Danny S. Tuckwell

The Collagen-binding A-domains of Integrins α1β1 and α2β1Recognize the Same Specific Amino Acid Sequence, GFOGER, in Native (Triple-helical) Collagens

We have previously assigned an integrin α2β1-recognition site in collagen I to the sequence, GFOGERGVEGPOGPA (O = Hyp), corresponding to residues 502–516 of the α1(I) chain and located in the fragment α1(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 α2β1-dependent cell adhesion and exhibits divalent cation-dependent binding of isolated α2β1 and recombinant α2A-domain, being at least as active as the parent collagen. Replacement of E by D causes loss of recognition. The same sequence binds integrin α1 A-domain and supports integrin α1β1-mediated cell adhesion. Triple-helical GFOGER completely inhibits α2 A-domain binding to collagens I and IV and α2β1-dependent adhesion of platelets and HT 1080 cells to these collagens. It also fully inhibits α1 A-domain binding to collagen I and strongly inhibits α1β1-mediated adhesion of Rugli cells to this collagen but has little effect on either α1 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 α2β1 and in collagen I for α1β1. Other high-affinity sites in collagen IV mediate its recognition of α1β1.

Identification and characterization of anaerobic gut fungi using molecular methodologies based on ribosomal ITS1 and 18S 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.

A structure prediction for the ligand-binding region of the integrin β 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 β 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.

Micromolar Ca2+ Concentrations Are Essential for Mg2+-dependent Binding of Collagen by the Integrin α2β1 in Human Platelets

Integrin receptor α2β1 requires micromolar Ca2+ to bind to collagen and to the peptide GPC(GPP)5GFOGER(GPP)5GPC (denoted GFOGER-GPP, where O represents hydroxyproline), which contains the minimum recognition sequence for the collagen-binding α2I-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 α2β1 in the presence of 2 mmMg2+. However, we show here that this interaction was abolished in the presence of 25 μm EGTA. Adhesion of Glanzmanns thrombasthenic platelets, which lack the fibrinogen receptor αIIbβ3, was also inhibited by micromolar EGTA. Mg2+-dependent adhesion of platelets was restored by the addition of 10 μmCa2+, but millimolar Ca2+ was inhibitory. Binding of isolated α2β1 to GFOGER-GPP was 70% inhibited by 50 μm EGTA but, as with intact platelets, was fully restored by the addition of micromolar Ca2+. 2 mm Ca2+ did not inhibit binding of isolated α2β1 to collagen or to GFOGER-GPP. Binding of recombinant α2 I-domain was not inhibited by EGTA, nor did millimolar Ca2+ inhibit binding. Our data suggest that high affinity Ca2+ binding to α2β1, outside the I-domain, is essential for adhesion to collagen. This is the first demonstration of a Ca2+ requirement in α2β1function.

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.

The alpha2beta1 integrin inhibitor rhodocetin binds to the A-domain of the integrin alpha2 subunit proximal to the collagen-binding site.

Rhodocetin is a snake venom protein that binds to alpha2beta1 integrin, inhibiting its interaction with its endogenous ligand collagen. We have determined the mechanism by which rhodocetin inhibits the function of alpha2beta1. The interaction of alpha2beta1 with collagen and rhodocetin differed: Ca(2+) ions and slightly acidic pH values increased the binding of alpha2beta1 integrin to rhodocetin in contrast with their attenuating effect on collagen binding, suggesting that rhodocetin preferentially binds to a less active conformation of alpha2beta1 integrin. The alpha2A-domain [von Willebrand factor domain A homology domain (A-domain) of the integrin alpha2 subunit] is the major site for collagen binding to alpha2beta1. Recombinant alpha2A-domain bound rhodocetin, demonstrating that the A-domain is also the rhodocetin-binding domain. Although the interaction of alpha2beta1 with rhodocetin is affected by altering divalent cations, the interaction of the A-domain was divalent-cation-independent. The rhodocetin-binding site on the alpha2A-domain was mapped first by identifying an anti-alpha2 antibody that blocked rhodocetin binding and then mapping the epitope of the antibody using human-mouse alpha2A-domain chimaeras; and secondly, by binding studies with alpha2A-domain, which bear point mutations in the vicinity of the mapped epitope. In this way, the rhodocetin-binding site was identified as the alpha3-alpha4 loop plus adjacent alpha-helices. This region is known to form part of the collagen-binding site, thus attaining a mainly competitive mode of inhibition by rhodocetin.

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.

Identification and analysis of collagen α1(XXI), a novel member of the FACIT collagen family

The FACIT collagens bind to the surface of collagen fibrils linking them with other matrix molecules. Bioinformatics analysis of cDNA clone DKFZp564B052 showed that it resembled the FACIT collagens and was therefore designated collagen alpha 1(XXI). Phylogenetic analyses of the N-terminal NC3 domains of alpha 1(XXI) and other FACIT collagens showed that (i) alpha 1(XXI) clustered with the FACIT collagens; (ii) collagen alpha 1(XXI) arose before the divergence of alpha 1(XII), alpha 1(XIV) and alpha 1(XX); (iii) collagen alpha 1(XIV) derived from the C-terminal region of the NC3 domain of a collagen alpha 1(XII)-like molecule; and (iv) collagen alpha 1(XX) derived from a collagen alpha 1(XIV)-like molecule. This study provides a framework for the evolution of the FACIT collagens which will be of value in linking NC3 domains with their functions.

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.

Identification of a novel class of annexin genes

The annexins are a family of calcium‐ and phospholipid‐binding proteins that have been widely studied in animals. Investigation of annexins in the fungus Aspergillus fumigatus identified a novel annexin‐like gene (ANXC4) as well as two conventional annexins (ANXC3.1 and ANXC3.2). The genes were initially identified by bioinformatics, and sequences were then determined experimentally. Reverse transcription polymerase chain reaction indicated that all three genes were expressed. ANXC4 lacked calcium‐binding consensus sequences and had a 553 residue N‐terminal tail. However, bioinformatics indicated that ANXC4 is an annexin and homologues were identified in other filamentous fungi. ANXC4 therefore represents a new grouping within the annexin family.