Tyrosine-sulfated dermatopontin shares multiple binding sites and recognition determinants on triple-helical collagens with proteins implicated in cell adhesion and collagen folding,fibrillogenesis, cross-linking,and degradation

Dermatopontin (DPT), a small extracellular matrix protein that stimulates collagen fibrillogenesis, contains sulfotyrosine residues but neither its level of sulfation nor its binding sites on fibrillar collagens are known. Here, we discovered that DPT is present in a relatively high mass concentration (~ 0.02%) in porcine corneal stroma, from which we purified five DPT charge variants (A-E) containing up to six sulfations. The major variant (C), containing four sulfotyrosine residues, was used to locate binding sites for DPT on triple-helical collagens II and III using the Collagen Toolkits. DPT-binding loci included the triple helix crosslinking sites and collagenase cleavage site. We find that strong DPT-binding sites on triple-helical collagen comprise an arginine-rich, positively-charged sequence that also contains hydrophobic residues. This collagen-binding signature of DPT is similar to that of the chaperone HSP47. Thus, we propose that DPT assumes the role of HSP47 as a collagen chaperone during and after the secretion. Peptide II-44, harbouring the conserved collagenase cleavage site, shows the strongest DPT-binding of the Collagen Toolkit II peptides. Substituting any of the three arginine residues (R) with alanine in the sequence GLAGQRGIVGLOGQRGER of II-44 resulted in almost complete loss of DPT binding. Since osteogenesis imperfecta, spondyloepiphyseal dysplasia, and spondyloepimetaphyseal dysplasia congenita are associated with missense mutations that substitute the corresponding arginine residues in collagens alpha-1(I) and alpha-1(II), we suggest that disrupted DPT binding to fibrillar collagens may contribute to these connective tissue disorders. In conclusion, the present work provides a cornerstone for further elucidation of the role of DPT.     

Characterization of high affinity binding motifs for the discoidin domain receptor DDR2 in collagen

The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by native triple-helical collagen. Here we have located three specific DDR2 binding sites by screening the entire triple-helical domain of collagen II, using the Collagen II Toolkit, a set of overlapping triple-helical peptides. The peptide sequence that bound DDR2 with highest affinity interestingly contained the sequence for the high affinity binding site for von Willebrand factor in collagen III. Focusing on this sequence, we used a set of truncated and alanine-substituted peptides to characterize the sequence GVMGFO (O is hydroxyproline) as the minimal collagen sequence required for DDR2 binding. Based on a recent NMR analysis of the DDR2 collagen binding domain, we generated a model of the DDR2-collagen interaction that explains why a triple-helical conformation is required for binding. Triple-helical peptides comprising the DDR2 binding motif not only inhibited DDR2 binding to collagen II but also activated DDR2 transmembrane signaling. Thus, DDR2 activation may be effected by single triple-helices rather than fibrillar collagen.     

Substrate conformation modulates aggrecanase (ADAMTS-4) affinity and sequence specificity. Suggestion of a common topological specificity for functionally diverse proteases

Protease-substrate interactions are governed by a variety of structural features. Although the substrate sequence specificities of numerous proteases have been established, "topological specificities," whereby proteases may be classified based on recognition of distinct three-dimensional structural motifs, have not. The aggrecanase members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family cleave a variety of proteins but do not seem to possess distinct sequence specificities. In the present study, the topological substrate specificity of ADAMTS-4 (aggrecanase-1) was examined using triple-helical or single-stranded poly(Pro) II helical peptides. Substrate topology modulated the affinity and sequence specificity of ADAMTS-4 with K(m) values indicating a preference for triple-helical structure. In turn, non-catalytic ADAMTS-4 domains were critical for hydrolysis of triple-helical and poly(Pro) II helical substrates. Comparison of ADAMTS-4 with MMP-1 (collagenase 1), MMP-13 (collagenase 3), trypsin, and thermolysin using triple-helical peptide (THP) and single-stranded peptide (SSP) substrates demonstrated that all five proteases possessed efficient "triple-helical peptidase" activity and fell into one of two categories: (k(cat)/K(m))(SSP) > (k(cat)/K(m))(THP) (thermolysin, trypsin, and MMP-13) or (k(cat)/K(m))(THP) > or = (k(cat)/K(m))(SSP) and (K(m))(SSP) > (K(m))(THP) (MMP-1 and ADAMTS-4). Overall these results suggest that topological specificity may be a guiding principle for protease behavior and can be utilized to design specific substrates and inhibitors. The triple-helical and single-stranded poly(Pro) II helical peptides represent the first synthetic substrates successfully designed for aggrecanases.     

The Streptococcal Collagen-binding Protein CNE Specifically Interferes with ��V��3-mediated Cellular Interactions with Triple Helical Collagen

Collagen fibers expose distinct domains allowing for specific interactions with other extracellular matrix proteins and cells. To investigate putative collagen domains that govern integrin α_Vβ₃-mediated cellular interactions with native collagen fibers we took advantage of the streptococcal protein CNE that bound native fibrillar collagens. CNE specifically inhibited α_Vβ₃-dependent cell-mediated collagen gel contraction, PDGF BB-induced and α_Vβ₃-mediated adhesion of cells, and binding of fibronectin to native collagen. Using a Toolkit composed of overlapping, 27-residue triple helical segments of collagen type II, two CNE-binding sites present in peptides II-1 and II-44 were identified. These peptides lack the major binding site for collagen-binding β₁ integrins, defined by the peptide GFOGER. Peptide II-44 corresponds to a region of collagen known to bind collagenases, discoidin domain receptor 2, SPARC (osteonectin), and fibronectin. In addition to binding fibronectin, peptide II-44 but not II-1 inhibited α_Vβ₃-mediated collagen gel contraction and, when immobilized on plastic, supported adhesion of cells. Reduction of fibronectin expression by siRNA reduced PDGF BB-induced α_Vβ₃-mediated contraction. Reconstitution of collagen types I and II gels in the presence of CNE reduced collagen fibril diameters and fibril melting temperatures. Our data indicate that contraction proceeded through an indirect mechanism involving binding of cell-produced fibronectin to the collagen fibers. Furthermore, our data show that cell-mediated collagen gel contraction does not directly depend on the process of fibril formation.     

Matrix metalloproteinase-1 takes advantage of the induced fit mechanism to cleave the triple-helical type I collagen molecule

The collagenases are members of the matrix metalloproteinase family (MMP) that degrade native triple-helical type I collagen. To understand the mechanism by which these enzymes recognize and cleave this substrate, we studied the substrate specificity of a modified form of MMP-1 (FC) in which its active site region (amino acids 212-254) had been replaced with that of MMP-9 (amino acids 395-437). Although this substitution increased the activity of the enzyme toward gelatin and the peptide substrate Mca-PLGL(Dpa)AR-NH2 by approximately 3- and approximately 11-fold, respectively, it decreased the type I collagenolytic activity of the enzyme to 0.13%. The replacement of Gly233, the only amino acid in this region of FC that is conserved in all collagenase family members, with the corresponding Glu residue in MMP-9 resulted in a substantial decrease in the type I collagenolytic activity of the enzyme without affecting its general proteolytic activities. The kinetic parameters of the FC/G233E mutant for the collagen substrate were similar to those of the chimeric enzyme. In addition, substituting Gly233 for Glu in the chimera increased the collagenolytic activity of the enzyme by 12-fold. Interestingly, replacing Glu415 in MMP-9 with Gly, its corresponding residue in FC, endowed the enzyme with type I collagenolytic activity. The catalytic activity of the MMP-9 mutant toward triple-helical type I collagen was 2-fold higher than that of the collagenase chimera. These data in conjunction with the X-ray crystal structure of FC indicate that Gly233 provides the flexibility necessary for the enzyme active site to change conformation upon substrate binding. The flexibility provided by the Gly residue is essential for type I collagenolytic activity.     

Defining requirements for collagenase cleavage in collagen type III using a bacterial collagen system

Degradation of fibrillar collagens is important in many physiological and pathological events. These collagens are resistant to most proteases due to the tightly packed triple-helical structure, but are readily cleaved at a specific site by collagenases, selected members of the matrix metalloproteinases (MMPs). To investigate the structural requirements for collagenolysis, varying numbers of GXY triplets from human type III collagen around the collagenase cleavage site were inserted between two triple helix domains of the Scl2 bacterial collagen protein. The original bacterial CL domain was not cleaved by MMP-1 (collagenase 1) or MMP-13 (collagenase 3). The minimum type III sequence necessary for cleavage by the two collagenases was 5 GXY triplets, including 4 residues before and 11 residues after the cleavage site (P4-P11'). Cleavage of these chimeric substrates was not achieved by the catalytic domain of MMP-1 or MMP-13, nor by full-length MMP-3. Kinetic analysis of the chimeras indicated that the rate of cleavage by MMP-1 of the chimera containing six triplets (P7-P11') of collagen III was similar to that of native collagen III. The collagenase-susceptible chimeras were cleaved very slowly by trypsin, a property also seen for native collagen III, supporting a local structural relaxation of the triple helix near the collagenase cleavage site. The recombinant bacterial-human collagen system characterized here is a good model to investigate the specificity and mechanism of action of collagenases.     

Matrix metalloproteinase triple-helical peptidase activities are differentially regulated by substrate stability

Matrix metalloproteinases (MMPs) are involved in physiological remodeling as well as pathological destruction of tissues. The turnover of the collagen triple-helical structure has been ascribed to several members of the MMP family, but the determinants for collagenolytic specificity have not been identified. The present study has compared the triple-helical peptidase activities of MMP-1 and MMP-14 (membrane-type 1 MMP; MT1-MMP). The ability of each enzyme to efficiently hydrolyze the triple helix was quantified using chemically synthesized fluorogenic triple-helical substrates that, via addition of N-terminal alkyl chains, differ in their thermal stabilities. One series of substrates was modeled after a collagenolytic MMP consensus cleavage site from types I-III collagen, while the other series had a single substitution in the P(1)' subsite of the consensus sequence. The substitution of Cys(4-methoxybenzyl) for Leu in the P(1)' subsite was greatly favored by MMP-14 but disfavored by MMP-1. An increase in substrate triple-helical thermal stability led to the decreased ability of the enzyme to cleave such substrates, but with a much more pronounced effect for MMP-1. Increased thermal stability was detrimental to enzyme turnover of substrate (k(cat)), but not binding (K(M)). Activation energies were considerably lower for MMP-14 hydrolysis of triple-helical substrates compared with MMP-1. Overall, MMP-1 was found to be less efficient at processing triple-helical structures than MMP-14. These results demonstrate that collagenolytic MMPs have subtle differences in their abilities to hydrolyze triple helices and may explain the relative collagen specificity of MMP-1.     

Collagen binding specificity of the discoidin domain receptors: Binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1

The discoidin domain receptors, DDR1 and DDR2 are cell surface receptor tyrosine kinases that are activated by triple-helical collagen. While normal DDR signalling regulates fundamental cellular processes, aberrant DDR signalling is associated with several human diseases. We previously identified GVMGFO (O is hydroxyproline) as a major DDR2 binding site in collagens I-III, and located two additional DDR2 binding sites in collagen II. Here we extend these studies to the homologous DDR1 and the identification of DDR binding sites on collagen III. Using sets of overlapping triple-helical peptides, the Collagen II and Collagen III Toolkits, we located several DDR2 binding sites on both collagens. The interaction of DDR1 with Toolkit peptides was more restricted, with DDR1 mainly binding to peptides containing the GVMGFO motif. Triple-helical peptides containing the GVMGFO motif induced DDR1 transmembrane signalling, and DDR1 binding and receptor activation occurred with the same amino acid requirements as previously defined for DDR2. While both DDRs exhibit the same specificity for binding the GVMGFO motif, which is present only in fibrillar collagens, the two receptors display distinct preferences for certain non-fibrillar collagens, with the basement membrane collagen IV being exclusively recognised by DDR1. Based on our recent crystal structure of a DDR2-collagen complex, we designed mutations to identify the molecular determinants for DDR1 binding to collagen IV. By replacing five amino acids in DDR2 with the corresponding DDR1 residues we were able to create a DDR2 construct that could function as a collagen IV receptor.     

Mapping of SPARC/BM-40/osteonectin-binding sites on fibrillar collagens

The 33-kDa matrix protein SPARC (BM-40, osteonectin) binds several collagen types with moderate affinity. The collagen-binding site resides in helix alphaA of the extracellular calcium-binding domain of SPARC and is partially masked by helix alphaC. Previously, we found that the removal of helix alphaC caused a 10-fold increase in the affinity of SPARC for collagen, and we identified amino acids crucial for binding by site-directed mutagenesis. In this study, we used rotary shadowing, CNBr peptides, and synthetic peptides to map binding sites of SPARC onto collagens I, II, and III. Rotary shadowing and electron microscopy of SPARC-collagen complexes identified a major binding site approximately 180 nm from the C terminus of collagen. SPARC binding was also detected with lower frequency near the matrix metalloproteinase cleavage site. These data fit well with our analysis of SPARC binding to CNBr peptides, denaturation of which abolished binding, indicating triple-helical conformation of collagen to be essential. SPARC binding was substantially decreased in two of seven alpha2(I) mutant procollagen I samples and after N-acetylation of Lys/Hyl side chains in wild-type collagen. Synthetic peptides of collagen III were used to locate the binding sites, and we found SPARC binding activity in a synthetic triple-helical peptide containing the sequence GPOGPSGPRGQOGVMGFOGPKGNDGAO (where O indicates 4-hydroxyproline), with affinity for SPARC comparable with that of procollagen III. This sequence is conserved among alpha chains of collagens I, II, III, and V. In vitro collagen fibrillogenesis was delayed in the presence of SPARC, suggesting that SPARC might modulate collagen fibril assembly in vivo.     

Hydrolysis of triple-helical collagen peptide models by matrix metalloproteinases

The matrix metalloproteinase (MMP) family has been implicated in the process of a variety of diseases such as arthritis, atherosclerosis, and tumor cell metastasis. To study the mechanisms of MMP action on collagenous substrates, we have constructed homotrimeric triple-helical peptide (THP) models of the collagenase cleavage sites in types I and II collagen. The THPs incorporate either the alpha1(I)772-786 or the alpha1(II)772-783 sequence. The alpha1(I)772-786 and alpha1(II)772-783 THPs were hydrolyzed by MMP-1 at the Gly-Ile and Gly-Leu bonds, respectively, analogous to the bonds cleaved in corresponding native collagens. Thus, the THPs contained all necessary information to direct MMP-1 binding and proteolysis. Subsequent investigations using the alpha1(I)772-786 THP showed hydrolysis by MMP-2, MMP-13, and a COOH-terminal domain-deleted MMP-1 (MMP-1(Delta(243-450))) but not by MMP-3 or a COOH-terminal domain-deleted MMP-3 (MMP-3(Delta(248-460))). Kinetic analyses showed a k(cat)/K(m) value of 1,808 s(-1) m(-1) for MMP-1 hydrolysis of alpha1(I)772-786 THP, approximately 10-fold lower than for type I collagen. The effect is caused primarily by relative K(m) values. MMP-2 and MMP-13 cleaved the THP more rapidly than MMP-1, but MMP-2 cleavage occurred at distinct multiple sites. Comparison of MMP-1 and MMP-1(Delta(243-450)) hydrolysis of alpha1(I)772-786 THP showed that both can cleave a triple-helical substrate with a slightly higher K(m) value for MMP-1(Delta(243-450)). We propose that the COOH-terminal domain of MMPs is necessary for orienting whole, native collagen molecules but may not be necessary for binding to and cleaving a THP. This proposal is consistent with the large distance between the MMP-1 catalytic and COOH-terminal domains observed by three-dimensional structural analysis and supports previous suggestions that the features of the catalytic domain contribute significantly toward enzyme specificity.     

Exosite interactions impact matrix metalloproteinase collagen specificities

Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. However, the substrate structural determinants that facilitate interaction with specific MMPs are not well defined. We hypothesized that type I-III collagen sequences located N- or C-terminal to the physiological cleavage site mediate substrate selectivity among MMP-1, MMP-2, MMP-8, MMP-13, and MMP-14/membrane-type 1 (MT1)-MMP. The enzyme kinetics for hydrolysis of three fluorogenic triple-helical peptides (fTHPs) was evaluated herein. The first fTHP contained consensus residues 769-783 from type I-III collagens, the second inserted α1(II) collagen residues 763-768 N-terminal to the consensus sequence, and the third inserted α1(II) collagen residues 784-792 C-terminal to the consensus sequence. Our analyses showed that insertion of the C-terminal residues significantly increased k(cat)/K(m) and k(cat) for MMP-1. MMP-13 showed the opposite behavior with a decreased k(cat)/K(m) and k(cat) and a greatly improved K(m) in response to the C-terminal residues. Insertion of the N-terminal residues enhanced k(cat)/K(m) and k(cat) for MMP-8 and MT1-MMP. For MMP-2, the C-terminal residues enhanced K(m) and dramatically decreased k(cat), resulting in a decrease in the overall activity. These changes in activities and kinetic parameters represented the collagen preferences of MMP-8, MMP-13, and MT1-MMP well. Thus, interactions with secondary binding sites (exosites) helped direct the specificity of these enzymes. However, MMP-1 collagen preferences were not recapitulated by the fTHP studies. The preference of MMP-1 for type III collagen appears to be primarily based on the flexibility of the hydrolysis site of type III collagen compared with types I and II. Further characterization of exosite determinants that govern interactions of MMPs with collagenous substrates should aid the development of pharmacotherapeutics that target individual MMPs.     

Collagenase unwinds triple-helical collagen prior to peptide bond hydrolysis

Breakdown of triple-helical interstitial collagens is essential in embryonic development, organ morphogenesis and tissue remodelling and repair. Aberrant collagenolysis may result in diseases such as arthritis, cancer, atherosclerosis, aneurysm and fibrosis. In vertebrates, it is initiated by collagenases belonging to the matrix metalloproteinase (MMP) family. The three-dimensional structure of a prototypic collagenase, MMP-1, indicates that the substrate-binding site of the enzyme is too narrow to accommodate triple-helical collagen. Here we report that collagenases bind and locally unwind the triple-helical structure before hydrolyzing the peptide bonds. Mutation of the catalytically essential residue Glu200 of MMP-1 to Ala resulted in a catalytically inactive enzyme, but in its presence noncollagenolytic proteinases digested collagen into typical 3/4 and 1/4 fragments, indicating that the MMP-1(E200A) mutant unwinds the triple-helical collagen. The study also shows that MMP-1 preferentially interacts with the alpha2(I) chain of type I collagen and cleaves the three alpha chains in succession. Our results throw light on the basic mechanisms that control a wide range of biological and pathological processes associated with tissue remodelling.     

Selective modulation of matrix metalloproteinase 9 (MMP-9) functions via exosite inhibition

Unregulated activities of the matrix metalloproteinase (MMP) family have been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of extracellular matrix components, such as collagen and laminin. However, clinical trials with small molecule MMP inhibitors have been largely unsuccessful, with a lack of selectivity considered particularly problematic. Enhanced selectivity could be achieved by taking advantage of differences in substrate secondary binding sites (exosites) within the MMP family. In this study, triple-helical substrates and triple-helical transition state analog inhibitors have been utilized to dissect the roles of potential exosites in MMP-9 collagenolytic behavior. Substrate and inhibitor sequences were based on either the alpha1(V)436-450 collagen region, which is hydrolyzed at the Gly (downward arrow) Val bond selectively by MMP-2 and MMP-9, or the Gly (downward arrow) Leu cleavage site within the consensus interstitial collagen sequence alpha1(I-III)769-783, which is hydrolyzed by MMP-1, MMP-2, MMP-8, MMP-9, MMP-13, and MT1-MMP. Exosites within the MMP-9 fibronectin II inserts were found to be critical for interactions with type V collagen model substrates and inhibitors and to participate in interactions with an interstitial (types I-III) collagen model inhibitor. A triple-helical peptide incorporating a fibronectin II insert-binding sequence was constructed and found to selectively inhibit MMP-9 type V collagen-based activities compared with interstitial collagen-based activities. This represents the first example of differential inhibition of collagenolytic activities and was achieved via an exosite-binding triple-helical peptide.     

The D2 period of collagen II contains a specific binding site for the human discoidin domain receptor, DDR2

The human discoidin domain receptors (DDRs), DDR1 and DDR2, are expressed widely and, uniquely among receptor tyrosine kinases, activated by the extracellular matrix protein collagen. This activation is due to a direct interaction of collagen with the DDR discoidin domain. Here, we localised a specific DDR2 binding site on the triple-helical region of collagen II. Collagen II was found to be a much better ligand for DDR2 than for DDR1. As expected, DDR2 binding to collagen II was dependent on triple-helical collagen and was mediated by the DDR2 discoidin domain. Collagen II served as a potent stimulator of DDR2 autophosphorylation, the first step in transmembrane signalling. To map the DDR2 binding site(s) on collagen II, we used recombinant collagen II variants with specific deletions of one of the four repeating D periods. We found that the D2 period of collagen II was essential for DDR2 binding and receptor autophosphorylation, whereas the D3 and D4 periods were dispensable. The DDR2 binding site on collagen II was further defined by recombinant collagen II-like proteins consisting predominantly of tandem repeats of the D2 or D4 period. The D2 construct, but not the D4 construct, mediated DDR2 binding and receptor autophosphorylation, demonstrating that the D2 period of collagen II harbours a specific DDR2 recognition site. The discovery of a site-specific interaction of DDR2 with collagen II gives novel insight into the nature of the interaction of collagen II with matrix receptors.     

Implications for Collagen Binding from the Crystallographic Structure of Fibronectin 6FnI1�C2FnII7FnI

Collagen and fibronectin (FN) are two abundant and essential components of the vertebrate extracellular matrix; they interact directly with cellular receptors and affect cell adhesion and migration. Past studies identified a FN fragment comprising six modules, ⁶FnI^1–2FnII^7–9FnI, and termed the gelatin binding domain (GBD) as responsible for collagen interaction. Recently, we showed that the GBD binds tightly to a specific site within type I collagen and determined the structure of domains ^8–9FnI in complex with a peptide from that site. Here, we present the crystallographic structure of domains ⁶FnI^1–2FnII⁷FnI, which form a compact, globular unit through interdomain interactions. Analysis of NMR titrations with single-stranded collagen peptides reveals a dominant collagen interaction surface on domains ²FnII and ⁷FnI; a similar surface appears involved in interactions with triple-helical peptides. Models of the complete GBD, based on the new structure and the ^8–9FnI·collagen complex show a continuous putative collagen binding surface. We explore the implications of this model using long collagen peptides and discuss our findings in the context of FN interactions with collagen fibrils.     

Kinetic analysis of matrix metalloproteinase activity using fluorogenic triple-helical substrates

Matrix metalloproteinase (MMP) family members are involved in the physiological remodeling of tissues and embryonic development as well as pathological destruction of extracellular matrix components. To study the mechanisms of MMP action on collagenous substrates, we have constructed homotrimeric, fluorogenic triple-helical peptide (THP) models of the MMP-1 cleavage site in type II collagen. The substrates were designed to incorporate the fluorophore/quencher pair of (7-methoxycoumarin-4-yl)acetyl (Mca) and N-2,4-dinitrophenyl (Dnp) in the P(5) and P(5)' positions, respectively. In addition, Arg was incorporated in the P(2)' and P(8)' positions to enhance enzyme activity and improve substrate solubility. The desired sequences were Gly-Pro-Lys(Mca)-Gly-Pro-Gln-Gly approximately Leu-Arg-Gly-Gln-Lys(Dnp)-Gly-Ile/Val-Arg. Two fluorogenic substrates were prepared, one using a covalent branching protocol (fTHP-1) and one using a peptide self-assembly approach (fTHP-3). An analogous single-stranded substrate (fSSP-3) was also synthesized. Both THPs were hydrolyzed by MMP-1 at the Gly approximately Leu bond, analogous to the bond cleaved in the native collagen. The individual kinetic parameters for MMP-1 hydrolysis of fTHP-3 were k(cat) = 0.080 s(-1) and K(M) = 61.2 microM. Subsequent investigations showed fTHP-3 hydrolysis by MMP-2, MMP-3, MMP-13, a C-terminal domain-deleted MMP-1 [MMP-1(Delta(243-450))], and a C-terminal domain-deleted MMP-3 [MMP-3(Delta(248-460))]. The order of k(cat)/K(M) values was MMP-13 > MMP-1 approximately MMP-1(Delta(243-450)) approximately MMP-2 > MMP-3 approximately MMP-3(Delta(248-460)). Studies on the effect of temperature on fTHP-3 and fSSP-3 hydrolysis by MMP-1 showed that the activation energies between these two substrates differed by 3.4-fold, similar to the difference in activation energies for MMP-1 hydrolysis of type I collagen and gelatin. This indicates that fluorogenic triple-helical substrates mimic the behavior of the native collagen substrate and may be useful for the investigation of collagenase triple-helical activity.     

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.     

Generation of glycosylated remnant epitopes from human collagen type II by gelatinase B

Gelatinase B/matrix metalloproteinase-9 (MMP-9) is an inflammatory mediator and effector. Considerable amounts of gelatinase B are released by neutrophils in the synovial cavity of patients with rheumatoid arthritis, and gelatinase B-deficient mice are resistant against antibody-induced arthritis. Native human collagen type II is susceptible to cleavage by various collagenases (MMP-1, MMP-8, and MMP-13), which cleave at a single position in the triple helix. Although the triple-helical structure may persist after this single cleavage, we show that gelatinase B degrades the resulting fragments into small remnant peptides. These were identified by mass spectrometry and Edman degradation. Localization of 31 cleavage sites shows that the immunodominant epitopes remain intact after cleavage and may become available, processed as antigens and presented in MHC-II molecules. Furthermore, most post-translational modifications were identified on the fragments, including nine glycosylation sites. In particular, it is shown for the first time by structural analysis that in natural human collagen II, lysines in the main immunodominant epitope are modified by partial hydroxylation and partial glycosylation. Determination of T-cell reactivity against such fragments indicates that, besides the two known main immunodominant epitopes, other glyco-epitopes may be present in collagen II. This reinforces the role of glycopeptide antigens in autoimmunity.     

Interaction of collagen-like peptide models of asymmetric acetylcholinesterase with glycosaminoglycans: spectroscopic studies of conformational changes and stability

The effect of heparin on the conformation and stability of triple-helical peptide models of the collagen tail of asymmetric acetylcholinesterase expands our understanding of heparin interactions with proteins and presents an opportunity for clarifying the nature of binding of ligands to collagen triple-helix domains. Within the collagen tail of AChE, there are two consensus sequences for heparin binding of the form BBXB, surrounded by additional basic residues. Circular dichroism studies were used to determine the effect of the addition of increasing concentrations of heparin on triple-helical peptide models for the heparin binding domains, including peptides in which the basic residues within and surrounding the consensus sequence were replaced by alanine residues. The addition of heparin caused an increased triple-helix content with saturation properties for the peptide modeling the C-terminal site, while precipitation, with no increased helix content resulted from heparin addition to the peptide modeling the N-terminal site. The results suggest that the two binding sites with a similar triple-helical conformation have distinctive ways of interacting with heparin, which must relate to small differences in the consensus sequence (GRKGR vs GKRGK) and in the surrounding basic residues. Addition of heparin increased the thermal stability of all peptides containing the consensus sequence. Heparan sulfate produced conformational and stabilization effects similar to those of heparin, while chondroitin sulfate led to a cloudy solution, loss of circular dichroism signal, and a smaller increase in thermal stability. Thus, specificity in both the sequence of the triple helix and the type of glycosaminoglycan is required for this interaction.     

Cleavage specificity of type IV collagenase (gelatinase) from human skin. Use of synthetic peptides as model substrates

Type IV collagenase (gelatinase) has a marked substrate specificity for denatured collagen (gelatin). Cleavage site specificity of type IV collagenase from human skin was determined using small collagenous peptides with varied sequences around Gly-Leu or Gly-Ile. Type IV collagenase showed essentially the same order of preference for the peptide substrates as did interstitial collagenase. Both required a peptide with a minimum of six amino acid residues to demonstrate significant gelatinolytic activity and were able to cleave uncharged molecules more rapidly than charged molecules. the repeating Gly-X-Y-Gly sequence of collagen is not an absolute requirement for either enzyme since both digested AcPro-Leu-Gly-Ile-Leu-Ala-Ala-OC2H5 at 70% of the rate of the best substrate peptide, AcPro-Leu-Gly-Leu-Leu-Gly-OC2H5. Km and kcat (Vmax) values were determined for several of the peptides and for the native substrate. Turnover numbers with type IV collagenase were similar to those with interstitial collagenase (Weingarten, H., Martin, R., and Feder, J. (1985) Biochemistry 24, 6730-6734). However, the Km for all peptides investigated was approximately 10-fold lower for type IV collagenase than for interstitial collagenase. Because type IV collagenase does not cleave helical interstitial collagens, the data support the conclusion that secondary structure determines whether the peptide bond can be hydrolyzed at any potential cleavage site.