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

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.

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

“…Considering these achievements, it is reasonable to predict that this approach would allow us to identify many more compounds originating from animal venoms, provided that the primary screening is enlarged to encompass more fractionated venoms. Overall, this work adds a new component to the growing list of molecules that have been characterized from the black desert cobra W. aegyptia venom: actiflagelin, a first sperm modulating peptide from this venom [ 52 ], two neurotoxins [ 53 ], a phospholipase A2 [ 54 ], and acetylcholinesterase [ 55 ]. Besides providing pro-fertility compounds, the W. aegyptia venom also appears to possess interesting antitumor compounds: (i) its potential was established in vitro in human breast carcinoma cell lines [ 56 ] and (ii) it induces apoptosis of human prostate cancer cells [ 57 ] and mouse models of multiple myeloma [ 58 ].…”

Biomedical applications of snake venom: from basic science to autoimmunity and rheumatology

“…More than 600 TFTs have been described since then, and several biological effects have been found with biomedical potential [ 130 ]. Among the biological effects are impacts on neurological signal transduction, due to its action on voltage-gated sodium channels [ 131 ], on GABA receptors [ [132] , [133] , [134] ] or on α-adrenergic antagonists [ 135 ]; effects on coagulation by inhibiting platelet aggregation [ 136 ] or inhibition of factor X [ 137 ]; insulinotropic activity [ 138 ] and effects on sperm motility [ 139 , 140 ].…”

Snake Venoms in Drug Discovery: Valuable Therapeutic Tools for Life Saving

“…Amino acid sequences of snake venom non-enzymatic proteins and peptides. Sequence alignment of two proteins for each non-enzymatic family [82,86,87,88,89,90,91,92,93,94,95,96]. All cysteine residues are shaded in grey.…”