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

Author(s)

Zelanis, Andre ^{[1, 2]} ; Oliveira, Ana K. ^{[3, 1]} ; Prudova, Anna ^{[4, 5]} ; Huesgen, Pitter F. ^{[4, 6]} ; Tashima, Alexandre K. ^{[1, 7]} ; Kizhakkedathu, Jayachandran ^{[4, 8]} ; Overall, Christopher M. ^{[4, 5]} ; Serrano, Solange M. T. ^[1]

Affiliation

^[1] Inst Butantan, Ctr Toxins Immune Response & Cell Signaling CeTIC, Lab Especial Toxinol Aplicada, BR-05503000 Sao Paulo, SP - Brazil

^[2] Fed Univ Sao Paulo ICT UNIFESP, Dept Sci & Technol, BR-12231280 Sao Jose Dos Campos, SP - Brazil

^[3] Brazilian Ctr Res Energy & Mat CNPEM, Brazilian Biosci Natl Lab LNBio, Campinas, SP - Brazil

^[4] Univ British Columbia, Ctr Blood Res, Vancouver, BC V6T 1Z3 - Canada

^[5] Univ British Columbia, Fac Dent, Dept Oral Biol & Med Sci, Vancouver, BC V6T 1Z3 - Canada

^[6] Forschungszentrum Julich, Cent Inst Engn Elect & Analyt, ZEA 3, D-52425 Julich - Germany

^[7] Fed Univ Sao Paulo EPM UNIFESP, Escola Paulista Med, Dept Biochem, Sao Paulo - Brazil

^[8] Univ British Columbia, Dept Pathol & Lab Med, Vancouver, BC V6T 1Z3 - Canada

• Document type: Journal article
• Source: JOURNAL OF PROTEOME RESEARCH; v. 18, n. 9, p. 3419-3428, SEP 2019.
• Web of Science Citations: 0

Abstract

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 (PIGS), 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. (AU)

• FAPESP's process: 13/07467-1 - CeTICS - Center of Toxins, Immune-Response and Cell Signaling
• Grantee: Hugo Aguirre Armelin
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC
• FAPESP's process: 11/23403-8 - Study of the degradome of HF3, a PIII-class snake venom metalloproteinase from Bothrops jararaca venom, on cultured fibroblasts
• Grantee: André Zelanis Palitot Pereira
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• FAPESP's process: 11/08514-8 - Study of the degradome of HF3, a PIII-class snake venom metalloproteinase from Bothrops jararaca venom, on cultured fibroblasts
• Grantee: André Zelanis Palitot Pereira
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 10/17328-0 - Comparative proteomic characterization of platelet aggregation induced by thrombin and PA-BJ, a serine proteinase from the venom of Bothrops jararaca.
• Grantee: Ana Karina de Oliveira
• Support Opportunities: Scholarships in Brazil - Doctorate