Rapid ligand fishing for identification of acetylcholinesterase-binding peptides in snake venom reveals new properties of dendrotoxins

Kenia Lourenço Vanzolini, Stuart Ainsworth, Ben Bruyneel, Volker Herzig, Mitchell G.L. Seraus, Govert W. Somsen, Nicholas R. Casewell, Quezia Bezerra Cass, Jeroen Kool*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review


Acetylcholinesterase (AChE) from Electrophorus electricus (eel) was immobilized on the surface of amino-modified paramagnetic beads to serve as a model for the development, validation and application of a new affinity-based ligand-fishing assay for the discovery of bioactive peptides from complex protein mixtures such as venoms. Nano liquid chromatography-mass spectrometry (nanoLC-MS) was used for the analysis of trapped peptides. Using enzyme-functionalized beads, the ligand-fishing assay was evaluated and optimized using a peptide reference mixture composed of one acetylcholinesterase binder (fasciculin-II) and five non-binders (mambalgin-1, angiotensin-II, bradykinin, cardiotoxin and α-bungarotoxin). As proof of concept, snake venom samples spiked with fasciculin-II demonstrated assay selectivity and sensitivity, fishing the peptide binder from complex venom solutions at concentrations as low as 1.0 μg/mL. As negative controls for method validation, venoms of four different snake species, not known to harbor AChE binding peptides, were screened and no AChE binders were detected. The applicability of the ligand fishing assay was subsequently demonstrated with venom from the black mamba, Jameson's mamba and western green mamba (Dendroaspis spp.), which have previously been reported to contain the AChE binding fasciculins. Unknown peptides (i.e. not fasciculins) with affinity to AChE were recovered from all mamba venoms tested. Tryptic digestion followed by nano-LC-MS analysis of the material recovered from black mamba venom identified the peptide with highest AChE-binding affinity as dendrotoxin-I, a pre-synaptic neurotoxin previously not known to interact with AChE. Co-incubation of AChE with various dendrotoxins in vitro revealed reduced inactivation of AChE activity over time, thus demonstrating that these toxins stabilize AChE.

Original languageEnglish
Pages (from-to)1-8
Number of pages8
Early online date7 Jul 2018
Publication statusPublished - 15 Sept 2018


This work was funded by a research grant 2013/01710-1 and also by a BEPE grant 2015/18504-0 from São Paulo State Research Foundation ( FAPESP ). Prof. Dr. Glenn King is thanked for critically reviewing the manuscript. The authors also thank the National Council for Scientific and Technological Development (CNPq) and National Institute of Science and Technology (INCT)−Controle Biorracional de InsetosPraga (CBIP). Appendix A

FundersFunder number
Fundação de Amparo à Pesquisa do Estado de São Paulo
Ulsan National Institute of Science and Technology
Conselho Nacional de Desenvolvimento Científico e Tecnológico


    • Acetylcholinesterase-binding peptides
    • Affinity-based protein assay
    • Dendrotoxin
    • Ligand fishing assay
    • Mamba
    • Mass spectrometry
    • Snake venoms


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