Different Collagenase Gene Products Have Different Roles in Degradation of Type I Collagen

Abstract

Vertebrate collagenases, matrix metalloproteinases (MMPs), cleave type I collagen at a single helical locus. We show here that rodent interstitial collagenases (MMP-13), but not human fibroblast collagenase (MMP-1), cleave type I collagen at an additional aminotelopeptide locus. Collagenase cDNAs and chimeric constructs in pET-3d, juxtaposing MMP-13 sequences amino-terminal to the active site in the catalytic domain and MMP-1 sequences carboxyl-terminal and vice versa, were expressed in Escherichia coli. Assays utilized collagen from wild type (؉/؉) mice or mice that carry a targeted mutation (r/r) that encodes substitutions in ␣1(I) chains that prevent collagenase cleavage at the helical locus. MMP-13 and chimeric molecules that contained the MMP-13 sequences amino-terminal to the active site cleaved (؉/؉) collagen at the helical locus and cleaved cross-linked (r/r) collagen in the aminotelopeptide (␤ components converted to ␣ chains). Human MMP-1 and chimeric MMP-1/MMP-13 with MMP-1 sequences aminoterminal to the active site cleaved collagen at the helical locus but not in the aminotelopeptide. All activities were inhibited by TIMP-1, 1,10-phenanthroline, and EDTA. Sequences in the distal two-thirds of the catalytic domain determine the aminotelopeptide-degrading capacity of MMP-13.When bone and other connective tissues are remodeled, components of the extracellular matrix are degraded and removed, and new components are synthesized and deposited. Three main classes of enzymes have been implicated in the degradation of collagen, the most abundant component of the extracellular matrix. The first includes members of the matrix metalloproteinase (MMP) 1 family, the second, lysosomal cysteine proteinases such as cathepsins B and L which cleave collagens in the telopeptide domain, and the third, serine proteinases such as plasmin, generated through the plasminogen activator system (1-6). Plasmin and cathepsin B have been implicated in the activation of the MMP zymogens (7). It is likely that the MMPs have a role in the physiological resorption of collagen in the uterus, in embryonic development and postnatal remodeling, and in pathological processes such as local invasion by malignant tumors, resorption of periodontal structures in periodontal disease, or the destruction of joints in rheumatoid arthritis (1-5).The MMPs or matrixins are members of a large subfamily of proteinases and have many common structural features (2, 3, 5). Included in the MMP subfamily (8) are genes that encode at least three collagenases, three stromelysins and several gelatinases among which are cell-bound forms with a transmembrane domain (9). Although there is considerable conservation of amino acid sequences and sequence motifs among the human MMPs, only the collagenases ("fibroblast" collagenase (MMP-1 or collagenase-1), "neutrophil" collagenase (MMP-8 or collagenase-2), and the rodent-type interstitial collagenase which is homologous to the recently described human collagenase-3 or MMP-13 (10)) can cleave native, undenatured, "interst...

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