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• W2025642009 abstract "Previously identified high affinity integrin-binding motifs in collagens, GFOGER and GLOGER, are not present in type III collagen. Here, we first characterized the binding of recombinant I domains from integrins α1 and α2 (α1I and α2I) to fibrillar collagen types I-III and showed that each I domain bound to the three types of collagens with similar affinities. Using rotary shadowing followed by electron microscopy, we identified a high affinity binding region in human type III collagen recognized by α1I and α2I. Examination of the region revealed the presence of two sequences that contain the critical GER motif, GROGER and GAOGER. Collagen-like peptides containing these two motifs were synthesized, and their triple helical nature was confirmed by circular dichroism spectroscopy. Experiments show that the GROGER-containing peptide was able to bind both α1I and α2I with high affinity and effectively inhibit the binding of α1I and α2I to type III and I collagens, whereas the GAOGER-containing peptide was considerably less effective. Furthermore, the GROGER-containing peptide supported adhesion of human lung fibroblast cells when coated on a culture dish. Thus, we have identified a novel high affinity binding sequence for the collagen-binding integrin I domains. Previously identified high affinity integrin-binding motifs in collagens, GFOGER and GLOGER, are not present in type III collagen. Here, we first characterized the binding of recombinant I domains from integrins α1 and α2 (α1I and α2I) to fibrillar collagen types I-III and showed that each I domain bound to the three types of collagens with similar affinities. Using rotary shadowing followed by electron microscopy, we identified a high affinity binding region in human type III collagen recognized by α1I and α2I. Examination of the region revealed the presence of two sequences that contain the critical GER motif, GROGER and GAOGER. Collagen-like peptides containing these two motifs were synthesized, and their triple helical nature was confirmed by circular dichroism spectroscopy. Experiments show that the GROGER-containing peptide was able to bind both α1I and α2I with high affinity and effectively inhibit the binding of α1I and α2I to type III and I collagens, whereas the GAOGER-containing peptide was considerably less effective. Furthermore, the GROGER-containing peptide supported adhesion of human lung fibroblast cells when coated on a culture dish. Thus, we have identified a novel high affinity binding sequence for the collagen-binding integrin I domains. Collagen is a major component of the extracellular matrix (ECM). 2The abbreviations used are: ECM, extracellular matrix; MIDAS, metal ion-dependent adhesion site; SPR, surface plasmon resonance; ELISA, enzyme-linked immunosorbent assay; Fmoc, N-(9-fluorenyl)methoxycarbonyl; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; BSA, bovine serum albumin; EM, electron microscopy.2The abbreviations used are: ECM, extracellular matrix; MIDAS, metal ion-dependent adhesion site; SPR, surface plasmon resonance; ELISA, enzyme-linked immunosorbent assay; Fmoc, N-(9-fluorenyl)methoxycarbonyl; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; BSA, bovine serum albumin; EM, electron microscopy. At least 27 genetically different collagen types have been identified, each containing at least one dominant collagenous domain (1Ramachandran G.N. Int. J. Pept. Protein Res. 1988; 31: 1-16Crossref PubMed Scopus (104) Google Scholar). These collagenous domains have a characteristic triple helical structure formed by repeating Gly-X-Y sequences in each participating polypeptide, where X often is proline and Y hydroxyproline. The collagen monomers often assemble into more complex structures of varying organizations, such as fibrils (types I-III, V, and XI), networks (types IV, VIII, and X), and beaded filaments (type VI) (2Hulmes D.J. Essays Biochem. 1992; 27: 49-67PubMed Google Scholar). The fibrillar collagen types I and III are the major structural components of the ECM of skin, cardiac, and vascular tissues, whereas type II collagen is a major component of cartilage. In addition to contributing to the structural integrity of the tissues, collagens also affect cell behavior through interactions with other matrix proteins and cellular receptors (3Prockop D.J. Kivirikko K.I. Annu. Rev. Biochem. 1995; 64: 403-434Crossref PubMed Scopus (1355) Google Scholar, 4Kuivaniemi H. Tromp G. Prockop D.J. Hum. Mutat. 1997; 9: 300-315Crossref PubMed Scopus (276) Google Scholar, 5Gelse K. Poschl E. Aigner T. Adv. Drug Deliv. Rev. 2003; 55: 1531-1546Crossref PubMed Scopus (1502) Google Scholar, 6Myllyharju J. Kivirikko K.I. Ann. Med. 2001; 33: 7-21Crossref PubMed Scopus (534) Google Scholar). The integrins are a family of heterodimeric cell surface receptors involved in cell-cell and cell-substrate adhesion. They act as bridging molecules that link intracellular signaling molecules to the ECM, controlling cell behavior and tissue architecture through bi-directional signaling (7Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8941) Google Scholar). Four integrins, α1β1, α2β1, α10β1, and α11β1, have been shown to bind collagens (8Kramer R.H. Marks N. J. Biol. Chem. 1989; 264: 4684-4688Abstract Full Text PDF PubMed Google Scholar, 9Camper L. Hellman U. Lundgren-Akerlund E. J. Biol. Chem. 1998; 273: 20383-20389Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 10Velling T. Kusche-Gullberg M. Sejersen T. Gullberg D. J. Biol. Chem. 1999; 274: 25735-25742Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Of these, the α1β1 and α2β1 integrins have been studied in more detail compared with the others. Collagen-integrin interactions play a role in normal and pathological physiology; these interactions directly affect cell adhesion, migration, proliferation, and differentiation, as well as angiogenesis, platelet aggregation, and ECM assembly (11Gullberg D.E. Lundgren-Akerlund E. Prog. Histochem. Cytochem. 2002; 37: 3-54Crossref PubMed Scopus (78) Google Scholar). The precise molecular events that lead to these activities are not understood. It is possible that different sites in collagens are recognized by different integrins and/or are capable of activating different signaling pathways. Consequently, detailed studies investigating the specificity of the collagen-integrin interactions are essential to further our understanding of these interactions in different biological processes. Collagen binding by the four integrins is mediated by the inserted (I) domain, a ∼200-amino acid-long segment found between blades 2 and 3 of the β-propeller domain of the α chains. All four I domains (α1I, α2I, α10I, and α11I) contain a metal ion-dependent adhesion site (MIDAS) that is required for coordinating a divalent cation and is essential for collagen binding. Synthetic collagen peptides containing the type I collagen derived sequences GFOGER or GLOGER have been reported to bind with a high affinity to α1I, α2I, and α11I; furthermore, synthetic peptides containing these sequences inhibit the binding of I domains to intact collagens (12Knight C.G. Morton L.F. Onley D.J. Peachey A.R. Messent A.J. Smethurst P.A. Tuckwell D.S. Farndale R.W. Barnes M.J. J. Biol. Chem. 1998; 273: 33287-33294Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 13Zhang W.M. Kapyla J. Puranen J.S. Knight C.G. Tiger C.F. Pentikainen O.T. Johnson M.S. Farndale R.W. Heino J. Gullberg D. J. Biol. Chem. 2003; 278: 7270-7277Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 14Siljander P.R. Hamaia S. Peachey A.R. Slatter D.A. Smethurst P.A. Ouwehand W.H. Knight C.G. Farndale R.W. J. Biol. Chem. 2004; 279: 47763-47772Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The crystal structures of apo-α2I and α2I in complex with a collagen peptide containing the GFOGER sequence have been solved and show that the apo-α2I adopts an inactive “closed” conformation and the ligand-bound α2I, an active “open” conformation (15Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 101: 47-56Abstract Full Text Full Text PDF PubMed Scopus (818) Google Scholar, 16Lee J.O. Bankston L.A. Arnaout M.A. Liddington R.C. Structure (Lond.). 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). The Glu residue in the collagen peptide was shown in the structure of the complex to directly interact with a Mg2+ ion coordinated by the MIDAS motif, and the Arg residue forms a salt bridge with D219 in α2I. The importance of the GER sequence in collagen for integrin binding was confirmed by mutagenesis studies showing that replacing the collagen peptide Glu with an Asp residue completely abolished integrin binding, whereas replacing the Arg with a Lys residue reduced binding by 50% (17Knight C.G. Morton L.F. Peachey A.R. Tuckwell D.S. Farndale R.W. Barnes M.J. J. Biol. Chem. 2000; 275: 35-40Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar). The Phe residue in the collagen sequence appeared to participate in hydrophobic interactions with α2I and presumably can be replaced by a Leu residue. However, changing the Phe residue to a Met or an Ala in the collagen peptide reduced the apparent affinity of the I domains (14Siljander P.R. Hamaia S. Peachey A.R. Slatter D.A. Smethurst P.A. Ouwehand W.H. Knight C.G. Farndale R.W. J. Biol. Chem. 2004; 279: 47763-47772Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). GASGER was also reported to be recognized by the I domains but bind with lower affinity than GFOGER and GLOGER (13Zhang W.M. Kapyla J. Puranen J.S. Knight C.G. Tiger C.F. Pentikainen O.T. Johnson M.S. Farndale R.W. Heino J. Gullberg D. J. Biol. Chem. 2003; 278: 7270-7277Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 14Siljander P.R. Hamaia S. Peachey A.R. Slatter D.A. Smethurst P.A. Ouwehand W.H. Knight C.G. Farndale R.W. J. Biol. Chem. 2004; 279: 47763-47772Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 18Xu Y. Gurusiddappa S. Rich R.L. Owens R.T. Keene D.R. Mayne R. Hook A. Hook M. J. Biol. Chem. 2000; 275: 38981-38989Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). Therefore, GFOGER and GLOGER are the only known collagen-derived sequence motifs that support high affinity binding by the collagen-binding I domains. However, these two motifs are absent in some collagens such as human type III collagen. A previous study showed that Chinese hamster ovary cells expressing α1β1 and α2β1 could adhere to and spread on human type III collagen; furthermore, the recombinant proteins of α1I and α2I could bind to this collagen type (19Nykvist P. Tu H. Ivaska J. Kapyla J. Pihlajaniemi T. Heino J. J. Biol. Chem. 2000; 275: 8255-8261Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). We now have examined α1I and α2I binding to human type III collagen in some detail and identified a previously unrecognized high affinity integrin-binding site. Recombinant I Domains—Recombinant I domains of integrin α1 and α2 subunits were generated and isolated as previously described (18Xu Y. Gurusiddappa S. Rich R.L. Owens R.T. Keene D.R. Mayne R. Hook A. Hook M. J. Biol. Chem. 2000; 275: 38981-38989Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 20Rich R.L. Deivanayagam C.C. Owens R.T. Carson M. Hook A. Moore D. Symersky J. Yang V.W. Narayana S.V. Hook M. J. Biol. Chem. 1999; 274: 24906-24913Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Purified recombinant proteins were examined by SDS-PAGE followed by staining with Coomassie blue. Purification of Recombinant Procollagen—Frozen yeast cells expressing recombinant type I and III procollagens were generously provided by FibroGen (San Francisco, CA). The yeast cells express both genes encoding human collagen and prolyl 4-hydroxylase enabling formation of hydroxyproline residues and thermally stable triple helical collagen. The cells were thawed in an ambient temperature water bath and resuspended in Start Buffer (0.1 m Tris, 0.4 m NaCl, 25 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 μm pepstatin, pH 7.5). The cells were lysed using a French press and the lysate was centrifuged at 30,000 × g for 30 min at 4 °C. The supernatant was then filtered through a 0.45-μm membrane, and the pH of the filtrate was adjusted to 7.5. An affinity column was prepared by coupling a recombinant collagen-binding MSCRAMM from Staphylococcus aureus, CNA (21Patti J.M. Jonsson H. Guss B. Switalski L.M. Wiberg K. Lindberg M. Hook M. J. Biol. Chem. 1992; 267: 4766-4772Abstract Full Text PDF PubMed Google Scholar), to CNBr-activated Sepharose 4B (Amersham Biosciences). The supernatant was applied to the column and incubated overnight at 4 °C. The column was washed with the Start Buffer, and bound material was eluted with 0.5 m acetic acid. Fractions were examined by SDS-PAGE (4%/8%) under reducing conditions followed by Coomassie Blue staining. Fractions with procollagen were pooled. The concentration of the procollagen was estimated by comparing its band intensity with that of a known amount of type I collagen (Vitrogen) in Coomassie Blue stained SDS-PAGE gels. Surface Plasmon Resonance (SPR) Measurements—For the analyses of interactions between recombinant I domains and fibrillar collagens, SPR measurements were carried out at ambient temperature using the BIAcore 3000 system (Biacore AB, Uppsala, Sweden) as described previously (20Rich R.L. Deivanayagam C.C. Owens R.T. Carson M. Hook A. Moore D. Symersky J. Yang V.W. Narayana S.V. Hook M. J. Biol. Chem. 1999; 274: 24906-24913Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) with the following modifications. First, purified recombinant human procollagen I, procollagen III (described above), or bovine mature collagen II (Sigma) were immobilized on the flow cells of a CM5 chip, resulting in 200-700 response units of immobilized protein. Different concentrations of the α1I and α2I proteins in HBS buffer (25 mm HEPES, 150 mm NaCl, pH 7.4) containing 5 mm β-mercaptoethanol, 1 mm MgCl2, and 0.05% octyl-d-glucopyranoside were passed over the immobilized collagen at 30 μl/min for 4 min. Regeneration of the collagen surfaces was achieved with 20 μl of HBS containing 0.01% SDS. Binding of α1I and α2I to a reference flow cell, which had been activated and deactivated without the coupling of collagen, was also measured and subtracted from the response to collagen-coated flow cells. SPR sensorgrams from different injections were overlaid using the BIAevaluation software. Data from the steady state portion of the sensorgrams were used to determine binding affinities. Based on the correlation between the SPR response and change in protein mass on the surfaces of flow cells, values for the binding ratio, νbound, and the concentration of free protein, [P]free, were calculated using the equations described previously (20Rich R.L. Deivanayagam C.C. Owens R.T. Carson M. Hook A. Moore D. Symersky J. Yang V.W. Narayana S.V. Hook M. J. Biol. Chem. 1999; 274: 24906-24913Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Scatchard analysis was performed by plotting νbound/[P]free against νbound in which the negative reciprocal of the slope is the dissociation constant, KD. Nonlinear regression was also performed by plotting νbound against [P]free and fitted with the one-binding class or the two-binding class models using the GraphPad Prism™ software (GraphPad Software Inc., San Diego, CA). Results from the two models were compared with respect to the value of R2 and the degree of freedom of the curve fit. The model that gave KD values outside the experimental data range was excluded. Experimental results were reproducibly performed with at least three independent protein preparations. SPR measurements of the interactions between I domains and a synthetic collagen peptide were carried out at 15 °C using the BIAcore 3000 system. The synthetic collagen peptide was immobilized onto a flow cell of a CM5 chip, and various concentrations of recombinant I domains were passed over the coated surface at 50 μl/min. Responses on a reference flow cell were subtracted from responses of the peptide-coated flow cell. The BIAevaluation software was used to determine the association rate (kon), dissociation rates (koff), and KD with a 1:1 binding model. Rmax of fitting was similar to calculated Rmax. The χ2 of each fitting was less than 2. Competition Enzyme-linked Immunosorbent Assay (ELISA)—Microtiter wells (Immulon 4, Thermo Labsystems) were coated with 1 μg of mature bovine type I collagen (Vitrogen) or purified human type III procollagen in HBS for 2 h at room temperature. The wells were washed with HBS and incubated with a blocking buffer (HBS containing 0.1% w/v ovalbumin and 0.05% v/v Tween 20) overnight at 4 °C. Varying concentrations of peptides were mixed with fixed concentrations of each recombinant I domain in the blocking buffer containing 1 mm MgCl2 and 5 mm β-mercaptoethanol and then added to the wells. After incubation at 4 °C for 3 h with gentle shaking, the wells were extensively washed with HBS containing 0.05% Tween 20 and 1 mm MgCl2. Bound α1I or α2I was detected by incubation with an anti-His monoclonal antibody (Amersham Biosciences) diluted 1:3000 in the blocking buffer containing 1 mm MgCl2 for 1 h at room temperature, followed by incubation with goat anti-mouse IgG (H+L)-alkaline phosphatase conjugate (Bio-Rad) diluted 1:3000 in the blocking buffer containing 1 mm MgCl2 for 1 h at room temperature. Bound antibodies were quantified by adding 100 μl of 1.3 m diethanolamine, pH 9.8, containing 1 mm MgCl2, and 1 mg/ml p-nitrophenyl phosphate (Southern Biotechnology Associates, Birmingham, AL) to each well and measuring the absorbance at 405 nm (A405 nm) after 20-40 min of incubation at room temperature. Background binding to the wells was determined by incubating the I domains in wells that had been pretreated with blocking buffer alone. These values were subtracted from the values generated in the collagen-coated wells to determine collagen specific binding. Data were presented as the mean value ± S.E. of A405 nm (n = 3) from a representative experiment. Rotary Shadowing and Electron Microscopy—Rotary shadowing and electron microscopy of I domain-collagen complexes were performed as described previously (18Xu Y. Gurusiddappa S. Rich R.L. Owens R.T. Keene D.R. Mayne R. Hook A. Hook M. J. Biol. Chem. 2000; 275: 38981-38989Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). Each binding event was measured from the C-terminal end of type III collagen, i.e. from the base of the globular domain, and to the middle of the binding spot. The binding events were then binned for every 10 nm along the collagen strand. The percentage of the number of events in each bin over total events counted was calculated and plotted against the length of the collagen strand. Synthesis and Purification of Collagen Peptides—Peptides were synthesized by a solid phase method on a TentaGel R RAM resin (RAPP Polymere GmbH, Tubingen, Germany) using Fmoc chemistry and a model 396 MBS Multiple Peptide Synthesizer from Advanced ChemTech Inc. (Louisville, KY). Fmoc amino acids were purchased from Novabiochem (San Diego, CA). Coupling of amino acids was carried out twice using diisopropylcarbodiimide/1-hydroxybenzotriazole for 60 min. Fmoc deprotection was carried out using a mixture of 2% (v/v) piperidine and 2% (v/v) 1,8-diazabicyclo-[5.4.0]undec-7-ene in dimethylformamide followed by treatment with 25% piperidine in dimethylformamide. Side chains were protected with the following groups: t-butyl (Glu, Ser, and hydroxy-Pro), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Arg), and trityl (Gln). After completion of the synthesis, peptide resins were washed thoroughly with dimethylformamide, ethanol, and ether and then dried in a vacuum desiccator. Peptides were released from the resin by treatment with a mixture of trifluoroacetic acid, thioanisole, ethanedithiol, and triethylsilane (90:5:2.5:2.5 by volume) for 8 h. The resins were filtered, and the peptides were precipitated with cold anhydrous ether. The precipitate was washed with anhydrous ether three times and dried. The cleaved peptides were analyzed by reverse phase high pressure liquid chromatography on a Waters 625 liquid chromatography system (Milford, MA) using a Waters Delta-Pak C18 column. CD Spectroscopy—Synthetic collagen peptides were analyzed by CD spectroscopy as described previously (18Xu Y. Gurusiddappa S. Rich R.L. Owens R.T. Keene D.R. Mayne R. Hook A. Hook M. J. Biol. Chem. 2000; 275: 38981-38989Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar) with the following modifications. Peptides were dissolved in HBS to a concentration of 50 μm. CD spectra were collected on a Jasco J720 spectropolarimeter (Tokyo, Japan) from 190 to 240 nm, with a bandwidth of 1 nm and integrated for 1 s at 0.2 nm intervals. Samples were measured at room temperature using cuvettes with a 0.02-cm path length. For temperature-dependent denaturation analysis, peptides (30 μm) were added to a thermostatically controlled cuvette with a 0.5-cm path length. Thermal transition profiles were recorded at 225 nm as described above with a temperature slope of 20 °C/h. To calculate the temperature melting points, the thermal transition profiles were fitted with a Boltzmann sigmoidal model using the GraphPad Prism™ software. Reagents and Cell Culture—The human recombinant mature type III collagen used for cell attachment assays was purchased from FibroGen. All cell culture media components were obtained from Invitrogen. The human lung fibroblast cell line MRC-5 was purchased from American Type Culture Collection (ATCC) (Manassas, VA). The cells were cultured and passaged in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 unit/ml penicillin, and 100 μg/ml streptomycin. The cells were grown to subconfluence and passaged every 2-3 days. Cell Attachment Assay—MRC-5 cells were starved overnight in serum-deficient DMEM containing penicillin and streptomycin, then detached using 1 mm EDTA and 0.025% trypsin at 37 °C for 2 min. The cells were washed with PBS and resuspended in DMEM containing 0.2% BSA supplemented with 2 mm MgCl2. The cell suspension (100 μl containing ∼1.5 × 104 cells) were added to the microtiter wells coated with different concentrations of collagen or collagen peptides and blocked with PBS containing 0.5% (w/v) BSA. After incubation at room temperature for 45 min, the wells were washed with PBS and the attached cells were fixed with 3% p-formaldehyde for 10 min at room temperature. Following washing with cold Tris-buffered saline, pH 7.4, cells were fixed again in 20% methanol for 10 min and stained with 0.5% crystal violet for 5 min. The wells were thoroughly washed with distilled water and air-dried. Sodium citrate (0.1 m) was then added to the wells to dissolve the dye and the absorbance at 590 nm was measured. The maximum cell attachment on type III collagen was set to 100%, and residual attachment on BSA was set to 0%. Computer Modeling—The coordinates of the crystal structure of α2I in complex with a synthetic collagen peptide were obtained from the Protein Data Bank (code 1dzi) and used as a template for the model studies. First, the Phe residues in both the middle and trailing strands were replaced by Arg residues. Then, the local minimization was carried out in sizes of 5 Å for the best fit. Several basic components (i.e. hydrogen bond, van der Waals, and electrostatic interactions) contributing to the binding energy between α2I and the mutated collagen peptide were analyzed. The molecular modeling experiment was carried out under ECEPP/3 force field by using the ICM software (Molsoft, La Jolla, CA). Characterization of the Binding of α1I and α2I to Type I, II, and III Collagens—The interactions between the two I domains and fibrillar collagens (types I-III) were examined by SPR. Solutions of 1 μm α1I or α2I were passed over chips containing immobilized collagen types I-III in the presence of 1 mm MgCl2. Both α1I and α2I showed binding to all three types of collagen (Fig. 1), consistent with a previous report (19Nykvist P. Tu H. Ivaska J. Kapyla J. Pihlajaniemi T. Heino J. J. Biol. Chem. 2000; 275: 8255-8261Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). To determine the dissociation constants (KD) for the interactions between the I domains and each collagen, increasing concentrations of recombinant I domains (0.01-50 μm) were passed over the collagen surfaces. In our previous SPR studies using the BIAcore 1000 system, we showed two classes of binding sites in type I collagen with different affinities for α1I (KD1 = 0.26 ± 0.01 μm, and KD2 = 13.9 ± 3.0 μm), whereas α2I appeared to have one class of binding sites (∼10 μm) (18Xu Y. Gurusiddappa S. Rich R.L. Owens R.T. Keene D.R. Mayne R. Hook A. Hook M. J. Biol. Chem. 2000; 275: 38981-38989Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 20Rich R.L. Deivanayagam C.C. Owens R.T. Carson M. Hook A. Moore D. Symersky J. Yang V.W. Narayana S.V. Hook M. J. Biol. Chem. 1999; 274: 24906-24913Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The BIAcore 3000 system used in this study has a higher sensitivity of detection, which enables us to examine the interactions between α2I and collagens within a submicromolar concentration range. Analyses using the SPR responses in the steady state portion of the sensorgrams, which indicates the equilibrium condition, showed that both I domains have at least two classes of binding sites in the three types of collagen. The dissociation constants (KD) of these interactions are summarized in TABLE ONE. α1I binds all three types of collagen with similar affinities (KD1 = ∼0.15-0.32 μm, and KD2 = ∼5.5-7.3 μm). The binding affinities of α2I to the three types of collagen appeared to be slightly more variable. The KD values for the high affinity binding class range from ∼0.3 μm for types I and III collagen to 1.75 μm for type II collagen, whereas the KD values for the low affinity binding class range from ∼4 μm for type I collagen to ∼16.5 μm and ∼14.5 μm for type II and III collagen, respectively.TABLE ONESummary of the binding affinities of α1I and α2I to fibrillar collagens (types I-III)KDaKD was calculated by equilibrium analysis. Data are presented as mean value ± S.E. of three independent studiesα1Iα2IμmCollagen I0.32 ± 0.100.26 ± 0.085.5 ± 1.463.99 ± 0.82Collagen II0.15 ± 0.031.75 ± 0.097.28 ± 1.1616.5 ± 3.89Collagen III0.19 ± 0.030.33 ± 0.036.15 ± 0.9514.5 ± 3.41a KD was calculated by equilibrium analysis. Data are presented as mean value ± S.E. of three independent studies Open table in a new tab The two recombinant I domains also exhibited different binding kinetics to the collagens as indicated by the shape of the corresponding SPR sensorgrams (Fig. 1). Comparison of the shapes of the SPR sensorgrams of α1I with those of α2I indicates a much slower association and dissociation rate of α1I compared with α2I, in agreement with previous reports (18Xu Y. Gurusiddappa S. Rich R.L. Owens R.T. Keene D.R. Mayne R. Hook A. Hook M. J. Biol. Chem. 2000; 275: 38981-38989Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 20Rich R.L. Deivanayagam C.C. Owens R.T. Carson M. Hook A. Moore D. Symersky J. Yang V.W. Narayana S.V. Hook M. J. Biol. Chem. 1999; 274: 24906-24913Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). However, there was no dramatic difference between each I domain and type I, II, or type III collagen. Thus, the binding characteristics of the interactions between α1I/α2I and type III collagen are similar to those of the interactions between α1I/α2I and type I/II collagen. Localization of a High Affinity α1I/α2I Binding Region in Type III Procollagen—Two sequence motifs, GFOGER and GLOGER, are identified as high affinity binding sites in triple helical collagen for α1I, α2I, and α11I (13Zhang W.M. Kapyla J. Puranen J.S. Knight C.G. Tiger C.F. Pentikainen O.T. Johnson M.S. Farndale R.W. Heino J. Gullberg D. J. Biol. Chem. 2003; 278: 7270-7277Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 14Siljander P.R. Hamaia S. Peachey A.R. Slatter D.A. Smethurst P.A. Ouwehand W.H. Knight C.G. Farndale R.W. J. Biol. Chem. 2004; 279: 47763-47772Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 18Xu Y. Gurusiddappa S. Rich R.L. Owens R.T. Keene D.R. Mayne R. Hook A. Hook M. J. Biol. Chem. 2000; 275: 38981-38989Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). The fact that these sequences are present in type I and II collagen, but not in type III collagen, suggests the presence of at least one novel high affinity binding site in type III collagen. To locate the high affinity binding region(s) in type III collagen, we examined collagen and I domain complexes by rotary shadowing followed by electron microscopy (EM). Type III procollagen was used in these experiments because it contains a globular-shaped C-terminal propeptide that allows us to determine the orientation of collagen molecules in EM. Type III procollagen was incubated with α1I or α2I under binding conditions and the complexes were then subjected to rotary shadowing and EM. The helical portion of the majority of the collagen molecules was found to be ∼ 300 nm long, indicating that these molecules are mostly intact, full-length molecules. Multiple binding sites in the helical portion of type III collagen were observed for both α1I and α2I (Fig. 2); however, one region at 270-300 nm from th" @default.
• W2025642009 created "2016-06-24" @default.
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• W2025642009 date "2005-09-01" @default.
• W2025642009 modified "2023-10-18" @default.
• W2025642009 title "A Novel Binding Site in Collagen Type III for Integrins α1β1 and α2β1" @default.
• W2025642009 cites W1498888463 @default.
• W2025642009 cites W1540209636 @default.
• W2025642009 cites W1546476211 @default.
• W2025642009 cites W1972207659 @default.
• W2025642009 cites W1976336086 @default.
• W2025642009 cites W1976772770 @default.
• W2025642009 cites W1979221090 @default.
• W2025642009 cites W1988757615 @default.
• W2025642009 cites W2002100378 @default.
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