Substrate Specificity of Human Collagenase 3 Assessed Using a Phage-displayed Peptide Library

Deng, Su-Jun; Bickett, D. Mark; Mitchell, Justin; Lambert, Millard H.; Blackburn, R. Kevin; Carter, H. Luke; Neugebauer, Jennifer M.; Pahel, Gregory; Weiner, Michael P.; Moss, Marcia L.

doi:10.1074/jbc.m004538200

Cited by 122 publications

(92 citation statements)

References 35 publications

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“…Significant differences are also observed at P 2Ј . In the group I MMP-9 substrates, 23 of 28 substrates have Ser or Thr at P 2Ј , but neither residue is prevalent at this subsite in the substrates selected for the other MMPs (32,47). These observations support the contention that subsite interactions other than P 3 and P 1Ј have a significant impact on substrate selectivity among the MMP family.…”

Section: Discussionsupporting

confidence: 77%

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Substrate Hydrolysis by Matrix Metalloproteinase-9*

Kridel

Chen

Kotra

et al. 2001

Journal of Biological Chemistry

173

140

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The catalytic clefts of all matrix metalloproteinases (MMPs) have a similar architecture, raising questions about the redundancy in substrate recognition across the protein family. In the present study, an unbiased phage display strategy was applied to define the substrate recognition profile of MMP-9. Three groups of substrates were identified, each occupying a distinct set of subsites within the catalytic pocket. The most prevalent motif contains the sequence Pro-X-X-Hy-(Ser/Thr) at P 3 through P 2 . This sequence is similar to the MMP cleavage sites within the collagens and is homologous to substrates the have been selected for other MMPs. Despite this similarity, most of the substrates identified here are selective for MMP-9 over MMP-7 and MMP-13. This observation indicates that substrate selectivity is conferred by key subsite interactions at positions other than P 3 and P 1 . This study shows that MMP-9 has a unique preference for Arg at both P 2 and P 1 , and a preference for Ser/Thr at P 2 . Substrates containing the consensus MMP-9 recognition motif were used to query the protein data bases. A surprisingly limited list of putative physiologic substrates was identified. The functional implications of these proteins lead to testable hypotheses regarding physiologic substrates for MMP-9.Matrix metalloproteinase-9 (MMP-9) 1 is a member of the matrixin family of metallo-endopeptidases (1-3). MMP-9 is historically referred to as gelatinase B because of its ability to cleave gelatin, a denatured form of collagen, in vitro. Along with MMP-2, MMP-9 differs from other MMPs because it contains three fibronectin type II repeats that have high binding affinity for collagen. These repeats are thought to mediate the binding of MMP-2 and -9 to collagen (1, 2). This binding interaction brings the catalytic pocket of the MMP in proximity to collagen, thereby enhancing its rate of hydrolysis. Despite these well characterized biochemical interactions, it is now clear that MMP-9 is also able to cleave a number of other proteins and may have a rather wide range of physiologic substrates (4 -8).Much of our understanding of the biological function of MMP-9 comes from the study of mice lacking this gene. For example, MMP-9-deficient mice have impaired ossification of the skeletal growth plate, a defect that has been partially attributed to poor vascularization of developing bone (9). Studies on these mice also show that MMP-9 is essential for the recruitment of osteoclasts into developing bones (10). Other work indicates that MMP-9-deficient mice are resistant to dermal blistering in a bullous pemphigoid model, an effect that has been attributed to the inability of these mice to cleave the SERPIN ␣1-proteinase inhibitor (5). Finally, recent studies in the RIP1-Tag2 transgenic mouse model of multistage carcinogenesis indicate that MMP-9 is part of the angiogenic "switch" that is essential for tumor growth (11,12). Other reports suggests that MMP-9 may play a role in inflammation in the nervous system. MMP-9 is elevated in enceph...

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Section: Discussionsupporting

confidence: 77%

“…Nearly one-third of all group I substrates for MMP-9 contain Arg at P 2 . Although Arg can also be found at P 2 in peptide substrates for MMP-13, its frequency is much lower than in the MMP-9 substrates we selected (47). Furthermore, Arg is rarely, if ever, found at P 2 in MMP-3 or -7 peptide substrates (32).…”

Section: Discussionmentioning

confidence: 96%

Substrate Hydrolysis by Matrix Metalloproteinase-9*

Kridel

Chen

Kotra

et al. 2001

Journal of Biological Chemistry

173

140

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“…As anticipated, non-selective substrates comprised primarily of the Pro-X-X-2-X Hy sequence were identified. These substrates are collagen-like and have emerged as a canonical, and generally non-selective, MMP recognition motif (21)(22)(23). We also identified substrates that are recognized by MT1-MMP and both gelatinases (MMP-2 and MMP-9).…”

mentioning

confidence: 84%

“…Interestingly, all of the Pro residues were expressed at the same site in the random peptides. Upon first inspection, these substrates appear similar to the Pro-X-X-2-Hy motif that is commonly cleaved by many MMPs (21)(22)(23). This motif represents a canonical collagen-like MMP recognition motif (9).…”

Section: Selection Of Peptide Substrates For Mt1-mmp-mentioning

confidence: 99%

A Unique Substrate Binding Mode Discriminates Membrane Type-1 Matrix Metalloproteinase from Other Matrix Metalloproteinases

Kridel

Sawai

Ratnikov

et al. 2002

Journal of Biological Chemistry

106

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In our study, we characterized the substrate recognition properties of membrane type-1 matrix metalloproteinase (MT1-MMP; also known as MMP-14), a key enzyme in tumor cell invasion and metastasis. A panel of optimal peptide substrates for MT1-MMP was identified using substrate phage display. The substrates can be segregated into four groups based on their degree of selectivity for MT1-MMP. Substrates with poor selectivity for MT1-MMP are comprised predominately of the Pro-X-X-2-X Hy motif that is recognized by a number of MMPs. Highly selective substrates lack the characteristic Pro at the P 3 position; instead they contain an Arg at the P 4 position. This P 4 Arg is essential for efficient hydrolysis and for selectivity for MT1-MMP. Molecular modeling indicates that the selective substrates adopt a linear conformation that extends along the entire catalytic pocket of MT1-MMP, whereas non-selective substrates are kinked at the conserved P 3 Pro residue. Importantly, the selective substrates can be made nonselective by insertion of a proline kink at P 3 , without significantly reducing overall k cat /K m values. Altogether the study provides a structural basis for selective and non-selective substrate recognition by MT1-MMP. The findings in this report are likely to explain several aspects of MT1-MMP biology.Matrix metalloproteinases are a family of zinc-dependent proteases that function in a number of physiologic remodeling events including wound healing, mammary gland involution, and bone resorption (1-3). The MMPs 1 are also linked to pathophysiologic conditions like tumor progression, inflammation, and arthritis (4, 5). Consequently, the MMPs have been explored aggressively as drug targets.The MMP family can be segregated into two groups, the soluble type and the membrane type. The membrane-type MMPs are tethered to the plasma membrane by either a transmembrane domain or a glycosylphosphatidylinositol linkage (6, 7). Membrane type-1 matrix metalloproteinase (MT1-MMP; also known as MMP -14) is the prototypic member of the MTMMPs, and its expression has been associated with a variety of cellular and developmental processes, as well as multiple pathophysiologic conditions. Similar to most other MMPs, MT1-MMP cleaves a number of matrix proteins including collagen, fibronectin, and vitronectin (8 -10). Other work shows that MT1-MMP has substrates that extend beyond extracellular matrix proteins. MT1-MMP is known to convert pro-MMP-2 to active protease (6, 11-13), apparently through its trimolecular complex with tissue inhibitor of metalloproteinase-2 and the ␣ v ␤ 3 integrin (14, 15). Recent work shows that cleavage of cell surface molecules such as CD44, pro-␣ v integrin, and transglutaminase by MT1-MMP modulates cell migration (16 -18). The importance of the broad substrate recognition specificity of MT1-MMP is consistent with the complex phenotype of the MT1-MMP-deficient mice. These mice exhibit overt dwarfism, but close inspection also showed several additional defects including arthritis, delayed ossificatio...

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“…These phage can then be amplified and further enriched to determine the optimal substrates for the protease of interest. This technique has been used to map the cleavage sites of subtilisin (Matthews and Wells, 1993), furin (Matthews et al, 1994) and several matrix metalloproteinases (MMPs) Deng et al, 2000;Kridel et al, 2001;Kridel et al, 2002;Smith et al, 1995). Not only protease substrates can be found using phage display.…”

Section: Applications Of Peptide Displaymentioning

confidence: 99%