Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil ; Programa de Pós-Graduação em Ciências Biológicas: Fisiologia e Farmacologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
PLoS Negl Trop Dis. 2014 Feb 13;8(2):e2693. doi: 10.1371/journal.pntd.0002693. eCollection 2014 Feb.
Scorpionism is a public health problem in Brazil, and Tityus serrulatus (Ts) is primarily responsible for severe accidents. The main toxic components of Ts venom are low-molecular-weight neurotoxins; however, the venom also contains poorly characterized high-molecular-weight enzymes. Hyaluronidase is one such enzyme that has been poorly characterized.
We examined clones from a cDNA library of the Ts venom gland and described two novel isoforms of hyaluronidase, TsHyal-1 and TsHyal-2. The isoforms are 83% identical, and alignment of their predicted amino acid sequences with other hyaluronidases showed conserved residues between evolutionarily distant organisms. We performed gel filtration followed by reversed-phase chromatography to purify native hyaluronidase from Ts venom. Purified native Ts hyaluronidase was used to produce anti-hyaluronidase serum in rabbits. As little as 0.94 µl of anti-hyaluronidase serum neutralized 1 LD50 (13.2 µg) of Ts venom hyaluronidase activity in vitro. In vivo neutralization assays showed that 121.6 µl of anti-hyaluronidase serum inhibited mouse death 100%, whereas 60.8 µl and 15.2 µl of serum delayed mouse death. Inhibition of death was also achieved by using the hyaluronidase pharmacological inhibitor aristolochic acid. Addition of native Ts hyaluronidase (0.418 µg) to pre-neutralized Ts venom (13.2 µg venom+0.94 µl anti-hyaluronidase serum) reversed mouse survival. We used the SPOT method to map TsHyal-1 and TsHyal-2 epitopes. More peptides were recognized by anti-hyaluronidase serum in TsHyal-1 than in TsHyal-2. Epitopes common to both isoforms included active site residues.
Hyaluronidase inhibition and immunoneutralization reduced the toxic effects of Ts venom. Our results have implications in scorpionism therapy and challenge the notion that only neurotoxins are important to the envenoming process.
蝎螫伤是巴西的一个公共卫生问题，而 Tityus serrulatus（Ts）是导致严重事故的主要原因。Ts 毒液的主要毒性成分是低分子量神经毒素，但毒液中还含有特征较差的高分子量酶。透明质酸酶就是这样一种酶，其特征尚未得到充分描述。
我们检查了 Ts 毒腺 cDNA 文库中的克隆，并描述了两种新型透明质酸酶同工型，TsHyal-1 和 TsHyal-2。同工型的同源性为 83%，它们的预测氨基酸序列与其他透明质酸酶的比对显示出进化上相距甚远的生物体之间保守的残基。我们进行凝胶过滤后，再进行反相色谱法，从 Ts 毒液中纯化天然透明质酸酶。从 Ts 毒液中纯化的天然 Ts 透明质酸酶被用于在兔子中产生抗透明质酸酶血清。仅 0.94μl 的抗透明质酸酶血清即可在体外中和 1LD50（13.2μg）的 Ts 毒液透明质酸酶活性。体内中和试验表明，121.6μl 的抗透明质酸酶血清可 100%抑制小鼠死亡，而 60.8μl 和 15.2μl 的血清则延迟了小鼠死亡。使用透明质酸酶药理学抑制剂马兜铃酸也可达到抑制死亡的效果。向预先中和的 Ts 毒液（13.2μg 毒液+0.94μl 抗透明质酸酶血清）中加入天然 Ts 透明质酸酶（0.418μg）可逆转小鼠的存活。我们使用 SPOT 方法对 TsHyal-1 和 TsHyal-2 的表位进行了作图。在 TsHyal-1 中，有更多的肽被抗透明质酸酶血清识别，而在 TsHyal-2 中则较少。两个同工型共有的表位包括活性位点残基。
透明质酸酶抑制和免疫中和减少了 Ts 毒液的毒性作用。我们的研究结果对蝎螫伤治疗具有重要意义，同时也挑战了仅神经毒素对毒液作用过程很重要的观点。
