Lima Priscila C, Bordon Karla C F, Pucca Manuela B, Cerni Felipe A, Zoccal Karina F, Faccioli Lucia H, Arantes Eliane C
Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP Brazil.
Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP Brazil.
J Venom Anim Toxins Incl Trop Dis. 2015 Dec 1;21:49. doi: 10.1186/s40409-015-0051-6. eCollection 2015.
The yellow scorpion Tityus serrulatus (Ts) is responsible for the highest number of accidents and the most severe scorpion envenoming in Brazil. Although its venom has been studied since the 1950s, it presents a number of orphan peptides that have not been studied so far. The objective of our research was to isolate and identify the components present in the fractions VIIIA and VIIIB of Ts venom, in order to search for a novel toxin. The major isolated toxins were further investigated for macrophage modulation.
The fractions VIIIA and VIIIB, obtained from Ts venom cation exchange chromatography, were rechromatographed on a C18 column (4.6 × 250 mm) followed by a reversed-phase chromatography using another C18 column (2.1 × 250 mm). The main eluted peaks were analyzed by MALDI-TOF and Edman's degradation and tested on macrophages.
The previously described toxins Ts2, Ts3-KS, Ts4, Ts8, Ts8 propeptide, Ts19 Frag-II and the novel peptide Ts19 Frag-I were isolated from the fractions VIIIA and VIIIB. Ts19 Frag-I, presenting 58 amino acid residues, a mass of 6,575 Da and a theoretical pI of 8.57, shares high sequence identity with potassium channel toxins (KTx). The toxins Ts4, Ts3-KS and the partially purified Ts19 Frag-I did not produce cytotoxic effects on macrophage murine cells line (J774.1). On the other hand, Ts19 Frag-I induced the release of nitric oxide (NO) by macrophages, while Ts4 and Ts3-KS did not affect the NO production at the tested concentration (50 μg/mL). At the same concentration, Ts19 Frag-I and Ts3-KS increased the production of interleukin-6 (IL-6). Ts19 Frag-I and Ts4 did not induce the release of IL-10, IL-1β or tumor necrosis factor-α by macrophage cells using the tested concentration (50 μg/mL).
We partially purified and determined the complete sequence and chemical/physical parameters of a new β-KTx, denominated Ts19 Frag-I. The toxins Ts4, Ts3-KS and Ts19 Frag-I showed no cytotoxicity toward macrophages and induced IL-6 release. Ts19 Frag-I also induced the release of NO, suggesting a pro-inflammatory activity.
黄蝎锯齿脂鲤蝎（Tityus serrulatus，Ts）在巴西导致的中毒事故数量最多，蝎毒中毒情况也最为严重。尽管自20世纪50年代以来对其毒液进行了研究，但仍存在一些迄今尚未研究的孤儿肽。我们研究的目的是分离并鉴定Ts毒液中VIIIA和VIIIB组分中的成分，以寻找一种新型毒素。对主要分离出的毒素进行了巨噬细胞调节方面的进一步研究。
从Ts毒液阳离子交换色谱法获得的VIIIA和VIIIB组分，先在C18柱（4.6×250 mm）上重新色谱分离，然后使用另一根C18柱（2.1×250 mm）进行反相色谱分离。对主要洗脱峰进行基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）和埃德曼降解分析，并在巨噬细胞上进行测试。
从VIIIA和VIIIB组分中分离出了先前描述的毒素Ts2、Ts3-KS、Ts4、Ts8、Ts8前体肽、Ts19片段-II和新型肽Ts19片段-I。Ts19片段-I含有58个氨基酸残基，质量为6575 Da，理论等电点为8.57，与钾通道毒素（KTx）具有高度序列同一性。毒素Ts4、Ts3-KS和部分纯化的Ts19片段-I对巨噬细胞小鼠细胞系（J774.1）没有产生细胞毒性作用。另一方面，Ts19片段-I可诱导巨噬细胞释放一氧化氮（NO），而Ts4和Ts3-KS在测试浓度（50 μg/mL）下不影响NO的产生。在相同浓度下，Ts19片段-I和Ts3-KS可增加白细胞介素-6（IL-6）的产生。在测试浓度（50 μg/mL）下，Ts19片段-I和Ts4不会诱导巨噬细胞释放IL-10、IL-1β或肿瘤坏死因子-α。
我们部分纯化并确定了一种新的β-KTx（命名为Ts19片段-I）的完整序列以及化学/物理参数。毒素Ts4、Ts3-KS和Ts19片段-I对巨噬细胞无细胞毒性，并可诱导IL-6释放。Ts19片段-I还可诱导NO释放，表明其具有促炎活性。
