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Biomedical subjects

J A Eble

Publications and source records attributed to J A Eble.

12 recordsLinked to original sources
PubMed2001

Rhodocytin (aggretin) activates platelets lacking alpha(2)beta(1) integrin, glycoprotein VI, and the ligand-binding domain of glycoprotein Ibalpha.

Although alpha(2)beta(1) integrin (glycoprotein Ia/IIa) has been established as a platelet collagen receptor, its role in collagen-induced platelet activation has been controversial. Recently, it has been demonstrated that rhodocytin (also termed aggretin), a snake venom toxin purified from the venom of Calloselasma rhodostoma, induces platelet activation that can be blocked by monoclonal antibodies against alpha(2)beta(1) integrin. This finding suggested that clustering of alpha(2)beta(1) integrin by rhodocytin is sufficient to induce platelet activation and led to the hypothesis that collagen may activate platelets by a similar mechanism. In contrast to these findings, we provided evidence that rhodocytin does not bind to alpha(2)beta(1) integrin. Here we show that the Cre/loxP-mediated loss of beta(1) integrin on mouse platelets has no effect on rhodocytin-induced platelet activation, excluding an essential role of alpha(2)beta(1) integrin in this process. Furthermore, proteolytic cleavage of the 45-kDa N-terminal domain of glycoprotein (GP) Ibalpha either on normal or on beta(1)-null platelets had no significant effect on rhodocytin-induced platelet activation. Moreover, mouse platelets lacking both alpha(2)beta(1) integrin and the activating collagen receptor GPVI responded normally to rhodocytin. Finally, even after additional proteolytic removal of the 45-kDa N-terminal domain of GPIbalpha rhodocytin induced aggregation of these platelets. These results demonstrate that rhodocytin induces platelet activation by mechanisms that are fundamentally different from those induced by collagen.

Animals↗
PubMed2001

Contraction-dependent apoptosis of normal dermal fibroblasts.

The mechanisms underlying the contraction-dependent apoptosis of primary fibroblasts are of prime importance in understanding anchorage-dependent survival/apoptosis of dermal fibroblasts. As integrins are essential extracellular matrix receptors in fibroblasts, their role in anchorage-dependent apoptosis/survival of fibroblasts was analyzed. Primary human fibroblasts displayed a marked reduction of apoptosis in mechanically relaxed collagen matrices in the presence of adhesion-blocking antibodies against alpha1beta1 or alpha2beta1. Anti-alphavbeta3 antibodies had a considerably weaker effect. In additional experiments RD cells, which lack alpha2 integrin, displayed no apoptosis in mechanically relaxed collagen matrices. Their susceptibility to apoptosis was restored after transfection with functional alpha2 integrin, and it could be blocked again by adhesion-blocking antibodies against alpha2beta1 integrin. Therefore we conclude that apoptosis of human primary fibroblasts in contractile collagen matrices is - at least in part - inhibited by adhesion-blocking anti-integrin antibodies, suggesting that the mode of apoptosis in this case is different from anoikis. Further, apoptosis in a mechanically relaxed collagen matrix could be abrogated by depolymerization of F-actin using cytochalasin D and also by disturbing actin-myosin interaction using 2,3-butanedione monoxime, indicating a possible dependence of apoptosis on mechanical forces and/or cell shape.

Antibodies↗
PubMed2001

Urokinase receptors promote beta1 integrin function through interactions with integrin alpha3beta1.

The urokinase receptor (uPAR) is linked to cellular migration through its capacity to promote pericellular proteolysis, regulate integrin function, and mediate cell signaling in response to urokinase (uPA) binding. The mechanisms for these activities remain incompletely defined, although uPAR was recently identified as a cis-acting ligand for the beta2 integrin CD11b/CD18 (Mac-1). Here we show that a major beta1 integrin partner for uPAR/uPA signaling is alpha3. In uPAR-transfected 293 cells uPAR complexed (>90%) with alpha3beta1 and antibodies to alpha3 blocked uPAR-dependent vitronectin (Vn) adhesion. Soluble uPAR bound to recombinant alpha3beta1 in a uPA-dependent manner (K(d) < 20 nM) and binding was blocked by a 17-mer alpha3beta1 integrin peptide (alpha325) homologous to the CD11b uPAR-binding site. uPAR colocalized with alpha3beta1 in MDA-MB-231 cells and uPA (1 nM) enhanced spreading and focal adhesion kinase phosphorylation on fibronectin (Fn) or collagen type I (Col) in a pertussis toxin- and alpha325-sensitive manner. A critical role of alpha3beta1 in uPA signaling was verified by studies of epithelial cells from alpha3-deficient mice. Thus, uPAR preferentially complexes with alpha3beta1, promoting direct (Vn) and indirect (Fn, Col) pathways of cell adhesion, the latter a heterotrimeric G protein-dependent mechanism of signaling between alpha3beta1 and other beta1 integrins.

Binding Sites↗
PubMed2000

alpha 2beta 1 integrin is not recognized by rhodocytin but is the specific, high affinity target of rhodocetin, an RGD-independent disintegrin and potent inhibitor of cell adhesion to collagen.

We have recombinantly expressed a soluble form of human alpha(2)beta(1) integrin that lacks the membrane-anchoring transmembrane domains as well as the cytoplasmic tails of both integrin subunits. This soluble alpha(2)beta(1) integrin binds to its collagen ligands the same way as the wild-type alpha(2)beta(1) integrin. Furthermore, like the wild-type form, it can be activated by manganese ions and an integrin-activating antibody. However, it does not bind to rhodocytin, a postulated agonist of alpha(2)beta(1) integrin from the snake venom of Calloselasma rhodostoma, which elicits platelet aggregation. Taking advantage of the recombinantly expressed, soluble alpha(2)beta(1) integrin, an inhibition assay was established in which samples can be tested for their capability to inhibit binding of soluble alpha(2)beta(1) integrin to immobilized collagen. Thus, by scrutinizing the C. rhodostoma snake venom in this protein-protein interaction assay, we found a component of the snake venom that inhibits the interaction of soluble alpha(2)beta(1) integrin to type I collagen efficiently. N-terminal sequences identified this inhibitor as rhodocetin, a recently published antagonist of collagen-induced platelet aggregation. We could demonstrate that its inhibitory effect bases on its strong and specific binding to alpha(2)beta(1) integrin, proving that rhodocetin is a disintegrin. Standing apart from the growing group of RGD-dependent snake venom disintegrins, rhodocetin interacts with alpha(2)beta(1) integrin in an RGD-independent manner. Furthermore, its native conformation, which is stabilized by disulfide bridges, is indispensibly required for its inhibitory activity. Rhodocetin does not contain any major collagenous structure despite its high affinity to alpha(2)beta(1) integrin, which binds to collagenous molecules much more avidly than to noncollagenous ligands, such as laminin. Blocking alpha(2)beta(1) integrin as the major collagen receptor on platelets, rhodocetin is responsible for hampering collagen-induced, alpha(2)beta(1) integrin-mediated platelet activation, leading to hemorrhages and bleeding disorders of the snakebite victim. Moreover, having a widespread tissue distribution, alpha(2)beta(1) integrin also mediates cell adhesion, spreading, and migration. We showed that rhodocetin is able to inhibit alpha(2)beta(1) integrin-mediated adhesion of fibrosarcoma cells to type I collagen completely.

Amino Acid Sequence↗
PubMed2000

The spatial orientation of the essential amino acid residues arginine and aspartate within the alpha1beta1 integrin recognition site of collagen IV has been resolved using fluorescence resonance energy transfer.

The interaction of collagen IV with cells is mediated mainly by the integrin alpha1beta1. The recognition site has been located to a segment of the triple-helical domain 100 nm away from the N terminus of the collagen molecule. The three essential amino acid residues of the alpha1beta1 binding site, arginine alpha2(IV)461 and the two aspartate residues alpha1(IV)461, are all located on different chains. Since the spatial array of the three residues depends on the stagger of the chains within the triple helix, the stagger has been elucidated using fluorescence resonance energy transfer with phenylalanine alpha1(IV)473 and tryptophan alpha2(IV)479 as the fluorescent donor/acceptor pair. The distance R between phenylalanine and tryptophan was determined by analysis of the energy transfer efficiency, E, and the orientation factor, kappa(2). In parallel, distance R and orientation factor, kappa(2 )were also calculated from the coordinates of the triple helix. Comparison of the calculated and empirically determined values unequivocally showed the stagger to be alpha1'alpha1alpha2. This arrangement of the three alpha chains describes the conformation of the alpha1beta1 integrin recognition site, that is the distinct orientation of the side-chains of the essential residues aspartate and arginine in respect to the helix axis.

Amino Acid Sequence↗
PubMed2000

Collagen XVII is destabilized by a glycine substitution mutation in the cell adhesion domain Col15.

Collagen XVII is a hemidesmosomal transmembrane molecule important for epithelial adhesion in the skin. It exists in two forms, as a full-length protein and as a soluble ectodomain that is shed from the keratinocyte surface by furin-mediated proteolysis. To obtain information on the conformation and the functions of this unusual collagen, its largest collagenous domain, Col15, was expressed in a eukaryotic episomal expression system and purified by DEAE and fast protein liquid- Mono S chromatography. The protein was triple-helical (T(m) of 26.5 degrees C) when produced in cultures containing ascorbic acid. When the vitamin supply was limited, the 4-hydroxyproline content was reduced from 74 to 9%, which, in turn, resulted in a drastic reduction of the stability of the triple helix. The glycine substitution mutation G627V associated with junctional epidermolysis bullosa, a human blistering skin disease, also had a striking effect on thermal stability of rCol15 causing partial unfolding already at 4 degrees C. Col15 promoted cell adhesion of epithelial and fibroblastic cell lines with a beta1 integrin-mediated mechanism. In concert with this, in acquired autoimmune blistering skin diseases, circulating IgG and IgA autoantibodies were found to target rCol15r.

Amino Acid Substitution↗
PubMed2000

A multi-domain protein for beta1 integrin-targeted DNA delivery.

The development of effective receptor-targeted nonviral vectors for use in vivo is complicated by a number of technical problems. One of these is the low efficiency of the conjugation procedures used to couple protein ligands to the DNA condensing carrier molecules. We have made and characterized a multi-domain protein (SPKR)4inv, that is designed to target plasmid DNA to beta1 integrins in remodeling tissue. It contains a nonspecific DNA-binding domain (SPKR)4, a rigid alpha-helical linker, and the C-terminal beta1 integrin binding domain (aa 793-987) of the Yersinia pseudotuberculosis invasin protein. (SPKR)4inv could be purified at high yields using a bacterial expression system. We show that (SPKR)4inv binds with high affinity to both plasmid DNA and beta1 integrins. In a cell attachment assay, the apparent affinity of (SPKR)4inv for beta1 integrins is three orders of magnitude higher than that of the synthetic peptide integrin ligand RGDS. (SPKR)4inv-plasmid complexes are not active in an in vitro transfection assay. However, transfection efficiencies of plasmid complexes with a cationic lipid micelle (DOTAP/Tween-20) or a cationic polymer (polyethylenimine), are significantly increased in combination with (SPKR)4inv. (SPKR)4inv-mediated transfection can be inhibited by a soluble form of beta1 integrin, which is evidence for its receptor specificity. In conclusion, (SPKR)4inv allows beta1 integrin-specific targeting of plasmid-carrier complexes, while avoiding inefficient and cumbersome coupling chemistry. The modular design of the expression vector allows production of similar multi-domain proteins with a different affinity. The further development of such complexes for use in vivo is discussed.

Antigen-Antibody Reactions↗
PubMed1998

Differential effects of integrin alpha chain mutations on invasin and natural ligand interaction.

To determine if recognition of the Yersinia pseudotuberculosis invasin protein and natural substrates requires identical integrin residues, a region of the human alpha3 integrin chain predicted to be involved in substrate adhesion was targeted for mutation. One point mutation located in a region of the third N-terminal repeat of the alpha3 chain, alpha3-W220A, failed to promote adhesion to the natural alpha3 beta1 substrate epiligrin but maintained near wild type levels of adhesion to invasin. A second nearby mutation, alpha3-Y218A, which showed no detectable adhesion to epiligrin, was only partially attenuated for invasin binding as well as invasin-mediated bacterial uptake. A third substitution, alpha3-D154A, predicted to be in the second N-terminal repeat not known to be implicated in cell adhesion, was competent for invasin-promoted adhesion events and appeared to encode a receptor of increased activity, as it had a higher efficiency than wild type receptor for adhesion to epiligrin. Cell lines expressing this derivative were not recognized by a function blocking anti-alpha3 antibody, indicating that the second and third repeats of the alpha3 chain are either closely linked in space or the second repeat can modulate activity of the third. Differential effects on substrate adhesion do not appear to be associated with all integrin alpha chain mutations, as alpha4 chain mutations affecting the divalent cation binding domains depressed adhesion to invasin to a significant extent.

Adhesins, Bacterial↗
PubMed1998

Recombinant soluble human alpha 3 beta 1 integrin: purification, processing, regulation, and specific binding to laminin-5 and invasin in a mutually exclusive manner.

Using insect cells, we expressed large quantities of soluble human integrin alpha 3 beta 1 ectodomain heterodimers, in which cytoplasmic and transmembrane domains were replaced by Fos and Jun dimerization motifs. In direct ligand binding assays, soluble alpha 3 beta 1 specifically bound to laminin-5 and laminin-10, but not to laminin-1, laminin-2, fibronectin, various collagens, nidogen, thrombospondin, or complement factors C3 and C3b. Soluble alpha 3 beta1 integrin also bound to invasin, a bacterial surface protein, that mediates entry of Yersinia species into the eukaryotic host cell. Invasin completely displaced laminin-5 from the alpha 3 beta 1 integrin, suggesting sterically overlapping or identical binding sites. In the presence of 2 mM Mg2+, alpha 3 beta 1's binding affinity for invasin (Kd = 3.1 nM) was substantially greater than its affinity for laminin-5 (Kd > 600 nM). Upon addition of 1 mM Mn2+, or activating antibody 9EG7, binding affinity for both laminin-5 and invasin increased by about 10-fold, whereas the affinity decreased upon addition of 2 mM Ca2+. Thus, functional regulation of the purified soluble integrin alpha 3 beta 1 ectodomain heterodimer resembles that of wild-type membrane-anchored beta 1 integrins. The integrin alpha 3 subunit was entirely cleaved into disulfide-linked heavy and light chains, at a newly defined cleavage site located C-terminal of a tetrabasic RRRR motif. Within the alpha 3 light chain, all potential N-glycosylation sites bear N-linked mannose-rich carbohydrate chains, suggesting an important structural role of these sugar residues in the stalk-like region of the integrin heterodimer. In conclusion, studies of our recombinant alpha 3 beta 1 integrin have provided new insights into alpha 3 beta1 structure, ligand binding function, specificity, and regulation.

Adhesins, Bacterial↗
PubMed1996

The recognition sites of the integrins alpha1beta1 and alpha2beta1 within collagen IV are protected against gelatinase A attack in the native protein.

The susceptibility of three different solubilized forms of type IV collagen to gelatinase A cleavage and the concomitant effects on cell and integrin binding have been assessed. Dithiothreitol-solubilized Engelbreth-Holm Swarm (EHS) type IV collagen with disrupted intramolecular disulfide bonds in the CB3[IV] region was cleaved N-terminally to the CB3[IV] region into the two characteristic 100-300-nm fragments at 30 degrees C and was totally degraded at 37 degrees C. This was reflected in the partial or total loss of the alpha1beta1 and alpha2beta1 integrin binding sites within this region. The ability of gelatinase A to cleave EHS type IV collagen preparations with intact interchain disulfide bonds in CB3[IV] only occurred at higher temperatures. Furthermore, no effect on binding of cells or isolated integrins to the gelatinase-treated collagen could be detected after treatment at 37 degrees C. Dimeric collagen IV of human placenta with intact disulfide bonds in the CB3[IV] region was not degraded at all by gelatinase A at 37 degrees C.

Amino Acid Sequence↗
PubMed1993

The alpha 1 beta 1 integrin recognition site of the basement membrane collagen molecule [alpha 1(IV)]2 alpha 2(IV).

Cells interact with type IV collagen mainly via the integrins alpha 1 beta 1 and alpha 2 beta 1. A triple helical CNBr derived fragment CB3[IV], which contains the recognition sites for both integrins, was isolated from type IV collagen. Trypsin treatment of CB3[IV] gave rise to four smaller fragments, F1-F4, of which the smallest one, F4, contained the recognition site for alpha 1 beta 1. Further fragmentation of F4 by thermolysin treatment at 50 degrees C led to fragment TL1, which represents the C-terminal half of F4, and which was no longer able to interact with alpha 1 beta 1. Therefore the recognition site of alpha 1 beta 1 had to be located within the N-terminal half of F4, a position which was verified by electron micrographs of a crosslinked F2-alpha 1 beta 1 complex. Modification of the Arg and Asp residues, which abolished the binding activity of F4, led to the identification of Arg (461) within the alpha 2(IV) and Asp (461) within the alpha 1 (IV) chain as essential residues for the alpha 1 beta 1. The array of these two residues on the surface of the triple helix is discussed.

Amino Acid Sequence↗