372회 Identification and Characterization of MMP Substrates

372회

연사 :  이 승 택, 연세대학교 자연과학대학 생화학과

제목:  Identification and Characterization of MMP Substrates

Abstract

Matrix metalloproteinases (MMPs), which are a family of zinc-containing endopeptidases, cleave extracellular matrix components as well as a variety of functional proteins. Here we describe a "degradomics" method to identify MMP substrates in a complex protein mixture using a proteomic approach. Plasma proteins that were incubated in the presence and the absence of the MMP-14 catalytic domain were displayed in two-dimensional gels. By comparison of the gels, theprotein spots reproducibly showing a disparity were selected. Upon in-gel digestion, mass determination, and peptide mass fingerprinting, 15 different proteins were identified. These proteins included 6 known substrates and 9 potential substrates of MMP-14. Therefore, this method would provide a powerful tool to identify substrates for MMPs and other proteases from a variety of highly complex protein mixtures and allow us to understand new biological roles of these enzymes.

One of the putative MMP-14 substrates was plasma gelsolin, which is known for a major component of the extracellular actin scavenging system. Comparing proteolytic activities of various MMPs to plasma gelsolin, we found that MMP-3 can cleave plasma gelsolin the most efficiently. The 90-kDa plasma gelsolin was digested to several fragments of 48-43 kDa by MMP-3. The cleavage sites were determined by specific labeling of all C-termini derived from in-gel digestion using a buffer containing 16O and 18O (1:1) and peptide mass mapping along with N-terminal amino acid sequencing of the MMP-3-digested fragments. It was determined that plasma gelsolin was cleaved at Asn389-Val390, Ser24-Met25, and Gln409-His410 by MMP-3. In addition, proteolytic cleavage of plasma gelsolin by MMP-3 resulted in a considerable loss of actin-severing activity of plasma gelsolin. These results demonstrate that MMPs weaken the extracellular actin-scavenging system by cleavage of plasma gelsolin and thus involve in pathological conditions induced by extracellular actin such as endothelial injury, respiratory distress syndrome, hepatic necrosis, and septic shock.

Another putative MMP-14 substrate was apolipoprotein C-II (apoC-II). ApoC-II is a cofactor of lipoprotein lipase (LPL) and a component of very-low density lipoprotein (VLDL). Using in vitro cleavage assay we have shown that the 9.8-kDa apoC-II was cleaved into smaller fragments about 4.9 kDa and 3.9 kDa by MMP-14. By measuring molecular masses of the fragments using MALDI-TOF, it was found that apoC-II was cleaved at Asn35-Leu36 by MMP-14.Cleavage of apoC-II with various MMPs demonstrated that apoC-II is the most susceptible for MMP-14 among the tested MMPs. Cleavage of apoC-II by MMP-14 would impair activity of LPL and thus fail to hydrolyze triglycerides in plasma and transfer the fatty acids to tissues. Our result suggests that cleavage of apoC-II by MMP-14 would be important for development of hyperlipidemia and atherosclerosis.