C G Knight

University of Cambridge, Cambridge, ENG, United Kingdom

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Publications (43)204.63 Total impact

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    ABSTRACT: Collagen-related peptide is a selective agonist for the platelet collagen receptor Glycoprotein VI. The triple helical peptide contains ten GPO triplets/strand (single letter amino acid nomenclature, where O is hydroxyproline) and so over-represents GPO compared with native collagen sequence. To investigate the ability of Glycoprotein VI to recognize GPO triplets in a setting more representative of the collagens, we synthesized a set of triple helical peptides containing fewer GPO triplets, varying their number and spacing within an inert (GPP)n backbone. The adhesion of recombinant human Glycoprotein VI ectodo-main, like that of human platelets, to these peptides increased with their GPO content, and platelet adhesion was abolished by the specific anti-Glycoprotein VI-blocking antibody, 10B12. Platelet aggregation and protein tyrosine phosphorylation were induced only by cross-linked peptides and only those that contained two or more GPO triplets. Such peptides were less potent than cross-linked collagen-related peptide. Our data suggest that both the sequences GPOGPO and GPO.........GPO represent functional Glycoprotein VI recognition motifs within collagen. Furthermore, we propose that the (GPO)4 motif can support simultaneous binding of two glycoprotein VI molecules, in either a parallel or anti-parallel stacking arrangement, which could play an important role in activation of signaling.
    Journal of Biological Chemistry 02/2007; 282(2):1296-304. · 4.60 Impact Factor
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    ABSTRACT: We report the development of an expression system for the production of soluble, calmodulin (CaM)-tagged proteins in Drosophila Schneider S2 cells and the subsequent use of these proteins for the selection of phage displayed antibodies. The CaM-tag permitted the purification of recombinant protein to >90% purity in a single step at yields of >20 mg/l. Using platelet glycoprotein VI (GP6) as a model, we demonstrated that the recombinant CaM-tagged protein was post-translationally N-glycosylated and had identical ligand specificity to native protein. A novel selection strategy, exploiting the CaM tag, was then used to isolate four single chain Fv fragments (scFvs) specific for GP6 from a non-immune phage display library. In contrast to other selection methods, which can result in antibodies that do not recognise native protein, all of the scFvs we selected bound cell surface expressed GP6. In conclusion, the production of CaM-tagged proteins in Drosophila Schneider S2 cells and the selection strategy reported here offer advantages over previously published methods, including simple culture conditions, rapid protein purification, specific elution of phage antibodies and preferential selection of phage antibodies that recognise native, cell surface expressed protein.
    Journal of Immunological Methods 10/2006; 316(1-2):75-83. · 2.01 Impact Factor
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    ABSTRACT: Only three recognition motifs, GFOGER, GLOGER, and GASGER, all present in type I collagen, have been identified to date for collagen-binding integrins, such as alpha(2)beta(1). Sequence alignment was used to investigate the occurrence of related motifs in other human fibrillar collagens, and located a conserved array of novel GER motifs within their triple helical domains. We compared the integrin binding properties of synthetic triple helical peptides containing examples of such sequences (GLSGER, GMOGER, GAOGER, and GQRGER) or the previously identified motifs. Recombinant inserted (I) domains of integrin subunits alpha(1), alpha(2) and alpha(11) all bound poorly to all motifs other than GFOGER and GLOGER. Similarly, alpha(2)beta(1) -containing resting platelets adhered well only to GFOGER and GLOGER, while ADP-activated platelets, HT1080 cells and two active alpha(2)I domain mutants (E318W, locked open) bound all motifs well, indicating that affinity modulation determines the sequence selectivity of integrins. GxO/SGER peptides inhibited platelet adhesion to collagen monomers with order of potency F >/= L >/= M > A. These results establish GFOGER as a high affinity sequence, which can interact with the alpha(2)I domain in the absence of activation and suggest that integrin reactivity of collagens may be predicted from their GER content.
    Journal of Biological Chemistry 11/2004; 279(46):47763-72. · 4.60 Impact Factor
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    ABSTRACT: Glycoprotein (GP) VI is the major receptor responsible for platelet activation by collagen, but the collagen-binding surface of GPVI is unknown. To address this issue we expressed, from insect cells, the immunoglobulin (Ig)-like ectodomains (residues 1-185) of human and murine GPVI, called hD1D2 and mD1D2, respectively. Both proteins bound specifically to collagen-related peptide (CRP), a GPVI-specific ligand, but hD1D2 bound CRP more strongly than did mD1D2. Molecular modeling and sequence comparison identified key differences between hD1D2 and mD1D2. Ten mutant hD1D2s were expressed, of which 4 had human residues replaced by their murine counterpart, and 6 had replacements by alanine. CRP binding studies with these mutants demonstrated that the exchange of lysine at position 59 for the corresponding murine glutamate substantially reduced binding to CRP. The position of lysine59 on the apical surface of GPVI suggests a mode of CRP binding analogous to that used by the related killer cell Ig-like receptors to bind HLA. This surface was confirmed as critical for collagen binding by epitope mapping of an inhibitory phage antibody against GPVI. This anti-GPVI, clone 10B12, gave dose-dependent inhibition of the hD1D2-collagen interaction. Clone 10B12 inhibited activation of platelets by CRP and collagen in aggregometry and thrombus formation by the latter in whole blood perfusion. Antibody 10B12 showed significantly reduced binding to the hD1D2-E59, and, on that basis, the GPVI:10B12 interface was modeled.
    Blood 03/2004; 103(3):903-11. · 9.78 Impact Factor
  • Methods in Molecular Biology 02/2004; 273:349-64. · 1.29 Impact Factor
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    ABSTRACT: We have determined the 1.8A crystal structure of a triple helical integrin-binding collagen peptide (IBP) with sequence (Gly-Pro-Hyp)(2)-Gly-Phe-Hyp-Gly-Glu-Arg-(Gly-Pro-Hyp)(3). The central GFOGER hexapeptide is recognised specifically by the integrins alpha2beta1, alpha1beta1, alpha10beta1 and alpha11beta1. These integrin/collagen interactions are implicated in a number of key physiological processes including cell adhesion, cell growth and differentiation, and pathological states such as thrombosis and tumour metastasis. Comparison of the IBP structure with the previously determined structure of an identical collagen peptide in complex with the integrin alpha2-I domain (IBP(c)) allows the first detailed examination of collagen in a bound and an unbound state. The IBP structure shows a direct and a water-mediated electrostatic interaction between Glu and Arg side-chains from adjacent strands, but no intra-strand interactions. The interactions between IBP Glu and Arg side-chains are disrupted upon integrin binding. A comparison of IBP and IBP(c) main-chain conformation reveals the flexible nature of the triple helix backbone in the imino-poor GFOGER region. This flexibility could be important to the integrin-collagen interaction and provides a possible explanation for the unique orientation of the three GFOGER strands observed in the integrin-IBP(c) complex crystal structure.
    Journal of Molecular Biology 02/2004; 335(4):1019-28. · 3.96 Impact Factor
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    ABSTRACT: We have determined the 1.8 Å crystal structure of a triple helical integrin-binding collagen peptide (IBP) with sequence (Gly-Pro-Hyp)2-Gly-Phe-Hyp-Gly-Glu-Arg-(Gly-Pro-Hyp)3. The central GFOGER hexapeptide is recognised specifically by the integrins α2β1, α1β1, α10β1 and α11β1. These integrin/collagen interactions are implicated in a number of key physiological processes including cell adhesion, cell growth and differentiation, and pathological states such as thrombosis and tumour metastasis. Comparison of the IBP structure with the previously determined structure of an identical collagen peptide in complex with the integrin α2-I domain (IBPc) allows the first detailed examination of collagen in a bound and an unbound state. The IBP structure shows a direct and a water-mediated electrostatic interaction between Glu and Arg side-chains from adjacent strands, but no intra-strand interactions. The interactions between IBP Glu and Arg side-chains are disrupted upon integrin binding. A comparison of IBP and IBPc main-chain conformation reveals the flexible nature of the triple helix backbone in the imino-poor GFOGER region. This flexibility could be important to the integrin–collagen interaction and provides a possible explanation for the unique orientation of the three GFOGER strands observed in the integrin-IBPc complex crystal structure.
    Journal of Molecular Biology 01/2004; · 3.96 Impact Factor
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    ABSTRACT: Integrin alpha2beta1 is the principal adhesive receptor for collagen but platelets also adhere through glycoprotein VI (GPVI). Integrin alphaIIbbeta3 may augment platelet adhesion. We have shown that disulfide exchange is necessary for platelet adhesion to fibrinogen, fibronectin, and collagen. However 2 questions remained: (1) Can activated alphaIIbbeta3 explain the observed role of disulfide exchange in adhesion to collagen, or is this role common to other integrins? (2) Is disulfide dependence specific to the integrin receptors or shared with GPVI? To discriminate adhesive functions of alpha2beta1 from those of alphaIIbbeta3 we used Glanzmann platelets and alphaIIbbeta3-specific antibodies applied to normal platelets. To resolve adhesive events mediated by alpha2beta1 from those of GPVI we used synthetic peptides specific to each receptor. We addressed direct integrin ligation using purified alpha2beta1 and recombinant I domain. We observed the following: adhesion to the alpha2beta1-specific peptide was disulfide-exchange dependent and protein disulfide isomerase (PDI) mediated; membrane-impermeant thiol blockers inhibited alpha2beta1, but not GPVI mediated, adhesion; direct blockade of PDI revealed that it is involved in adhesion through alpha2beta1 but not GPVI; and purified alpha2beta1, but not recombinant I domain, depended on free thiols for ligation. These data suggest that the enzymatically catalyzed adhesion-associated reorganization of disulfide bonds is common to members of the integrin family and specific to this family.
    Blood 10/2003; 102(6):2085-92. · 9.78 Impact Factor
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    ABSTRACT: Three mammalian ADAMTS enzymes, ADAMTS-1, -4 and -5, are known to cleave aggrecan at certain glutamyl bonds and are considered to be largely responsible for cartilage aggrecan catabolism observed during the development of arthritis. We have previously reported that certain catechins, polyphenolic compounds found in highest concentration in green tea (Camellia sinensis), are capable of inhibiting cartilage aggrecan breakdown in an in vitro model of cartilage degradation. We have now cloned and expressed recombinant human ADAMTS-1, -4 and -5 and report here that the catechin gallate esters found in green tea potently inhibit the aggrecan-degrading activity of these enzymes, with submicromolar IC50 values. Moreover, the concentration needed for total inhibition of these members of the ADAMTS group is approximately two orders of magnitude lower than that which is needed to partially inhibit collagenase or ADAM-10 activity. Catechin gallate esters therefore provide selective inhibition of certain members of the ADAMTS group of enzymes and could constitute an important nutritional aid in the prevention of arthritis as well as being part of an effective therapy in the treatment of joint disease and other pathologies involving the action of these enzymes.
    European Journal of Biochemistry 07/2003; 270(11):2394-403. · 3.58 Impact Factor
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    ABSTRACT: The integrins alpha(1)beta(1), alpha(2)beta(1), alpha(10)beta(1), and alpha(11)beta(1) are referred to as a collagen receptor subgroup of the integrin family. Recently, both alpha(1)beta(1) and alpha(2)beta(1) integrins have been shown to recognize triple-helical GFOGER (where single letter amino acid nomenclature is used, O = hydroxyproline) or GFOGER-like motifs found in collagens, despite their distinct binding specificity for various collagen subtypes. In the present study we have investigated the mechanism whereby the latest member in the integrin family, alpha(11)beta(1), recognizes collagens using C2C12 cells transfected with alpha(11) cDNA and the bacterially expressed recombinant alpha(11) I domain. The ligand binding properties of alpha(11)beta(1) were compared with those of alpha(2)beta(1). Mg(2+)-dependent alpha(11)beta(1) binding to type I collagen required micromolar Ca(2+) but was inhibited by 1 mm Ca(2+), whereas alpha(2)beta(1)-mediated binding was refractory to millimolar concentrations of Ca(2+). The bacterially expressed recombinant alpha(11) I domain preference for fibrillar collagens over collagens IV and VI was the same as the alpha(2) I domain. Despite the difference in Ca(2+) sensitivity, alpha(11)beta(1)-expressing cells and the alpha(11) I domain bound to helical GFOGER sequences in a manner similar to alpha(2)beta(1)-expressing cells and the alpha(2) I domain. Modeling of the alpha I domain-collagen peptide complexes could partially explain the observed preference of different I domains for certain GFOGER sequence variations. In summary, our data indicate that the GFOGER sequence in fibrillar collagens is a common recognition motif used by alpha(1)beta(1), alpha(2)beta(1), and also alpha(11)beta(1) integrins. Although alpha(10) and alpha(11) chains show the highest sequence identity, alpha(2) and alpha(11) are more similar with regard to collagen specificity. Future studies will reveal whether alpha(2)beta(1) and alpha(11)beta(1) integrins also show overlapping biological functions.
    Journal of Biological Chemistry 03/2003; 278(9):7270-7. · 4.60 Impact Factor
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    ABSTRACT: The collagen-platelet interaction is central to haemostasis and may be a critical determinant of arterial thrombosis, where subendothelium is exposed after rupture of atherosclerotic plaque. Recent research has capitalized on the cloning of an important signalling receptor for collagen, glycoprotein VI, which is expressed only on platelets, and on the use of collagen-mimetic peptides as specific tools for both glycoprotein VI and integrin alpha 2 beta 1. We have identified sequences, GPO and GFOGER (where O denotes hydroxyproline), within collagen that are recognized by the collagen receptors glycoprotein VI and integrin alpha 2 beta 1 respectively, allowing their signalling properties and specific functional roles to be examined. Triple-helical peptides containing these sequences were used to show the signalling potential of integrin alpha 2 beta 1, and to confirm its important contribution to platelet adhesion. Glycoprotein VI appears to operate functionally on the platelet surface as a dimer, which recognizes GPO motifs that are separated by four triplets of collagen sequence. These advances will allow the relationship between the structure of collagen and its haemostatic activity to be established.
    Biochemical Society Symposium 02/2003; · 2.74 Impact Factor
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    ABSTRACT: ADAMs (a disintegrin and metalloprotease domains) are metalloprotease and disintegrin domain-containing transmembrane glycoproteins with proteolytic, cell adhesion, cell fusion, and cell signaling properties. ADAM8 was originally cloned from monocytic cells, and its distinct expression pattern indicates possible roles in both immunology and neuropathology. Here we describe our analysis of its biochemical properties. In transfected COS-7 cells, ADAM8 is localized to the plasma membrane and processed into two forms derived either by prodomain removal or as remnant protein comprising the extracellular region with the disintegrin domain at the N terminus. Proteolytic removal of the ADAM8 propeptide was completely blocked in mutant ADAM8 with a Glu(330) to Gln exchange (EQ-A8) in the Zn(2+) binding motif (HE(330)LGHNLGMSHD), arguing for autocatalytic prodomain removal. In co-transfection experiments, the ectodomain but not the entire MP domain of ADAM8 was able to remove the prodomain from EQ-ADAM8. With cells expressing ADAM8, cell adhesion to a substrate-bound recombinant ADAM8 disintegrin/Cys-rich domain was observed in the absence of serum, blocked by an antibody directed against the ADAM8 disintegrin domain. Soluble ADAM8 protease, consisting of either the metalloprotease domain or the complete ectodomain, cleaved myelin basic protein and a fluorogenic peptide substrate, and was inhibited by batimastat (BB-94, IC(50) approximately 50 nm) but not by recombinant tissue inhibitor of matrix metalloproteinases 1, 2, 3, and 4. Our findings demonstrate that ADAM8 processing by autocatalysis leads to a potential sheddase and to a form of ADAM8 with a function in cell adhesion.
    Journal of Biological Chemistry 01/2003; 277(50):48210-9. · 4.60 Impact Factor
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    ABSTRACT: The ADAM family of proteases are type I transmembrane proteins with both metalloproteinase and disintegrin containing extracellular domains. ADAMs are implicated in the proteolytic processing of membrane-bound precursors and involved in modulating cell-cell and cell-matrix interactions. ADAM8 (MS2, CD156) has been identified in myeloid and B cells. In this report we demonstrate that soluble ADAM8 is an active metalloprotease in vitro and is able to hydrolyse myelin basic protein and a variety of peptide substrates based on the cleavage sites of membrane-bound cytokines, growth factors and receptors which are known to be processed by metalloproteinases. Interestingly, although ADAM8 was inhibited by a number of peptide analogue hydroxamate inhibitors, it was not inhibited by the tissue inhibitors of metalloproteinases (TIMPs). We also demonstrate that the activity of recombinant soluble ADAM9 (meltrin-gamma, MDC9) lacks inhibition by the TIMPs, but can be inhibited by hydroxamate inhibitors. The lack of TIMP inhibition of ADAM8 and 9 contrasts with other membrane-associated metalloproteinases characterised to date in this respect (ADAM10, 12, 17, and the membrane-type metalloproteinases) which have been implicated in protein processing at the cell surface.
    FEBS Letters 08/2002; 524(1-3):154-8. · 3.34 Impact Factor
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    ABSTRACT: The binding of certain growth factors and cytokines to components of the extracellular matrix can regulate their local availability and modulate their biological activities. We show that mesenchymal cell-derived keratinocyte growth factor (KGF), a key stimulator of epithelial cell proliferation during wound healing, preferentially binds to collagens I, III, and VI. Binding is inhibited in a dose-dependent manner by denatured single collagen chains and collagen cyanogen bromide peptides. This interaction is saturable with dissociation constants of approximately 10(-8) to 10(-9) m and estimated molar ratios of up to three molecules of KGF bound to one molecule of triple helical collagen. Furthermore, collagen-bound KGF stimulated the proliferation of transformed keratinocyte or HaCaT cells. Ligand blotting of collagen-derived peptides points to a limited set of collagenous consensus sequences that bind KGF. By using synthetic collagen peptides, we defined the consensus sequence (Gly-Pro-Hyp)(n) as the collagen binding motif. We conclude that the preferential binding of KGF to the abundant collagens leads to a spatial pattern of bioavailable KGF that is dictated by the local organization of the collagenous extracellular matrix. The defined collagenous consensus peptide or its analogue may be useful in wound healing by increasing KGF bioactivity and thus modulating local epithelial remodeling and regeneration.
    Journal of Biological Chemistry 08/2002; 277(30):26872-8. · 4.60 Impact Factor
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    ABSTRACT: Collagen fibers or a glycoprotein VI-specific collagen-related peptide (CRP-XL) stimulated tyrosine phosphorylation of the focal adhesion kinase, p125(fak) (FAK), in human platelets. An integrin alpha(2)beta(1)-specific triple-helical peptide ligand, containing the sequence GFOGER (single-letter nomenclature, O = Hyp) was without effect. Antibodies to the alpha(2) and beta(1) integrin subunits did not inhibit platelet FAK tyrosine phosphorylation caused by either collagen fibers or CRP-XL. Tyrosine phosphorylation of FAK caused by CRP-XL or thrombin, but not that caused by collagen fibers, was partially inhibited by GR144053F, an antagonist of integrin alpha(IIb)beta(3). The intracellular Ca(2+) chelator, BAPTA, and the protein kinase C inhibitor, Ro31-8220, were each highly effective inhibitors of the FAK tyrosine phosphorylation caused by collagen or CRP-XL. These data suggest that, in human platelets, 1) occupation or clustering of the integrin alpha(2)beta(1) is neither sufficient nor necessary for activation of FAK, 2) the fibrinogen receptor alpha(IIb)beta(3) is not required for activation of FAK by collagen fibers, and 3) both intracellular Ca(2+) and protein kinase C activity are essential intermediaries of FAK activation.
    Journal of Biological Chemistry 03/2001; 276(5):3167-74. · 4.60 Impact Factor
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    ABSTRACT: 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.
    Journal of Biological Chemistry 09/2000; 275(32):24560-4. · 4.60 Impact Factor
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    ABSTRACT: Convulxin (CVX), a potent platelet aggregating protein from the venom of the snake Crotalus durissus terrificus, is known to bind to the platelet collagen receptor, glycoprotein VI (GPVI). CVX binding to human platelets was investigated by flow cytometry, using fluorescein labeled convulxin (FITC-CVX). Scatchard analysis indicated high and low affinity binding sites with Kd values of 0.6 and 4 nM and Bmax values of 1200 and 2000 binding sites per platelet. FITC-CVX binding was inhibited by collagen related peptides (CRPs) comprising a repeated GPO sequence, namely GCO(GPO)(10)GCOGNH(2) and GKO(GPO)(10)GKOGNH(2), which also bind to receptor GPVI. These peptides (monomeric or cross-linked forms) gave a high affinity inhibition of 10-20% for concentrations between 10 ng/ml and 5 microg/ml, followed by a second phase of inhibition at concentrations greater than 5 microg/ml. It was shown also that the inhibition of FITC-CVX binding by CRPs was independent on the time of preincubation of platelets with CRPs, and the same percentage of inhibition was seen with various concentrations of convulxin. Confocal microscopy of the distribution of FITC-CVX binding sites on platelets showed an homogeneous distribution of FITC-CVX bound to GPVI, although some limited clustering may exist.
    Biochemical and Biophysical Research Communications 07/2000; 273(1):246-50. · 2.28 Impact Factor
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    ABSTRACT: A recombinant soluble form of the catalytic domain of human ADAM-10 was expressed as an Fc fusion protein from myeloma cells. The ADAM-10 was catalytically active, cleaving myelin basic protein and peptides based on the previously described 'metallosheddase' cleavage sites of tumour necrosis factor alpha, CD40 ligand and amyloid precursor protein. The myelin basic protein degradation assay was used to demonstrate that hydroxamate inhibitors of matrix metalloproteinases (MMPs) were also inhibitors of ADAM-10. The natural MMP inhibitors, TIMP-2 and TIMP-4 were unable to inhibit ADAM-10, but TIMP-1 and TIMP-3 were inhibitory. Using a quenched fluorescent substrate assay and ADAM-10 we obtained approximate apparent inhibition constants of 0.1 nM (TIMP-1) and 0.9 nM (TIMP-3). The TIMP-1 inhibition of ADAM-10 could therefore prove useful in distinguishing its activity from that of TACE, which is only inhibited by TIMP-3, in cell based assays.
    FEBS Letters 06/2000; 473(3):275-9. · 3.34 Impact Factor
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    ABSTRACT: We have determined the crystal structure of a complex between the I domain of integrin α2β1 and a triple helical collagen peptide containing a critical GFOGER motif. Three loops on the upper surface of the I domain that coordinate a metal ion also engage the collagen, with a collagen glutamate completing the coordination sphere of the metal. Comparison with the unliganded I domain reveals a change in metal coordination linked to a reorganization of the upper surface that together create a complementary surface for binding collagen. Conformational changes propagate from the upper surface to the opposite pole of the domain, suggesting both a basis for affinity regulation and a pathway for signal transduction. The structural features observed here may represent a general mechanism for integrin–ligand recognition.
    Cell 03/2000; 101(1):47-56. · 33.12 Impact Factor
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    ABSTRACT: 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).
    Journal of Biological Chemistry 01/2000; 275(1):35-40. · 4.60 Impact Factor

Publication Stats

3k Citations
204.63 Total Impact Points


  • 1998–2007
    • University of Cambridge
      • Department of Biochemistry
      Cambridge, ENG, United Kingdom
    • The University of Manchester
      Manchester, England, United Kingdom
    • University of Münster
      • Department of Internal Medicine
      Münster, North Rhine-Westphalia, Germany
  • 2004
    • University of Leicester
      • Department of Biochemistry
      Leicester, ENG, United Kingdom
  • 2003
    • Rabin Medical Center
      Tell Afif, Tel Aviv, Israel
  • 1999
    • University Medical Center Utrecht
      • Department of Hematology
      Utrecht, Provincie Utrecht, Netherlands
    • Maastricht University
      • Biochemie
      Maastricht, Provincie Limburg, Netherlands