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Degradation of fibrillar collagens is important in many physiological and pathological events. These collagens are resistant to most proteases due to the tightly packed triple-helical structure, but are readily cleaved at a specific site by collagenases, selected members of the matrix metalloproteinases (MMPs). To investigate the structural requirements for collagenolysis, varying numbers of GXY triplets from human type III collagen around the collagenase cleavage site were inserted between two triple helix domains of the Scl2 bacterial collagen protein. The original bacterial CL domain was not cleaved by MMP-1 (collagenase 1) or MMP-13 (collagenase 3). The minimum type III sequence necessary for cleavage by the two collagenases was 5 GXY triplets, including 4 residues before and 11 residues after the cleavage site (P4-P11'). Cleavage of these chimeric substrates was not achieved by the catalytic domain of MMP-1 or MMP-13, nor by full-length MMP-3. Kinetic analysis of the chimeras indicated that the rate of cleavage by MMP-1 of the chimera containing six triplets (P7-P11') of collagen III was similar to that of native collagen III. The collagenase-susceptible chimeras were cleaved very slowly by trypsin, a property also seen for native collagen III, supporting a local structural relaxation of the triple helix near the collagenase cleavage site. The recombinant bacterial-human collagen system characterized here is a good model to investigate the specificity and mechanism of action of collagenases.

Original publication

DOI

10.1074/jbc.m112.348979

Type

Journal article

Journal

The Journal of biological chemistry

Publication Date

06/2012

Volume

287

Pages

22988 - 22997

Addresses

Department of Biochemistry and Center for Advanced Biotechnology and Medicine, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.

Keywords

Humans, Escherichia coli, Collagen Type III, Collagenases, Trypsin, Escherichia coli Proteins, Recombinant Fusion Proteins, Extracellular Matrix Proteins, Amino Acid Sequence, Catalytic Domain, Protein Structure, Tertiary, Substrate Specificity, Molecular Sequence Data, Matrix Metalloproteinase 13, HEK293 Cells