High-throughput immuno-profiling of mamba (Dendroaspis) venom toxin epitopes using high-density peptide microarrays.

Mikael Engmark,Federico De Masi,Eric Sullivan,Bruno Lomonte,Jigar Patel,Mikael R Andersen,Ole Lund,Jens V Kringelum,José María Gutiérrez,Christian S Hansen,Andreas H Laustsen

doi:10.1038/srep36629

Abstract

Snakebite envenoming is a serious condition requiring medical attention and administration of antivenom. Current antivenoms are antibody preparations obtained from the plasma of animals immunised with whole venom(s) and contain antibodies against snake venom toxins, but also against other antigens. In order to better understand the molecular interactions between antivenom antibodies and epitopes on snake venom toxins, a high-throughput immuno-profiling study on all manually curated toxins from Dendroaspis species and selected African Naja species was performed based on custom-made high-density peptide microarrays displaying linear toxin fragments. By detection of binding for three different antivenoms and performing an alanine scan, linear elements of epitopes and the positions important for binding were identified. A strong tendency of antivenom antibodies recognizing and binding to epitopes at the functional sites of toxins was observed. With these results, high-density peptide microarray technology is for the first time introduced in the field of toxinology and molecular details of the evolution of antibody-toxin interactions based on molecular recognition of distinctive toxic motifs are elucidated.

Highlights

Venoms in immunisation Dendroaspis angusticeps D. jamesoni D. polylepis Naja annulifera N. melanoleuca N. mossambica N. nivea Bitis arietans B. gabonica
High-density peptide microarray technology is for the first time exploited for identification of linear epitope elements in snake venom toxins
The results obtained clearly demonstrate the power of this high-throughput approach compared to similar methods based on SPOT synthesis[6,7,12] and mutation analysis[11]

Summary

Introduction

Venoms in immunisation Dendroaspis angusticeps D. jamesoni D. polylepis Naja annulifera N. melanoleuca N. mossambica N. nivea Bitis arietans B. gabonica. Studies involving immunoassay quantification of antivenom binding to immobilised synthetic peptides (SPOT synthesis), corresponding to individual segments of the amino acid sequence of a given toxin, have added valuable molecular insight by elucidating which sequences contain linear elements of epitopes recognised by given antivenoms[6,7,12,13,14] These kind of meticulous epitope mapping experiments have been performed on toxins from Tityus scorpions[15,16,17] and Loxosceles spiders[18]. Mamba envenomings are known for their rapid onset of neurotoxicity[19,20], which may manifest itself already after 15 min via clinical signs such as flaccid paralysis, dyspnea due to respiratory muscle paralysis, and involuntary skeletal muscle contractions or fasciculations[19] These effects are explained by the venom compositions, which are dominated by potent small neurotoxins belonging to the three-finger toxin family and Kunitz-type serine protease inhibitor family. Overlapping 12-mer peptides, covering the entire primary sequence of each neurotoxin, were prepared by light-directed solid-phase synthesis. 21

Methods

Results

Conclusion