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

Autor(es)

Vanzolini, Kenia Lourenco ^[1] ; Ainsworth, Stuart ^[2] ; Bruyneel, Ben ^[3] ; Herzig, Volker ^[4] ; Seraus, Mitchell G. L. ^[3] ; Somsen, Govert W. ^[3] ; Casewell, Nicholas R. ^{[2, 5]} ; Cass, Quezia Bezerra ^[1] ; Kool, Jeroen ^[3]

Afiliação do(s) autor(es)

^[1] Univ Fed Sao Carlos, Dept Quim, SEPARARE Nuclo Pesquisa Cromatog, Caixa Postal 676, BR-13565905 Sao Carlos, SP - Brazil

^[2] Univ Liverpool Liverpool Sch Trop Med, Parasitol Dept, Alistair Reid Venom Res Unit, Pembroke Pl, Liverpool L3 5QA, Merseyside - England

^[3] Vrije Univ Amsterdam, Fac Sci, Dept Chem & Pharmaceut Sci, Div BioAnalyt Chem, De Boelelaan 1085, NL-1081 HV Amsterdam - Netherlands

^[4] Univ Queensland, Inst Mol Biosci, Bldg 80, St Lucia, Qld 4072 - Australia

^[5] Univ Liverpool Liverpool Sch Trop Med, Res Ctr Drugs & Diagnost, Pembroke Pl, Liverpool L3 5QA, Merseyside - England

• Tipo de documento: Artigo Científico
• Fonte: Toxicon; v. 152, p. 1-8, SEP 15 2018.
• Citações Web of Science: 3

Resumo

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 alpha-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 mu g/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. (AU)

• Processo FAPESP: 15/18504-0 - Triagem e identificação rápida de peptídeos como ligantes da acetilcolineserase em toxinas
• Beneficiário: Kenia Lourenço Vanzolini
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Pós-Doutorado
• Processo FAPESP: 13/01710-1 - Ligantes enzimáticos: novos modelos de triagem
• Beneficiário: Quezia Bezerra Cass
• Modalidade de apoio: Auxílio à Pesquisa - Temático