Comparison of proteomic profiles of the venoms of two of the 'Big Four' snakes of India, the Indian cobra (Naja naja) and the common krait (Bungarus caeruleus), and analyses of their toxins

Abstract

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 A₂ 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.

Keywords: ESI-LC-MS/MS; Toxin profile; Toxins; Tryptic digestion; Venom variation; Venomics.