Identification, Characterization and Synthesis of Walterospermin, a Sperm Motility Activator from the Egyptian Black Snake Walterinnesia aegyptia Venom.

Tarek Mohamed Abd El-Aziz,Philippe Bulet,Lucie Jaquillard,Sandrine Bourgoin-Voillard,Christophe Arnoult,Michel Seve,Stéphanie Combemale,Rémy Beroud,Michel De Waard,Sylvie Michelland,Mathilde Triquigneaux,Claude Zoukimian,Sawsan Al Khoury,Guillaume Martinez,Jessica Escoffier,Jean-Pascal Hograindleur

doi:10.3390/ijms21207786

Abstract

Animal venoms are small natural mixtures highly enriched in bioactive components. They are known to target at least two important pharmacological classes of cell surface receptors: ion channels and G protein coupled receptors. Since sperm cells express a wide variety of ion channels and membrane receptors, required for the control of cell motility and acrosome reaction, two functions that are defective in infertility issues, animal venoms should contain interesting compounds capable of modulating these two essential physiological functions. Herein, we screened for bioactive compounds from the venom of the Egyptian black snake Walterinnesia aegyptia (Wa) that possess the property to activate sperm motility in vitro from male mice OF1. Using RP-HPLC and cation exchange chromatography, we identified a new toxin of 6389.89 Da (termed walterospermin) that activates sperm motility. Walterospermin was de novo sequenced using a combination of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF MS/MS) and liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF MS/MS) following reduction, alkylation, and enzymatic proteolytic digestion with trypsin, chymotrypsin or V8 protease. The peptide is 57 amino acid residues long and contains three disulfide bridges and was found to be identical to the previously cloned Wa Kunitz-type protease inhibitor II (Wa Kln-II) sequence. Moreover, it has strong homology with several other hitherto cloned Elapidae and Viperidae snake toxins suggesting that it belongs to a family of compounds able to regulate sperm function. The synthetic peptide shows promising activation of sperm motility from a variety of species, including humans. Its fluorescently-labelled analog predominantly marks the flagellum, a localization in agreement with a receptor that controls motility function.

Highlights

Snake venoms are complex chemical mixtures of pharmacologically active compounds such as enzymes, peptides and proteins [1,2], as well as non-protein components
Separation of 2 mg of W. aegyptia venom by analytical C18 RP-HPLC led to 24 fractions with most venom material segregating mainly between fractions 8 and 18 (Figure 1A)
All these fractions were probed against male OF1 sperm using the Computer-Assisted Semen Analysis (CASA) system to examine two motility parameters: VCL, the curvilinear velocity, and ALH, the amplitude of the lateral head displacement (Figure 1B)

Summary

Introduction

Snake venoms are complex chemical mixtures of pharmacologically active compounds such as enzymes, peptides and proteins [1,2], as well as non-protein components (carbohydrates, lipids and metal ions). Eptifibatide or Integrilin, a six-residue cyclic peptide, was first identified from the southeastern pygmy rattlesnake (Sistrurus miliarus barbouri) venom and targets the αIIIbβ integrin for the treatment of acute coronary syndrome [23,24]. It received FDA approval a year earlier in 1998. Batroxobin, a 231 amino acid protein from the lancehead snake venom that targets fibrinogen is used outside the USA for the treatment of perioperative bleeding Besides these approved drugs, many other snake venom components are involved in preclinical trials for a variety of therapeutic applications [25,26]

Methods

Results

Conclusion