Identification of peptide substrates for human MMP-11 (stromelysin-3) using phage display.
The MMP-11 proteinase, also known as stromelysin-3, probably plays an important role in human cancer because MMP-11 is frequently overexpressed in human tumors and MMP-11 levels affect tumorogenesis in mice. Unlike other MMPs, however, human MMP-11 does not cleave extracellular matrix proteins, such as collagen, laminin, fibronectin, and elastin. To help identify physiologic MMP-11 substrates, a phage display library was used to find peptide substrates for MMP-11. One class of peptides containing 26 members had the consensus sequence A(A/Q)(N/A) downward arrow (L/Y)(T/V/M/R)(R/K), where downward arrow denotes the cleavage site. This consensus sequence was similar to that for other MMPs, which also cleave peptides containing Ala in position 3, Ala in position 1, and Leu/Tyr in position 1', but differed from most other MMP substrates in that proline was rarely found in position 3 and Asn was frequently found in position 1. A second class of peptides containing four members had the consensus sequence G(G/A)E downward arrow LR. Although other MMPs also cleave peptides with these residues, other MMPs prefer proline at position 3 in this sequence. In vitro assays with MMP-11 and representative peptides from both classes yielded modest kcat/Km values relative to values found for other MMPs with their preferred peptide substrates. These reactions also showed that peptides with proline in position 3 were poor substrates for MMP-11. A structural basis for the lower kcat/Km values of human MMP-11, relative to other MMPs, and poor cleavage of position 3 proline substrates by MMP-11 is provided. Taken together, these findings explain why MMP-11 does not cleave most other MMP substrates and predict that MMP-11 has unique substrates that may contribute to human cancer.
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08534, USA.
Weijun
W
Pan
Marc
M
Arnone
Marvin
M
Kendall
Robert H
RH
Grafstrom
Steven P
SP
Seitz
Zelda R
ZR
Wasserman
Charles F
CF
Albright
D001665:Binding Sites; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D005109:Extracellular Matrix / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D053507:Matrix Metalloproteinase 11; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D008958:Models, Molecular; D009369:Neoplasms / Q000378:metabolism; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D019151:Peptide Library; D010455:Peptides; D011392:Proline; D053507:Matrix Metalloproteinase 11; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases