Yue Yang, Yu Huahua, Li Rongfeng, Xing Ronge, Liu Song, Li Kecheng, Wang Xueqin, Chen Xiaolin, Li Pengcheng
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China.
University of the Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100039, China.
Toxins (Basel). 2017 Jan 26;9(2):47. doi: 10.3390/toxins9020047.
Medusozoans utilize explosively discharging penetrant nematocysts to inject venom into prey. These venoms are composed of highly complex proteins and peptides with extensive bioactivities, as observed in vitro. Diverse enzymatic toxins have been putatively identified in the venom of jellyfish, and , through examination of their proteomes and transcriptomes. However, functional examination of putative enzymatic components identified in proteomic approaches to elucidate potential bioactivities is critically needed.
In this study, enzymatic toxins were functionally identified using a combined approach consisting of zymography and liquid chromatography tandem mass spectrometry (LC-MS/MS). The potential roles of metalloproteinases and lipases in hemolytic activity were explored using specific inhibitors.
Zymography indicated that nematocyst venom possessed protease-, lipase- and hyaluronidase-class activities. Further, proteomic approaches using LC-MS/MS indicated sequence homology of proteolytic bands observed in zymography to extant zinc metalloproteinase-disintegrins and astacin metalloproteinases. Moreover, pre-incubation of the metalloproteinase inhibitor batimastat with . nematocyst venom resulted in an approximate 62% reduction of hemolysis compared to venom exposed sheep erythrocytes, suggesting that metalloproteinases contribute to hemolytic activity. Additionally, species within the molecular mass range of 14-18 kDa exhibited both egg yolk and erythrocyte lytic activities in gel overlay assays.
For the first time, our findings demonstrate the contribution of jellyfish venom metalloproteinase and suggest the involvement of lipase species to hemolytic activity. Investigations of this relationship will facilitate a better understanding of the constituents and toxicity of jellyfish venom.
水母纲动物利用可爆发性释放的穿刺刺丝囊将毒液注入猎物。这些毒液由具有广泛生物活性的高度复杂蛋白质和肽组成，这在体外实验中已得到证实。通过对水母毒液的蛋白质组和转录组进行研究，已推测鉴定出多种酶毒素。然而，迫切需要对蛋白质组学方法中鉴定出的假定酶成分进行功能检测，以阐明其潜在的生物活性。
在本研究中，采用凝胶酶谱法和液相色谱串联质谱法（LC-MS/MS）相结合的方法对酶毒素进行功能鉴定。使用特异性抑制剂探讨金属蛋白酶和脂肪酶在溶血活性中的潜在作用。
凝胶酶谱法表明刺丝囊毒液具有蛋白酶、脂肪酶和透明质酸酶类活性。此外，使用LC-MS/MS的蛋白质组学方法表明，凝胶酶谱中观察到的蛋白水解条带与现存的锌金属蛋白酶-解整合素和虾红素金属蛋白酶具有序列同源性。此外，与暴露于毒液的绵羊红细胞相比，金属蛋白酶抑制剂batimastat与刺丝囊毒液预孵育后，溶血率降低了约62%，这表明金属蛋白酶有助于溶血活性。此外，在凝胶覆盖试验中，分子量范围为14-18 kDa的物质表现出蛋黄和红细胞溶解活性。
我们的研究结果首次证明了水母毒液金属蛋白酶的作用，并表明脂肪酶与溶血活性有关。对这种关系的研究将有助于更好地理解水母毒液的成分和毒性。
