EMD - Calbiochem: Matrix Metalloproteinases (MMPs)

		Technical Resources

		Technical Information

		Calbiochem Information

		Signal Transduction Resource

Matrix Metalloproteinases (MMPs)

�

Technical Overview I Figure: Structural Domains of Matrix Metalloproteinases
Activators I Inhibitors I Kits I Substrates I Related Products I Related Links

�

Matrix Metalloproteinases (MMPs) are a family of zinc metalloendopeptidases secreted by cells, and are responsible for much of the turnover of matrix components. They are included in the “MB clan” of metallopeptidases, containing the motif HEXXHXXGXXH as the zinc-binding active site. The MB clan members are generically referred to as “Metzincins” since they all contain a conserved methionine that forms a turn eight residues downstream from the active site. Clan MB contains a number of families, and MMPs are in family M10. Family M10 is further divided into subfamilies A and B, and MMPs are in subfamily A, also known as the “matrixins.” MMPs from vertebrate species are given MMP numbers (i.e. MMP1, MMP2, etc.), and MMPs from invertebrates rely on trivial names. The MMP family consists of at least 26 members, all of which share a common catalytic core with a zinc molecule in the active site.

The MMPs are produced as zymogens, with a signal sequence and propeptide segment that must be removed during activation. The propeptide domain contains a conserved cysteine, which chelates the active zinc site. An exception is MMP-23 which lacks this conserved cysteine, and has a very different propeptide domain. The generally conserved sequence PRCGVP around the chelating cysteine has been called the “cysteine switch.” A subset of MMPs, including the membrane bound MMPs (MT-MMPs), as well as MMP- 11, MMP-21, MMP-23, and MMP-28, contain a basic prohormone convertase cleavage sequence (RRKR, RRRR, RKRR, etc.), which is thought to be cleaved by the PACE/Furin family enzymes. Two of the MMPs (MMP-2 and MMP-9) contain a fibronectin-like domain inserted into the catalytic domain, presumably to enhance substrate binding. MMP-9 also contains a collagen type-V-like domain, which may enhance substrate binding and specificity. MMP-21 from Xenopus also contains a vitronectin-like region just after the cysteine switch, presumably to assist in substrate interaction.

�

The catalytic domain of the MMPs is similar to more ancient proteinases such as thermolysin and Clostridium collagenase (in the MA clan). The active site forms a long groove that divides the domain, giving it a “pacman” appearance. The propeptide domain lies in reverse orientation in this groove, with the cysteine switch in close proximity to the active site zinc molecule. The MMPs differ in the geography of the active site groove, allowing for different substrate and inhibitor specificities. The different depth of the S1′ pocket of some MMPs has allowed generation of synthetic inhibitors that selectively bind the MMPs, and contain zinc chelators to inhibit the active site. These peptidomimetic MMP inhibitors are being used in a number of clinical trials to inhibit MMP function in different diseases.

All but two MMPs (MMP-7 and MMP-26) contain a regulatory subunit, the hemopexin domain, separated from the catalytic domain by a variable hinge region. The hemopexin domain is thought to confer much of the substrate specificity to the MMPs, and is involved in activation as well as inhibition of the MMPs. The hinge region also confers specificity to the MMPs, either by direct binding or by setting the orientation of the hemopexin domain and the catalytic domain. The hemopexin domain is in the vitronectin family, and is known to bind heparin. Heparin has been shown to potentiate some MMP activities, and MMPs are often found associated with heparin sulfate glycosaminoglycans on the cell surface. The overall three dimensional structure of the hemopexin domain is a fourbladed propeller, with a calcium binding site nestled in the folds. Calcium seems to be required for some MMP/substrate interactions, and not others. Final activation of the MMPs often includes shedding of the hemopexin domain, and the isolated hemopexin domain has been shown to inhibit intact MMPs. This hemopexin domain feedback might lead to direct down-regulation of MMP activity, or may keep MMPs from associating with ECM or substrate.

The MT-MMPs (Membrane Type Matrix Metalloproteinases) are localized to the cell surface. Four of the MT-MMPs contain hydrophobic transmembrane domains (MMP-14, MMP-15, MMP-16, MMP-24), followed by a cytoplasmic domain. The other two MTMMPs (MMP-17, MMP-25) lack cytoplasmic domains and are thought to be GPI-anchored to the cell surface. The cytoplasmic domain is thought to be involved in cyto-skeletal signaling cascades, and may be directly phosphorylated by various kinase cascades. MMP-23 is believed to contain a unique transmembrane domain in the propeptide region, which may allow for a distinctly different attachment and activation cascade than the other MMPs.

MMPs are controlled by endogenous inhibitors, the Tissue Inhibitors of Metallo-Proteinases (TIMPs). There are currently four known TIMPs, and they operate with different inhibition efficiencies against the different MMPs. The four TIMPs are also differentially expressed in tissues, and temporally follow the influx of MMPs. TIMP-2 is constitutively produced, as is MMP-2, with which it is normally paired. This pairing is in fact required for the on-demand activation of MMP-2. The TIMPs are slow, tight-binding inhibitors with low nanomolar inhibition constants. TIMP-3 is localized in the extracellular matrix (ECM), and TIMP-4 is localized mostly in vascular tissue. The TIMPs have also been shown to inhibit the ADAMs (A Disintegrin And Metalloproteinase), but with a much greater range of efficacy. Thus far, only TIMP-3 has been shown to be an effective ADAMs inhibitor, with inhibition constants similar to those for MMPs. Since there are more than forty known ADAMs family members, and no other endogenous inhibitor has been identified, the TIMPs may be responsible for inhibiting a large family of proteinases in vivo.

The MMPs are involved in a wide range of proteolytic events, in normal and pathological circumstances. Normal physiological roles for the MMPs include neurite growth, cell migration, bone elongation, wound healing, angiogenesis, ovulation, sperm maturation, uterine involution, menstruation, enamel formation, antigen processing and presentation, mammary gland development, hair follicle development, and embryo implantation. Pathological processes involving MMPs include tumor growth and migration, fibrosis, arthritis, glaucoma, lupus, scleroderma, cirrhosis, multiple sclerosis, aortic aneurysms, infertility, and many more diseases. It is fair to say that a proteolytic event is key to a wide range of biological processes, including tissue remodeling and also modification or release of biological factors.

Substrate specificity for the MMPs is not yet fully characterized. Known substrates include most of the ECM components (fibronectin, vitronectin, laminin, entactin, tenascin, aggrecan, myelin basic protein, etc). The collagens (Types I, II, III, IV, V, VI, VII, VIII, IX, X, XIV) have all been shown to be substrates of different MMPs, with greatly different efficacies. In addition to connective tissue and ECM components, proteinase inhibitors such as a₁-proteinase inhibitor, antithrombin-III and a₂-macroglobulin are selectively cleaved by MMPs. Growth factors such as IL-1a and pro-TNF-a are cleaved, as are IGF binding protein-3 and IGFBP-5. The most common substrates used to study MMP activity are casein and gelatin. While gelatin (heat denatured collagen) might be considered a valid substrate for the gelatinases (MMP-2 and MMP-9), casein is not likely to be a physiologically relevant substrate. Casein is used as a generic proteinase substrate because it is digested by a wide range of proteinases. Intact Type I collagen has also been used as a substrate for some MMPs. The Type I collagen is cleaved at the same site on all three strands, releasing � and � length fragments. MMP-1 cleaves intact triple helical collagen efficiently, but does not work well on other substrates. Other MMPs, such as MMP-3 and MMP-7, cleave a broad range of substrates. For most of the MMPs, the substrate specificity in vivo is not yet defined.

�

Selected Reviews on Matrix Metalloproteinases:
Bonomi, P. 2002. Semin. Oncol. 29(1 Suppl 4), 78.
Brinckerhoff, C.E., and Matrisian, L.M. 2002. Nat. Rev. Mol. Cell. Biol. 3, 207.
Coussens, L.M., 2002. Science 295, 2387.
Egeblad, M., and Werb, Z. 2002. Nat. Rev. Cancer 2, 161.
Vihinen, P., and Kahari, V.M. 2002. Int. J. Cancer 99, 157.
Werner, J.A., et al. 2002. Clin. Exp. Metastasis 19, 275.
Cox, G., and O’Byrne, K.J. 2001. Anticancer Res. 21, 4207.
Steffensen, B., et al. 2001. Crit. Rev. Oral Biol. Med. 12, 373.
Heath, E.I, and Grochow, L.B. 2000. Drugs 59, 1043.
Denis, L.J., and Verweij, J. 1997. Invest New Drugs 15, 175.

�

Matrix Metalloproteinase Activator

�

Matrix Metalloproteinase Antibody

�

Matrix Metalloproteinase Inhibitors

�

Matrix Metalloproteinase Substrates

�

Matrix Metalloproteinase & TIMP Assays

�

Matrix Metalloproteinase & TIMP Proteins, Enzymes, and Related Products

�

top of page