Substrate specificity of human collagenase 3 assessed using a phage-displayed peptide library

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

Research output: Contribution to journalArticle

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Abstract

The substrate specificity of human collagenase 3 (MMP-13), a member of the matrix metalloproteinase family, is investigated using a phage-displayed random hexapeptide library containing 2 x 108 independent recombinants. A total of 35 phage clones that express a peptide sequence that can be hydrolyzed by the recombinant catalytic domain of human collagenase 3 are identified. The translated DNA sequence of these clones reveals highly conserved putative P1, P2, P3 and P1', P2', and P3' subsites of the peptide substrates. Kinetic analysis of synthetic peptide substrates made from human collagenase 3 selected phage clones reveals that some of the substrates are highly active and selective. The most active substrate, 2,4-dinitrophenyl-GPLGM-RGL-NH2 (CP), has a k(cat)/K(m) value of 4.22 x 106 M-1 S-1 for hydrolysis by collagenase 3. CP was synthesized as a consensus sequence deduced from the preferred subsites of the aligned 35 phage clones. Peptide substrate CP is 1300-, 11-, and 820-fold selective for human collagenase 3 over the MMPs stromelysin-1, gelatinase B, and collagenase 1, respectively. In addition, cleavage of CP is 37-fold faster than peptide NF derived from the major MMP-processing site in aggrecan. Phage display screening also selected five substrate sequences that share sequence homology with a major MMP cleavage sequence in aggrecan and seven substrate sequences that share sequence homology with the primary collagenase cleavage site of human type II collagen. In addition, putative cleavage sites similar to the consensus sequence are found in human type IV collagen. These findings support previous observations that human collagenase 3 can degrade aggrecan, type II and type IV collagens.

Original languageEnglish (US)
Pages (from-to)31422-31427
Number of pages6
JournalJournal of Biological Chemistry
Volume275
Issue number40
StatePublished - Oct 6 2000
Externally publishedYes

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Matrix Metalloproteinase 13
Peptide Library
Bacteriophages
Substrate Specificity
Matrix Metalloproteinases
Aggrecans
Substrates
Clone Cells
Peptides
Collagen Type IV
Collagen Type II
Consensus Sequence
Sequence Homology
Matrix Metalloproteinase 3
Matrix Metalloproteinase 9
Collagenases
DNA sequences
Libraries
Catalytic Domain
Hydrolysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Deng, S. J., Bickett, D. M., Mitchell, J. L., Lambert, M. H., Blackburn, R. K., Carter, H. L., ... Moss, M. L. (2000). Substrate specificity of human collagenase 3 assessed using a phage-displayed peptide library. Journal of Biological Chemistry, 275(40), 31422-31427.

Substrate specificity of human collagenase 3 assessed using a phage-displayed peptide library. / Deng, Su Jun; Bickett, D. Mark; Mitchell, Justin L.; Lambert, Millard H.; Blackburn, R. Kevin; Carter, H. Luke; Neugebauer, Jennifer; Pahel, Gregory; Weiner, Michael P.; Moss, Marcia L.

In: Journal of Biological Chemistry, Vol. 275, No. 40, 06.10.2000, p. 31422-31427.

Research output: Contribution to journalArticle

Deng, SJ, Bickett, DM, Mitchell, JL, Lambert, MH, Blackburn, RK, Carter, HL, Neugebauer, J, Pahel, G, Weiner, MP & Moss, ML 2000, 'Substrate specificity of human collagenase 3 assessed using a phage-displayed peptide library', Journal of Biological Chemistry, vol. 275, no. 40, pp. 31422-31427.
Deng SJ, Bickett DM, Mitchell JL, Lambert MH, Blackburn RK, Carter HL et al. Substrate specificity of human collagenase 3 assessed using a phage-displayed peptide library. Journal of Biological Chemistry. 2000 Oct 6;275(40):31422-31427.
Deng, Su Jun ; Bickett, D. Mark ; Mitchell, Justin L. ; Lambert, Millard H. ; Blackburn, R. Kevin ; Carter, H. Luke ; Neugebauer, Jennifer ; Pahel, Gregory ; Weiner, Michael P. ; Moss, Marcia L. / Substrate specificity of human collagenase 3 assessed using a phage-displayed peptide library. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 40. pp. 31422-31427.
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AU - Carter, H. Luke

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