Collagen-binding proteins: insights from the Collagen Toolkits.

The Collagen Toolkits are libraries of 56 and 57 triple-helical synthetic peptides spanning the length of the collagen II and collagen III helices. These have been used in solid-phase binding assays to locate sites where collagen receptors and extracellular matrix components bind to collagens. Truncation and substitution allowed exact binding sites to be identified, and corresponding minimal peptides to be synthesised for use in structural and functional studies. 170 sites where over 30 proteins bind to collagen II have been mapped, providing firm conclusions about the amino acid distribution within such binding sites. Protein binding to collagen II is not random, but displays a periodicity of approximately 28 nm, with several prominent nodes where multiple proteins bind. Notably, the vicinity of the collagenase-cleavage site in Toolkit peptide II-44 is highly promiscuous, binding over 20 different proteins. This may reflect either the diverse chemistry of that locus or its diverse function, together with the interplay between regulatory binding partners. Peptides derived from Toolkit studies have been used to determine atomic level resolution of interactions between collagen and several of its binding partners and are finding practical application in tissue engineering

Structural constraints on the evolution of the collagen fibril: convergence on a 1014-residue COL domain.

Type I collagen is the fundamental component of the extracellular matrix. Its α1 gene is the direct descendant of ancestral fibrillar collagen and contains 57 exons encoding the rod-like triple-helical COL domain. We trace the evolution of the COL domain from a primordial collagen 18 residues in length to its present 1014 residues, the limit of its possible length. In order to maintain and improve the essential structural features of collagen during evolution, exons can be added or extended only in permitted, non-random increments that preserve the position of spatially sensitive cross-linkage sites. Such sites cannot be maintained unless the twist of the triple helix is close to 30 amino acids per turn. Inspection of the gene structure of other long structural proteins, fibronectin and titin, suggests that their evolution might have been subject to similar constraints.The work was funded by a project grant to D.A.S. and R.W.F. from British Heart Foundation (PG/08/011/24416). R.W.F. was supported by BHF Programme grant nos. (RG/09/003/27122 and RG/15/4/31268).This is the final version of the article. It first appeared from Royal Society Publishing via http://dx.doi.org/10.1098/rsob.14022

Integrins in GtoPdb v.2023.1

Integrins are unusual signalling proteins that function to signal both from the extracellular environment into the cell, but also from the cytoplasm to the external of the cell. The intracellular signalling cascades associated with integrin activation focus on protein kinase activities, such as focal adhesion kinase and Src. Based on this association between extracellular signals and intracellular protein kinase activity, we have chosen to include integrins in the 'Catalytic receptors' section of the database until more stringent criteria from NC-IUPHAR allows precise definition of their classification.Integrins are heterodimeric entities, composed of α and β subunits, each 1TM proteins, which bind components of the extracellular matrix or counter-receptors expressed on other cells. One class of integrin contains an inserted domain (I) in its α subunit, and if present (in α1, α2, α10, α11, αD, αE, αL, αM and αX), this I domain contains the ligand binding site. All β subunits possess a similar I-like domain, which has the capacity to bind ligand, often recognising the RGD motif. The presence of an α subunit I domain precludes ligand binding through the β subunit. Integrins provide a link between ligand and the actin cytoskeleton (through typically short intracellular domains). Integrins bind several divalent cations, including a Mg2+ ion in the I or I-like domain that is essential for ligand binding. Other cation binding sites may regulate integrin activity or stabilise the 3D structure. Integrins regulate the activity of particular protein kinases, including focal adhesion kinase and integrin-linked kinase. Cellular activation regulates integrin ligand affinity via inside-out signalling and ligand binding to integrins can regulate cellular activity via outside-in signalling.Several drugs that target integrins are in clinical use including: (1) abciximab (αIIbβ3) for short term prevention of coronary thrombosis, (2) vedolizumab (α4β7) to reduce gastrointestinal inflammation, and (3) natalizumab (α4β1) in some cases of severe multiple sclerosis

Integrins (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Integrins are unusual signalling proteins that function to signal both from the extracellular environment into the cell, but also from the cytoplasm to the external of the cell. The intracellular signalling cascades associated with integrin activation focus on protein kinase activities, such as focal adhesion kinase and Src. Based on this association between extracellular signals and intracellular protein kinase activity, we have chosen to include integrins in the 'Catalytic receptors' section of the database until more stringent criteria from NC-IUPHAR allows precise definition of their classification.Integrins are heterodimeric entities, composed of α and β subunits, each 1TM proteins, which bind components of the extracellular matrix or counter-receptors expressed on other cells. One class of integrin contains an inserted domain (I) in its α subunit, and if present (in α1, α2, α10, α11, αD, αE, αL, αM and αX), this I domain contains the ligand binding site. All β subunits possess a similar I-like domain, which has the capacity to bind ligand, often recognising the RGD motif. The presence of an α subunit I domain precludes ligand binding through the β subunit. Integrins provide a link between ligand and the actin cytoskeleton (through typically short intracellular domains). Integrins bind several divalent cations, including a Mg2+ ion in the I or I-like domain that is essential for ligand binding. Other cation binding sites may regulate integrin activity or stabilise the 3D structure. Integrins regulate the activity of particular protein kinases, including focal adhesion kinase and integrin-linked kinase. Cellular activation regulates integrin ligand affinity via inside-out signalling and ligand binding to integrins can regulate cellular activity via outside-in signalling.Several drugs that target integrins are in clinical use including: (1) abciximab (αIIbβ3) for short term prevention of coronary thrombosis, (2) vedolizumab (α4β7) to reduce gastrointestinal inflammation, and (3) natalizumab (α4β1) in some cases of severe multiple sclerosis

The tyrosine kinase inhibitors methyl 2,5-dihydroxycinnamate and genistein reduce thrombin-evoked tyrosine phosphorylation and Ca2+ entry in human platelets

AbstractPlatelet activation is associated with the phosphorylation of a number of platelet proteins at tyrosine residues. The significance of this is unknown. Here we have investigated the effects of two tyrosine kinase inhibitors, methyl 2,5-dihydroxycinnamate and genistein, on thrombin-evoked protein tyrosine phosphorylation and Ca2+ signal generation in fura-2-loaded human platelets. Both compounds inhibited thrombin-evoked tyrosine phosphorylation and reduced the elevation of [Ca2+], in the presence, but not the absence, of external Ca2+. This suggested a selective inhibition of thrombin-evoked Ca2+ entry but not release from internal stores. Both compounds also reduced thrombin-evoked Mn2+ entry. In contrast, selective blockade of protein kinase C with Ro 31/8220-002 potentiated the thrombin-evoked Ca2+ signal. These data are compatible with a role for protein tyrosine phosphorylation contributing to thrombin-evoked Ca2+ entry in human platelets

Activation-induced changes in platelet surface receptor expression and the contribution of the large-platelet subpopulation to activation.

Objective:Platelet surface receptors are also present subcellularly in organelle membranes and can be expressed on the surface upon platelet activation. However, some receptors were reported to be decreased after activation. We analyzed the mechanism of activation-dependent expression for different receptors. Methods:Flow cytometry using platelet-rich plasma or washed platelets was used to analyze receptor-expression changes after platelet activation by glycoprotein (GP) VI-specific agonists, crosslinked collagen-related peptide (CRP-XL) and convulxin (Cvx), and thrombin. Platelets prelabeled with fluorescent antibody specific for a receptor were allowed to adhere on immobilized collagen or fibrinogen and post-stained with antibody against the same receptor labeled with another fluorophore, allowing us to differentiate preexisting receptors from newly expressed receptors. Results:Surface expression of αIIbβ3 increased in CRP-XL-, Cvx-, or thrombin-stimulated platelets, but GPIb decreased due to shedding and internalization. Both total and dimeric GPVI increased in thrombin-induced platelets, but decreased in platelets stimulated by Cvx, as a result of internalization. The larger platelets showed a greater increase in surface receptor (α2β1, αIIbβ3, GPVI, GPIb) expression upon activation compared to the smaller ones. Pre- and postlabeling with antibody specific for the same receptor, but conjugated with different fluorophores, allowed us to differentiate the receptors expressed on the surface of resting platelets from receptors newly exposed to the surface upon platelet activation. Conclusions:Increased receptor expressions after activation are mainly manifested in the larger platelets. On platelets adhered on fibrinogen, the newly expressed receptors, especially GPVI, are localized in the lamellipodia of the spread platelets

Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase.

The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.SK and KR were supported by grants from the Swedish Cancer Foundation, the Swedish Research Council, the Alfred Österlund Foundation, the Crafoord Foundation, the Magnus Bergvall Foundation, and the Åke Wiberg Foundation; AB, DB and RWF by grants from the Wellcome Trust (094470/Z/10/Z) and British Heart Foundation (RG/15/4/31268).This is the final version of the article. It first appeared from the American Society for Biochemistry and Molecular Biology] via http://dx.doi.org/10.1074/jbc.M115.69340

Structural and functional analysis of two small leucine-rich repeat proteoglycans, fibromodulin and chondroadherin.

The small leucine-rich proteoglycans (SLRPs) are important regulators of extracellular matrix assembly and cell signalling. We have determined crystal structures at ~2.2Å resolution of human fibromodulin and chondroadherin, two collagen-binding SLRPs. Their overall fold is similar to that of the prototypical SLRP, decorin, but unlike decorin neither fibromodulin nor chondroadherin forms a stable dimer. A previously identified binding site for integrin α2β1 maps to an α-helix in the C-terminal cap region of chondroadherin. Interrogation of the Collagen Toolkits revealed a unique binding site for chondroadherin in collagen II, and no binding to collagen III. A triple-helical peptide containing the sequence GAOGPSGFQGLOGPOGPO (O is hydroxyproline) forms a stable complex with chondroadherin in solution. In fibrillar collagen I and II, this sequence is aligned with the collagen cross-linking site KGHR, suggesting a role for chondroadherin in cross-linking

Structural studies of the MMP-3 interaction with triple-helical collagen introduce new roles for the enzyme in tissue remodelling

Abstract: Matrix metalloproteinase-3 (MMP-3) participates in normal extracellular matrix turnover during embryonic development, organ morphogenesis and wound healing, and in tissue-destruction associated with aneurysm, cancer, arthritis and heart failure. Despite its inability to cleave triple-helical collagens, MMP-3 can still bind to them, but the mechanism, location and role of binding are not known. We used the Collagen Toolkits, libraries of triple-helical peptides that embrace the entire helical domains of collagens II and III, to map MMP-3 interaction sites. The enzyme recognises five sites on collagen II and three sites on collagen III. They share a glycine-phenylalanine-hydroxyproline/alanine (GFO/A) motif that is recognised by the enzyme in a context-dependent manner. Neither MMP-3 zymogen (proMMP-3) nor the individual catalytic (Cat) and hemopexin (Hpx) domains of MMP-3 interact with the peptides, revealing cooperative binding of both domains to the triple helix. The Toolkit binding data combined with molecular modelling enabled us to deduce the putative collagen-binding mode of MMP-3, where all three collagen chains make contacts with the enzyme in the valley running across both Cat and Hpx domains. The observed binding pattern casts light on how MMP-3 could regulate collagen turnover and compete with various collagen-binding proteins regulating cell adhesion and proliferation

Impact of UV- and carbodiimide-based crosslinking on the integrin-binding properties of collagen-based materials.

Collagen constructs are widely used for tissue engineering. These are frequently chemically crosslinked, using EDC, to improve their stability and tailor their physical properties. Although generally biocompatible, chemical crosslinking can modify crucial amino acid side chains, such as glutamic acid, that are involved in integrin-mediated cell adhesion. Instead UV crosslinking modifies aromatic side chains. Here we elucidate the impact that EDC, in combination with UV, exerts on the activity of integrin-binding motifs. By employing a model cell line that exclusively utilises integrin α2β1, we found that whilst EDC crosslinking modulated cell binding, from cation-dependent to cation-independent, UV-mediated crosslinking preserved native-like cell binding, proliferation and surface colonisation. Similar results were observed using a purified recombinant I-domain from integrin α1. Conversely, binding of the I-domain from integrin α2 was sensitive to UV, particularly at low EDC concentrations. Therefore, from this in vitro study, it appears that UV can be used to augment EDC whist retaining a specific subset of integrin-binding motifs in the native collagen molecule. These findings, delineating the EDC- and UV-susceptibility of cell-binding motifs, permit controlled cell adhesion to collagen-based materials through specific integrin ligation in vitro. However, in vivo, further consideration of the potential response to UV wavelength and dose is required in the light of literature reports that UV initiated collagen scission may lead to an adverse inflammatory response. STATEMENT OF SIGNIFICANCE: Recently, there has been rapid growth in the use of extracellular matrix-derived molecules, and in particular collagen, to fabricate biomaterials that replicate the cellular micro-environment. Often chemical or physical crosslinkers are required to enhance the biophysical properties of these materials. Despite extensive use of these crosslinkers, the cell-biological consequences have not been ascertained. To address this, we have investigated the integrin-binding properties of collagen after chemically crosslinking with EDC and physically crosslinking with UV-irradiation. We have established that whilst EDC crosslinking abates all of the integrin binding sites in collagen, UV selectively inhibits interaction with integrin-α2 but not -α1. By providing a mechanistic model for this behaviour, we have, for the first time, defined a series of crosslinking parameters to systematically control the interaction of collagen-based materials with defined cellular receptors.The authors would like to thank the EPSRC [Fellowship EP/N019938/1] the ERC [Advanced Grant 320598 3D-E] and the British Heart Foundation [Special Project SP/15/7/31561] for providing financial support for this project. DVB was funded by the People's Programme of the EU 7th Framework Programme [RAE no: PIIF-GA-2013-624904]