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Insights into the Hypertensive Effects of Tityus serrulatus Scorpion Venom: Purification of an Angiotensin-Converting Enzyme-Like Peptidase.

ABSTRACT: The number of cases of envenomation by scorpions has grown significantly in Brazil since 2007, with the most severe cases being caused by the Tityus serrulatus scorpion. Although envenomed patients mostly suffer neurotoxic manifestations, other symptoms, such as hypertension, cannot be exclusively attributed to neurotoxins. Omics analyses have detected plentiful amounts of metalloproteases in T. serrulatus venom. However, the roles played by these enzymes in envenomation are still unclear. Endeavoring to investigate the functions of scorpion venom proteases, we describe here for the first time an Angiotensin I-Converting Enzyme-like peptidase (ACE-like) purified from T. serrulatus venom. The crude venom cleaved natural and fluorescent substrates and these activities were inhibited by captopril. Regarding the serum neutralization, the scorpion antivenom was more effective at blocking the ACE-like activity than arachnid antivenom, although neither completely inhibited the venom cleavage action, even at higher doses. ACE-like was purified from the venom after three chromatographic steps and its identity was confirmed by mass spectrometric and transcriptomic analyses. Bioinformatics analysis showed homology between the ACE-like transcript sequences from Tityus spp. and human testis ACE. These findings advance our understanding of T. serrulatus venom components and may improve treatment of envenomation victims, as ACE-like may contribute to envenomation symptoms, especially the resulting hypertension.

SUBMITTER: Cajado-Carvalho D

PROVIDER: S-EPMC5198543 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Insights into the Hypertensive Effects of Tityus serrulatus Scorpion Venom: Purification of an Angiotensin-Converting Enzyme-Like Peptidase.

Cajado-Carvalho Daniela D Kuniyoshi Alexandre Kazuo AK Duzzi Bruno B Iwai Leo Kei LK Oliveira Úrsula Castro de ÚC Junqueira de Azevedo Inácio de Loiola Meirelles IL Kodama Roberto Tadashi RT Portaro Fernanda Vieira FV

Toxins 20161124 12

The number of cases of envenomation by scorpions has grown significantly in Brazil since 2007, with the most severe cases being caused by the <i>Tityus serrulatus</i> scorpion. Although envenomed patients mostly suffer neurotoxic manifestations, other symptoms, such as hypertension, cannot be exclusively attributed to neurotoxins. Omics analyses have detected plentiful amounts of metalloproteases in <i>T. serrulatus</i> venom. However, the roles played by these enzymes in envenomation are still ...[more]

PMID: 27886129

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Project description:BACKGROUND:The yellow scorpion Tityus serrulatus (Ts) is responsible for the highest number of accidents and the most severe scorpion envenoming in Brazil. Although its venom has been studied since the 1950s, it presents a number of orphan peptides that have not been studied so far. The objective of our research was to isolate and identify the components present in the fractions VIIIA and VIIIB of Ts venom, in order to search for a novel toxin. The major isolated toxins were further investigated for macrophage modulation. METHODS:The fractions VIIIA and VIIIB, obtained from Ts venom cation exchange chromatography, were rechromatographed on a C18 column (4.6?×?250 mm) followed by a reversed-phase chromatography using another C18 column (2.1?×?250 mm). The main eluted peaks were analyzed by MALDI-TOF and Edman's degradation and tested on macrophages. RESULTS:The previously described toxins Ts2, Ts3-KS, Ts4, Ts8, Ts8 propeptide, Ts19 Frag-II and the novel peptide Ts19 Frag-I were isolated from the fractions VIIIA and VIIIB. Ts19 Frag-I, presenting 58 amino acid residues, a mass of 6,575 Da and a theoretical pI of 8.57, shares high sequence identity with potassium channel toxins (KTx). The toxins Ts4, Ts3-KS and the partially purified Ts19 Frag-I did not produce cytotoxic effects on macrophage murine cells line (J774.1). On the other hand, Ts19 Frag-I induced the release of nitric oxide (NO) by macrophages, while Ts4 and Ts3-KS did not affect the NO production at the tested concentration (50 ?g/mL). At the same concentration, Ts19 Frag-I and Ts3-KS increased the production of interleukin-6 (IL-6). Ts19 Frag-I and Ts4 did not induce the release of IL-10, IL-1? or tumor necrosis factor-? by macrophage cells using the tested concentration (50 ?g/mL). CONCLUSIONS:We partially purified and determined the complete sequence and chemical/physical parameters of a new ?-KTx, denominated Ts19 Frag-I. The toxins Ts4, Ts3-KS and Ts19 Frag-I showed no cytotoxicity toward macrophages and induced IL-6 release. Ts19 Frag-I also induced the release of NO, suggesting a pro-inflammatory activity.

Project description:Antimicrobial peptides (AMPs) are ubiquitous and multipotent components of the innate immune defense arsenal used by both prokaryotic and eukaryotic organisms. The search for new AMPs has increased in recent years, due to the growing development of microbial resistance to therapeutical drugs. In this work, we evaluate the effects of Tityus serrulatus venom (Tsv), its fractions and its major toxin Ts1, a beta-neurotoxin, on fungi growth. The fractions were obtained by ion-exchange chromatography of Tsv. The growth inhibition of 11 pathogenic and non-pathogenic filamentous fungi (Aspergillus fumigatus, A. nidulans, A. niger, A. terreus, Neurospora crassa, Penicillium corylophilum, P. ochrochloron, P. verrucosum, P. viridicatum, P. waksmanii, and Talaromyces flavus) was evaluated by quantitative microplate reader assay. Tsv (100 and 500 ?g/well, which correspond to 1 and 5 mg/mL, respectively, of total soluble protein) was active in inhibiting growth of A. nidulans, A. terreus, P. corylophilum, and P. verrucosum, especially in the higher concentration used and at the first 30 h. After this period, fungi might have used Tsv components as alternative sources of nutrients, and therefore, increased their growth tax. Only fractions IX, X, XI, XIIA, XIIB (3 and 7.5 ?g/well, which correspond to 30 and 75 ?g/mL, respectively, of total soluble protein) and Ts1 (1.5, 3, and 6 ?g/well, which correspond to 2.18, 4.36, and 8.72 ?M, respectively) showed antifungal activity. Ts1 showed to be a non-morphogenic toxin with dose-dependent activity against A. nidulans, inhibiting 100% of fungal growth from 3 ?g/well (4.36 ?M). The inhibitory effect of Ts1 against A. nidulans growth was accompanied by fungistatic effects and was not amended by 1 mM CaCl2 or tetrodotoxin (46.98 and 93.96 ?M). The structural differences between Ts1 and drosomycin, a potent cysteine-rich antifungal peptide, are discussed here. Our results highlight the antifungal potential of the first cysteine-containing scorpion toxin. Since Ts1 is a multifunctional toxin, we suggest that it could be used as a template in the design of engineered scorpion AMPs and in the search for new mechanisms of action of antifungal drugs.

Project description:BackgroundThe hyaluronidase enzyme is generally known as a spreading factor in animal venoms. Although its activity has been demonstrated in several organisms, a deeper knowledge about hyaluronidase and the venom spreading process from the bite/sting site until its elimination from the victim's body is still in need. Herein, we further pursued the goal of demonstrating the effects of inhibition of T. serrulatus venom (TsV) hyaluronidase on venom biodistribution.Methods and principal findingsWe used technetium-99m radiolabeled Tityus serrulatus venom (99mTc-TsV) to evaluate the venom distribution kinetics in mice. To understand the hyaluronidase's role in the venom's biodistribution, 99mTc-TsV was immunoneutralized with specific anti-T.serrulatus hyaluronidase serum. Venom biodistribution was monitored by scintigraphic images of treated animals and by measuring radioactivity levels in tissues as heart, liver, lungs, spleen, thyroid, and kidneys. In general, results revealed that hyaluronidase inhibition delays venom components distribution, when compared to the non-neutralized 99mTc-TsV control group. Scintigraphic images showed that the majority of the immunoneutralized venom is retained at the injection site, whereas non-treated venom is quickly biodistributed throughout the animal's body. At the first 30 min, concentration peaks are observed in the heart, liver, lungs, spleen, and thyroid, which gradually decreases over time. On the other hand, immunoneutralized 99mTc-TsV takes 240 min to reach high concentrations in the organs. A higher concentration of immunoneutralized 99mTc-TsV was observed in the kidneys in comparison with the non-treated venom. Further, in situ neutralization of 99mTc-TsV by anti-T.serrulatus hyaluronidase serum at zero, ten, and 30 min post venom injection showed that late inhibition of hyaluronidase can still affect venom biodistribution. In this assay, immunoneutralized 99mTc-TsV was accumulated in the bloodstream until 120 or 240 min after TsV injection, depending on anti-hyaluronidase administration time. Altogether, our data show that immunoneutralization of hyaluronidase prevents venom spreading from the injection site.ConclusionsBy comparing TsV biodistribution in the absence or presence of anti-hyaluronidase serum, the results obtained in the present work show that hyaluronidase has a key role not only in the venom spreading from the inoculation point to the bloodstream, but also in venom biodistribution from the bloodstream to target organs. Our findings demonstrate that hyaluronidase is indeed an important spreading factor of TsV and its inhibition can be used as a novel first-aid strategy in envenoming.

Project description:Background:Tityus serrulatus venom (Ts venom) is a complex mixture of several compounds with biotechnological and therapeutical potentials, which highlights the importance of the identification and characterization of these components. Although a considerable number of studies have been dedicated to the characterization of this complex cocktail, there is still a limitation of knowledge concerning its venom composition. Most of Ts venom studies aim to isolate and characterize their neurotoxins, which are small, basic proteins and are eluted with high buffer concentrations on cation exchange chromatography. The first and largest fraction from carboxymethyl cellulose-52 (CMC-52) chromatography of Ts venom, named fraction I (Fr I), is a mixture of proteins of high and low molecular masses, which do not interact with the cation exchange resin, being therefore a probable source of components still unknown of this venom. Thus, the present study aimed to perform the proteome study of Fraction I from Ts venom, by high resolution mass spectrometry, and its biochemical characterization, by the determination of several enzymatic activities. Methods:Fraction I was obtained by a cation exchange chromatography using 50 mg of crude venom. This fraction was subjected to a biochemical characterization, including determination of L-amino acid oxidase, phospholipase, hyaluronidase, proteases activities and inhibition of angiotensin converting enzyme (ACE) activity. Fraction I was submitted to reduction, alkylation and digestion processes, and the tryptic digested peptides obtained were analyzed in a Q-Exactive Orbitrap mass spectrometer. Data analysis was performed by PEAKS 8.5 software against NCBI database. Results:Fraction I exhibits proteolytic activity and it was able to inhibit ACE activity. Its proteome analysis identified 8 different classes of venom components, among them: neurotoxins (48%), metalloproteinases (21%), hypotensive peptides (11%), cysteine-rich venom protein (9%), antimicrobial peptides (AMP), phospholipases and other enzymes (chymotrypsin and lysozymes) (3%) and phosphodiesterases (2%). Conclusions:The combination of a proteomic and biochemical characterization strategies leads us to identify new components in the T. serrulatus scorpion venom. The proteome of venom´s fraction can provide valuable direction in the obtainment of components in their native forms in order to perform a preliminary characterization and, consequently, to promote advances in biological discoveries in toxinology.

Project description:Angioedema is a potentially life-threatening adverse effect of angiotensin-converting enzyme inhibitors. Bradykinin and substance P, substrates of angiotensin-converting enzyme, increase vascular permeability and cause tissue edema in animals. Studies indicate that amino-terminal degradation of these peptides, by aminopeptidase P and dipeptidyl peptidase IV, may be impaired in individuals with angiotensin-converting enzyme inhibitor-associated angioedema. This case-control study tested the hypothesis that dipeptidyl peptidase IV activity and antigen are decreased in sera of patients with a history of angiotensin-converting enzyme inhibitor-associated angioedema. Fifty subjects with a history of angiotensin-converting enzyme inhibitor-associated angioedema and 176 angiotensin-converting enzyme inhibitor-exposed control subjects were ascertained. Sera were assayed for angiotensin-converting enzyme activity, aminopeptidase P activity, aminopeptidase N activity, dipeptidyl peptidase IV activity, and antigen and the ex vivo degradation half-lives of bradykinin, des-Arg(9)-bradykinin, and substance P in a subset. The prevalence of smoking was increased and of diabetes decreased in case versus control subjects. Overall, dipeptidyl peptidase IV activity (26.6+/-7.8 versus 29.6+/-7.3 nmol/mL per minute; P=0.026) and antigen (465.8+/-260.8 versus 563.1+/-208.6 ng/mL; P=0.017) were decreased in sera from individuals with angiotensin-converting enzyme inhibitor-associated angioedema compared with angiotensin-converting enzyme inhibitor-exposed control subjects without angioedema. Dipeptidyl peptidase IV activity (21.5+/-4.9 versus 29.8+/-6.7 nmol/mL per minute; P=0.001) and antigen (354.4+/-124.7 versus 559.8+/-163.2 ng/mL; P=0.003) were decreased in sera from cases collected during angiotensin-converting enzyme inhibition but not in the absence of angiotensin-converting enzyme inhibition. The degradation half-life of substance P correlated inversely with dipeptidyl peptidase IV antigen during angiotensin-converting enzyme inhibition. Environmental or genetic factors that reduce dipeptidyl peptidase IV activity may predispose individuals to angioedema.

Dataset Information

Insights into the Hypertensive Effects of Tityus serrulatus Scorpion Venom: Purification of an Angiotensin-Converting Enzyme-Like Peptidase.

Publications

Insights into the Hypertensive Effects of Tityus serrulatus Scorpion Venom: Purification of an Angiotensin-Converting Enzyme-Like Peptidase.

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