Monoclonal antibodies identify residues 199-216 of the integrin alpha2 vWFA domain as a functionally important region within alpha2beta1.

Integrin alpha2beta1 is the major receptor for collagens in the human body, and the collagen-binding site on the alpha2 subunit von Willebrand factor A-type domain (vWFA domain) is now well defined. However, the biologically important conformational changes that are associated with collagen binding, and the means by which the vWFA domain is integrated into the whole integrin are not completely understood. We have raised monoclonal antibodies against recombinant alpha2 vWFA domain for use as probes of function. Three antibodies, JA202, JA215 and JA218, inhibited binding to collagen, collagen I C-propeptide and E-cadherin, demonstrating that their function is important for structurally diverse alpha2beta1 ligands. Cross-blocking studies grouped the epitopes into two clusters: (I) JA202, the inhibitory antibody, Gi9, and a non-inhibitory antibody, JA208; (II) JA215 and JA218. Both clusters were sensitive to events at the collagen binding site, as binding of Gi9, JA202, JA215 and JA218 were inhibited by collagen peptide, JA208 binding was enhanced by collagen peptide, and binding of JA202 was decreased after mutagenesis of the cation-binding residue Thr(221) to alanine. Binding of cluster I antibodies was inhibited by the anti-functional anti-beta1 antibody Mab13, and binding of Gi9 and JA218 to alpha2beta1 was inhibited by substituting Mn(2+) for Mg(2+), demonstrating that these antibodies were sensitive to changes initiated outside the vWFA domain. Mapping of epitopes showed that JA202 and Gi9 bound between residues 212-216, while JA208 bound between residues 199-216. We have therefore identified two epitope clusters with novel properties; i.e. they are intimately associated with the collagen-binding site, responsive to conformational changes at the collagen-binding site and sensitive to events initiated outside the vWFA domain.

Micromolar Ca2+ concentrations are essential for Mg2+-dependent binding of collagen by the integrin alpha 2beta 1 in human platelets.

Integrin receptor alpha(2)beta(1) requires micromolar Ca(2+) to bind to collagen and to the peptide GPC(GPP)(5)GFOGER(GPP)(5)GPC (denoted GFOGER-GPP, where O represents hydroxyproline), which contains the minimum recognition sequence for the collagen-binding alpha(2) I-domain (Knight, C. G., Morton, L. F., Peachey, A. R., Tuckwell, D. S., Farndale, R. W., and Barnes, M. J. (2000) J. Biol. Chem. 275, 35-40). Platelet adhesion to these ligands is completely dependent on alpha(2)beta(1) in the presence of 2 mm Mg(2+). However, we show here that this interaction was abolished in the presence of 25 microm EGTA. Adhesion of Glanzmann's thrombasthenic platelets, which lack the fibrinogen receptor alpha(IIb)beta(3), was also inhibited by micromolar EGTA. Mg(2+)-dependent adhesion of platelets was restored by the addition of 10 microm Ca(2+), but millimolar Ca(2+) was inhibitory. Binding of isolated alpha(2)beta(1) to GFOGER-GPP was 70% inhibited by 50 microm EGTA but, as with intact platelets, was fully restored by the addition of micromolar Ca(2+). 2 mm Ca(2+) did not inhibit binding of isolated alpha(2)beta(1) to collagen or to GFOGER-GPP. Binding of recombinant alpha(2) I-domain was not inhibited by EGTA, nor did millimolar Ca(2+) inhibit binding. Our data suggest that high affinity Ca(2+) binding to alpha(2)beta(1), outside the I-domain, is essential for adhesion to collagen. This is the first demonstration of a Ca(2+) requirement in alpha(2)beta(1) function.

Identification and characterization of anaerobic gut fungi using molecular methodologies based on ribosomal ITS1 and 185 rRNA.

The gut fungi are an unusual group of zoosporic fungi occupying a unique ecological niche, the anaerobic environment of the rumen. They exhibit two basic forms, with nuclear migration throughout the hyphal mass for polycentric species and with concentration of nuclear material in a zoosporangium for monocentric species. Differentiation between isolates of these fungi is difficult using conventional techniques. In this study, DNA-based methodologies were used to examine the relationships within and between two genera of monocentric gut fungi gathered from various geographical locations and host animals. The ribosomal ITS1 sequence from 16 mono- and 4 polycentric isolates was PCR-amplified and sequenced; the sequences obtained were aligned with published sequences and phylogenetic analyses were performed. These analyses clearly differentiate between the two genera and reflect the previously published physiological conclusions that Neocallimastix spp. constitute a more closely related genus than the relatively divergent genus Piromyces. The analyses place two type species N. frontalis and N. hurleyensis together but, contrary to a recent suggestion in the literature, place them apart from the other agreed species N. patriciarum. In situ hybridization and slot-blotting were investigated as potential methods for detection of and differentiation between monocentric gut fungi. DNA slot-blot analysis using ribosomal sequences is able to differentiate between gut fungal genera and thus has considerable potential for use in ecological studies of these organisms.

The collagen-binding A-domains of integrins alpha(1)beta(1) and alpha(2)beta(1) recognize the same specific amino acid sequence, GFOGER, in native (triple-helical) collagens.

We have previously assigned an integrin alpha(2)beta(1)-recognition site in collagen I to the sequence, GFOGERGVEGPOGPA (O = Hyp), corresponding to residues 502-516 of the alpha(1)(I) chain and located in the fragment alpha(1)(I)CB3 (Knight, C. G., Morton, L. F., Onley, D. J., Peachey, A. R., Messent, A. J., Smethurst, P. A., Tuckwell, D. S., Farndale, R. W., and Barnes, M. J. (1998) J. Biol. Chem. 273, 33287-33294). In this study, we show that recognition is entirely contained within the six-residue sequence GFOGER. This sequence, when in triple-helical conformation, readily supports alpha(2)beta(1)-dependent cell adhesion and exhibits divalent cation-dependent binding of isolated alpha(2)beta(1) and recombinant alpha(2) A-domain, being at least as active as the parent collagen. Replacement of E by D causes loss of recognition. The same sequence binds integrin alpha(1) A-domain and supports integrin alpha(1)beta(1)-mediated cell adhesion. Triple-helical GFOGER completely inhibits alpha(2) A-domain binding to collagens I and IV and alpha(2)beta(1)-dependent adhesion of platelets and HT 1080 cells to these collagens. It also fully inhibits alpha(1) A-domain binding to collagen I and strongly inhibits alpha(1)beta(1)-mediated adhesion of Rugli cells to this collagen but has little effect on either alpha1 A-domain binding or adhesion of Rugli cells to collagen IV. We conclude that the sequence GFOGER represents a high-affinity binding site in collagens I and IV for alpha(2)beta(1) and in collagen I for alpha(1)beta(1). Other high-affinity sites in collagen IV mediate its recognition of alpha(1)beta(1).

Identification of heparin as a ligand for the A-domain of Plasmodium falciparum thrombospondin-related adhesion protein.

Thrombospondin-related adhesion protein (TRAP) is a Plasmodium falciparum transmembrane protein that is expressed within the micronemes of sporozoites, and is implicated in host cell invasion and motility. Contained within the extracellular region of TRAP is an A-domain, a module found in a number of membrane, plasma and matrix proteins, that is often involved in ligand recognition. In order to determine the role of the TRAP A-domain, it has been expressed as a glutathione S-transferase fusion protein and its ligand binding compared with that of other characterised glutathione S-transferase A-domain fusion proteins. Using a solid phase assay to screen for binding to known A-domain ligands, the TRAP A-domain was found to bind heparin. Binding to heparin appeared to be specific as it was saturable, and was inhibited by soluble heparin, fucoidan and dextran sulfate, but not by other negatively charged sulfated glycosaminoglycans such as chondroitin sulfates. Furthermore, unlike some A-domain ligand interactions, the A-domain of both TRAP and the leukocyte integrin, Mac-1, bound to heparin in the absence of divalent cations. It has been shown previously that another domain within TRAP, which is homologous to region II-plus of circumsporozoite protein, binds to sulfatide and to heparan sulfate on the immortalised hepatocyte line HepG2. The TRAP A-domain also bound to sulfatide and to HepG2 cells. Thus the A-domain shares certain binding properties already attributed to the region II-plus-like domain of TRAP, and may contribute to the binding of TRAP to heparan sulfate on hepatocytes.

Identification in collagen type I of an integrin alpha2 beta1-binding site containing an essential GER sequence.

The collagen type I-derived fragment alpha1(I)CB3 is known to recognize the platelet collagen receptor integrin alpha2beta1 as effectively as the parent collagen, although it lacks platelet-aggregatory activity. We have synthesized the fragment as seven overlapping peptides that spontaneously assemble into triple helices. On the basis of their capacity to bind purified alpha2 beta1 and the recombinant alpha2 A-domain, and their ability to support alpha2 beta1-mediated cell adhesion, we identified two peptides, CB3(I)-5 and -6, which contain an alpha2 beta1 recognition site. Synthesis of the peptide CB3(I)-5/6, containing the overlap sequence between peptides 5 and 6, allowed us to locate the binding site within the 15-residue sequence, GFP*GERGVEGPP*GPA (where P* represents hydroxyproline), corresponding to residues 502-516 of the collagen type I alpha1 chain. The Glu and Arg residues in the GER triplet were found to be essential for recognition since substitution of either residue with Ala caused a loss of alpha2 A-domain binding. By contrast, substitution of the Glu in GVE did not reduce binding, but rather enhanced it slightly. We were unable to detect significant recognition of alpha2 beta1 by the peptide CB3(I)-2 containing the putative alpha2 beta1 recognition sequence DGEA. Peptides CB3(I)-1 to -6, together with peptide CB3(I)-5/6, exhibited good platelet-aggregatory activity, in some cases better than collagen. However, peptide CB3(I)-7 was inactive, suggesting the presence of an inhibitory element that might account for the lack of aggregatory activity of the parent alpha1(I)CB3 fragment.

Effects of collagenase-cleavage of type I collagen on alpha2beta1 integrin-mediated cell adhesion.

In this paper we show that collagenase-3 cleavage of type I collagen has a marked effect on alpha2beta1 integrin-mediated interactions with the collagen fragments generated. Isolated alpha2beta1 integrin and alpha2 integrin A-domain were found to bind to both native collagen and native 3/4 fragment and, to a lesser degree, native 1/4 fragment. Whole integrin and integrin A-domain binding were lost after heat denaturation of the collagen fragments. At physiological temperature, cell adhesion to triple-helical 3/4 fragment via alpha2beta1 integrin was still possible; however, no alpha2beta1 integrin-mediated adhesion to the 1/4 fragment was observed. Unwinding of the collagen fragment triple helices by heating to physiological temperatures prior to adsorption to plastic tissue culture plates resulted in total abrogation of HT1080 cell attachment to either fragment. These results provide significant evidence in support of a role for matrix-metalloproteinase cleavage of the extracellular matrix in modifying cell-matrix interactions.

Surface loops adjacent to the cation-binding site of the complement factor B von Willebrand factor type A module determine C3b binding specificity.

The interaction of factor B with C3b deposited on the surface of pathogens is the first step in the activation of the alternative complement pathway. The role of the von Willebrand factor type A (VWFA) module of factor B in this interaction has been investigated by generating three chimeras, Ch1-Ch3, in which surface loops of the VWFA module flanking the cation-binding residues were replaced by the corresponding sequences of C2, a factor B-like molecule which does not bind C3b. The location of the three loops was inferred from a homology model based on the structure of the integrin alphaM VWFA module [Ch1, betaA-alpha1 loop: Ch2, alpha3-alpha4 loop; and Ch3, betaD-alpha5 loop; Lee, J.-O., et al. (1995b) Cell 80, 631-638]. The function of the chimeras was studied by means of hemolytic assays and assays of the individual steps of the alternative complement pathway, i.e., binding to the C3b analogue cobra venom factor and factor D cleavage. These experiments showed that Ch1 and Ch3 define regions that are involved in C3b binding whereas Ch2 does not appear to be involved in binding specificity. The inability of Ch1 to register the enhancement of cobra venom factor binding normally seen after factor D cleavage suggested that the betaA-alpha1 loop mediates the conformational regulation of ligand binding affinity. Homology modeling of the chimeras has been used to visualize the surface structures which potentially define the C3b binding site.

Molecular characterisation of integrin-procollagen C-propeptide interactions.

The carboxyl-terminal propeptide of type I procollagen (CPP-I) plays a key role in regulation of collagen fibrillogenesis, and may exert feedback control of collagen biosynthesis. We have previously shown that CPP-I is a ligand for the integrin alpha2beta1 [Weston, S. A., Hulmes, D. J. S., Mould, A. P., Watson, R. B. & Humphries, M. J. (1994) Identification of the integrin alpha2beta1 as a cell surface receptor for the C-propeptide of type I procollagen, J. Biol. Chem. 269, 20982-20986] suggesting that some of the phenotypic effects of C-propeptides may be mediated by adhesion receptors. Here we have extended this work to study the molecular basis of this interaction. We have broadened the ligand range by demonstrating that the C-terminal propeptide of type II procollagen supports alpha2beta1-mediated binding of NHS human fibroblasts in cell attachment assays. Also, we have used function-blocking antibodies in cell attachment and solid-phase binding assays with purified integrin to expand the CPP-I receptor family, showing that integrin alpha1beta1 is also a receptor for CPP-I. Integrin alpha-subunit A-domains are known to be major ligand-binding sites and recombinant alpha1 and alpha2 subunit A-domains were able to bind CPP-I. Finally we have shown that peptides corresponding to potential integrin-binding sequences in CPP-I do not mediate integrin-CPP-I adhesion. Taken together, these studies indicate that the interactions between C-propeptides and integrins are more numerous than previously reported, that C-propeptides are a new class of molecule which bind to A-domains, and that the integrin-C-propeptide interaction does not utilise established peptide motifs.

The integrin alpha1 A-domain is a ligand binding site for collagens and laminin.

The integrin alpha1beta1 is a cell surface receptor for collagens and laminin. The alpha1 subunit contains an A-domain, and the A-domains of other integrins are known to mediate ligand binding. To determine the role of the alpha1 A-domain in ligand binding and the extent to which it reproduced the ligand binding activity and specificity of the parent molecule, we produced recombinant alpha1 A-domain and tested its ability to bind collagens and laminin. In solid phase assays, the A-domain from alpha1 was found to bind to collagen I, collagen IV, and laminin in a largely cation-dependent manner. The alpha2 A-domain, from the alpha2beta1 integrin, also bound to these ligands, but the binding hierarchy differed from that seen for alpha1. This is the first demonstration of laminin binding by A-domains. Specificity of A-domain-ligand binding was further investigated using the triple-helical proteolytic fragment of collagen IV, CB3, and its subfragments, F1 and F4. alpha1 A-domain bound to all three fragments, while the alpha2 A-domain bound CB3 less well and exhibited little binding to F1 and no binding to F4. These differences mirror previous reports of distinct integrin binding sites in collagen IV and for the first time identify a limited proteolytic fragment of a ligand that contains integrin A-domain binding activity. To gain insight into the contribution that the A-domain makes to ligand binding within the whole integrin heterodimer, we measured binding constants for A-domain-collagen interactions using surface plasmon resonance biosensor technology. The values obtained were similar to those reported for intact integrin binding, suggesting that the A-domain is the major collagen binding site in the alpha1beta1 and alpha2beta1 integrins.

A structure prediction for the ligand-binding region of the integrin beta subunit: evidence for the presence of a von Willebrand factor A domain.

The integrins are a family of cell surface receptors that mediate biologically important adhesive interactions. Integrin-ligand binding has been extensively studied because of the potential for the development of anti-adhesive therapies, but the molecular basis of this interaction is still poorly understood. A conserved region near the N-terminus of the beta subunit appears to be of particular importance in ligand binding, but to date this domain has not been expressed in isolation. As a prelude to expression and potential structure determination, we have performed a detailed structure prediction for this region. Primary, secondary and tertiary structure analyses indicate that the region folds into a von Willebrand factor A-domain, thereby potentially placing a previously characterised module at the centre of a key functional region.

The A-domain of integrin alpha 2 binds specifically to a range of collagens but is not a general receptor for the collagenous motif.

Integrin alpha 2 beta 1 is a major cellular receptor for collagens, but the molecular basis of its function is unknown. The alpha 2 subunit contains a von Willebrand factor A-domain (I-domain) in its N-terminal region, and it has been demonstrated recently that this domain binds specifically to collagen I. This interaction requires divalent cations (e.g., Mg2+) and native collagen conformation, as does binding of the parent integrin to collagen. The alpha 2 A-domain therefore has a number of functional similarities to the parent integrin, alpha 2 beta 1. However, while sequence specificity has been demonstrated for the parent integrin, no such observations have been made for the A-domain. In particular, it is not known whether the A-domain is responsible for sequence-specific recognition of collagens or whether it binds to the genetic collagenous motif. To investigate the ligand specificity of the alpha 2 A-domain, its binding to a range of collagenous ligands has been studied, with cation dependence, collagen triple-helicity, and inhibition by function-blocking antibodies as criteria for specificity. Binding of the parent integrin was examined for comparison. The alpha 2 A-domain was found to bind specifically to collagens I, II, IV and XI. The complement component C1q has a collagenous domain but this was unable to support specific binding of alpha 2 A-domain or alpha 2 beta 1. Furthermore, synthetic triple-helical collagenous peptides failed to act as specific ligands. In conclusion, the alpha 2 A-domain binds specifically to a range of extracellular matrix collagens, but it is not a receptor for all collagenous triple helices. By inference, these findings indicate the existence of an integrin-specific sequence motif within collagenous ligands recognised by the alpha 2 A-domain.

Protein secondary structure prediction by the analysis of variation and conservation in multiple alignments.

A number of methods exist for the prediction of protein secondary structure from primary sequence. One method identifies variable charged and conserved hydrophobic residues within large multiple alignments as a means of indicating outside and inside sites respectively in the protein structure. These sites are then manually fitted to secondary structure templates to generate a secondary structure prediction. Using the existing theoretical bases of this method, we present an algorithm (STAMA) which automatically carries out the initial variation/conservation analysis of the alignment. We also test the accuracy of complete predictions carried out by manual fitting of the STAMA-derived assignments to structure templates, using five large multiple alignments each including a protein of known structure. The method was found on average to predict only 57% of residues in the correct secondary structure, and was only as accurate as predictions carried out using the established and automated method of Garnier, Osguthorpe and Robson (1978) applied to a single sequence. When used in conjunction with other secondary structure prediction methods, however, the resulting consensus predictions were found to be very accurate, with 78% of the elements (alpha helices or beta strands) for which a consensus could be obtained being predicted correctly. The algorithm presented here, plus the assessment of the accuracy of prediction generated by this method, should enable this predictive approach to receive informed general use.

Effect of beta-mercaptoethanol on the detection of biotinylated proteins.

Biotinylated proteins were visualized by enhanced chemiluminescence (ECL) or conventional autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein transfer onto nitrocellulose. Soaking polyacrylamide gels run under nonreducing conditions in beta-mercaptoethanol (2-ME) prior to protein transfer onto nitrocellulose resulted in a 2- to 10-fold augmentation of the resultant signal. This enhancement was observed for both disulfide- and nondisulfide-bonded proteins. Furthermore, 2-ME had no effect on either the activity of the extravidin-horse-radish peroxidase conjugate, used to detect biotin moieties, or the net protein transfer onto nitrocellulose. Thus, we propose that amplification of either ECL or gamma emission following 2-ME treatment is due to its ability to modify protein conformation, which in turn provides greater access of avidin to biotin.

A secondary structure model of the integrin alpha subunit N-terminal domain based on analysis of multiple alignments.

The integrins are alpha/beta heterodimeric proteins which mediate cell-matrix and cell-cell interactions. Current data indicate that the N-terminal moiety of the alpha subunit is involved in ligand binding. This region of the receptor is made up of a seven-fold repeated sequence of unknown structure which contains EF-hand-like putative divalent cation-binding sites. Recent studies have shown that multiple sequence alignments can be analysed to yield secondary structure predictions. Therefore, to obtain a model structure for the integrin alpha subunit N-terminal domain repeat, a large alignment of the seven repeats from sixteen integrin sequences was generated. Two methods of analysis were used: First, Chou and Fasman and Garnier, Osguthorpe and Robson predictions were carried out for individual sequences and the consensus predictions derived. Consensus hydrophobicity and chain flexibility data were also used to provide additional data. Second, sites of conservation and variation were analysed by a computer program STAMA (STructure After Multiple Alignment) to yield a secondary structure prediction. The two analyses gave essentially the same predicted structure: undefined region, loop, alpha-helix, beta-strand, divalent cation-binding loop, beta-strand, putative turn, loop, beta-strand. This is the first model structure to be presented for an integrin domain. Its implications for integrin function are discussed.

Conformation dependence of integrin-type II collagen binding. Inability of collagen peptides to support alpha 2 beta 1 binding, and mediation of adhesion to denatured collagen by a novel alpha 5 beta 1-fibronectin bridge.

The mechanism of interaction of chondrocytic cells with cartilage-specific type II collagen has been examined using HCS-2/8 human chondrosarcoma cells as a model system. By the criteria of specific collagen secretion and integrin expression profile, HCS-2/8 have a similar differentiated phenotype to normal chondrocytes and are therefore a good model system. HCS-2/8 cells were able to attach and spread on both native and heat-denatured pepsinised type II collagen, and assays using denatured cyanogen bromide fragments apparently localised the major cell binding site to the CB10 fragment. However, when they were used as soluble inhibitors, cyanogen bromide fragments were found to block adhesion to denatured collagen, but had no effect on either attachment or spreading on the native molecule. The inability of cyanogen bromide fragments to reproduce the cell binding site of native collagen demonstrated a strict dependence on collagen conformation. This was also reflected in the receptors that were employed by HCS-2/8 cells for binding to type II collagen: binding to native collagen was mediated by the integrin alpha 2 beta 1 while binding to denatured collagen was mediated by a novel alpha 5 beta 1-fibronectin bridge. The identification of this bridge adds to the mechanisms by which cells can bind to denatured collagens. The previously characterised KDGEA active site peptide from type I collagen was found to be inactive as an inhibitor of type II collagen-mediated adhesion. The implications of these findings for the strategies used to identify adhesive active sites within collagens are discussed. In particular, these data suggest that, unlike other integrin ligands, a synthetic peptide-based approach is not suitable for the identification of collagen active sites.