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

 

Tarek Mohamed Abd El-Aziz^1  2  3, Sawsan Al Khoury^1  2, Lucie Jaquillard⁴, Mathilde Triquigneaux⁴, Guillaume Martinez^5  6, Sandrine Bourgoin-Voillard^5 7 8, Michel Sève^5 7 8, Christophe Arnoult^5 6, Rémy Beroud⁴, Michel De Waard^1 2 4

 

1 Institute of Thorax, INSERM UMR 1087/CNRS UMR 6291, LabEx Ion Channels, Science and Therapeutics, 8 Quai Moncousu, BP 70721, 44007 Nantes Cedex 1, France

2 University of Nantes, 44007 Nantes, France

3 Zoology Department, Faculty of Science, Minia University, El-Minia 61519, Egypt

4 Smartox Biotechnology, 570 Rue de la Chimie, 38400 Saint Martin d'Hères, France

5 University Grenoble Alpes, PROMETHEE proteomic Platform, 38000 Grenoble, France

6 INSERM 1209, CNRS UMR 5309, Equipe “Génétique, Epigénétique et Thérapies de l'Infertilité", 38000 Grenoble, France

7 Institut de Biologie et de Pathologie, CHU de Grenoble, PROMETHEE proteomic Platform, 38000 Grenoble, France

8 Inserm U1055, LBFA and BEeSy, Saint Martin d'Hères, France

 

ABSTRACT

Background

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. 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.

 

Methods

Herein, 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.

 

Results

Using 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-C₈and 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.

 

Conclusions

This report demonstrates the feasibility of identifying profertility compounds that may be of therapeutic potential for infertility cases where motility is an issue.

 

Keywords Snake venom; Walterinnesia aegyptia; Bioactive compounds; Fertility; Sperm motility; Venomics; Tandem mass spectrometry; De novo sequencing; Edman degradation

 

Received: July 18, 2017.

Accepted: January 02, 2018.

 

Correspondence: michel.dewaard@univ-nantes.fr

 

Authors’ contributions

^§TMAEA and SAK contributed equally to this work. TMAEA, SAK and GM performed toxin screening experiments and sperm motility assays. TMAEA, LJ, MT and SBV did MS analyses. MS, CA, RB and MDW did experimental supervision. MDW wrote the manuscript. All authors read and approved the final manuscript.

 

Competing interests

The authors declare that they have no competing interests.