expressed. The nucleotide sequence coding for this GPIIb domain was generated by polymerase chain reaction, cloned into the pTG1924 expression vector, and expressed in Escherichia coli strain TGE901. The recombinant protein was purified by gel exclusion chromatography and used in equilibrium dialysis experiments. The results demonstrate that the four binding sites can be occupied by Ca2+. Two classes of binding sites can be detected, including two sites with a Kd of 30 microns and two sites of lower affinity with a Kd of 120 microns. Interaction of Ca2+ with these two classes of sites was inhibited by a large excess of Mg2+ or Mn2+, suggesting that these cations are competitive for the same sites on GPIIb. Thus, the four Ca(2+)-binding sites of GPIIb are not similar and exhibit different affinities for divalent ions. To verify the functional implication of these Ca(2+)-binding sites, the effect of Ca2+ on the binding of fibrinogen to the recombinant protein was analyzed using a solid-phase assay. The results indicate that optimal fibrinogen binding occurs when the four calcium-binding sites are occupied and establish the functional importance of this Ca(2+)-binding domain in the ligand-binding activity of GPIIb.

Amino Acid Sequence↗
PubMed1991

A highly conserved sequence of the Arg-Gly-Asp-binding domain of the integrin beta 3 subunit is sensitive to stimulation.

The Arg-Gly-Asp (RGD)-binding domain of GPIIb-IIIa has been localized in a fragment of the GPIIIa subunit that includes the sequence between amino acids 109 and 171. To examine, in a platelet membrane environment, the activated versus nonactivated status of this domain, we have produced a monoclonal antibody against a synthetic peptide (residues 109-128) located within the RGD-binding region on GPIIIa. This kappa-IgM, named AC7, was specific for GPIIIa peptide 109-128 and interacted only with activated platelets. Fibrinogen, RGDF peptide, and the fibrinogen phi chain decapeptide LGGAKQAGDV inhibited the binding of AC7 to ADP-stimulated platelets. AC7 IgM and "small fragments" inhibited fibrinogen binding and platelet aggregation in a dose-dependent fashion. Induction of AC7 binding by D33C, a monoclonal antibody recognizing the GPIIb 426-437 sequence and stimulating fibrinogen binding, indicated that the GPIIb 426-437 and the GPIIIa 109-128 sequences were both involved in a stimulation-dependent conformational modification of the receptor. AC7 was able to recognize beta subunits other than GPIIIa on leucocyte surfaces but only after cell fixation with glutaraldehyde. The results are consistent with the implication of the RGD-binding domain in receptor ligand interaction on the platelet surface and its conformational modification and exposure upon receptor induction.

Amino Acid Sequence↗
PubMed1984

Molecular characterization of an abnormal fibrinogen by two-dimensional electrophoresis.

We examined normal and abnormal fibrinogen (fibrinogen "Grenoble") by two-dimensional gel electrophoresis to obtain data on possible defects at the molecular level. Fibrinogen Grenoble is characterized by an abnormal rate of fibrin monomer aggregation. The electrophoretic analysis revealed the presence of abnormal gamma chains. Two kinds of gamma chains can be detected in fibrinogen Grenoble: (a) normal gamma chains and (b) gamma chains Grenoble (gamma G) with a greater molecular mass but no modification in isoelectric point. The latter chain can be detected in whole plasma by two-dimensional gel electrophoresis. Metrological analysis was performed in an attempt to quantify observed differences between normal fibrinogen and fibrinogen Grenoble. On use of gels stained either with Coomassie Brilliant Blue or with silver, the partly qualified evaluation gives about 60% normal gamma chain and 40% gamma chain Grenoble.

Blood Coagulation Disorders↗
PubMed1977

Soluble fibrin complexes: separation as a function of pH and characterization.

1. The influence of the pH on the separation of high molecular weight derivatives obtained by a limited action of thrombin on fibrinogen was studied by agarose gel chromatography. When the pH of the elution buffer was 8.5, non crosslinked associations were easily separated in two peaks eluted prior to the fibrinogen peak: one contained a dimer, the other several high polymers. At pH 6.5, only the fibrinogen peak appeared: the fibrinogen molecule proteolysed by thrombin formed no stable associations at this pH and was eluted with the intact fibrinogen molecule. In the presence of factor XIII and Ca++, numerous associations were obtained which are independant of the pH. 2. The polypeptide chain composition of the different species separated at pH 8.5 was studied by SDS-polyacrylamide gel electrophoresis. This technic showed Aalpha, Bbeta and gamma chains in the fibrinogen peak, whereas in the chromatographic fractions containing the dimer four bands corresponding to Aalpha, alpha, Bbeta and gamma chains were found. In the peak containing the high polymers, only the presence of alpha, Bbeta and gamma chains was demonstrated. 3. These experimental results concerning the effect of pH on the formation of soluble complexes showed that the presence of fibrin monomers in fibrinogen solution was not sufficient to promote any associations. The formation of such derivatives is strongly dependent on the pH of the solution. This obviously can be explained by an influence of the pH either on the ionization of polymerisation sites and the intermolecular bonds between the complex units or on the unmasking of the polymerisation sites by a hypothetical pH induced conformational change of the fibrinogen molecule.

Animals↗