title: Defining requirements for collagenase cleavage in collagen type III using a bacterial collagen system.
abstract: 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.
authors.affiliation: Department of Biochemistry and Center for Advanced Biotechnology and Medicine, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
authors.forename: Zhuoxin
authors.initials: Z
authors.lastname: Yu
authors.forename: Robert
authors.initials: R
authors.lastname: Visse
authors.forename: Masayori
authors.initials: M
authors.lastname: Inouye
authors.forename: Hideaki
authors.initials: H
authors.lastname: Nagase
authors.forename: Barbara
authors.initials: B
authors.lastname: Brodsky
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000502:physiology; D024061:Collagen Type III / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017364:Collagenases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D053509:Matrix Metalloproteinase 13 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
chemical_list: D024061:Collagen Type III; D029968:Escherichia coli Proteins; D016326:Extracellular Matrix Proteins; D011993:Recombinant Fusion Proteins; D014357:Trypsin; D017364:Collagenases; D053509:Matrix Metalloproteinase 13; C417249:collagenase 1