Laboratório de Química e Função de Proteínas e Peptídeos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Avenida Alberto Lamego, 2000, CEP 28013-602, Campos dos Goytacazes, RJ, Brazil.
Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Toxinologia, Rio de Janeiro, RJ, Brazil.
Pestic Biochem Physiol. 2022 Jun;184:105129. doi: 10.1016/j.pestbp.2022.105129. Epub 2022 May 26.
Callosobruchus maculatus is the main pest cowpea (Vigna unguiculata). Given its relevance as an insect pest, studies have focused in finding toxic compounds which could prevent its predatory action towards the seeds. Clitoria fairchildiana is a native Amazon species, whose seeds are refractory to insect predation. This characteristic was the basis of our interest in evaluating the toxicity of its seed proteins to C. maculatus larvae. Seed proteins were fractioned, according to their solubility, to albumins (F1), globulins (F2), kaphyrins (F3), glutelins (F4), linked kaphyrins (F5) and cross-linked glutelins (F6). The fractionated proteins were quantified, analysed by tricine-SDS-PAGE and inserted into the diet of this insect pest in order to evaluate their insecticidal potential. The most toxic fraction to C. maculatus, the propanol soluble F3, was submitted to molecular exclusion chromatography and all of the peaks obtained, F3P1, F3P2, F3P3, caused a reduction of larval mass, especially F3P1, seen as a major ~12 kDa electrophoretic band. This protein was identified as a vicilin-like protein by mass spectrometry and BLAST analysis. The alignment of the Cfvic (C. fairchildiana vicilin) peptides with a V. unguiculata vicilin sequence, revealed that Cfvic has at least five peptides (ALLTLVNPDGR, AILTLVNPDGR, NFLAGGKDNV, ISDINSAMDR, NFLAGEK) which lined up with two chitin binding sites (ChBS). This finding was corroborated by chitin affinity chromatography and molecular docking of chitin-binding domains for N-Acetyl-D-glucosamine and by the reduction of Cfvic chitin affinity after chemical modification of its Lys residues. In conclusion, Cfvic is a 12 kDa vicilin-like protein, highly toxic to C. maculatus, acting as an insect toxin through its ability to bind to chitin structures present in the insect midgut.
斑蝥是豇豆（豇豆）的主要害虫。鉴于其作为昆虫害虫的重要性，研究集中在寻找可防止其对种子捕食作用的有毒化合物上。Clitoria fairchildiana 是一种原产于亚马逊的物种，其种子不易受到昆虫捕食。这种特性是我们评估其种子蛋白对 C 的毒性的基础。斑蝥幼虫。种子蛋白根据其溶解度进行分级，得到白蛋白（F1），球蛋白（F2），卡皮林（F3），麦醇溶蛋白（F4），连接卡皮林（F5）和交联麦醇溶蛋白（F6）。对分级蛋白进行定量分析，通过三嗪 SDS-PAGE 分析，并将其插入该昆虫的饮食中，以评估其杀虫潜力。对 C 最毒的部分。斑蝥，丙醇可溶 F3，经分子排阻色谱分离，得到的所有峰，F3P1，F3P2，F3P3，均导致幼虫质量降低，尤其是 F3P1，可见一个主要的~12 kDa 电泳带。通过质谱和 BLAST 分析，这种蛋白质被鉴定为类菜豆球蛋白。Cfvic（C. fairchildiana vicilin）肽与 V. unguiculata vicilin 序列的比对显示，Cfvic 至少有五个肽（ALLTLVNPDGR，AILTLVNPDGR，NFLAGGKDNV，ISDINSAMDR，NFLAGEK）与两个几丁质结合位点（ChBS）对齐。几丁质亲和色谱和 N-乙酰-D-葡萄糖胺几丁质结合结构域的分子对接以及 Cfvic 几丁质亲和力在其赖氨酸残基化学修饰后的降低证实了这一发现。总之，Cfvic 是一种 12 kDa 的类菜豆球蛋白，对 C 具有高度毒性。斑蝥，通过与昆虫中肠中存在的几丁质结构结合来发挥其作为昆虫毒素的作用。
