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.
Highlights
Scorpion accidents have been the main cause of human envenomation by animals in Brazil since 2007 [1]
The observation that the venom of Tityus serrulatus contains metallopeptidases acting on essential human neuropeptides—dynorphin A (1-13), neuropeptide Y, peptide YY and pancreatic polypeptide—was the motivation for the development of the present study, since the neurotoxic syndromes are the main symptoms presented by victims of accidents and these molecules are broadly distributed in the body, being found mainly in the adrenal gland, peripheral nervous system and in immune cells [25,26,27,28]
The mass spectrometry analysis of the F3-4 identified 14 unique peptides from the metalloserrulase 3 sequence, covering around 19% of the zymogen molecule and 57% of its mature form (Figure 2, panel D)
Summary
Scorpion accidents have been the main cause of human envenomation by animals in Brazil since 2007 [1]. Little is known about their proteolytic components and their role during the envenomation process This could be partially explained by the lack of information about the effects of scorpion venom peptidases, or even due to the difficulty of obtaining ideal amounts of these venoms for the isolation of such molecules. The observation that the venom of Tityus serrulatus contains metallopeptidases acting on essential human neuropeptides—dynorphin A (1-13), neuropeptide Y, peptide YY and pancreatic polypeptide—was the motivation for the development of the present study, since the neurotoxic syndromes are the main symptoms presented by victims of accidents and these molecules are broadly distributed in the body, being found mainly in the adrenal gland, peripheral nervous system and in immune cells [25,26,27,28]. Here we describe the isolation and biochemical characterization of metalloserrulases 3 and 4, TsMS 3 and TsMS 4, from the T. serrulatus venom, and their in vitro proteolytic activities on human neuropeptides
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