Supplementary Figures 1-6 from Carcinomas Contain a Matrix Metalloproteinase–Resistant Isoform of Type I Collagen Exerting Selective Support to Invasion

Supplementary Table 1 from Carcinomas Contain a Matrix Metalloproteinase–Resistant Isoform of Type I Collagen Exerting Selective Support to Invasion

Cleavage of collagen by collagenases such as matrix metalloproteinase 1 (MMP-1) is a key step in development, tissue remodeling, and tumor proliferation. The abundant heterotrimeric type I collagen composed of two α1(I) chains and one α2(I) chain is efficiently cleaved by MMP-1 at a unique site in the triple helix, a process which may be initiated by local unfolding within the peptide chains. Atypical homotrimers of the α1(I) chain, found in embryonic and cancer tissues, are very resistant to MMP cleavage. To investigate MMP-1 cleavage, recombinant homotrimers were constructed with sequences from the MMP cleavage regions of human collagen chains inserted into a host bacterial collagen protein system. All triple-helical constructs were cleaved by MMP-1, with α2(I) homotrimers cleaved efficiently at a rate similar to that seen for α1(II) and α1(III) homotrimers, while α1(I) homotrimers were cleaved at a much slower rate. The introduction of destabilizing Gly to Ser mutations within the human collagenase susceptible region of the α2(I) chain did not interfere with MMP-1 cleavage. Molecular dynamics simulations indicated a greater degree of transient hydrogen bond breaking in α2(I) homotrimers compared with α1(I) homotrimers at the MMP-1 cleavage site, and showed an extensive disruption of hydrogen bonding in the presence of a Gly to Ser mutation, consistent with chymotrypsin digestion results. This study indicates that α2(I) homotrimers are susceptible to MMP-1, proves that the presence of an α1(I) chain is not a requirement for α2(I) cleavage, and supports the importance of local unfolding of α2(I) in collagenase cleavage.

Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a central inhibitor of matrix-degrading and sheddase families of metalloproteinases. Extracellular levels of the inhibitor are regulated by the balance between its retention on the extracellular matrix and its endocytic clearance by the scavenger receptor low density lipoprotein receptor-related protein 1 (LRP1). Here, we used molecular modeling to predict TIMP-3 residues potentially involved in binding to LRP1 based on the proposed LRP1 binding motif of 2 lysine residues separated by about 21 Å and mutated the candidate lysine residues to alanine individually and in pairs. Of the 22 mutants generated, 13 displayed a reduced rate of uptake by HTB94 chondrosarcoma cells. The two mutants (TIMP-3 K26A/K45A and K42A/K110A) with lowest rates of uptake were further evaluated and found to display reduced binding to LRP1 and unaltered inhibitory activity against prototypic metalloproteinases. TIMP-3 K26A/K45A retained higher affinity for sulfated glycosaminoglycans than K42A/K110A and exhibited increased affinity for ADAMTS-5 in the presence of heparin. Both mutants inhibited metalloproteinase-mediated degradation of cartilage at lower concentrations and for longer than wild-type TIMP-3, indicating that their increased half-lives improved their ability to protect cartilage. These mutants may be useful in treating connective tissue diseases associated with increased metalloproteinase activity.

It is well established that the expression profiles of multiple and possibly redundant matrix-remodeling proteases (e.g., collagenases) differ strongly in health, disease, and development. Although enzymatic redundancy might be inferred from their close similarity in structure, their in vivo activity can lead to extremely diverse tissue-remodeling outcomes. We observed that proteolysis of collagen-rich natural extracellular matrix (ECM), performed uniquely by individual homologous proteases, leads to distinct events that eventually affect overall ECM morphology, viscoelastic properties, and molecular composition. We revealed striking differences in the motility and signaling patterns, morphology, and gene-expression profiles of cells interacting with natural collagen-rich ECM degraded by different collagenases. Thus, in contrast to previous notions, matrix-remodeling systems are not redundant and give rise to precise ECM-cell crosstalk. Because ECM proteolysis is an abundant biochemical process that is critical for tissue homoeostasis, these results improve our fundamental understanding its complexity and its impact on cell behavior.

Matrix metalloproteinase 13 (MMP-13) degrades collagenous extracellular matrix and its aberrant activity associates with diseases such as arthritis, cancer, atherosclerosis and fibrosis. The wide range of MMP-13 proteolytic capacity suggests that it is a powerful, potentially destructive proteinase and thus it has been believed that MMP-13 is not produced in most adult human tissues in the steady state. Present study has revealed that human chondrocytes isolated from healthy adults constitutively express and secrete MMP-13, but that it is rapidly endocytosed and degraded by chondrocytes. Both pro- and activated MMP-13 bind to clusters II and III of low-density lipoprotein (LDL) receptor-related protein 1 (LRP1). Domain deletion studies indicated that the hemopexin domain is responsible for this interaction. Binding competition between MMP-13 and ADAMTS-4, -5 or TIMP-3, which also bind to cluster II, further shown that the MMP-13 binding site within cluster II is different from those of ADAMTS-4, -5 or TIMP-3. MMP-13 is therefore co-endocytosed with ADAMTS-5 and TIMP-3 by human chondrocytes. These findings indicate that MMP-13 may play a role on physiological turnover of cartilage extracellular matrix and that LRP1 is a key modulator of extracellular levels of MMP-13 and its internalization is independent of the levels of ADAMTS-4, -5 and TIMP-3.

Background: MMP-13 recognizes poorly-defined sequences in collagens. Results: MMP-13 binds key residues in the canonical cleavage site and another site near the collagen N terminus. Conclusion: MMP-1 and MMP-13 differ in their recognition and cleavage of collagen, which is regulated primarily through the Hpx domain of MMP-13. Significance: Our data explain the preference of MMP-13 for collagen II. Remodeling of collagen by matrix metalloproteinases (MMPs) is crucial to tissue homeostasis and repair. MMP-13 is a collagenase with a substrate preference for collagen II over collagens I and III. It recognizes a specific, well-known site in the tropocollagen molecule where its binding locally perturbs the triple helix, allowing the catalytic domain of the active enzyme to cleave the collagen α chains sequentially, at Gly775–Leu776 in collagen II. However, the specific residues upon which collagen recognition depends within and surrounding this locus have not been systematically mapped. Using our triple-helical peptide Collagen Toolkit libraries in solid-phase binding assays, we found that MMP-13 shows little affinity for Collagen Toolkit III, but binds selectively to two triple-helical peptides of Toolkit II. We have identified the residues required for the adhesion of both proMMP-13 and MMP-13 to one of these, Toolkit peptide II-44, which contains the canonical collagenase cleavage site. MMP-13 was unable to bind to a linear peptide of the same sequence as II-44. We also discovered a second binding site near the N terminus of collagen II (starting at helix residue 127) in Toolkit peptide II-8. The pattern of binding of the free hemopexin domain of MMP-13 was similar to that of the full-length enzyme, but the free catalytic subunit bound none of our peptides. The susceptibility of Toolkit peptides to proteolysis in solution was independent of the very specific recognition of immobilized peptides by MMP-13; the enzyme proved able to cleave a range of dissolved collagen peptides.

Remodeling of collagen by matrix metalloproteinases (MMPs) is crucial to tissue homeostasis and repair. MMP-13 is a collagenase with a substrate preference for collagen II over collagens I and III. It recognizes a specific, well-known site in the tropocollagen molecule where its binding locally perturbs the triple helix, allowing the catalytic domain of the active enzyme to cleave the collagen α chains sequentially, at Gly(775)-Leu(776) in collagen II. However, the specific residues upon which collagen recognition depends within and surrounding this locus have not been systematically mapped. Using our triple-helical peptide Collagen Toolkit libraries in solid-phase binding assays, we found that MMP-13 shows little affinity for Collagen Toolkit III, but binds selectively to two triple-helical peptides of Toolkit II. We have identified the residues required for the adhesion of both proMMP-13 and MMP-13 to one of these, Toolkit peptide II-44, which contains the canonical collagenase cleavage site. MMP-13 was unable to bind to a linear peptide of the same sequence as II-44. We also discovered a second binding site near the N terminus of collagen II (starting at helix residue 127) in Toolkit peptide II-8. The pattern of binding of the free hemopexin domain of MMP-13 was similar to that of the full-length enzyme, but the free catalytic subunit bound none of our peptides. The susceptibility of Toolkit peptides to proteolysis in solution was independent of the very specific recognition of immobilized peptides by MMP-13; the enzyme proved able to cleave a range of dissolved collagen peptides.

Matrix metalloproteinase (MMP)-13 is one of the mammalian collagenases that play key roles in tissue remodelling and repair and in progression of diseases such as cancer, arthritis, atherosclerosis, and aneurysm. For collagenase to cleave triple helical collagens, the triple helical structure has to be locally unwound before hydrolysis, but this process is not well understood. We report crystal structures of catalytically inactive full-length human MMP-13(E223A) in complex with peptides of 14-26 aa derived from the cleaved prodomain during activation. Peptides are bound to the active site of the enzyme by forming an extended -strand with Glu(40) or Tyr(46) inserted into the S-1 specificity pocket. The structure of the N-terminal part of the peptides is variable and interacts with different parts of the catalytic domain. Those areas are designated substrate-dependent exosites, in that they accommodate different peptide structures, whereas the precise positioning of the substrate backbone is maintained in the active site. These modes of peptide-MMP-13 interactions have led us to propose how triple helical collagen strands fit into the active site cleft of the collagenase.Stura, E. A., Visse, R., Cuniasse, P., Dive, V., Nagase, H. Crystal structure of full-length human collagenase 3 (MMP-13) with peptides in the active site defines exosites in the catalytic domain.

Osteoarthritis (OA) is the leading cause of joint pain and disability in middle-aged and elderly patients, and is characterized by progressive loss of articular cartilage. Among the various matrix metalloproteinases (MMPs), MMP-13 is specifically expressed in the cartilage of human OA patients and is not present in normal adult cartilage. Thus, MMP-13-selective inhibitors are promising candidates in osteoarthritis therapy. Recently, we designed an N-isopropoxy-arylsulfonamide-based hydroxamate inhibitor, which showed low nanomolar activity and high selectivity for MMP-13. In parallel to further studies aiming to assess the in vivo activity of our compound, we screened the Life Chemicals database through computational docking to seek for novel scaffolds as zinc-chelating non-hydroxamate inhibitors. Experimental evaluation of 20 selected candidate compounds verified five novel leads with IC(50) in the low μM range. These newly discovered inhibitors are structurally unrelated to the ones known so far and provide useful scaffolds to develop compounds with more desirable properties. Finally, a first round of structure-based optimization on lead 1 was accomplished and led to an increase in potency of more than 5 fold.

Collagenases of the matrix metalloproteinase (MMP) family play major roles in morphogenesis, tissue repair, and human diseases, but how they recognize and cleave the collagen triple helix is not fully understood. Here, we report temperature-dependent binding of a catalytically inactive MMP-1 mutant (E200A) to collagen through the cooperative action of its catalytic and hemopexin domains. Contact between the two molecules was mapped by screening the Collagen Toolkit peptide library and by hydrogen/deuterium exchange. The crystal structure of MMP-1(E200A) bound to a triple-helical collagen peptide revealed extensive interactions of the 115-Å–long triple helix with both MMP-1 domains. An exosite in the hemopexin domain, which binds the leucine 10 residues C-terminal to the scissile bond, is critical for collagenolysis and represents a unique target for inhibitor development. The scissile bond is not correctly positioned for hydrolysis in the crystallized complex. A productive binding mode is readily modeled, without altering the MMP-1 structure or the exosite interactions, by axial rotation of the collagen homotrimer. Interdomain flexing of the enzyme and a localized excursion of the collagen chain closest to the active site, facilitated by thermal loosening of the substrate, may lead to the first transition state of collagenolysis.

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.Background: Structural requirements of triple-helical collagen for collagenolysis are not fully understood.Results: Recombinant bacterial collagens with human collagen III sequence insertions defined the minimum sequence for cleavage by human collagenases.Conclusion: Susceptibility of bacterial-human collagen chimeras to collagenases mimicked that of human collagen III.Significance: This recombinant system is useful to investigate biological functions of collagen segments in a triple-helical context.

Homodimerization is an essential step for membrane type 1 matrix metalloproteinase (MT1-MMP) to activate proMMP-2 and to degrade collagen on the cell surface. To uncover the molecular basis of the hemopexin (Hpx) domain-driven dimerization of MT1-MMP, a crystal structure of the Hpx domain was solved at 1.7 Å resolution. Two interactions were identified as potential biological dimer interfaces in the crystal structure, and mutagenesis studies revealed that the biological dimer possesses a symmetrical interaction where blades II and III of molecule A interact with blades III and II of molecule B. The mutations of amino acids involved in the interaction weakened the dimer interaction of Hpx domains in solution, and incorporation of these mutations into the full-length enzyme significantly inhibited dimer-dependent functions on the cell surface, including proMMP-2 activation, collagen degradation, and invasion into the three-dimensional collagen matrix, whereas dimer-independent functions, including gelatin film degradation and two-dimensional cell migration, were not affected. These results shed light on the structural basis of MT1-MMP dimerization that is crucial to promote cellular invasion.

The degradation of interstitial collagens I, II and III is an integral part of many biological events such as embryonic development, organ morphogenesis, tissue remodelling and repair, and in diseases such as arthritis, cancer, atherosclerosis, aneurysm and fibrosis, but these collagens are resistant to most proteolytic enzymes due to their triple helical structures. In vertebrates, the enzymes that degrade interstitial collagens are collagenases that are members of the matrix metalloproteinase (MMP) family, and cathepsin K, a lysosomal cysteine proteinase. Non-vertebrate collagenases include collagenolytic serine proteinases in crustacea and Clostridium histolyticum collagenases, also called "bacterial collagenases." This chapter describes the unique properties of these collagenolytic proteinases and the current proposal as to how they recognize triple helical structures and cleave them. Potential enzymatic pathways that may be involved in the degradation of collagen fibrils are also discussed.

Homodimerization is an essential step for membrane type 1 matrix metalloproteinase (MT1-MMP) to activate proMMP-2 and to degrade collagen on the cell surface. To uncover the molecular basis of the hemopexin (Hpx) domain-driven dimerization of MT1-MMP, a crystal structure of the Hpx domain was solved at 1.7 Å resolution. Two interactions were identified as potential biological dimer interfaces in the crystal structure, and mutagenesis studies revealed that the biological dimer possesses a symmetrical interaction where blades II and III of molecule A interact with blades III and II of molecule B. The mutations of amino acids involved in the interaction weakened the dimer interaction of Hpx domains in solution, and incorporation of these mutations into the full-length enzyme significantly inhibited dimer-dependent functions on the cell surface, including proMMP-2 activation, collagen degradation, and invasion into the three-dimensional collagen matrix, whereas dimer-independent functions, including gelatin film degradation and two-dimensional cell migration, were not affected. These results shed light on the structural basis of MT1-MMP dimerization that is crucial to promote cellular invasion.

Abstract Collagen fibers affect metastasis in two opposing ways, by supporting invasive cells but also by generating a barrier to invasion. We hypothesized that these functions might be performed by different isoforms of type I collagen. Carcinomas are reported to contain α1(I)3 homotrimers, a type I collagen isoform normally not present in healthy tissues, but the role of the homotrimers in cancer pathophysiology is unclear. In this study, we found that these homotrimers were resistant to all collagenolytic matrix metalloproteinases (MMP). MMPs are massively produced and used by cancer cells and cancer-associated fibroblasts for degrading stromal collagen at the leading edge of tumor invasion. The MMP-resistant homotrimers were produced by all invasive cancer cell lines tested, both in culture and in tumor xenografts, but they were not produced by cancer-associated fibroblasts, thereby comprising a specialized fraction of tumor collagen. We observed the homotrimer fibers to be resistant to pericellular degradation, even upon stimulation of the cells with proinflammatory cytokines. Furthermore, we confirmed an enhanced proliferation and migration of invasive cancer cells on the surface of homotrimeric versus normal (heterotrimeric) type I collagen fibers. In summary, our findings suggest that invasive cancer cells may use homotrimers for building MMP-resistant invasion paths, supporting local proliferation and directed migration of the cells whereas surrounding normal stromal collagens are cleaved. Because the homotrimers are universally secreted by cancer cells and deposited as insoluble, MMP-resistant fibers, they offer an appealing target for cancer diagnostics and therapy. Cancer Res; 70(11); 4366–74. ©2010 AACR.

Collagen forms the main connective tissue in the body. Collagen turnover is intimately linked with healing of wounds, embryo development and tissue regeneration. Furthermore, the breakdown of collagen in various pathologies such as inflammatory arthritis and cancer is linked to disease progression and is accompanied by profound changes in its structure and mechanical response. Therefore, there has been increased interest in the study of mechanical properties of single collagen fibrils in the past decade. Nevertheless, the influence of the metalloproteases, which degrade collagen fibrils both in healthy tissue and a number of disease conditions, on mechanical properties of collagen has never been studied. In this work we present an investigation of the influence of MMP1 on the bending of type I collagen fibrils. Angular distribution of the segments of single collagen fibrils can be used to characterize the flexibility of collagen fibrils. High resolution images of the type I collagen fibrils were acquired by atomic force microscope (AFM) under ambient conditions in tapping mode. Angular distributions of segments of each collagen fibril were evaluated by an image recognition program from the resulting images. These distributions were compared and analyzed for native collagen type 1 fibrils and after treatment with matrix metalloprotease 1 (MMP1).

We have reported previously that reactive-site mutants of N-TIMP-3 [N-terminal inhibitory domain of TIMP-3 (tissue inhibitor of metalloproteinases 3)] modified at the N-terminus, selectively inhibited ADAM17 (a disintegrin and metalloproteinase 17) over the MMPs (matrix metalloproteinases). The primary aggrecanases ADAMTS (ADAM with thrombospondin motifs) -4 and -5 are ADAM17-related metalloproteinases which are similarly inhibited by TIMP-3, but are poorly inhibited by other TIMPs. Using a newly developed recombinant protein substrate based on the IGD (interglobular domain) of aggrecan, gst-IGD-flag, these reactive-site mutants were found to similarly inhibit ADAMTS-4 and ADAMTS-5. Further mutations of N-TIMP-3 indicated that up to two extra alanine residues can be attached to the N-terminus before the Ki (app) for ADAMTS-4 and ADAMTS-5 increased to over 100 nM. No other residues tested at the [−1] position produced inhibitors as potent as the alanine mutant. The mutants N-TIMP-3(T2G), [−1A]N-TIMP-3 and [−2A]N-TIMP-3 were effective inhibitors of aggrecan degradation, but not of collagen degradation in both IL-1α (interleukin-1α)-stimulated porcine articular cartilage explants and IL-1α with oncostatin M-stimulated human cartilage explants. Molecular modelling studies indicated that the [−1A]N-TIMP-3 mutant has additional stabilizing interactions with the catalytic domains of ADAM17, ADAMTS-4 and ADAMTS-5 that are absent from complexes with MMPs. These observations suggest that further mutation of the residues of N-TIMP-3 which make unique contacts with these metalloproteinases may allow discrimination between them.

Type I collagen cleavage is crucial for tissue remodeling, but its homotrimeric isoform is resistant to all collagenases. The homotrimers occur in fetal tissues, fibrosis, and cancer, where their collagenase resistance may play an important physiological role. To understand the mechanism of this resistance, we studied interactions of α1(I)3 homotrimers and normal α1(I)2α2(I) heterotrimers with fibroblast collagenase (MMP-1). Similar MMP-1 binding to the two isoforms and similar cleavage efficiency of unwound α1(I) and α2(I) chains suggested increased stability and less efficient unwinding of the homotrimer triple helix at the collagenase cleavage site. The unwinding, necessary for placing individual chains inside the catalytic cleft of the enzyme, was the rate-limiting cleavage step for both collagen isoforms. Comparative analysis of the homo- and heterotrimer cleavage kinetics revealed that MMP-1 binding promotes stochastic helix unwinding, resolving the controversy between different models of collagenase action.