Acta NaturaeActa NaturaeActa Naturae2075-8251Acta Naturae Ltd2759010.32607/actanaturae.27590Research ArticlesЭкспериментальные статьиResearch ArticleRecombinant production, species-specific activity at the TRPA1 channel, and significance of the N-terminal residue of ProTx-I toxin from Thrixopelma pruriens tarantula venomРекомбинантная продукция, видоспецифическая активность на канале TRPA1 и важная роль N-концевого остатка токсина ProTx-I из яда тарантула Thrixopelma pruriensShulepkoMikhail A.ШулепкоМихаил Анатольевич
China

Faculty of Biology

Биологический факультет

mikhailshulepko@yandex.ruZhangMendiДжанМенди
China

Faculty of Biology

Биологический факультет

zmd010816@163.comZhivovEugenii A.ЖивовЕвгений Андреевич
Russian Federation
evgeniy-kovalenko2016@mail.ruKulbatskiiDmitrii S.КульбацкийДмитрий Сергеевич
Russian Federation
d.kulbatskiy@gmail.comhttps://orcid.org/0000-0003-3614-560XParamonovAlexander S.ПарамоновАлександр Сергеевич
Russian Federation
apar@nmr.ruCheYuqiЧэЮйци
China

Faculty of Biology

Биологический факультет

cheyuqi1997@163.comKuznetsovA. V.КузнецовАлександр В.
China

Faculty of Biology

Биологический факультет

kuznetsov.a25@icloud.comPopovA. V.ПоповА. В.
Russian Federation
lyukmanova_ekaterina@smbu.edu.cnKirpichnikovMikhail P.КирпичниковМихаил Петрович
Russian Federation

Interdisciplinary Scientific and Educational School “Molecular Technologies of the Living Systems and Synthetic Biology”, Faculty of Biology, Lomonosov Moscow State University

Междисциплинарная научно-образовательная школа «Молекулярные технологии живых систем и синтетическая биология», биологический факультет Московского государственного университета им. М.В. Ломоносова

kirpichnikov@inbox.ruShenkarevZakhar O.ШенкаревЗахар Олегович
Russian Federation
zakhar-shenkarev@yandex.ruLyukmanovaEkaterina N.ЛюкмановаЕкатерина Назымовна
China

Faculty of Biology, Shenzhen MSU-BIT University; Interdisciplinary Scientific and Educational School “Molecular Technologies of the Living Systems and Synthetic Biology”, Faculty of Biology, Lomonosov Moscow State University

Биологический факультет, Шэньчжэньский МГУ-ППИ Университет; Междисциплинарная научно-образовательная школа «Молекулярные технологии живых систем и синтетическая биология», биологический факультет Московского государственного университета им. М.В. Ломоносова

lyukmanova_ekaterina@smbu.edu.cnShenzhen MSU-BIT UniversityШэньчжэньский МГУ-ППИ УниверситетShemyakin–Ovchinnikov Institute of Bioorganic ChemistryФедеральное государственное бюджетное учреждение науки Государственный научный центр Российской Федерации Институт биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова Российской академии наукMoscow Center for Advanced StudiesМосковский центр перспективных исследованийKurchatov Medical Primatology Center of National Research Center “Kurchatov Institute”Курчатовский комплекс медицинской приматологии НИЦ «Курчатовский институт»Lomonosov Moscow State UniversityМосковский государственный университет им. М.В. Ломоносова041220251741211291112202403072025Copyright ©; 2025, Shulepko M.A., Zhang M., Zhivov E.A., Kulbatskii D.S., Paramonov A.S., Che Y., Kuznetsov A.V., Popov A.V., Kirpichnikov M.P., Shenkarev Z.O., Lyukmanova E.N.Copyright ©; 2025, Шулепко М.А., Джан М., Живов Е.А., Кульбацкий Д.С., Парамонов А.С., Чэ Ю., Кузнецов А.В., Попов А.В., Кирпичников М.П., Шенкарев З.О., Люкманова Е.Н.2025Shulepko M.A., Zhang M., Zhivov E.A., Kulbatskii D.S., Paramonov A.S., Che Y., Kuznetsov A.V., Popov A.V., Kirpichnikov M.P., Shenkarev Z.O., Lyukmanova E.N.Шулепко М.А., Джан М., Живов Е.А., Кульбацкий Д.С., Парамонов А.С., Чэ Ю., Кузнецов А.В., Попов А.В., Кирпичников М.П., Шенкарев З.О., Люкманова Е.Н.https://creativecommons.org/licenses/by/4.0https://actanaturae.ru/2075-8251/article/view/27590

The ProTx-I toxin from Thrixopelma pruriens tarantula venom inhibits voltage-gated sodium (NaV), potassium, and calcium channels, as well as the chemosensitive TRPA1 ion channel, affecting the activating processes of these channels. Due to its action at the NaV1.7, NaV1.8, and TRPA1 channels involved in pain perception and propagation, ProTx-I may be used as a model for the development of next-generation analgesics. ProTx-I consists of 35 amino acid residues, with three disulfide bonds forming an inhibitor cystine knot (ICK) motif, which challenges its heterologous production. An efficient ProTx-I production system is necessary to study, at the molecular level, the mechanism by which the toxin acts. In this study, we tested several approaches for bacterial production of disulfide-containing toxins. Cytoplasmic expression of ProTx-I fused with either thioredoxin or glutathione-S-transferase failed to yield a correctly folded toxin. However, the natively folded ProTx-I was successfully obtained by “direct” expression in the form of cytoplasmic inclusion bodies, followed by renaturation, as well as by secretion into the periplasmic space via fusion with maltose-binding protein. The activity of the recombinant ProTx-I was studied by electrophysiology in X. laevis oocytes expressing rat and human TRPA1 channels. The toxin proved to be more active on the rat channel than on the human channel (IC50 = 250 ± 85 and 840 ± 190 nM, respectively). The presence of an additional N-terminal methionine residue in the toxin obtained through “direct” expression significantly attenuated its activity.

Токсин ProTx-I из яда тарантула Thrixopelma pruriens ингибирует потенциал-зависимые натриевые (NaV), калиевые и кальциевые каналы, а также хемочувствительный канал TRPA1, влияя на процессы их активации. Благодаря активности в отношении каналов NaV1.7, NaV1.8 и TRPA1, участвующих в восприятии и распространении болевых сигналов, ProTx-I может рассматриваться в качестве модели для создания анальгетиков нового поколения. ProTx-I состоит из 35 аминокислотных остатков, при этом три дисульфидные связи в его структуре образуют мотив ингибиторного цистинового узла, что затрудняет рекомбинантную продукцию токсина. Разработка эффективной системы продукции ProTx-I необходима для изучения механизма действия токсина на молекулярном уровне. В представленной работе мы сравнили ряд подходов к бактериальной продукции дисульфидсодержащих токсинов. Цитоплазматическая экспрессия ProTx-I в составе слитого растворимого белка с тиоредоксином или глутатион-S-трансферазой не позволила получить правильно свернутый токсин. В то же время ProTx-I с природной структурой был получен в ходе «прямой» экспрессии в виде цитоплазматических телец включения с последующей ренатурацией, а также при секреции в периплазматическое пространство в слитой конструкции с мальтозосвязывающим белком. Активность рекомбинантного ProTx-I была изучена электрофизиологическими методами на ооцитах Xenopus laevis, экспрессирующих каналы TRPA1 крысы и человека. Токсин показал большую активность на канале крысы, чем на канале человека (IC50 = 250 ± 70 и 840 ± 190 нМ соответственно). Также было обнаружено, что наличие дополнительного N-концевого остатка метионина у токсина, полученного в ходе «прямой» экспрессии, значительно ослабляет активность ProTx-I.

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