Ramírez-Carreto Santos, Pérez-García Erick I, Salazar-García Sandra I, Bernáldez-Sarabia Johanna, Licea-Navarro Alexei, Rudiño-Piñera Enrique, Pérez-Martínez Leonor, Pedraza-Alva Gustavo, Rodríguez-Almazán Claudia
Universidad Nacional Autónoma de México, Instituto de Biotecnología, Departamento de Medicina Molecular y Bioprocesos, Av. Universidad 2001, Cuernavaca, Morelos, México.
Centro de Investigación Científica y de Educación Superior de Ensenada, Departamento de Innovación Biomédica, Baja California, México.
J Venom Anim Toxins Incl Trop Dis. 2019 Feb 11;25:e147418. doi: 10.1590/1678-9199-JVATITD-1474-18. eCollection 2019.
Pore-forming proteins (PFP) are a class of toxins abundant in the venom of sea anemones. Owing to their ability to recognize and permeabilize cell membranes, pore-forming proteins have medical potential in cancer therapy or as biosensors. In the present study, we showed the partial purification and sequencing of a pore-forming protein from Verrill (1869). 17.
Cytolytic activity of Verrill (1869) venom was determined via hemolysis assay in the erythrocytes of four mammals (sheep, goat, human and rabbit). The cytotoxic activity was analyzed in the human adherent lung carcinoma epithelial cells (A549) by the cytosolic lactate dehydrogenase (LDH) assay, and trypan blue staining. The venom was fractionated via ammonium sulfate precipitation gradient, dialysis, and ion exchange chromatography. The presence of a pore-forming protein in purified fractions was evaluated through hemolytic and cytotoxic assays, and the activity fraction was analyzed using the percent of osmotic protections after polyethylene glycol (PEG) treatment and mass spectrometry. 18.
The amount of protein at which the venom produced 50% hemolysis (HU) was determined in hemolysis assays using erythrocytes from sheep (HU = 10.7 ± 0.2 μg), goat (HU = 13.2 ± 0.3 μg), rabbit (HU = 34.7 ± 0.5 μg), and human (HU = 25.6 ± 0.6 μg). The venom presented a cytotoxic effect in A549 cells and the protein amount present in the venom responsible for producing 50% death (IC) was determined using a trypan blue cytotoxicity assay (1.84 ± 0.40 μg/mL). The loss of membrane integrity in the A549 cells caused by the venom was detected by the release of LDH in proportion to the amount of protein. The venom was fractionated; and the fraction with hemolytic and cytotoxic activities was analyzed by mass spectrometry. A pore-forming protein was identified. The cytotoxicity in the A549 cells produced by the fraction containing the pore-forming protein was osmotically protected by PEG-3350 Da molecular mass, which corroborated that the loss of integrity in the plasma membrane was produced via pore formation. 19. Conclusion: Verrill (1869) venom contains a pore-forming protein suitable for designing new drugs for cancer therapy.
成孔蛋白（PFP）是一类在海葵毒液中大量存在的毒素。由于其识别细胞膜并使其通透的能力，成孔蛋白在癌症治疗或作为生物传感器方面具有医学潜力。在本研究中，我们展示了从1869年的韦里尔海葵中分离出的一种成孔蛋白的部分纯化及测序结果。
通过对四种哺乳动物（绵羊、山羊、人类和兔子）红细胞进行溶血试验，测定1869年韦里尔海葵毒液的溶细胞活性。通过胞质乳酸脱氢酶（LDH）测定法和台盼蓝染色法，分析人贴壁肺癌上皮细胞（A549）中的细胞毒性活性。毒液通过硫酸铵沉淀梯度、透析和离子交换色谱进行分级分离。通过溶血和细胞毒性试验评估纯化级分中是否存在成孔蛋白，并使用聚乙二醇（PEG）处理后的渗透保护百分比和质谱分析活性级分。
在使用绵羊（溶血单位[HU]=10.7±0.2μg）、山羊（HU=13.2±0.3μg）、兔子（HU=34.7±0.5μg）和人类（HU=25.6±0.6μg）红细胞进行的溶血试验中，测定了毒液产生50%溶血所需的蛋白量。毒液对A549细胞呈现细胞毒性作用，并使用台盼蓝细胞毒性试验测定毒液中导致50%细胞死亡（半数抑制浓度[IC]）的蛋白量（1.84±0.40μg/mL）。通过LDH释放量与蛋白量成比例，检测到毒液导致A549细胞的膜完整性丧失。毒液进行了分级分离；对具有溶血和细胞毒性活性的级分进行了质谱分析。鉴定出一种成孔蛋白。含成孔蛋白的级分对A549细胞产生的细胞毒性可被分子量为3350道尔顿的PEG渗透保护，这证实了质膜完整性的丧失是通过形成孔道产生的。
1869年韦里尔海葵毒液含有一种适合用于设计癌症治疗新药的成孔蛋白。
