Actiflagelin, a new sperm activator isolated from Walterinnesia aegyptia venom using phenotypic screening

Tarek Mohamed Abd El-Aziz,Guillaume Martinez,Sandrine Bourgoin-Voillard,Lucie Jaquillard,Michel Sève,Michel De Waard,Sawsan Al Khoury,Rémy Beroud,Christophe Arnoult,Mathilde Triquigneaux

doi:10.1186/s40409-018-0140-4

Abstract

BackgroundSperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. Conversely, animal venoms are enriched in bioactive compounds that primarily target those ion channels and cell surface receptors. We hypothesized, therefore, that animal venoms should be rich enough in sperm-modulating compounds for a drug discovery program. Our objective was to demonstrate this fact by using a sperm-based phenotypic screening to identify positive modulators from the venom of Walterinnesia aegyptia.MethodsHerein, as proof of concept that venoms contain interesting compounds for sperm physiology, we fractionated Walterinnesia aegyptia snake venom by RP-HPLC and screened for bioactive fractions capable of accelerating mouse sperm motility (primary screening). Next, we purified each compound from the positive fraction by cation exchange and identified the bioactive peptide by secondary screening. The peptide sequence was established by Edman sequencing of the reduced/alkylated compound combined to LC-ESI-QTOF MS/MS analyses of reduced/alkylated fragment peptides following trypsin or V8 protease digestion.ResultsUsing this two-step purification protocol combined to cell phenotypic screening, we identified a new toxin of 7329.38 Da (actiflagelin) that activates sperm motility in vitro from OF1 male mice. Actiflagelin is 63 amino acids in length and contains five disulfide bridges along the proposed pattern of disulfide connectivity C1-C5, C2-C3, C4-C6, C7-C8 and C9-C10. Modeling of its structure suggests that it belongs to the family of three finger toxins with a noticeable homology with bucandin, a peptide from Bungarus candidus venom.ConclusionsThis report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.

Highlights

Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction
In an earlier report using W. aegyptia venom, we illustrated that reversed-phase high performance liquid chromatography (RP-HPLC) remains a method of choice for a wellresolved separation of individual compounds [28]
Most compounds eluted between fractions 8 and 19 according to the absorbance measured at 214 nm. All these fractions were first lyophilized, resuspended in 100 μL of distilled H2O (dH2O), and 10 μL of each fraction was tested on OF1 male mouse sperm motility using the Computer assisted semen analysis (CASA) system as described in the Methods section

Summary

Introduction

Sperm contains a wealth of cell surface receptors and ion channels that are required for most of its basic functions such as motility and acrosome reaction. In spite of possessing a complete understanding of the cascade of events that preside over these sperm functions, it appears clear that subtle interplays between cell surface receptors and ion channels play essential contributing roles to these processes [6,7,8]. This holds true for sperm motility, which involves a coordinated movement of the flagellum [9,10,11]. As G-protein coupled receptors (GPCR) and ion channels alone account for the first and second most important targets, as witnessed by currently marketed drugs [12], it would come as no surprise that screening programs with libraries enriched in compounds active on these pharmacological targets should impact these sperm functions

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Conclusion