Matrix Metalloproteinase Cleavage Site Research Articles

Dissecting MMP P10′ and P11′ subsite sequence preferences, utilizing a positional scanning, combinatorial triple-helical peptide library

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that remodel the extracellular matrix environment and mitigate outside-in signaling. Loss of regulation of MMP activity plays a role in numerous pathological states. In particular, aberrant collagenolysis affects tumor invasion and metastasis, osteoarthritis, and cardiovascular and neurodegenerative diseases. To evaluate the collagen sequence preferences of MMPs, a positional scanning synthetic combinatorial library was synthesized herein and was used to investigate the P10' and P11' substrate subsites. The scaffold for the library was a triple-helical peptide mimic of the MMP cleavage site in types I-III collagen. A FRET-based enzyme activity assay was used to evaluate the sequence preferences of eight MMPs. Deconvolution of the library data revealed distinct motifs for several MMPs and discrimination among closely related MMPs. On the basis of the screening results, several individual peptides were designed and evaluated. A triple-helical substrate incorporating Asp-Lys in the P10'-P11' subsites offered selectivity between MMP-14 and MMP-15, whereas Asp-Lys or Trp-Lys in these subsites discriminated between MMP-2 and MMP-9. Future screening of additional subsite positions will enable the design of selective triple-helical MMP probes that could be used for monitoring in vivo enzyme activity and enzyme-facilitated drug delivery. Furthermore, selective substrates could serve as the basis for the design of specific triple-helical peptide inhibitors targeting only those MMPs that play a detrimental role in a disease of interest.

Matrix Metalloproteinase (MMP) Proteolysis of the Extracellular Loop of Voltage-gated Sodium Channels and Potential Alterations in Pain Signaling

Congenital insensitivity to pain (CIP) or congenital analgesia is a rare monogenic hereditary condition. This disorder is characterized by the inability to perceive any form of pain. Nonsense mutations in Nav.1.7, the main pain signaling voltage-gated sodium channel, lead to its truncations and, consequently, to the inactivation of the channel functionality. However, a non-truncating homozygously inherited missense mutation in a Bedouin family with CIP (Nav1.7-R907Q) has also been reported. Based on our currently acquired in-depth knowledge of matrix metalloproteinase (MMP) cleavage preferences, we developed the specialized software that predicts the presence of the MMP cleavage sites in the peptide sequences. According to our in silico predictions, the peptide sequence of the exposed extracellular unstructured region linking the S5-S6 transmembrane segments in the DII domain of the human Nav1.7 sodium channel is highly sensitive to MMP-9 proteolysis. Intriguingly, the CIP R907Q mutation overlaps with the predicted MMP-9 cleavage site sequence. Using MMP-9 proteolysis of the wild-type, CIP, and control peptides followed by mass spectrometry of the digests, we demonstrated that the mutant sequence is severalfold more sensitive to MMP-9 proteolysis relative to the wild type. Because of the substantial level of sequence homology among sodium channels, our data also implicate MMP proteolysis in regulating the cell surface levels of the Nav1.7, Nav1.6, and Nav1.8 channels, but not Nav1.9. It is likely that the aberrantly accelerated MMP-9 proteolysis during neurogenesis is a biochemical rational for the functional inactivation in Nav1.7 and that the enhanced cleavage of the Nav1.7-R907Q mutant is a cause of CIP in the Bedouin family.

High-Throughput Multiplexed Peptide-Centric Profiling Illustrates Both Substrate Cleavage Redundancy and Specificity in the MMP Family

Matrix metalloproteinases (MMPs) play incompletely understood roles in health and disease. Knowing the MMP cleavage preferences is essential for a better understanding of the MMP functions and design of selective inhibitors. To elucidate the cleavage preferences of MMPs, we employed a high-throughput multiplexed peptide-centric profiling technology involving the cleavage of 18,583 peptides by 18 proteinases from the main sub-groups of the MMP family. Our results enabled comparison of the MMP substrates on a global scale, leading to the most efficient and selective substrates. The data validated the accuracy of our cleavage prediction software. This software allows us and others to locate, with nearly 100% accuracy, the MMP cleavage sites in the peptide sequences. In addition to increasing our understanding of both the selectivity and the redundancy of the MMP family, our study generated a roadmap for the subsequent MMP structural-functional studies and efficient substrate and inhibitor design.

Complete sequencing and characterization of equine aggrecan.

To fully sequence and characterize equine aggrecan and confirm conservation of major aggrecanase, calpain and matrix metalloproteinase (MMP) cleavage sites. Reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends were used to generate clones that encompassed the complete equine aggrecan sequence. Clones were sequenced and compared with the equine genome database to determine intron-exon boundaries. The aggrecan gene spans over 61 kb on chromosome 1 and is encoded by 17 exons. Two major variants of aggrecan were cloned; one containing 8187 bp (2728 amino acids) and a second sequence of 8061 nucleotides (2686 amino acids). The variation was due to a CS1 domain polymorphism. Both sequences are substantially larger than predicted by the genomic database; 11 CS1 repeat elements are absent in the database sequence. The equine amino acid sequence was compared with human, bovine and murine sequences. Globular domains 1, 2 and 3 are highly conserved (overall identity over 80%). Equine CS1 is considerably larger than in other species and, therefore, is the least conserved domain (an overall amino acid identity of 22%). Previously defined aggrecanase, calpain and MMP cleavage sites were identified. Western blotting of chondrocyte culture samples showed complex post-secretion processing. The complete equine aggrecan sequence will support more in-depth research on aggrecan processing and degradation in equine articular cartilage and other musculoskeletal tissues.

A quicker degradation rate is yielded by a novel kind of transgenic silk fibroin consisting of shortened silk fibroin heavy chains fused with matrix metalloproteinase cleavage sites.

Degradation performance of silk fibroin is an important property for its medical applications. Herein we constructed a shortened silk fibroin heavy chain protein fused with a matrix metalloproteinase cleavage site (SSFH-MMP) along with a glutathione S-transferase tag ahead. The digestion assay shows it can be cut by matrix metalloproteinase-2 (MMP-2) at its MMP cleavage site. Furthermore, we introduced the SSFH-MMP into silk fibroin by genetic modification of silkworms in order to increase the degradation rate of the silk fibroin. After acquisition of a race of transgenic silkworms with the coding sequence of the MMP cleavage site in their genomic DNA, we tested some properties of their silk fibroin designated TSF-MMP. The results show that the TSF-MMP has MMP cleavage sites and yields a quicker degradation rate during dilution in MMP-2 enzyme buffer or implantation into tumor tissues compared with that of normal silk fibroin. Moreover, the TSF-MMP is in vitro non-toxic to humanbone marrow mesenchymal stem cells (hBM-MSCs) indicating that the TSF-MMP may become a biomaterial with a quicker degradation rate for its medical applications.

The Role of Collagen Charge Clusters in the Modulation of Matrix Metalloproteinase Activity

Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. Several substrate structural features that direct MMP collagenolysis have been identified. The present study evaluated the role of charged residue clusters in the regulation of MMP collagenolysis. A series of 10 triple-helical peptide (THP) substrates were constructed in which either Lys-Gly-Asp or Gly-Asp-Lys motifs replaced Gly-Pro-Hyp (where Hyp is 4-hydroxy-L-proline) repeats. The stabilities of THPs containing the two different motifs were analyzed, and kinetic parameters for substrate hydrolysis by six MMPs were determined. A general trend for virtually all enzymes was that, as Gly-Asp-Lys motifs were moved from the extreme N and C termini to the interior next to the cleavage site sequence, kcat/Km values increased. Additionally, all Gly-Asp-Lys THPs were as good or better substrates than the parent THP in which Gly-Asp-Lys was not present. In turn, the Lys-Gly-Asp THPs were also always better substrates than the parent THP, but the magnitude of the difference was considerably less compared with the Gly-Asp-Lys series. Of the MMPs tested, MMP-2 and MMP-9 most greatly favored the presence of charged residues with preference for the Gly-Asp-Lys series. Lys-Gly-(Asp/Glu) motifs are more commonly found near potential MMP cleavage sites than Gly-(Asp/Glu)-Lys motifs. As Lys-Gly-Asp is not as favored by MMPs as Gly-Asp-Lys, the Lys-Gly-Asp motif appears advantageous over the Gly-Asp-Lys motif by preventing unwanted MMP hydrolysis. More specifically, the lack of Gly-Asp-Lys clusters may diminish potential MMP-2 and MMP-9 collagenolytic activity. The present study indicates that MMPs have interactions spanning the P23-P23' subsites of collagenous substrates.

Expression pattern analysis and activity determination of matrix metalloproteinase derived from human macrophage subsets.

The polarization behavior of macrophages determines the clinical outcome after implantation of biomaterials. Formation of classically activated macrophages (CAM) may result in cell fusion to form foreign body giant cells, which induce and support uncontrolled inflammatory responses and can cause undesired material degradation. In contrast, polarization into alternatively activated macrophages (AAM) is assumed to support healing processes and implant integration. The expression of matrix metalloproteinases (MMP) by the different macrophage subsets might play a crucial role for inflammatory and wound healing processes and may subsequently influence the implant integration. Therefore, it is of importance to characterize the MMP expression pattern by the different macrophage subsets. This knowledge could support the design of biomaterials in which specific MMP cleavage sites are incorporated allowing a controlled cell-mediated degradation of the material. However, it needs to be considered that the pure expression levels may not correlate with the enzymatic activity of the MMP, which depends on a variety of different parameters such as additional co-factors. For this reason, the differential MMP expression levels and the overall enzymatic activity of in vitro generated human non-polarized macrophages (M0), CAM, and AAM are analyzed in this study. While MMP-1, MMP-3, and MMP-10 showed the highest expression levels in CAM, MMP-12 was most strongly expressed by AAM. Interestingly, although various MMP were expressed at high levels in CAM, the enzymatic MMP activity was increased in supernatants of AAM cultures. The data presented here illustrate the importance to combine the measurement of MMP expression levels with the analysis of the enzymatic activity. The observed MMP-12 expression in combination with the higher enzymatic activity detected in AAM supernatants might motivate the design of biomaterials, whose structure could be modified by MMP-12 catalyzed reactions leading to interactive polymers.

Latency can be conferred to a variety of cytokines by fusion with latency-associated peptide from TGF-β

Objectives:Targeting cytokines to sites of disease has clear advantages because it increases their therapeutic index. We designed fusion proteins of the latent-associated peptide (LAP) derived from TGF-β with various cytokines via a matrix metalloproteinase (MMP) cleavage site. This design confers latency, increased half-life and targeting to sites of inflammation. The aim of this study is to determine whether this approach can be applied to cytokines of different molecular structures and sizes.Methods:Mature cytokines cloned downstream of LAP and a MMP cleavage site were expressed in 293T cells and assessed for latency and biological activity by Western blotting and bioassay.Results:We demonstrate here that fusion proteins of TGF-β, erythropoietin, IL-1ra, IL-10, IL-4, BMP-7, IGF1 and IL-17 were rendered latent by fusion to LAP, requiring cleavage to become active in respective bioassays. As further proof of principle, we also show that delivery of engineered TGF-β can inhibit experimental autoimmune encephalomyelitis and that this approach can be used to efficiently deliver cytokines to the brain and spinal cord in mice with this disease.Conclusions: The latent cytokine approach can be successfully applied to a range of molecules, including cytokines of different molecular structure and mass, growth factors and a cytokine antagonist.

Overexpression of Membrane-Bound Fas Ligand (CD95L) Exacerbates Autoimmune Disease and Renal Pathology in Pristane-Induced Lupus

Loss-of-function mutations in the Fas death receptor or its ligand result in a lymphoproliferative syndrome and exacerbate clinical disease in most lupus-prone strains of mice. One exception is mice injected with 2,6,10,14-tetramethylpentadecane (TMPD), a hydrocarbon oil commonly known as pristane, which induces systemic lupus erythematosus-like disease. Although Fas/Fas ligand (FasL) interactions have been strongly implicated in the activation-induced cell death of both lymphocytes and other APCs, FasL can also trigger the production of proinflammatory cytokines. FasL is a transmembrane protein with a matrix metalloproteinase cleavage site in the ectodomain. Matrix metalloproteinase cleavage inactivates membrane-bound FasL and releases a soluble form reported to have both antagonist and agonist activity. To better understand the impact of FasL cleavage on both the proapoptotic and proinflammatory activity of FasL, its cleavage site was deleted through targeted mutation to produce the deleted cleavage site (ΔCS) mouse line. ΔCS mice express higher levels of membrane-bound FasL than do wild-type mice and fail to release soluble FasL. To determine to what extent FasL promotes inflammation in lupus mice, TMPD-injected FasL-deficient and ΔCS BALB/c mice were compared with control TMPD-injected BALB/c mice. We found that FasL deficiency significantly reduced the early inflammatory exudate induced by TMPD injection. In contrast, ΔCS mice developed a markedly exacerbated disease profile associated with a higher frequency of splenic neutrophils and macrophages, a profound change in anti-nuclear Ab specificity, and markedly increased proteinuria and kidney pathology compared with controls. These results demonstrate that FasL promotes inflammation in TMPD-induced autoimmunity, and its cleavage limits FasL proinflammatory activity.

A comparative study of matrix metalloproteinase and aggrecanase mediated release of latent cytokines at arthritic joints

BackgroundLatent cytokines are engineered by fusing the latency associated peptide (LAP) derived from transforming growth factor-β (TGF-β) with the therapeutic cytokine, in this case interferon-β (IFN-β), via an inflammation-specific matrix...

Mapping of matrix metalloproteinase cleavage sites on syndecan‐1 and syndecan‐4 ectodomains

Syndecans are transmembrane heparan sulfate proteoglycans with roles in cell proliferation, differentiation, adhesion, and migration. They have been associated with multiple functions in tumour progression, through their ability to interact with a wide range of ligands as well as other receptors, which makes them key effectors in the pericellular microenvironment. Extracellular shedding of syndecans by tumour-associated matrix metalloproteinases (MMPs) may have an important role in tumour progression. Such ectodomain shedding generates soluble ectodomains that may function as paracrine or autocrine effectors, or as competitive inhibitors of the intact proteoglycan. Tumour-associated MMPs are shown here to cleave the ectodomains of human syndecan-1 and syndecan-4. Two membrane proximal regions of both syndecan-1 and syndecan-4 are favoured MMP cleavage sites, six and 15 residues from the transmembrane domain. Other sites are 35-40 residues C-terminal from the heparan sulfate chain substitution sites in both syndecans. The MT1-MMP cleavage sites in syndecan-1 and syndecan-4 were confirmed by site-directed mutagenesis. These findings provide insights into the characteristics of syndecan shedding.

Insight into the Degradation of Type-I Collagen Fibrils by MMP-8

Although a number of studies have shed light on the mechanism of collagen degradation in solution, the precise mechanism of collagenolysis in the native fibrillar state remains unclear. To gain insight into the mechanism of fibrillar degradation, we calculated the conformational free-energy landscape for unfolding regions of the α2 chain of type-I collagen within the context of the microfibril. Our data suggest that, relatively, imino-rich sequences maintain the canonical triple-helical structure at body temperature. By contrast, the unique MMP (matrix metalloproteinase) cleavage site adopts conformations where the α2 chain is dissociated from the rest of the fibril—behavior that is similar to what was observed in unfolding simulations of isolated collagen-like model peptides in solution. However, the dissociated cleavage site does not fit within the catalytic site of MMP-8, a representative fibrillar collagenase. Additional free-energy simulations suggest that the presence of the catalytic domain leads to a reorientation of the α2 chain such that it adopts a pose that is complementary to the enzyme's active site. These observations argue that, in the fibrillar state, there is a synergy between the normal thermal fluctuations of the substrate when the enzyme is absent and the fluctuations of the substrate when the enzyme is present. More precisely, our findings suggest that thermal fluctuations serve as the driving force for a degradative process that requires both an unfolded cleavage site and the presence of the enzyme.

Substrate-Guided Proteomics Enhances Degradome Resolution

High-throughput technologies offer unrecognized insights into the genesis of human disease, propelling innovation across a spectrum of biomedical specialties.1 The evolution of molecular diagnostics continues to shape deployment of modern therapies, including identification of markers of disease prognosis, predictors of therapeutic response, and determinants of optimal clinical management, all recognized cornerstones of modern medical practice.2 In the postgenomic era, proteomic paradigms have demonstrated particular utility in advancing the practice of personalized disease prediction, diagnosis, and therapy,3 with systems-based approaches facilitating functional deconvolution of proteomes despite their inherent biological complexity.4 The ever-expanding proteomics toolkit encompasses an array of sophisticated technologies with the flexibility to interrogate a totality of putative protein targets, thereby enabling systematic resolution of proteome structures and functions. Indeed, mapping and decoding of proteome landscapes in health and disease provide the foundation to iteratively collate, integrate, and prioritize large-scale raw data.5 Thus, high-throughput proteomics offers a robust, adaptable technological foundation from which to systematically comprehend the underpinnings of (patho)physiological processes. Article see p 106 Innovative proteomic methods are designed to separate and identify proteins with high fidelity and to assess protein abundance, structure, posttranslational modifications, and interactions, enabling charting of the global proteome (ie, the protein complement of a genome; Figure). Progress in proteomic research has benefitted from the application of stringent criteria in assigning peptide and protein identities for comprehensive proteome cartography, such that perturbed or modified subproteomes within reconstituted protein networks can now be extracted at unprecedented rates for interpretation of their biological relevance. Expansion of proteomic studies has also been propelled by the concomitant development of high-resolution, high-mass-accuracy instrumentation, together with next-generation bioinformatics platforms for data analysis and systems interpretation. Existing approaches, however, may fall short of offering adequate experimental solutions arising from particular protein physicochemical constraints, limiting the otherwise far-reaching …

MMP proteolysis of the human extracellular matrix protein aggrecan is mainly a process of normal turnover

Although it has been shown that aggrecanases are involved in aggrecan degradation, the role of MMP (matrix metalloproteinase) aggrecanolysis is less well studied. To investigate MMP proteolysis of human aggrecan, in the present study we used neoepitope antibodies against MMP cleavage sites and Western blot analysis to identify MMP-generated fragments in normal and OA (osteoarthritis/osteoarthritic) cartilage, and in normal, knee injury and OA and SF (synovial fluid) samples. MMP-3 in vitro digestion showed that aggrecan contains six MMP cleavage sites, in the IGD (interglobular domain), the KS (keratan sulfate) region, the border between the KS region and CS (chondroitin sulfate) region 1, the CS1 region, and the border between the CS2 and the G3 domain, and kinetic studies showed a specific order of digestion where the cleavage between CS2 and the G3 domain was the most preferred. In vivo studies showed that OA cartilage contained (per dry weight) 3.4-fold more MMP-generated FFGV fragments compared with normal cartilage, and although aggrecanase-generated SF-ARGS concentrations were increased 14-fold in OA and knee-injured patients compared with levels in knee-healthy reference subjects, the SF-FFGV concentrations did not notably change. The results of the present study suggest that MMPs are mainly involved in normal aggrecan turnover and might have a less-active role in aggrecan degradation during knee injury and OA.

Molecular engineering of short half-life small peptides (VIP, αMSH and γ3MSH) fused to latency-associated peptide results in improved anti-inflammatory therapeutics

ObjectiveTo facilitate the targeting to inflammation sites of small anti-inflammatory peptides, with short half-lives, by fusion with the latency-associated peptide (LAP) of transforming growth factor β1 through a cleavable matrix...

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.

Potential Relation of Aberrant Proteolysis of Human Protein Tyrosine Kinase 7 (PTK7) chuzhoi by Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) to Congenital Defects

Membrane PTK7 pseudo-kinase plays an essential role in planar cell polarity and the non-canonical Wnt pathway in vertebrates. Recently, a new N-ethyl-N-nitrosourea-induced mutant named chuzhoi (chz) was isolated in mice. chz embryos have severe birth defects, including a defective neural tube, defective heart and lung development, and a shortened anterior-posterior body axis. The chz mutation was mapped to the Ala-Asn-Pro tripeptide insertion into the junction region between the fifth and the sixth Ig-like domains of PTK7. Unexpectedly, chz reduced membrane localization of the PTK7 protein. We hypothesized and then proved that the chz mutation caused an insertion of an additional membrane type 1 matrix metalloproteinase cleavage site in PTK7 and that the resulting aberrant proteolysis of chz affected the migratory parameters of the cells. It is likely that aberrations in the membrane type 1 matrix metalloproteinase/PTK7 axis are detrimental to cell movements that shape the body plan and that chz represents a novel model system for increasing our understanding of the role of proteolysis in developmental pathologies, including congenital defects.

Managing the Quality of Life for Proteins: Focusing on the Exit Path

Managing the Quality of Life for Proteins: Focusing on the Exit Path

Development of an attenuated interleukin-2 fusion protein that can be activated by tumour-expressed proteases

The ability to alter the cytokine microenvironment has the potential to shape immune responses in many physiological settings, including the immunotherapy of tumours. We set out to develop a general approach in which cytokines could be functionally attenuated until activated. We report the development and initial characterization of fusion proteins in which human or mouse interleukin-2 (IL-2), a potent growth factor for immune cells, is joined to a specific IL-2 inhibitory binding component separated by a protease site. The rationale is that upon cleavage by a protease the cytokine is free to dissociate from the inhibitory component and becomes biologically more available. We describe the successful development of two attenuation strategies using specific binding: the first uses the mouse IL-2 receptor alpha chain as the inhibitory binding component whereas the second employs a human antibody fragment (scFv) reactive with human IL-2. We demonstrated that the fusion proteins containing a prostate-specific antigen or a matrix metalloproteinase (MMP) protease cleavage site are markedly attenuated in the intact fusion protein but had enhanced bioactivity of IL-2 in vitro when cleaved. Further, we showed that a fusion protein composed of the IL-2/IL-2 receptor alpha chain with an MMP cleavage site reduced tumour growth in vivo in a peritoneal mouse tumour model. This general strategy should be applicable to other proteases and immune modulators allowing site-specific activation of immunomodulators while reducing unwanted side-effects.

Matrix Metalloproteinase Proteolysis of the Myelin Basic Protein Isoforms Is a Source of Immunogenic Peptides in Autoimmune Multiple Sclerosis

BackgroundMatrix metalloproteinases (MMPs) play a significant role in the fragmentation of myelin basic protein (MBP) and demyelination leading to autoimmune multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The classic MBP isoforms are predominantly expressed in the oligodendrocytes of the CNS. The splice variants of the single MBP gene (Golli-MBP BG21 and J37) are widely expressed in the neurons and also in the immune cells. The relative contribution of the individual MMPs to the MBP cleavage is not known.Methodology/Principal FindingsTo elucidate which MMP plays the primary role in cleaving MBP, we determined the efficiency of MMP-2, MMP-8, MMP-9, MMP-10, MMP-12, MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP and MT6-MMP in the cleavage of the MBP, BG21 and J37 isoforms in the in vitro cleavage reactions followed by mass-spectroscopy analysis of the cleavage fragments. As a result, we identified the MMP cleavage sites and the sequence of the resulting fragments. We determined that MBP, BG21 and J37 are highly sensitive to redundant MMP proteolysis. MT6-MMP (initially called leukolysin), however, was superior over all of the other MMPs in cleaving the MBP isoforms. Using the mixed lymphocyte culture assay, we demonstrated that MT6-MMP proteolysis of the MBP isoforms readily generated, with a near quantitative yield, the immunogenic N-terminal 1–15 MBP peptide. This peptide selectively stimulated the proliferation of the PGPR7.5 T cell clone isolated from mice with EAE and specific for the 1–15 MBP fragment presented in the MHC H-2U context.Conclusions/SignificanceIn sum, our biochemical observations led us to hypothesize that MT6-MMP, which is activated by furin and associated with the lipid rafts, plays an important role in MS pathology and that MT6-MMP is a novel and promising drug target in MS especially when compared with other individual MMPs.