Elsevier

Biochimie

Volume 162, July 2019, Pages 33-45
Biochimie

Research paper
Biochemical and molecular characterization of the hyaluronidase from Bothrops atrox Peruvian snake venom

https://doi.org/10.1016/j.biochi.2019.03.022Get rights and content

Highlights

  • Hyaluronidase from the venom of Peruvian Bothrops atrox (Hyal-Ba) was functional and structural characterized.

  • Hyal-Ba specifically hydrolyze hyaluronic acid and acts as Bothrops atrox venom'diffusor.

  • Hyal-Ba enzymatic activity is optimal at pH 6.0 and 40 °C and is positively regulated by Na+ and K+.

  • Hyal-Ba is a glycoprotein and its cDNA shares identity with other Bothrops hyaluronidases.

Abstract

Snake venoms are a rich source of enzymes such as metalloproteinases, serine proteinases phospholipases A2 and myotoxins, that have been well characterized structurally and functionally. However, hyaluronidases (E.C.3.2.1.35) have not been studied extensively. In this study, we describe the biochemical and molecular features of a hyaluronidase (Hyal-Ba) isolated from the venom of the Peruvian snake Bothrops atrox. Hyal-Ba was purified by a combination of ion-exchange and gel filtration chromatography. Purified Hyal-Ba is a 69-kDa (SDS-PAGE) monomeric glycoprotein with an N-terminal amino acid sequence sharing high identity with homologous snake venom hyaluronidases. Detected associated carbohydrates were hexoses (16.38%), hexosamines (2.7%) and sialic acid (0.69%). Hyal-Ba selectively hydrolyzed only hyaluronic acid (HA; specific activity = 437.5 U/mg) but it did not hydrolyze chondroitin sulfate or heparin. The optimal pH and temperature for maximum activity were 6.0 and 40 °C, respectively, and its Km was 0.31 μM. Its activity was inhibited by EDTA, iodoacetate, 2-mercaptoethanol, TLCK and dexamethasone. Na+ and K+ (0.2 M) positively affect hyaluronidase activity; while Mg2+, Br2+, Ba2+, Cu2+, Zn2+, and Cd2+ reduced catalytic activity. Hyal-Ba potentiates the hemorrhagic and hemolytic activity of whole venom, but decreased subplantar edema caused by an l-amino acid oxidase (LAAO). The Hyal-Ba cDNA sequence (2020 bp) encodes 449 amino acid residues, including the catalytic site residues (Glu135, Asp133, Tyr206, Tyr253 and Trp328) and three functional motifs for N-linked glycosylation, which are conserved with other snake hyaluronidases. Spatial modeling of Hyal-Ba displayed a TIM-Barrel (α/β) fold and an EGF-like domain in the C-terminal portion. The phylogenetic analysis of Hyal-Ba with other homologous Hyals showed the monophyly of viperids. Further, Hyal-Ba studies may extend our knowledge of B. atrox toxinology and provides insight to improve the neutralizing strategies of therapeutic antivenoms.

Introduction

Snakebite envenomation constitutes a serious health issue, justifying its recent classification into category A of the neglected tropical diseases by WHO [1]. Annually, hundreds of thousands to several million ophidian accidents occur worldwide, and are mainly reported in impoverished tropical and sub-tropical rural areas of developing Asian, African and South American countries [2].

Peru, is a country with a large venomous ophidian fauna (33 formally reported species) [3], and snakebite envenomation represent a relevant public health issue [3,4]. The pitviper Bothrops atrox is by far the main causative agent of the vast majority (∼87.6%) of reported snakebites in the low lands east of the Andes and in tropical rain forests up to 1200 m [4]. As happen with the majority of viperid snakes, envenoming by B. atrox is characterized by a complex pathophysiology including local tissue damage (hemorrhage, erythema, edema, inflammation, necrosis and ECM degradation) as well as systemic hemostatic disfunctions resulting in multi-organ bleeding, renal alterations and coagulopathies, with consumption of fibrinogen and other blood coagulation factors [4,5]. Thereby, the severity of the envenomation symptoms correlates with venom features, such as amount, composition and systemic distribution via the bloodstream and appropriate therapeutic intervention by using specific antivenoms [[4], [5], [6]].

Despite the recognized medical relevance of accidents caused by B. atrox in Peru, only recently for the last few years, studies to elucidate the venom composition and its pharmacological properties have received more attention, including proteomic elucidation [6,7] as well as the identification and detailed characterization of important compounds/toxins. Among them are metalloproteinases which induce hemorrhage and coagulopathies [8], l-amino acid oxidase (L-AAO) [9] that triggers autophagy, apoptosis and necrosis in human keratinocytes.

Hyaluronidase (Hyal) enzyme (E.C. 3.2.1.35), commonly known as “spreading factor”, is another venom component with limited in vitro and in vivo studies [10]. Although the Hyal is claimed to be non-toxic, it degrades hyaluronic acid (HA) of connective tissue, thereby facilitating the diffusion of toxic components of the venom throughout the victim's organism and blood vessels, thus accelerating the envenoming process [[10], [11], [12]]. Thus, the synergy of its action with other venom component may cause a fast and more severe collapse of the prey physiology. Therefore, its mode of action is similar in many venomous snakes [12]. Due to the role in venom induced toxic effects by its spreading property of venom toxins throughout the victim's body, Hyal has received special attention regarding the efficacy of therapeutic antivenoms [10].

The main limitations for studying Hyals are related to the complex isolation process due to their high instability, rapid degradation and low concentration in the venom [13,14]. Despite these disadvantages, several snake venom Hyals have been characterized from the venoms of Naja naja [15], Agkistrodon contortrix contortrix [16], Cerastes cerastes [17], Daboia ruselli [18], Crotalus durissus terrificus [12] and Lachesis muta rhombeata [19]. From Bothrops genus, homologous Hyals have been previously reported from B. moojeni [20] and B. pauloensis [14]. Our studies highlight the fact that the earlier administration of specific antivenom, in conjunction with the rapid spread of venom toxins promoted by Hyal are key aspects to take in consideration for better management of snakebites.

The present work reports the structural and functional characterization of a hyaluronidase (Hyal-Ba) from the venom of the Peruvian pitviper B. atrox, which was previously identified in our laboratory [21]. Furthermore, we describe the cDNA and predicted the amino acid sequence of Hyal-Ba. We believe that our results provide important information on the structure-function relationship of Hyal-Ba, emphasizing the importance of the spreading effect of the enzyme on snakebite envenomation. This, may assist understanding of pathophysiological processes involving Hyal with the aim to improve the anti-venom therapy caused by B. atrox or other pitviper species.

Section snippets

Venom and reagents

Bothrops atrox venom was obtained from specimens captured in several localities of Perú. Table 1, shows the localities where the snakes were captured, (Alto Marañón (3), Pucallpa (5) Tingo María (1), Junín (2) y La Merced (1)), and the hyaluronidase specific activity. B. atrox specimens were kept in captivity at the Serpentarium “Oswaldo Meneses”, located in the Museo de Historia Natural, Universidad Nacional Mayor de San Marcos. Snakes were milked by applying mechanical pressure on the venom

Enzymatic stability

Hyaluronidase activity of B. atrox venom was more stable at pH 5.0 for 40 h, with a slight decrease after 160 h incubation (Fig. 1A). In addition, NaCl (0.15 M) enhanced its activity within the first 20 h, but slightly decreased at 24 h- (Fig. 1B). Geographic variation in hyaluronidase activity of B. atrox venom samples was not detected, but individual differences were identified (Table 1). Venom samples from Alto Marañon locality were used to purify Hyal-Ba.

Purification of Hyal-Ba

Hyal-Ba was purified to homogeneity

Discussion

This paper reports the purification (36-fold) and its molecular and biological characterization of a hyaluronidase (Hyal-Ba) from the venom of the Peruvian Bothrops atrox snake. Hyals are endoglycosidases which primarily degrade hyaluronan, a glycosaminoglycan (GAG) of ECM [15,16], and are widely distributed in nature, including in snake venoms. Unlike to other toxic compounds found in snake venoms e.g. SVMPs, PLA2s, l-amino acid oxidases, myotoxins amongst others, Hyals are apparently the

Conclusion

Hyals are important compounds during the envenoming process caused by viper snakes and despite its great value available information on it is still limited. A Hyal from the Peruvian Bothrops atrox venom (Hyal-Ba) was purified and characterized at the biochemical and molecular level. Hyal-Ba was considered as a spreading factor that promotes distribution of lethal toxins from B. atrox venom and thus, contribute to pathophysiological effects of snake bite victims. For these reasons,

Conflict of interest

The authors declare no conflict of interest.

Author contribution

Study design: D.E.V.R., J.D.,

Experiment conduction: E.G.K., J.D., D.E.V.R., E.R., G.S, F.L.

Data analysis: D.E.V.R., E.F.S., J.D. C.CH.O., A.Y. P.M.P.

Manuscript writing and revision: D.E.V.R. E.F.S., P.M.P., E.G., A.Y.

Acknowledgements

We thank Prof. J.A. Eble from Münster University, Germany for critical reading the manuscript. This work was supported by Vicerrectorado de Investigación y Posgrado – UNMSM (Proyecto N° B17101271), Programa Nacional de Innovación para la Competitividad y Productividad - Innovate Perú (Contrato N° 131-FINCyT-IB-2013) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais, FAPEMIG, Brazil (Proceso N° HVT-00069-17). Part of this report is a dissertation for Master Degree of Julio Delgadillo

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