The collagenolytic action of MMP-1 is regulated by the interaction between the catalytic domain and the hinge region

The role of the hinge region in the unwinding and cleavage of type I collagen by interstitial collagenase (MMP-1) has been studied at 37 °C and pH 7.3. The collagenolytic processing by MMP-1 displays a very similar overall rate for both chains of collagen I, even though the affinity is higher for the α-1 chain and the cleavage rate is faster for the α-2 chain. MMP-1 binding to collagen I brings about a significant unwinding of the triple-helical arrangement only after the first cleavage step of the α-1 and α-2 chains. The proteolytic processing by wild-type MMP-1 on a synthetic substrate and collagen I has been compared with that observed for site-directed mutants obtained either by truncating the hinge region (∆255–272) or by individually replacing the conserved amino acids Val268, Gly272, and Lys277 of the hinge region with residues observed for the corresponding position in stromelysin-1 (MMP-3), a noncollagenolytic metalloproteinase. The ∆256–272 mutant has no collagenolytic activity, clearly demonstrating the crucial role of this region for the enzymatic processing of collagen I. However, among various mutants investigated, only Gly272Asp shows a dramatically reduced enzymatic activity both on the synthetic substrate and on collagen I. This effect, however, is clearly related to the substituting residue, since substitution of Ala or Asn for Gly272 does not have any effect on the kinetic properties of MMP-1. These data suggest that the substrate specificity of MMP-1 is dictated by the reciprocal structural relationships between the catalytic domain and the carboxy-terminal domain through the conformational arrangement of the hinge region.     

Characterization of the mechanisms by which gelatinase A, neutrophil collagenase, and membrane-type metalloproteinase MMP-14 recognize collagen I and enzymatically process the two alpha-chains

The turnover of native collagen has been ascribed to different members of the matrix metalloproteinase (MMP) family. Here, the mechanisms by which neutrophil collagenase (MMP-8), gelatinase A (MMP-2), and the ectodomain of MT1-MMP (ectMMP-14) degrade fibrillar collagen were examined. In particular, the hydrolysis of type I collagen at 37 degrees C was investigated to identify functional differences in the processing of the two alpha-chain types of fibrillar collagen. Thermodynamic and kinetic parameters were used for a quantitative comparison of the binding, unwinding, and hydrolysis of triple helical collagen. We demonstrate that the MMP family has developed at least two distinct mechanisms for collagen unwinding and cleavage. MMP-8 and ectMMP-14 display a similar mechanism (although with different catalytic parameters), which is characterized by binding (likely through the hemopexin-like domain) and cleavage of alpha-1 and/or alpha-2 chains without distinguishing between them and keeping the gross conformation of the triple helix (at least during the first cleavage step). On the other hand, MMP-2 binds preferentially the alpha-1 chains (likely through the fibronectin-like domain, which is not present in MMP-8 and ectMMP-14), grossly altering the whole triple helical arrangement of the collagen molecule and cleaving preferentially the alpha-2 chain. These distinctive mechanisms underly a drastically different mode of interaction with triple helical fibrillar collagen I, according to which the MMP domain is involved in binding. These findings can be related to the different role exerted by these MMPs on collagen homeostasis in the extracellular matrix.     

Mechanical forces-induced human osteoblasts differentiation involves MMP-2/MMP-13/MT1-MMP proteolytic cascade

Matrix metalloproteinase (MMP) family proteins play diverse roles in many aspects of cellular processes such as osteoblastic differentiation. Besides, mechanical forces that occur in 3D collagen gel promote the osteoblastic phenotype and accelerate matrix mineralization. Although MMPs have been involved in bone differentiation, the proteolytic cascades triggered by mechanical forces are still not well characterized. In this study, we have investigated the contribution of both proteolytic cascades, MMP-3/MMP-1 and MMP-2/MMP-13/MT1-MMP in the differentiation of human osteoblasts cultured in a floating type I collagen lattice (FL) versus an attached collagen lattice (AL). Compared to AL, contraction of human osteoblasts-populated FL led to a fast (1 day) induction of alkaline phosphatase (ALP), bone sialoprotein (BSP), osteoprotegerin (OPG), and Runx-2 expression. At day 4, osteocalcin (OC) overexpression preceded the formation of calcium-containing nodule formation as assessed by X-ray analyses. MMP-1 and MMP-3 were produced to similar extent by cells cultured in FL and AL, whereas contraction of collagen lattices triggered both mRNA overexpression of MMP-2, MMP-13, and MT1-MMP (i.e., MMP-14), and their activation as evidenced by Western blotting or zymographic analyses. Down-regulating MT1-MMP expression or activity either by siRNA transfection or supplementation of culture medium with TIMP-1 or TIMP-2 highlighted the contribution of that enzyme in OC, ALP, and OPG expression. MMP-2 and MMP-13 were more directly involved in BSP expression. So, these results suggest that the main proteolytic cascade, MMP-2/MMP-13/MT1-MMP, and more particularly, its initial regulator MT1-MMP is involved in osteoblast differentiation through mechanical forces.     

Effect of fibroblast activation protein and alpha2-antiplasmin cleaving enzyme on collagen types I, III, and IV

The circulating enzyme, α₂-antiplasmin cleaving enzyme (APCE), has very similar sequence homology and proteolytic specificity as fibroblast activation protein (FAP), a membrane-bound proteinase. FAP is expressed on activated fibroblasts associated with rapid tissue growth as in embryogenesis, wound healing, and epithelial-derived malignancies, but not in normal tissues. Its presence on stroma suggests that FAP functions to remodel extracellular matrix (ECM) during neoplastic growth. Precise biologic substrates have not been defined for FAP, although like APCE, it cleaves α₂-antiplasmin to a derivative more easily cross-linked to fibrin. While FAP has been shown to cleave gelatin, evidence for cleavage of native collagen, the major ECM component, remains indistinct. We examined the potential proteolytic effects of FAP or APCE alone and in concert with selected matrix metalloproteinases (MMPs) on collagens I, III, and IV. SDS-PAGE analyses demonstrated that neither FAP nor APCE cleaves collagen I. Following collagen I cleavage by MMP-1, however, FAP or APCE digested collagen I into smaller peptides. These peptides were analogous to, yet different from, those produced by MMP-9 following MMP-1 cleavage. Amino-terminal sequencing and mass spectrometry analyses of digestion mixtures identified several peptide fragments within the sequences of the two collagen chains. The proteolytic synergy of APCE in the cleavage of collagen I and III was not observed with collagen IV. We conclude that FAP works in synchrony with other proteinases to cleave partially degraded or denatured collagen I and III as ECM is excavated, and that derivative peptides might function to regulate malignant cell growth and motility.     

Isolation and partial characterization of bovine gingival AB collagen

Summary Limited proteolysis with pepsin solubilized 25% of the insoluble gingival matrix as mainly soluble collagenous material. Fractional salt precipication at neutral pH resulted in the separation of types III and I at 1.8 and 2.6 M NaCl, respectively. In addition, a collagenous fraction accounting for 2% of the solubilized collagen and precipitating at 4.5 M NaCl was shown to be identical with type V collagen. Isolation and partial characterization of the constituent-α-chains of the 4.5 M PPT by gel filtration, ion exchange and hydroxylapatite chromatography as well as disc electrophoresis showed that gingival type V collagen contains αA and αB chains in a ratio αB/αA of 1.73–1.8. Electron microscopic examination of ATP-precipitates showed that this collagen type gave only one kind of SLS aggregates with asymmetric band pattern characteristically different from that of type I collagen. The data provide evidence that gingival AB collagen is a heteropolymer in which the αA and αB chains are assembled in the same macromolecule in a 1∶2 ratio.     

Matrix metalloproteinases (MMP-2,9) and their tissue inhibitors (TIMP-1,2) as novel markers of stress response and atherogenesis in children with chronic kidney disease (CKD) on conservative treatment

The system of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) may play a key role in atherogenesis of chronic kidney disease (CKD) patients by its impact on matrix accumulation. Connections with inflammation, stress, or endothelial dysfunction are also probable. However, the data on correlations between these parameters in CKD patients are scarce in adults and absent in children. The aim of our study was to evaluate serum concentrations of MMP-2, MMP-9, TIMP-1, and TIMP-2, as well as their correlations with markers of stress response (Hsp90-α, anti-Hsp60), endothelial dysfunction (sE-selectin), and inflammation (high-sensitivity C-reactive protein) in CKD children treated conservatively. Thirty-seven patients were divided into two groups according to the CKD stage (gr.CKDI, 19 children with CKD stages 2–3; gr.CKDII, 18 subjects with CKD stages 4–5). Twenty-four age-matched healthy subjects served as controls. Serum concentrations of MMP-2, MMP-9, TIMP-1, TIMP-2, Hsp90-α, anti-Hsp60, and sE-selectin were assessed by ELISA. Median values of MMP-2, MMP-9, TIMP-1, and TIMP-2 were significantly higher in all CKD children vs. controls and were increased in patients with CKD stages 4–5 vs. CKD stages 2–3. Hsp90-α, anti-Hsp60, sE-selectin, and glomerular filtration rate predicted the values of MMPs and TIMPs. Chronic kidney disease in children is characterized by MMP/TIMP system dysfunction, aggravated by the progression of renal failure. Correlations between examined parameters, heat shock proteins, and markers of endothelial damage suggest the possibility of MMP/TIMP application as indicators of stress response and atherogenesis in children with CKD on conservative treatment.     

Molecular Design of a Highly Selective and Strong Protein Inhibitor against Matrix Metalloproteinase-2 (MMP-2)

Synthetic inhibitors of matrix metalloproteinases (MMPs), designed previously, as well as tissue inhibitors of metalloproteinases (TIMPs) lack enzyme selectivity, which has been a major obstacle for developing inhibitors into safe and effective MMP-targeted drugs. Here we designed a fusion protein named APP-IP-TIMP-2, in which the ten amino acid residue sequence of APP-derived MMP-2 selective inhibitory peptide (APP-IP) is added to the N terminus of TIMP-2. The APP-IP and TIMP-2 regions of the fusion protein are designed to interact with the active site and the hemopexin-like domain of MMP-2, respectively. The reactive site of the TIMP-2 region, which has broad specificity against MMPs, is blocked by the APP-IP adduct. The recombinant APP-IP-TIMP-2 showed strong inhibitory activity toward MMP-2 (K_i^app = 0.68 pm), whereas its inhibitory activity toward MMP-1, MMP-3, MMP-7, MMP-8, MMP-9, or MT1-MMP was six orders of magnitude or more weaker (IC₅₀ > 1 μm). The fusion protein inhibited the activation of pro-MMP-2 in the concanavalin A-stimulated HT1080 cells, degradation of type IV collagen by the cells, and the migration of stimulated cells. Compared with the decapeptide APP-IP (t_½ = 30 min), APP-IP-TIMP-2 (t_½ ≫ 96 h) showed a much longer half-life in cultured tumor cells. Therefore, the fusion protein may be a useful tool to evaluate contributions of proteolytic activity of MMP-2 in various pathophysiological processes. It may also be developed as an effective anti-tumor drug with restricted side effects.     

Exposure to pro-inflammatory cytokines upregulates MMP-9 synthesis by mesenchymal stem cells-derived osteoprogenitors

An intimate interplay exists between the bone and the immune system, which has been recently termed osteoimmunology. The activity of immune cells affects the intrinsic balance of bone mineralization and resorption carried out by the opposing actions of osteoblasts and osteoclasts. The aim of this study was to determine the possible interaction between inflammatory-induced conditions and matrix metalloproteinases-2,-9 (MMP-2,-9) synthesis and secretion by bone marrow-derived osteoprogenitor cells during advanced stages of osteogenesis. Rat bone marrow-derived mesenchymal stem cells (MSCs) were cultured in the presence of osteogenic supplements in order to direct the cells towards the osteogenic differentiation lineage. At the late stages of osteogenesis, assessed by histochemistry, immunohistochemistry and RT-PCR, cultures were exposed to pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin-1alpha (IL-1α). Biochemical, histochemical and molecular biology techniques were used to discern the influence of pro-inflammatory cytokines on MMP-2,-9 synthesis and secretion. Results indicated that MMP-9 synthesis and secretion were significantly induced after exposure to the cytokines (TNF-α, IL-1α) treatment, while MMP-2 levels remained unchanged. These results indicate that in response to inflammatory processes, osteoblasts, in addition to osteoclasts, can also be involved and contribute to the process of active bone resorption by secretion and activation of MMPs.     

Regulation of cell invasion and morphogenesis in a three-dimensional type I collagen matrix by membrane-type matrix metalloproteinases 1, 2, and 3

During tissue-invasive events, migrating cells penetrate type I collagen-rich interstitial tissues by mobilizing undefined proteolytic enzymes. To screen for members of the matrix metalloproteinase (MMP) family that mediate collagen-invasive activity, an in vitro model system was developed wherein MDCK cells were stably transfected to overexpress each of ten different MMPs that have been linked to matrix remodeling states. MDCK cells were then stimulated with scatter factor/hepatocyte growth factor (SF/HGF) to initiate invasion and tubulogenesis atop either type I collagen or interstitial stroma to determine the ability of MMPs to accelerate, modify, or disrupt morphogenic responses. Neither secreted collagenases (MMP-1 and MMP-13), gelatinases (gelatinase A or B), stromelysins (MMP-3 and MMP-11), or matrilysin (MMP-7) affected SF/HGF-induced responses. By contrast, the membrane-anchored metalloproteinases, membrane-type 1 MMP, membrane-type 2 MMP, and membrane-type 3 MMP (MT1-, MT2-, and MT3-MMP) each modified the morphogenic program. Of the three MT-MMPs tested, only MT1-MMP and MT2-MMP were able to directly confer invasion-incompetent cells with the ability to penetrate type I collagen matrices. MT-MMP-dependent invasion proceeded independently of proMMP-2 activation, but required the enzymes to be membrane-anchored to the cell surface. These findings demonstrate that MT-MMP-expressing cells can penetrate and remodel type I collagen-rich tissues by using membrane-anchored metalloproteinases as pericellular collagenases.     

In situ hybridization reveals that type I and III collagens are produced by pericytes in the anterior pituitary gland of rats

Type I and III collagens widely occur in the rat anterior pituitary gland and are the main components of the extracellular matrix (ECM). Although ECM components possibly play an important role in the function of the anterior pituitary gland, little is known about collagen-producing cells. Type I collagen is a heterotrimer of two α1(I) chains (the product of the col1a1 gene) and one α2(I) chain (the product of the col1a2 gene). Type III collagen is a homotrimer of α1(III) chains (the product of the col3a1 gene). We used in situ hybridization with digoxigenin-labeled cRNA probes to examine the expression of col1a1, col1a2, and col3a1 mRNAs in the pituitary gland of adult rats. mRNA expression for these collagen genes was clearly observed, and cells expressing col1a1, col1a2, and col3a1 mRNA were located around capillaries in the gland. We also investigated the possible double-staining of collagen mRNA and pituitary hormones, S-100 protein (a marker of folliculo-stellate cells), or desmin (a marker of pericytes). Col1a1 and col3a1 mRNA were identified in desmin-immunopositive cells. Thus, only pericytes produce type I and III collagens in the rat anterior pituitary gland.     

Characterization of stromelysin 1 (MMP-3), matrilysin (MMP-7), and membrane type 1 matrix metalloproteinase (MT1-MMP) derived fibrin(ogen) fragments D-dimer and D-like monomer: NH2-terminal sequences of late-stage digest fragments.

Matrix metalloproteinases (MMPs) participate in physiological remodeling of the extracellular matrix. Recently we determined that both fibrinogen (Fg) and cross-linked fibrin (XL-Fb) are substrates for selected MMPs. Specifically, XL-Fb clots were solubilized by MMP-3 (stromelysin 1) by cleavage at gamma Gly 404-Ala 405, resulting in a D-like monomer fragment. Similarly, MMP-7 (matrilysin) and MT1-MMP (membrane type 1 matrix metalloproteinase) solubilized XL-Fb clots. However, the molecular mass of fragment D-dimer, obtained after MMP-7 and MT1-MMP degradation of XL-Fb, is similar to that of fragment D-dimer from plasmin degradation ( approximately 186 kDa). In contrast, fragment D-like monomer, from MMP-3 degradation of both fibrinogen (Fg) and XL-Fb, is similar to fragment D from plasmin degradation of Fg ( approximately 94 kDa). Reduced chains from MMP-3, MMP-7, and MT1-MMP digests of Fg and XL-Fb were subjected to direct sequence analyses and D/D-dimer alpha-chain showed cleavage at both alpha Asp 97-Phe 98 and alpha Asn 102-Asn 103. Degradation of the beta-chain resulted in microheterogeneity of cleavage sites at beta Asp 123-Leu 124, beta Asn 137-Val 138, and beta Glu 141-Tyr 142, whereas all three enzymes cleaved the gamma-chain at gamma Thr 83-Leu 84. In both Fg and XL-Fb, several cleavage sites obtained by proteolysis with MMP-3, MMP-7, and MT1-MMP were found to be in very close proximity to those obtained by plasmin on these same substrates. That does not occur with other MMPs such as MMP-1, -2, and -9 and MT2-MMP. The degradation of XL-Fb by MMPs suggests both plasmin-dependent and independent mechanisms of fibrinolysis that might be relevant in inflammation, angiogenesis, arthritis, and atherosclerosis.     

Characterization of the distinct collagen binding, helicase and cleavage mechanisms of matrix metalloproteinase 2 and 14 (gelatinase A and MT1-MMP): the differential roles of the MMP hemopexin c domains and the MMP-2 fibronectin type II modules in collagen triple helicase activities

Matrix metalloproteinase-2 (MMP-2, gelatinase A) and membrane type (MT)1-MMP (MMP-14) are cooperative dynamic components of a cell surface proteolytic axis involved in regulating the cellular signaling environment and pericellular collagen homeostasis. Although MT1-MMP exhibits type I collagenolytic but poor gelatinolytic activities, MMP-2 is a potent gelatinase with weak type I collagenolytic behavior. Recombinant linker/hemopexin C domain (LCD) of MT1-MMP binds native type I collagen, blocks MT1-MMP collagenolytic activity in trans, and by circular dichroism spectroscopy, induces localized structural perturbation in the collagen. These changes were reflected by enhanced cleavage of the MT1-LCD-bound collagen by the collagenases MMP-1 and MMP-8 but not by trypsin or MMP-7. Thus, the MT1-LCD alone can initiate triple helicase activity. In contrast, the native and denatured collagen binding properties of MMP-2 reside in the fibronectin type II modules, accordingly termed the collagen binding domain (CBD). Recombinant CBD (but not the MMP-2 LCD) also changed the circular dichroism spectra leading to increased MMP-1 and -8 cleavage of native collagen. However, recombinant CBD reduced gelatin and collagen cleavage by MMP-2 in trans as did CBD23, which comprises the second and third fibronectin type II modules, but not the CBD23 mutant W316A/W374A, which neither binds gelatin nor collagen. This indicates that MMP-2 and MT1-MMP bind collagen at a different site than MMP-1 and MMP-8. Thus, MMP-2 utilizes the CBD in cis for collagen binding and triple helicase activity, which compensates for the lack of collagen binding by the MMP-2 LCD. Hence, the MMP family has evolved two distinct mechanisms for collagen triple helicase activity using two structurally distinct domains, with triple helicase activity occurring independent of alpha-chain hydrolysis.     

Estrogen effects on MMP-13 and MMP-14 regulation of left ventricular mass in Dahl salt-induced hypertension

Background:Female Dahl salt-sensitive (DS) rats fed a low-salt diet develop hypertension at 6 months of age. Ovariectomy at 2 months of age accelerates the development of hypertension, and estrogen replacement delays it. Although acute pressure overload induces structural changes in the left ventricle (LV) further effects of gradual hypertension on LV remodeling have not been examined in the DS rat model.Objective: The purpose of this study was to test the hypothesis that aging and estrogen loss in hypertensive DS rats are accompanied by changes in LV remodeling.Methods: Four groups of DS rats were examined: young intact, middle-aged (MA) intact, MA ovariectomized (MA-OVX), and MA-OVX with 17β-eestradiol (E₂) supplementation (MA-OVX+E²). Myocardial matrix metalloproteinases (MMPs),tissue inhibitors of metalloproteinases (TIMPs),and extracellular matrix (ECM) proteins were assessed by immunoblotting.Results: Each of the 4 groups comprised 6 animals. Mean (SEM) LV mass was significantly greater in the MA-intact and the MA-OVX groups (1257 [31] mg and 1199 [25] mg, respectively; both, P < 0.05) compared with the young-intact group (697 [6] mg). LV mass in the MA-OVX+E₂ group was significantly lower compared with the MA-intact and MA-OVX groups (both, P < 0.05), suggesting that estrogen may attenuate LV remodeling.Fibronectin and collagen III and IV concentrations increased significantly in the MA-intact and MA-OOVX groups (all, P < 0.05),indicating increased fibrosis. Multiple MMPs also increased in the MA-intact an nd MA-OVX rats, including MMP-3, -7, -99, -113, and -114, and all TIMPs. In contrast, estrogen attenuated fibrosis by increasing MMP-8 concentrations and increasing collagen III fragments. From good-fit regression modeling, MMP-13 and MMP-14 concentrations correlated positively with LV mass for the MA-intact and MA-OVX groups, respectively.Conclusions: Gradual hypertension stimulated ECM turnover by increasing both MMP/TIMP production and ECM degradation.Estrogen loss or gain resulted in a shift in MMP profiles, suggesting that MMP-13 and MMP-14 may be differentially regulated in postmenopausal hypertension.     

Heterogeneity and subunit composition of the haemoglobins of five tilapiine species (Teleostei, Cichlidae) of the genera Oreochromis and Sarotherodon

Haemoglobins of five tilapiine species of the genera Oreochromis and Sarotherodon were investigated. By gel filtration chromatography a molecular weight of 67–69 kDa was determined for the tetrameric molecules which remained stable between pH 5.0 and pH 9.1. When subjected to sodium dodecyl sulphate-Urea-polyacrylamide gel electrophoresis (PAGE), haemoglobins of all species each were split into monomers of three different molecular weights ranging between 16.3 kDA and 17.6 kDa. Subsequently, isoelectric focusing separated haemolysates into about 23 differently charged tetrameric haemoglobins that were arranged in species-specific patterns. This diversity was shown to result from the occurrence of different types of globin chains. By acidic urea PAGE a total of seven major α-globins and five major β-globins were detected and species-characteristic chain variants were identified. To determine the globin chain composition of particular haemoglobin tetramers, 26 bands were isolated by isoelectric focusing and analysed by acidic urea PAGE. Tetramers consisted of doublets of identical α- and identical β-chains (α₂β₂, symmetric tetramers), or combinations of three (α₂ββ*; αα*β₂) or four (αα*ββ*) distinct chains (asymmetric tetramers). Finally, globin chains of Oreochromis niloticus were subjected to partial N-terminal amino acid sequencing. Differences in the composition of the three major β-chains could be shown, whereas the α-chains were N-terminally blocked. Accepted: 12 September 1997     

Identification of collagen binding domain residues that govern catalytic activities of matrix metalloproteinase-2 (MMP-2)

An innovative approach to enhance the selectivity of matrix metalloproteinase (MMP) inhibitors comprises targeting these inhibitors to catalytically required substrate binding sites (exosites) that are located outside the catalytic cleft. In MMP-2, positioning of collagen substrate molecules occurs via a unique fibronectin-like domain (CBD) that contains three distinct modular collagen binding sites. To characterize the contributions of these exosites to gelatinolysis by MMP-2, seven MMP-2 variants were generated with single, or concurrent double and triple alanine substitutions in the three fibronectin type II modules of the CBD. Circular dichroism spectroscopy verified that recombinant MMP-2 wild-type (WT) and variants had the same fold. Moreover, the MMP-2 WT and variants had the same activity on a short FRET peptide substrate that is hydrolyzed independently of CBD binding. Among single-point variants, substitution in the module 3 binding site had greatest impact on the affinity of MMP-2 for gelatin. Simultaneous substitutions in two or three CBD modules further reduced gelatin binding. The rates of gelatinolysis of MMP-2 variants were reduced by 20–40% following single-point substitutions, by 60–75% after double-point modifications, and by > 90% for triple-point variants. Intriguingly, the three CBD modules contributed differentially to cleavage of dissociated α-1(I) and α-2(I) collagen chains. Importantly, kinetic analyses (k_cat/K_m) revealed that catalysis of a triple-helical FRET peptide substrate by MMP-2 relied primarily on the module 3 binding site. Thus, we have identified three collagen binding site residues that are essential for gelatinolysis and constitute promising targets for selective inhibition of MMP-2.     

The Recognition of Collagen and Triple-helical Toolkit Peptides by MMP-13: SEQUENCE SPECIFICITY FOR BINDING AND CLEAVAGE*

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.     

The alpha 2 chain of collagen type VI sequesters latent proforms of matrix-metalloproteinases and modulates their activation and activity

The extracellular matrix (ECM) attracts increasing attention as a store of biologically active molecules and as a reservoir of potent cell signalling molecules released by proteolytic action. Both, cytokines and proteases mediating such release are sequestered in the ECM. Here, we found matrix metalloproteinase (MMP) proforms closely associated with collagenous septae in fibrotic liver tissue, and we screened immobilized human placenta-derived collagen chains and other ECM proteins for MMP-binding activity. Following the establishment of a novel highly-efficient two-step chromatography procedure for the isolation of the purified α-chains of the pepsin-resistant triple-helical CVI fragment (CVI/PR) solid phase and surface plasmon resonance binding studies were performed. We identified the triple-helical domain of the α2 chain of microfilamentous CVI α2(VI) as having nanomolar affinity for the collagenases proMMP-1, -8 , -13 and stromelysin-1 (MMP-3), thus extending the repertoire of pericellular and substrate-based interactions of MMPs. Enzymatic activity assays enabled the correlation of MMP activity with CVI binding, in that α2(VI) chain-mediated inhibition of enzymatic activity is accompanied by increased binding. Similar results were shown for the gelatinase proMMP-9, whereas for proMMP-2, the α2(VI) chain at low concentrations seems to interfere with prodomain binding resulting in enhanced activity without scission of the prodomain. Stable complexes of proMMP-2 and α2(VI) chain competed with gelatinase binding to the preferred ligand, collagen type I. In conclusion, the α2(VI) chain modulates MMP availability by sequestering proMMPs in the ECM, blocking proteolytic activity. Therefore, CVI and especially its α2(VI) chain might serve as a lead structure for MMP-based therapeutics which modulates the action of these matrix components, e.g. in fibrosis and cancer.