摘要
Integrins α1β1 and α2β1 are two major collagen receptors on the surface of eukaryotic cells. Binding to collagen is primarily due to an A-domain near the N terminus of the α chains. Previously, we reported that recombinant A-domain of α1β1(α1A) had at least two affinity classes of binding sites in type I collagen (Rich, R. L., et al. (1999)J. Biol. Chem. 274, 24906–24913). Here, we compared the binding of the recombinant A-domain of α2β1 (α2A) to type I collagen with that of α1A using surface plasmon resonance and showed that α2A exhibited only one detectable class of binding sites in type I collagen, with a KD of ∼10 μm at ∼3 binding sites per collagen molecule. We further demonstrated that α1A and α2A competed with each other for binding to type I collagen in enzyme-linked immunosorbent assay (ELISA), suggesting that the binding sites in collagen for the two A-domains overlap or are adjacent to each other. By using rotary shadowing, the complexes of α1A- and α2A-procollagen were visualized. Morphometric analyses indicated three major binding regions (near the N terminus, in the central part, and near the C terminus) along the type I procollagen molecule for both A-domains. The positions of the respective binding regions for α1A and α2A were overlapping with or adjacent to each other, consistent with the ELISA results. Analysis of the sequences of type I collagen revealed that GER or GER-like motifs are present at each of the binding regions, and notably, the central region contains the GFOGER sequence, which was previously identified as a high affinity site for both α1A and α2A (Knight, C. G., et al. (2000) J. Biol. Chem. 275, 35–40). Peptides containing GLOGERGRO (peptide I, near the N terminus), GFOGERGVQ (peptide II, central), and GASGERGPO (peptide III, near the C terminus) were synthesized. Peptides I and II effectively inhibited the binding of α1A and α2A to type I collagen, while peptide III did so moderately. The N-terminal site in type I collagen has the sequence GLOGER in all three chains. Thus, it seems that peptide I represents a newly discovered native high affinity site for α1A and α2A. Integrins α1β1 and α2β1 are two major collagen receptors on the surface of eukaryotic cells. Binding to collagen is primarily due to an A-domain near the N terminus of the α chains. Previously, we reported that recombinant A-domain of α1β1(α1A) had at least two affinity classes of binding sites in type I collagen (Rich, R. L., et al. (1999)J. Biol. Chem. 274, 24906–24913). Here, we compared the binding of the recombinant A-domain of α2β1 (α2A) to type I collagen with that of α1A using surface plasmon resonance and showed that α2A exhibited only one detectable class of binding sites in type I collagen, with a KD of ∼10 μm at ∼3 binding sites per collagen molecule. We further demonstrated that α1A and α2A competed with each other for binding to type I collagen in enzyme-linked immunosorbent assay (ELISA), suggesting that the binding sites in collagen for the two A-domains overlap or are adjacent to each other. By using rotary shadowing, the complexes of α1A- and α2A-procollagen were visualized. Morphometric analyses indicated three major binding regions (near the N terminus, in the central part, and near the C terminus) along the type I procollagen molecule for both A-domains. The positions of the respective binding regions for α1A and α2A were overlapping with or adjacent to each other, consistent with the ELISA results. Analysis of the sequences of type I collagen revealed that GER or GER-like motifs are present at each of the binding regions, and notably, the central region contains the GFOGER sequence, which was previously identified as a high affinity site for both α1A and α2A (Knight, C. G., et al. (2000) J. Biol. Chem. 275, 35–40). Peptides containing GLOGERGRO (peptide I, near the N terminus), GFOGERGVQ (peptide II, central), and GASGERGPO (peptide III, near the C terminus) were synthesized. Peptides I and II effectively inhibited the binding of α1A and α2A to type I collagen, while peptide III did so moderately. The N-terminal site in type I collagen has the sequence GLOGER in all three chains. Thus, it seems that peptide I represents a newly discovered native high affinity site for α1A and α2A. metal ion-dependent adhesion site microbial surface component recognizing adhesive matrix molecules surface plasmon resonance enzyme-linked immunosorbent assay polymerase chain reaction N-(9-fluorenyl)methoxycarbonyl Collagen is a major component of the extracellular matrix of all mammalian connective tissues. In addition to providing structural support, collagen can also affect cell behavior and gene expression through interactions with other matrix proteins and cellular receptors. We currently recognize 19 genetically distinct collagen types, and numerous other proteins have been described that contain collagenous domains. The collagen triple helix is formed by repeating GXY sequences within each chain, where X is often proline and Y is often hydroxyproline. Such molecules then interact to form higher structures of varying organization, such as fibrils (types I, II, III, V, and XI), networks (types IV, VIII, and X), and beaded filaments (type VI). Collagen type I, the most abundant collagen type, is a heterotrimer composed of two α1(I) and one α2(I) chains to give a molecular organization [α1(I)]2α2(I) (for review, see Ref. 1Prockop D.J. Kivirikko K.I. Annu. Rev. Biochem. 1995; 64: 403-434Crossref PubMed Scopus (1379) Google Scholar). The integrins are a family of heterodimeric cell surface receptors involved in cell-cell and cell-substrate adhesion, as well as in several signal transduction pathways (for reviews, see Refs 2Humphries M.J. Trends Pharmacol. Sci. 2000; 21: 29-32Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar and 3Hynes R.O. Lander A.D. Cell. 1992; 68: 303-322Abstract Full Text PDF PubMed Scopus (762) Google Scholar). So far, four different integrins (α1β1, α2β1, α10β1, and α11β1) have been shown to bind collagen (4Kramer R.H. Marks N. J. Biol. Chem. 1989; 264: 4684-4688Abstract Full Text PDF PubMed Google Scholar, 5Camper L. Hellman U. Lundgren-Akerlund E. J. Biol. Chem. 1998; 273: 20383-20389Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 6Velling T. Kusche-Gullberg M. Sejersen T. Gullberg D. J. Biol. Chem. 1999; 274: 25735-25742Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Of these, the α1β1 and α2β1 integrins have been most extensively studied. The interactions of both integrins with collagen involve a so-called von Willebrand factor A-like domain (A-domain), also called an inserted domain (I-domain), and require triple helical collagen structures. Furthermore, the presence of Mg2+ or Mn2+ is required for high affinity binding. Recombinant α1 and α2 A-domains (α1A and α2A) expressed in Escherichia coli were previously shown to bind to collagen type I and type IV in a concentration-dependent manner and to have the same cation dependence as the corresponding intact integrins (7Calderwood D.A. Tuckwell D.S. Ebles J. Kühn K. Humphries M.J. J. Biol. Chem. 1997; 272: 12311-12317Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 8Dickeson S.K. Walsh J.J. Santoro S.A. J. Biol. Chem. 1997; 272: 7661-7668Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). They also appeared to retain the ligand specificity reported for the intact receptors (see Refs. 7Calderwood D.A. Tuckwell D.S. Ebles J. Kühn K. Humphries M.J. J. Biol. Chem. 1997; 272: 12311-12317Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar and 9Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1994; 269: 9659-9663Abstract Full Text PDF PubMed Google Scholar, 10Kern A. Marcantonio E.E. J. Cell. Physiol. 1998; 176: 634-641Crossref PubMed Scopus (29) Google Scholar, 11Tuckwell D. Calderwood D.A. Green L.J. Humphries M.J. J. Cell Sci. 1995; 108: 1629-1637Crossref PubMed Google Scholar and for reviews see Refs. 12Dickeson S.K. Santoro S.A. Cell. Mol. Life Sci. 1998; 54: 556-566Crossref PubMed Scopus (69) Google Scholar and13Loftus J.C. Liddington R.C. J. Clin. Invest. 1997; 99: 2302-2306Crossref PubMed Google Scholar). The x-ray crystal structures of α1A and α2A have been solved (14Emsley J. King S.L. Bergelson J.M. Liddington R.C. J. Biol. Chem. 1997; 272: 28512-28517Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 15Nolte M. Pepinsky R.B. Venyaminov S.Y. Koteliansky V. Gotwals P.J. Karpusas M. FEBS Lett. 1999; 452: 379-385Crossref PubMed Scopus (57) Google Scholar, 16Rich R.L. Deivanayagam C.C.S. Owens R.T. Carson M. Höök A. Moore D. Symerski J. Yang V.W.-C. Narayana S.V.L. Höök M. J. Biol. Chem. 1999; 274: 24906-24913Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), and the polypeptides were shown to adopt the dinucleotide binding fold that is typical of all the A and A-like domain crystal structures solved to date (17Biekowska J. Cruz M. Atiemo A. Handin R. Liddington R. J. Biol. Chem. 1997; 272: 25162-25167Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 18Qu A. Leahy D.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10277-10281Crossref PubMed Scopus (290) Google Scholar, 19Lee J.-O. Bankston L.A. Arnaout M.A. Liddington R.C. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 20Lee J.-O. Rieu P. Arnaout M.A. Liddington R.C. Cell. 1995; 80: 631-635Abstract Full Text PDF PubMed Scopus (805) Google Scholar, 21Li R. Rieu P. Griffith D.L. Scott D. Arnaout M.A. J. Cell Biol. 1998; 143: 1523-1534Crossref PubMed Scopus (123) Google Scholar). The structures determined for α1A and α2A both contain a trench centered on a motif called the metal ion-dependent adhesion site (MIDAS).1 The results from site-directed mutagenesis of residues in and around the MIDAS as well as from the mapping of epitopes of antibodies that block the binding of α2β1 to collagen indicated that this area is critical for collagen binding (9Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1994; 269: 9659-9663Abstract Full Text PDF PubMed Google Scholar, 22Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar, 23Kamata T. Liddington R.C. Takada Y. J. Biol. Chem. 1999; 274: 32108-32111Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 24Käpylä J. Ivaska J. Riikonen R. Nykvist P. Pentikainen O. Johnson M. Heino J. J. Biol. Chem. 2000; 275: 3348-3354Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 25Dickeson S.K. Mathis N.L. Rahman M. Bergelson J.M. Santoro S. J. Biol. Chem. 1999; 274: 32182-32191Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 26Smith C. Estavillo D. Emsley J. Bankston L.A. Liddington R.C. Cruz M.A. J. Biol. Chem. 2000; 275: 4205-4209Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Computer modeling using structures of collagen peptides indicated that these triple helical peptides fit into the trenches of α1A and α2A and that a glutamate residue in the collagen could coordinate Mg2+ in the MIDAS site (14Emsley J. King S.L. Bergelson J.M. Liddington R.C. J. Biol. Chem. 1997; 272: 28512-28517Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 16Rich R.L. Deivanayagam C.C.S. Owens R.T. Carson M. Höök A. Moore D. Symerski J. Yang V.W.-C. Narayana S.V.L. Höök M. J. Biol. Chem. 1999; 274: 24906-24913Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Trench-containing structures have also been observed in the collagen-binding microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) CNA and ACE from the bacteriaStaphylococcus aureus and Enterococcus faecalis, respectively (16Rich R.L. Deivanayagam C.C.S. Owens R.T. Carson M. Höök A. Moore D. Symerski J. Yang V.W.-C. Narayana S.V.L. Höök M. J. Biol. Chem. 1999; 274: 24906-24913Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 27Symersky J. Patti J.M. Carson M. House-Pompeo K. Teale M. Moore D. Jin L. Schneider A. DeLucas L.J. Höök M. Narayana S.V.L. Nat. Struct. Biol. 1997; 4: 833-838Crossref PubMed Scopus (123) Google Scholar, 28Rich R.L. Kreikemeyer B. Owens R.T. LaBrenz S. Narayana S.V.L. Weinstock G.M. Murray B.E. Höök M. J. Biol. Chem. 1999; 274: 26939-26945Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), and have been implicated in ligand binding. Thus, integrins and MSCRAMMs appear to employ similar binding surfaces for their interactions with collagen. Other similarities in the collagen binding mechanisms of CNA-(30–531) and α1A have been observed. Both proteins bound to multiple sites in collagen type I with various affinities. The high affinity class of binding sites in type I collagen for α1A exhibited a KDof 0.09 ± 0.06 μm and occurred 2.5 ± 0.5 times per monomer, whereas CNA-(30–531) bound to 1.3 ± 0.1 sites per type I collagen monomer with highest affinity (KD = 0.21 ± 0.02 μm) (16Rich R.L. Deivanayagam C.C.S. Owens R.T. Carson M. Höök A. Moore D. Symerski J. Yang V.W.-C. Narayana S.V.L. Höök M. J. Biol. Chem. 1999; 274: 24906-24913Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Studies using CNBr or collagenase-generated fragments indicated that α1β1 and α2β1 bound to multiple sites in collagens (29Gullberg D. Gehlsen K.R. Turner D.C. Ahlen K. Zijenah L.S. Barnes M.J. Rubin K. EMBO J. 1992; 11: 3865-3873Crossref PubMed Scopus (207) Google Scholar, 30Morton L.F. Peachey A.R. Zijenah L.S. Goodall A.H. Humphries M.J. Barnes M.J. Biochem. J. 1994; 299: 791-797Crossref PubMed Scopus (87) Google Scholar, 31Saelman E.U.M. Horton L.F. Barnes M.J. Gralnik H.R. Hese K.M. Nieuwenhuis H.K. Groot P.G.D. Sixma J.J. Blood. 1993; 82: 3029-3033Crossref PubMed Google Scholar, 32Messent A.J. Tuckwell D.S. Knauper V. Humphries M.J. Murphy G. Gavrilovic J. J. Cell Sci. 1998; 111: 1127-1135Crossref PubMed Google Scholar). By using synthetic peptides that can adopt a triple helical conformation, Knight and colleagues (33Knight 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 (243) Google Scholar, 34Knight 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 (546) Google Scholar) identified the sequence GFOGER (O represents hydroxyproline) (residues 502–507) as a major binding site in collagen α1(I)CB3 for α1A and α2A as well as for the intact receptors. Replacement of residue Glu by Asp causes complete loss of recognition by α1A and α2A, whereas replacement of residue Arg by Lys causes substantial loss of recognition (34Knight 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 (546) Google Scholar). Recently, the GFOGER-containing collagen peptide was co-crystallized in complex with α2A (35Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 100: 47-56Abstract Full Text Full Text PDF Scopus (843) Google Scholar). Structural analyses of this complex revealed that residue Glu in the collagen peptide directly coordinates with the metal ion; residue Arg forms a salt bridge to an Asp residue in α2A, and residue Phe makes hydrophobic contact with α2A (35Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 100: 47-56Abstract Full Text Full Text PDF Scopus (843) Google Scholar). Thus, in addition to residues Glu and Arg, it seems important that the second residue in this 6-amino acid segment is a hydrophobic residue. Comparison between the collagen-bound structure and α2A without ligand revealed that the formation of a complementary surface for collagen binding involves major conformational changes of the A-domain. Although both α1β1 and α2β1 bind to collagen types I and IV, their relative affinities for the two collagen types differ. Thus, α1β1 binds to collagen type IV with higher affinity than to type I, whereas α2β1 has a higher affinity for collagen type I than for type IV (36Kern A. Eble J. Golbik R. Kühn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (181) Google Scholar). Similar differences have also been observed in the interaction of the α1A- and α2A-domains with these two collagen types (7Calderwood D.A. Tuckwell D.S. Ebles J. Kühn K. Humphries M.J. J. Biol. Chem. 1997; 272: 12311-12317Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 10Kern A. Marcantonio E.E. J. Cell. Physiol. 1998; 176: 634-641Crossref PubMed Scopus (29) Google Scholar, 24Käpylä J. Ivaska J. Riikonen R. Nykvist P. Pentikainen O. Johnson M. Heino J. J. Biol. Chem. 2000; 275: 3348-3354Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 25Dickeson S.K. Mathis N.L. Rahman M. Bergelson J.M. Santoro S. J. Biol. Chem. 1999; 274: 32182-32191Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Furthermore, α1A and α2A appear to bind to collagen type I with different affinities although results from different studies are not consistent (7Calderwood D.A. Tuckwell D.S. Ebles J. Kühn K. Humphries M.J. J. Biol. Chem. 1997; 272: 12311-12317Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 24Käpylä J. Ivaska J. Riikonen R. Nykvist P. Pentikainen O. Johnson M. Heino J. J. Biol. Chem. 2000; 275: 3348-3354Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 25Dickeson S.K. Mathis N.L. Rahman M. Bergelson J.M. Santoro S. J. Biol. Chem. 1999; 274: 32182-32191Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Comparison between the structures of α1A and α2A revealed that their trenches differ in dimensions, with the one of α1A being longer, wider, and deeper than that of α2A (16Rich R.L. Deivanayagam C.C.S. Owens R.T. Carson M. Höök A. Moore D. Symerski J. Yang V.W.-C. Narayana S.V.L. Höök M. J. Biol. Chem. 1999; 274: 24906-24913Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In both α1A and α2A, a so-called αC helix was found between β-strand E and helix α6 and this αC helix forms a wall of each trench. The αC helix in α2A is more protruding than that of α1A, contributing to the smaller dimensions of its trench. Mutations of residues in the αC helix affect collagen binding of α2A, but the reported results differ perhaps due to the introduction of different amino acids and/or to the different assay systems used (9Kamata T. Puzon W. Takada Y. J. Biol. Chem. 1994; 269: 9659-9663Abstract Full Text PDF PubMed Google Scholar, 22Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar, 23Kamata T. Liddington R.C. Takada Y. J. Biol. Chem. 1999; 274: 32108-32111Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 24Käpylä J. Ivaska J. Riikonen R. Nykvist P. Pentikainen O. Johnson M. Heino J. J. Biol. Chem. 2000; 275: 3348-3354Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 25Dickeson S.K. Mathis N.L. Rahman M. Bergelson J.M. Santoro S. J. Biol. Chem. 1999; 274: 32182-32191Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The MIDAS residues and their spatial arrangement are conserved between α1A and α2A, suggesting a possible common metal-coordinating mechanism in their interactions with collagen. The structural differences observed in the binding trenches, however, raise the possibility of differences in binding characteristics for α1A and α2A. We previously reported that α1A has at least two classes of binding sites in type I collagen with the highest affinity class having a KD of 0.09 ± 0.06 μmand approximately three binding sites. We now report on the characterizations of the binding of α2A to type I collagen using both surface plasmon resonance (SPR) and ELISAs. We show that α2A binds type I collagen in a slightly different manner compared with α1A. However, the two A-domains seem to target the same or overlapping sites in type I collagen. By using rotary shadowing followed by electron microscopy of mixtures of each A-domain and type I procollagen, we located these sites along the collagen molecule. Amino acid sequences within these regions were selected and incorporated into synthetic collagen peptides. Analyses of these triple helical peptides identified a new high affinity binding site in type I collagen for both α1A and α2A. A DNA fragment encoding the A-domain of α1 was amplified by PCR from a human hepatoma cDNA library and cloned in the expression vector pQE30 (Qiagen Inc., Chatsworth, CA) as described previously (16Rich R.L. Deivanayagam C.C.S. Owens R.T. Carson M. Höök A. Moore D. Symerski J. Yang V.W.-C. Narayana S.V.L. Höök M. J. Biol. Chem. 1999; 274: 24906-24913Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The A-domain of α2 was cloned by using a similar strategy. In this case, oligonucleotide primers 5′ CGG ATC CCC TGA TTT TCA GCT CTC AGC C and 5′ GCT GCA GTC AAA TGC TGA AAA TTT GTT CTC C were used to amplify by PCR the DNA fragment encoding α2A-domain from a human hepatoma cDNA library. AmpliTaq DNA polymerase (PerkinElmer Life Sciences) was used according to the manufacturer's instructions. The primers contained restriction sites for BamHI and PstI. The PCR product was purified, digested with BamHI and PstI, purified again, and ligated to pQE30 digested with the same enzymes and treated with calf intestine alkaline phosphatase (Life Technologies, Inc.). The ligation mixture was transformed into E. coli JM101. The construct was verified by restriction enzyme digestion and DNA sequencing. The encoded amino acid sequence of the α2A-domain was as published (37Takada Y. Hemler M.E. J. Cell Biol. 1989; 109: 397-407Crossref PubMed Scopus (252) Google Scholar). Large scale expression and purification of α1A and α2A proteins were as described previously using HiTrap Ni2+-chelating chromatography (16Rich R.L. Deivanayagam C.C.S. Owens R.T. Carson M. Höök A. Moore D. Symerski J. Yang V.W.-C. Narayana S.V.L. Höök M. J. Biol. Chem. 1999; 274: 24906-24913Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Protein concentrations were determined from the absorbance at 280 nm as measured on a Beckman DU-70 UV-visible spectrophotometer. The molar extinction coefficient at λ280 of each protein was calculated using the method of Pace et al. (38Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3452) Google Scholar). Previous analysis showed gradual precipitation of the recombinant A-domains within several days of post-purification. Addition of 5 mm β-mercaptoethanol delayed the precipitation for several weeks (16Rich R.L. Deivanayagam C.C.S. Owens R.T. Carson M. Höök A. Moore D. Symerski J. Yang V.W.-C. Narayana S.V.L. Höök M. J. Biol. Chem. 1999; 274: 24906-24913Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Analysis of immediately purified protein in buffer with or without 5 mmβ-mercaptoethanol gave the same results, indicating that 5 mm β-mercaptoethanol does not disturb the interaction. Thus, all analyses were performed in the presence of β-mercaptoethanol and within 2 weeks of purification. Analyses were carried out at ambient temperature using the BIACORE 1000 system (BIAcore AB, Uppsala, Sweden) as described previously (16Rich R.L. Deivanayagam C.C.S. Owens R.T. Carson M. Höök A. Moore D. Symerski J. Yang V.W.-C. Narayana S.V.L. Höök M. J. Biol. Chem. 1999; 274: 24906-24913Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Briefly, 3000–4000 response units of bovine type I collagen (Vitrogen 100, Collagen Biomaterials, Palo Alto, CA) or chicken type I procollagen were immobilized on one of the flow cells on a CM5 chip. The α1A and α2A proteins at the indicated concentrations in HBS (10 mm HEPES, 150 mmNaCl, pH 7.4) buffer containing 5 mm β-mercaptoethanol, 1 mm MgCl2, and 0.05% octyl-β-d-glucopyranoside were run over these surfaces at 5 μl/min for 4 min. Regeneration of the collagen surface was achieved by running 20 μl of a solution of 0.02% SDS, 5 mmβ-mercaptoethanol, and 0.05% octyl-β-d-glucopyranoside through the flow cell at 5 μl/min. Binding of α1A and α2A to reference flow cells, which had been activated and deactivated without the coupling of collagen, was also measured and was subtracted from the binding to collagen-coated chips. SPR sensorgrams from different injections were overlaid using the BIAevaluation 2.1 software (BIAcore AB). Data from the equilibrium portion of the sensorgrams were used for analysis. Based on the correlation between the SPR response and change in soluble A-domain protein binding to the immobilized collagen, values for the binding ratio, νbound, and the concentration of free protein, [P]free, were calculated using the equations described previously (28Rich R.L. Kreikemeyer B. Owens R.T. LaBrenz S. Narayana S.V.L. Weinstock G.M. Murray B.E. Höök M. J. Biol. Chem. 1999; 274: 26939-26945Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Scatchard analysis was performed by plotting νbound/[P]free against νboundin which the negative reciprocal of the slope is the dissociation constant, KD, and the X-intercept is the number of binding interactions, n. 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 R 2 and the degree of freedom of the curve fit. KD values outside the experimental data range were excluded. In the case when the two KD values given by the two-class equation were similar to each other, one-binding class equation was used to calculateKD and n. Experiments were performed with at least three independent protein preparations and three flow cells coated independently with bovine type I collagen. For chicken type I procollagen, two BIAcore runs were performed for each A-domain. The results were reproducible. EZ-LinkTM Sulfo-NHS-LC-Biotin (Pierce) was dissolved in distilled H2O to a concentration of 1 μg/μl. The solution was immediately added to the recombinant proteins in HBS with 5 mm β-mercaptoethanol at a ratio of 70 μl of biotin solution per mg of protein and incubated at room temperature for 1 h in an end-over-end shaker. The proteins were then dialyzed against HBS containing 5 mmβ-mercaptoethanol and 1 mm MgCl2 at 4 °C with three buffer changes. Final protein concentration was measured as described above. Microtiter wells (Immulon 4, Dynex Technologies, Chantilly, VA) were coated with 1 μg of bovine type I collagen or ovalbumin in HBS overnight at 4 °C. The wells were washed with HBS and incubated with blocking buffer (HBS containing 0.1% w/v ovalbumin and 0.05% v/v Tween 20) for 1 h at room temperature. Varying concentrations of the biotinylated recombinant proteins in blocking buffer containing 1 mmMgCl2 and 5 mm β-mercaptoethanol were added to the wells. After incubation at room temperature for 2 h with gentle shaking, the wells were extensively washed with HBS containing 0.05% Tween 20 and 1 mm MgCl2. Streptavidin-alkaline phosphatase conjugate (Roche Molecular Biochemicals) was diluted 10,000-fold with blocking buffer containing 1 mm MgCl2 and added to the wells. After incubation at room temperature for 45 min, the wells were washed with HBS containing 0.05% Tween 20 and 1 mm MgCl2. For color development, 100 μl of 1.3 m diethanolamine, pH 9.8, containing 1 mm MgCl2, and 1 mg/mlp-nitrophenyl phosphate (Southern Biotechnology Associates, Birmingham, AL) were added to the wells. Absorbance at 405 nm (A405 nm) was measured using a Thermomax microplate reader (Molecular Devices Corp., Menlo Park, CA) after 20–30 min of incubation at room temperature. Experiments were performed in triplicate and repeated with independently prepared protein preparations. Binding to ovalbumin-coated wells was considered background and therefore subtracted from binding to collagen. Data were presented as the mean value ± S.E. ofA405 nm from a representative experiment (n = 3). Apparent dissociation constants,KD(app), were calculated using the nonlinear regression method described above, withA405 nm versus [P], instead of νbound and [P]free, where [P] is the total protein concentration. Analysis of the results indicated that the one-binding class model rather than the two-binding class model was a better fit for both α1A and α2A. Thus the one-binding class model was used to calculate the apparent dissociation constants (KD(app)) of both the α1A and α2A interaction with collagen. Competition ELISAs were performed essentially as described above except that biotinylated proteins were mixed with unlabeled proteins in varying ratios and added to the wells. The binding to the blocking agent ovalbumin was subtracted from the binding to collagen. TheA405 nm of biotinylated protein in the absence of competitors was set to 100%. Data was presented as the mean value ± S.E. of percent binding from three independent experiments (n = 9). The concentration of protein required to compete for half the specific binding, IC50, was calculated based on the one-site competition equation using GraphPad PrismTM software (GraphPad Software Inc.). Purification of type I procollagen from 11-day embryonic chicken tendons was as described previously (39Swasdison S. Mayne P.M. Wright D.W. Accavitti M.A. Fitch J.M. Linsenmayer T.F. Mayne R. Matrix. 1992; 12: 56-65Crossref PubMed Scopus (16) Google Scholar). One part 4.9 μm α1A or 4.7 μmα2A was mixed with 1 part 0.7 μm type I procollagen in 0.1 μm(NH4)2CO3 buffer contai