Fractionation and proteomic analysis of the <i>Walterinnesia aegyptia</i> snake venom using OFFGEL and MALDI‐TOF‐MS techniques

El-Aziz, Tarek Mohamed Abd; Bourgoin-Voillard, Sandrine; Combemale, Stéphanie; Béroud, Rémy; Fadl, Mahmoud; Sève, Michel; Waard, Michel De

doi:10.1002/elps.201500207

Cited by 10 publications

(10 citation statements)

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“…RP-HPLC is a method of choice for separating venom components with a good resolution (little overlap in MS ion composition among adjacent fractions) and fewer losses of components [ 35 ]. In addition, desalting steps, that result in material loss, are not required for a phenotypic screening.…”

Section: Resultsmentioning

confidence: 99%

“…The initial screen enabled us to identify the venom of the Egyptian black snake Walterinnesia (W.) aegyptia as promising sperm motility enhancer. From our extensive beforehand experience on the fractionation techniques and mass spectrometry analyses of this venom type [ 35 ], we therefore performed a screening of venom components on OF1 mice sperm motility using a first step RP-HPLC separation procedure followed by a second step cation exchange separation technique. The combination of these two techniques led to the identification of a three disulfide-bridged component of 6389.89 Da (termed walterospermin) that was subsequently de novo sequenced using a combination of MALDI-TOF/TOF MS/MS and LC-ESI-QTOF MS/MS techniques after reduction, alkylation and proteases-mediated peptide digestion of the isolated compound.…”

Section: Introductionmentioning

confidence: 99%

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

El-Aziz

Jaquillard²,

Bourgoin-Voillard

et al. 2020

IJMS

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

El-Aziz

Jaquillard²,

Bourgoin-Voillard

et al. 2020

IJMS

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“…In an earlier report using W. aegyptia venom, we illustrated that RP-HPLC remains a method of choice for a well-resolved separation of individual compounds [ 28 ]. The venom was therefore separated by analytical C18 RP-HPLC and the venom compounds eluted with a gradient of acetonitrile.…”

Section: Resultsmentioning

confidence: 99%

“…While therapeutic potential of this venom has been illustrated by its antitumor efficacy in vitro on human breast carcinoma cell lines [ 27 ], it has never been used on human populations to treat infertility issues. For this study on sperm motility, we benefited from the experience built up on the best fractionation techniques to separate the compounds of this venom [ 28 ]. Herein, we report on the identification of actiflagelin, a new sperm motility activator using advanced techniques in venomics.…”

Section: Introductionmentioning

confidence: 99%

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

El-Aziz

Khoury

Jaquillard³

et al. 2018

J Venom Anim Toxins Incl Trop Dis

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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.

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“…This type of separation performs well in the case of animal toxins because a large majority of toxins have a positive net global charge, whereas the opposite is true for aptamers. This is, however, not a general reality as a significant number of acidic peptides/proteins have also been observed in an Elapidae snake venom [178], suggesting that other modes of collection may be needed for snake venom components. In the case of the aptamer/αC-conotoxin PrXA complexes, they could be collected within an elution window located between the elution of the peptide and that of the aptamer library.…”

Section: Dna Aptamers Against Cardiotoxin From Naja Atra Snake Venommentioning

confidence: 99%

Diagnostic and Therapeutic Value of Aptamers in Envenomation Cases

Ascoët

Waard

2020

IJMS

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It is now more than a century since Albert Calmette from the Institut Pasteur changed the world of envenomation by demonstrating that antibodies raised against animal venoms have the ability to treat human victims of previously fatal bites or stings. Moreover, the research initiated at that time effectively launched the discipline of toxicology, first leading to the search for toxic venom components, followed by the demonstration of venoms that also contained compounds of therapeutic value. Interest from pharmaceutical companies to treat envenomation is, however, declining, mainly for economic reasons, and hence, the World Health Organization has reclassified this public health issue to be a highest priority concern. While the production, storage, and safety of antivenom sera suffer from major inconveniences, alternative chemical and technological approaches to the problem of envenomation need to be considered that bypass the use of antibodies for toxin neutralization. Herein, we review an emerging strategy that relies on the use of aptamers and discuss how close—or otherwise—we are to finding a viable alternative to the use of antibodies for the therapy of human envenomation.

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Fractionation and proteomic analysis of the Walterinnesia aegyptia snake venom using OFFGEL and MALDI‐TOF‐MS techniques

Cited by 10 publications

References 67 publications

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

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

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

Diagnostic and Therapeutic Value of Aptamers in Envenomation Cases

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