Tokmina-Roszyk, Michal

Collagen is the major structural scaffold in the body and serves as barrier between tissues, and thus its turnover is tightly regulated. Collagen triple-helical structure renders it resistant to general proteolysis. Several proteases are capable of cleaving the triplehelical regions of collagen, including several mammalian matrix metalloproteinases (MMPs) and bacterial collagenases. MMP-mediated collagenolysis is associated with numerous diseases and some bacterial collagenases have found clinical application use due to their efficiency in the hydrolysis of the collagen triple-helix. A selective Förster resonance energy transfer triple-helical peptide (fTHP) probe for monitoring the activity of Clostridial collagenase has been developed. The fTHP [sequence: Gly-mep-Flp-(Glyvi Pro-Hyp)4-Gly-Lys(Mca)-Thr-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Lys(Dnp)-Ser-(Gly-Pro-Hyp)4-NH2] was stable at 37 °C and was efficiently hydrolyzed by bacterial collagenase (kcat/KM = 25,000 s -1 M-1) but not by clostripain, trypsin, neutral protease, thermolysin, or elastase. The bacterial collagenase fTHP assay can be utilized in applications where specific activity towards triple-helical collagen needs to be evaluated, such as isolation of cells from various tissues. An fTHP scaffold was also utilized to evaluate the sequence preferences of eight MMPs. Residues spanning from P3 to P11 investigated using a positional scanning synthetic combinatorial library. Deconvolution of the library data revealed distinct motifs for several MMPs and discrimination among closely related MMPs. The results of this study show that the P10 11 substrate play an important role in the collagenase-substrate interactions and that modifying these residues can drastically affect the affinity of MMPs towards THP substrates. The identified sequence preferences of MMPs will enable the design of selective triple-helical MMP probes that could be used for monitoring in vivo enzyme activity and enzyme-facilitated drug delivery.