Trackman, PhilpMochida, YoshiyukiAlnoury, Arwa2018-12-192018-12-192018https://hdl.handle.net/2144/32949Decorin is the most characterized member of Small Leucine-Rich Repeat Proteoglycan (SLRP) family. It has been previously shown that some SLRP members including decorin bind to collagen type I. Binding of decorin to collagen was reported to inhibit collagen degradation by collagenases, suggesting that decorin might limit the access of collagenases to their collagen cleavage site. The amino acid sequence SYIRIADTNIT in decorin was reported to be the binding region to collagen type I. The objective of the current study was to investigate the effect of the synthetic SYIRIADTNIT peptide derived from the binging site of decorin (SYI peptide, hereafter) to collagen on MMP1-mediated collagen degradation in vitro. SYI peptide and its scrambled peptide were prepared. Collagen type I samples were incubated with various concentrations of SYI peptide and degraded by MMP1. The samples were subjected to electrophoresis and Coomassie Brilliant Blue (CBB) staining. CBB- positive collagen bands were quantified to assess the extent of collagen degradation. Biotin affinity chromatography was used to identify the collagen fragment that the biotinylated SYI peptide binds to. Two collagen degrading enzymes; MMP1 and trypsin were each used to generate collagen fragments. The activity of MMP1 to degrade a substrate different from collagen in the presence or absence of SYI peptide was measured. The intensity of non-degraded collagen in the presence and absence of SYI peptide was compared using CBB-stained SDS-PAGE. There was no difference of collagen degradation in the presence or absence of SYI peptide. Collagen fibrils were retrieved from biotin-SYI peptide/streptavidin column, suggesting that collagen fibrils bind to SYI peptide. However, when collagen was treated with MMP1 or trypsin, the collagen fragment was not retrieved from biotin-SYI peptide/streptavidin column, suggesting that biotin-SYI peptide binds to collagen fibrils, but not fragments. Although there was no statistical difference, MMP1 activity was slightly reduced when SYI peptide or scrambled peptide was used at higher concentrations. In conclusion, there was no significant effect of SYI peptide on MMP1-mediated collagen degradation. SYI peptide did not act as a competitive substrate for MMP1.en-USBiologyThesis/Dissertation2018-10-24