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

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 (Km, kcat, 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.

Genetic and toxinological divergence among populations of Tityus trivittatus Kraepelin, 1898 (Scorpiones: Buthidae) inhabiting Paraguay and Argentina

“…However, component from the T. trivittatus Paraguayan population contained peptides similar to additional T. serrulatus metalloproteinases (metalloserrulases 1, 16, 18, 20) compared with the Argentinean population (metalloserrulases 18,20). Distinct metalloserrulases have a preference for cleaving neuropeptides with high specificity, implying that they are neuropeptidases with different biological targets and roles in the envenoming process [37]. Considering the differences in venom gelatin zymograms and electrophoretic mobility, it is feasible that proteolytic proteins with differential properties exist in these T. trivittatus populations that could influence their toxicity.…”

“…Tables 3 and 4 summarize the identified venom peptides. Significant sequence matches were found for peptides from both populations with putative venom metalloproteinases from T. bahiensis (identified through transcriptomics [35]), metalloserrulases from T. serrulatus (which are metalloproteases identified both at the molecular and functional levels [36,37]), and a hyaluronidase from T. bahiensis (UniProtKB A0A0C9RFM5) (identified at the transcript level [35]). Both T. trivittatus venoms shared all listed T. bahiensis putative proteases and T. serrulatus metalloserrulases 18 (UniProtKB A0A1S5QN52) and 20 (UniProtKB A0A1S5QN67).…”

Leptolysin, a Leptospira secreted metalloprotease of the pappalysin family with broad-spectrum activity

“…Inhibition of leptolysin by divalent cations may result from competition with the zinc ion present in the active site, causing its substitution and, thus, destabilizing the enzyme ( Dudev and Lim, 2001 ; Rawlings et al., 2018 ). The use of a greater number of substrates for a more accurate primary specificity study will be necessary, but the preliminary results presented here, using the cleavage points attained on five FRET peptides, which are used by our group to study metalloproteases ( Cajado-Carvalho et al., 2019 ), provided relevant information on leptolysin preferences for substrate hydrolysis. A high preference for arginine at the S1 subsite of leptolysin was observed.…”

Estudo das metalopeptidases presentes no veneno do escorpião amarelo, >i<Tityus serrulatus>/i<: busca de novos substratos

“…Além das neurotoxinas, o veneno de T. serrulatus é composto por nucleotídeos, muco, sais, aminoácidos livres, lipídeos, inibidores de serino e metalopeptidases, peptídeos potenciadores de bradicinina, hipotensinas e peptídeos antimicrobianos (COLOGNA et al, 2009;PUCCA et al, 2015;DUZZI et al, 2016;DE OLIVEIRA et al, 2018) (Figura 4). Entre os componentes de maior massa molecular encontram-se proteínas como fatores de crescimento semelhantes à insulina, proteínas secretadas ricas em cisteína e enzimas (lipases, fosfolipases, hialuronidases, serino peptidases, cisteíno peptidases e metalopeptidases) (CAJADO-CARVALHO et al, 2016;DE OLIVEIRA et al, 2018;AMORIM et al, 2019;CAJADO-CARVALHO et al, 2019). Análise dos componentes do veneno de T. serrulatus por abordagem transcriptômica.…”

“…OLIVEIRA et al, 2018). Apesar dessa evidência, ainda são poucos os relatos de isolamento e caracterização desses componentes do VTs (BERTAZZI, 2007;FLETCHER et al 2010;CAJADO-CARVALHO et al, 2016;CAJADO-CARVALHO et al, 2019).…”