Selective hydrolysis of triple-helical peptides by matrix metalloproteinases

Abstract

The matrix metalloproteinase (MMP) family plays an integral role in both normal and pathological connective tissue remodeling. This accelerated local turnover of the extracellular matrix can be found in such diverse diseases as arthritis, periodontal disease, and tumor cell metastasis [1,2]. More specifically, MMP-1 is believed to initiate interstitial collagen catabolism and participate in denatured collagen (gelatin) degradation. Three-dimensional structures of collagenases have indicated that the substrate binding groove in the catalytic domain is too narrow to accommodate the triple-helical collagen molecule unless it unwinds. Thus, a local unwinding of the triple-helical structure must occur before proteolysis of interstitial collagens. One approach for better defining the mechanism by which MMPs hydrolyze interstitial collagens is the utilization of triple-helical peptide (THP) models. Successful THP substrates should incorporate a sequence that could be cleaved in triple-helical conformation while having sufficient thermal stability to remain triple-helical under assay conditions. In addition, the ability to control thermal stability by adjusting chain length and composition gives THP substrates more flexibility than native collagen. The interstitial collagen sequences targeted by MMP-1, -2, -8, and -13 have been identified, and a model collagenase cleavage site has been proposed based on the combination of primary, secondary, and super-secondary structures of triple-helical collagen [3]. Our laboratory has developed two solid-phase THP synthetic methods, one which features a C-terminal Lys covalent branch [4], and one which features an N-terminally attached lipophilic molecule [5]. For the present study, we have constructed THP models of 1(I)772-786, the collagenase cleavage site in type I collagen. We have compared the susceptibility and kinetics of THPs for the MMP family members MMP-1, -2, -3, and -13.