A systemic view of active site specificity profiling of the snake venom metalloprotease HF3 using Proteomic Identification of Cleavage Sites and N-terminomics TAILS

ABSTRACT: Snake venom metalloproteinases play important roles in the pathological effects of Viperidae venoms including severe local tissue damage, hemorrhage and coagulopathy. Hemorrhagic Factor 3 (HF3), a metalloproteinase isolated from Bothrops jararaca venom, induces severe local hemorrhage. Previous proteomic studies have shown that HF3 targets important ECM components, including collagens and proteoglycans, and plasma proteins. However, the full substrate repertoire of this metalloproteinase is unknown. Using Proteomic Identification of Cleavage Sites (PICS), a tryptic library derived from THP-1 monocytic cells was used as substrate for identifying protease cleavage sites and sequence preferences in peptides. 1,252 cleavage sites were detected and revealed a clear preference for Leu at P1′ position. A Terminal Amine Isotopic Labeling of Substrates (TAILS) analysis of the incubation of HF3 with mouse embryonic fibroblasts (MEF) secretome resulted in the identification of more than 3,600 cleavage sites in proteins, and confirmed the predominance of Leu at P1′ position. Various substrates were detected including ECM and focal adhesion proteins, and the cysteine proteinase inhibitor, cystatin-C. Interestingly, 597 peptides matched to annotated cleavage sites in the TopFIND database, suggesting that these cleavages might have been generated by other proteases activated upon incubation with HF3, including caspases -3 and -7, cathepsins D and E, granzyme B, and MMPs 2 and 9. Taken together, these results greatly expand the known substrate degradome of HF3, and reveals potential new targets which may serve as basis to better elucidate the complex pathophysiology of snake envenomation.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Early Embryonic Cell

SUBMITTER: Andre Zelanis  

LAB HEAD: Solange M.T Serrano

PROVIDER: PXD009201 | Pride | 2019-07-29

REPOSITORIES: Pride