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 language | English (US) |
|---|---|
| Pages (from-to) | 31422-31427 |
| Number of pages | 6 |
| Journal | Journal of Biological Chemistry |
| Volume | 275 |
| Issue number | 40 |
| State | Published - Oct 6 2000 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
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 journal › Article
}
TY - JOUR
T1 - Substrate specificity of human collagenase 3 assessed using a phage-displayed peptide library
AU - Deng, Su Jun
AU - Bickett, D. Mark
AU - Mitchell, Justin L.
AU - Lambert, Millard H.
AU - Blackburn, R. Kevin
AU - Carter, H. Luke
AU - Neugebauer, Jennifer
AU - Pahel, Gregory
AU - Weiner, Michael P.
AU - Moss, Marcia L.
PY - 2000/10/6
Y1 - 2000/10/6
N2 - 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.
AB - 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.
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M3 - Article
C2 - 10906330
AN - SCOPUS:0034613174
VL - 275
SP - 31422
EP - 31427
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 40
ER -