Degradation of tropoelastin by matrix metalloproteinases - cleavage site specificities and release of matrikines

To provide a basis for the development of approaches to treat elastin-degrading diseases, the aim of this study was to investigate the degradation of the natural substrate tropoelastin by the elastinolytic matrix metalloproteinases MMP-7, MMP-9, and MMP-12 and to compare the cleavage site specificities of the enzymes using complementary MS techniques and molecular modeling. Furthermore, the ability of the three proteases to release bioactive peptides was studied. Tropoelastin was readily degraded by all three MMPs. Eighty-nine cleavage sites in tropoelastin were identified for MMP-12, whereas MMP-7 and MMP-9 were found to cleave at only 58 and 63 sites, respectively. Cleavages occurred predominantly in the N-terminal and C-terminal regions of tropoelastin. With respect to the cleavage site specificities, the study revealed that all three MMPs similarly tolerate hydrophobic and/or aliphatic amino acids, including Pro, Gly, Ile, and Val, at P1′. MMP-7 shows a strong preference for Leu at P1′, which is also well accepted by MMP-9 and MMP-12. Of all three MMPs, MMP-12 best tolerates bulky charged and aromatic amino acids at P1′. All three MMPs showed a clear preference for Pro at P3 that could be structurally explained by molecular modeling. Analysis of the generated peptides revealed that all three MMPs show a similar ability to release bioactive sequences, with MMP-12 producing the highest number of these peptides. Furthermore, the generated peptides YTTGKLPYGYGPGG, YGARPGVGVGGIP, and PGFGAVPGA, containing GxxPG motifs that have not yet been proven to be bioactive, were identified as new matrikines upon biological activity testing. Structured digital abstract •  MINT-7709630: MMP-7 (uniprotkb:P09237) cleaves (MI:0194) Tropoelastin (uniprotkb:P15502) by protease assay (MI:0435) •  MINT-7709668: MMP-9 (uniprotkb:P14780) cleaves (MI:0194) Tropoelastin (uniprotkb:P15502) by protease assay (MI:0435) •  MINT-7709289: MMP-12 (uniprotkb:P39900) cleaves (MI:0194) Tropoelastin (uniprotkb:P15502) by protease assay (MI:0435)