Choudhury Manisha, McCleary Ryan J R, Kesherwani Manish, Kini R Manjunatha, Velmurugan Devadasan
CAS in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai, 600025, India.
Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, Singapore, 117543, Singapore; Department of Biology, 5305 Old Main Hill, Utah State University, Logan, UT, 84322, USA.
Toxicon. 2017 Sep 1;135:33-42. doi: 10.1016/j.toxicon.2017.06.005. Epub 2017 Jun 8.
Snake venoms are mixtures of biologically-active proteins and peptides, and several studies have described the characteristics of some of these toxins. However, complete proteomic profiling of the venoms of many snake species has not yet been done. The Indian cobra (Naja naja) and common krait (Bungarus caeruleus) are elapid snake species that are among the 'Big Four' responsible for the majority of human snake envenomation cases in India. As understanding the composition and complexity of venoms is necessary for successful treatment of envenomation in humans, we utilized three different proteomic profiling approaches to characterize these venoms: i) one-dimensional SDS-PAGE coupled with in-gel tryptic digestion and electrospray tandem mass spectrometry (ESI-LC-MS/MS) of individual protein bands; ii) in-solution tryptic digestion of crude venoms coupled with ESI-LC-MS/MS; and iii) separation by gel-filtration chromatography coupled with tryptic digestion and ESI-LC-MS/MS of separated fractions. From the generated data, 81 and 46 different proteins were identified from N. naja and B. caeruleus venoms, respectively, belonging to fifteen different protein families. Venoms from both species were found to contain a variety of phospholipases A and three-finger toxins, whereas relatively higher numbers of snake venom metalloproteinases were found in N. naja compared to B. caeruleus venom. The analyses also identified less represented venom proteins including L-amino acid oxidases, cysteine-rich secretory proteins, 5'-nucleotidases and venom nerve growth factors. Further, Kunitz-type serine protease inhibitors, cobra venom factors, phosphodiesterases, vespryns and aminopeptidases were identified in the N. naja venom, while acetylcholinesterases and hyaluronidases were found in the B. caeruleus venom. We further analyzed protein coverage (Lys/Arg rich and poor regions as well as potential glycosylation sites) using in-house software. These studies expand our understanding of the proteomes of the venoms of these two medically-important species.
蛇毒是具有生物活性的蛋白质和肽的混合物，多项研究描述了其中一些毒素的特性。然而，许多蛇种毒液的完整蛋白质组分析尚未完成。印度眼镜蛇（Naja naja）和金环蛇（Bungarus caeruleus）是眼镜蛇科蛇种，属于在印度导致大多数人类蛇咬伤中毒病例的“四大毒蛇”。由于了解毒液的成分和复杂性对于成功治疗人类蛇咬伤中毒至关重要，我们采用了三种不同的蛋白质组分析方法来表征这些毒液：i）一维SDS-PAGE结合单个蛋白条带的胶内胰蛋白酶消化和电喷雾串联质谱（ESI-LC-MS/MS）；ii）粗毒液的溶液内胰蛋白酶消化结合ESI-LC-MS/MS；iii）凝胶过滤色谱分离结合分离级分的胰蛋白酶消化和ESI-LC-MS/MS。从生成的数据中，分别从印度眼镜蛇和金环蛇毒液中鉴定出81种和46种不同的蛋白质，属于15个不同的蛋白质家族。发现这两种蛇的毒液都含有多种磷脂酶A和三指毒素，而与金环蛇毒液相比，印度眼镜蛇毒液中发现的蛇毒金属蛋白酶数量相对较多。分析还鉴定出含量较少的毒液蛋白，包括L-氨基酸氧化酶、富含半胱氨酸的分泌蛋白、5'-核苷酸酶和毒液神经生长因子。此外，在印度眼镜蛇毒液中鉴定出了Kunitz型丝氨酸蛋白酶抑制剂、眼镜蛇毒因子、磷酸二酯酶、vespryns和氨肽酶，而在金环蛇毒液中发现了乙酰胆碱酯酶和透明质酸酶。我们使用内部软件进一步分析了蛋白质覆盖率（富含赖氨酸/精氨酸和缺乏赖氨酸/精氨酸的区域以及潜在的糖基化位点）。这些研究扩展了我们对这两种具有医学重要性的蛇种毒液蛋白质组的理解。
