Exploring the unknown activity of the Huwentoxin-II spider toxin family: A screening of Ap1 toxins from Acanthoscurria paulensis venom

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Exploring the unknown activity of the Huwentoxin-II spider toxin family: A screening of Ap1 toxins from Acanthoscurria paulensis venom

Abstract

Spider venoms are complex mixtures containing neuropeptides that act on ion channels and neurotransmitter receptors. In this study, we investigated Ap1a and Ap1b, two homologous peptides isolated from the venom of the Brazilian tarantula Acanthoscurria paulensis. Ap1a, the most abundant peptide in this venom, and Ap1b, its isoform differing by a single hydrophobic residue (Ile42 → Val), were classified within the Huwentoxin-II family and exhibit structural features consistent with the inhibitor cystine knot (ICK) motif. Circular dichroism (CD) analyses confirmed their high thermal stability and similar secondary structures, mainly composed of β-sheets, β-turns, and disordered regions. Insecticidal assays demonstrated dose-dependent paralysis in Spodoptera frugiperda larvae induced by Ap1b, with an ED₅₀ (9.09 ± 1.67 μg/g) value comparable to Ap1a (13 μg/g). Electrophysiological screening showed that Ap1a modulates human voltage-gated sodium channels (hNav1.1, hNav1.3, and hNav1.7), shifting activation to more hyperpolarized voltages and reducing current amplitude. No significant effects were observed on insect Nav channels (BgNav1, VdNav1), potassium channels (Shaker, Shab, Shal, Shaw, KQT1, Kv1.1, rKv4.2, hKv7), or on glutamate binding and glutamate-gated chloride channels, suggesting potential involvement of calcium signaling pathways. Comparative phylogenetic and structural analyses positioned Ap1a and Ap1b within a monophyletic group of Theraphosinae tarantula toxins, supporting an evolutionary divergence within the Huwentoxin-II family where both DDH and ICK structural motifs coexist. This study provides the first comprehensive electrophysiological and functional characterization of an ICK-containing Huwentoxin-II peptide, advancing our understanding of spider venom neuropeptide diversity and function.
Garcia, A. B., Mourão, C. B. R., Campos, L. A., Gómez-Lagunas, F., Peigneur, S., Carleer, K., Tytgat, J., Do Nascimento, J. M., Campos, G. A. A., Mortari, M. R., Schwartz, E. F., & Tibery, D. V. (2025). Exploring the unknown activity of the Huwentoxin-II spider toxin family: A screening of Ap1 toxins from Acanthoscurria paulensis venom. International Journal of Biological Macromolecules, 149037. https://doi.org/10.1016/j.ijbiomac.2025.149037