Purification and Biochemical Characterization of TsMS 3 and TsMS 4: Neuropeptide-Degrading Metallopeptidases in the Tityus serrulatus Venom

Author(s)

Cajado-Carvalho, Daniela ^[1] ; Fernandes da Silva, Cristiane Castilho ^[1] ; Kodama, Roberto Tadashi ^[1] ; Ceolin Mariano, Douglas Oscar ^[2] ; Pimenta, Daniel Carvalho ^[2] ; Duzzi, Bruno ^[1] ; Kuniyoshi, Alexandre Kazuo ^[1] ; Portaro, Fernanda Vieira ^[1]

Affiliation

^[1] Butantan Inst, Immunochem Lab, BR-05503900 Sao Paulo, SP - Brazil

^[2] Butantan Inst, Biochem & Biophys Lab, BR-05503900 Sao Paulo, SP - Brazil

• Document type: Journal article
• Source: TOXINS; v. 11, n. 4 APR 2019.
• Web of Science Citations: 1

Abstract

Although omics studies have indicated presence of proteases on the Tityus serrulatus venom (TsV), little is known about the function of these molecules. The TsV contains metalloproteases that cleave a series of human neuropeptides, including the dynorphin A (1-13) and the members of neuropeptide Y family. Aiming to isolate the proteases responsible for this activity, the metalloserrulase 3 and 4 (TsMS 3 and TsMS 4) were purified after two chromatographic steps and identified by mass spectrometry analysis. The biochemical parameters (pH, temperature and cation effects) were determined for both proteases, and the catalytic parameters (K-m, k(cat), cleavage sites) of TsMS 4 over fluorescent substrate were obtained. The metalloserrulases have a high preference for cleaving neuropeptides but presented different primary specificities. For example, the Leu-enkephalin released from dynorphin A (1-13) hydrolysis was exclusively performed by TsMS 3. Neutralization assays using Butantan Institute antivenoms show that both metalloserrulases were well blocked. Although TsMS 3 and TsMS 4 were previously described through cDNA library studies using the venom gland, this is the first time that both these toxins were purified. Thus, this study represents a step further in understanding the mechanism of scorpion venom metalloproteases, which may act as possible neuropeptidases in the envenomation process. (AU)

• FAPESP's process: 13/15343-0 - Purification and characterization of peptidases present in the venom of the scorpion Tityus serrulatus
• Grantee: Daniela Cajado de Oliveira Souza Carvalho
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 15/15364-3 - Toxic potential analysis of proteases and peptides present in scorpion Tityus serrulatus venom and the blockage capacity of commercial antivenoms: Enhancing the knowledge of venom and its mechanism of action.
• Grantee: Fernanda Calheta Vieira Portaro
• Support Opportunities: Regular Research Grants