Deep Profiling of the Cleavage Specificity and Human Substrates of Snake Venom Metalloprotease HF3 by Proteomic Identification of Cleavage Site Specificity (PICS) Using Proteome Derived Peptide Libraries and Terminal Amine Isotopic Labeling of Substrates (TAILS) N‑Terminomics

Snakebite
is a major medical concern in many parts of the world
with metalloproteases playing important roles in the pathological
effects of Viperidae venoms, including local tissue damage, hemorrhage,
and coagulopathy. Hemorrhagic Factor 3 (HF3), a metalloprotease from Bothrops jararaca venom, induces local hemorrhage and targets
extracellular matrix (ECM) components, including collagens and proteoglycans,
and plasma proteins. However, the full substrate repertoire of this
metalloprotease is unknown. We report positional proteomic studies
identifying >2000 N-termini, including neo-N-termini of HF3
cleavage
sites in mouse embryonic fibroblast secretome proteins. Terminal amine
isotopic labeling of substrates (TAILS) analysis identified a preference
for Leu at the P1′ position among candidate HF3 substrates
including proteins of the ECM and focal adhesions and the cysteine
protease inhibitor cystatin-C. Interestingly, 190 unique peptides
matched to annotated cleavage sites in the TopFIND N-termini database,
suggesting that these cleavages occurred at a site prone to cleavage
or 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. Using Proteomic identification of cleavage site
specificity (PICS), a tryptic library derived from THP-1 monocytic
cells was used as HF3 substrates for identifying protease cleavage
sites and sequence preferences in peptides. A total of 799 unique
cleavage sites were detected and, in accordance with TAILS analysis
using native secreted protein substrates of MEF cells, revealed a
clear preference for Leu at P1′. Taken together, these results
greatly expand the known substrate degradome of HF3 and reveal potential
new targets, which may serve as a basis to better elucidate the complex
pathophysiology of snake envenomation.