The Indian cobra reference genome and transcriptome enables comprehensive identification of venom toxins

Snakebite envenoming is a serious and neglected tropical disease that kills ~100,000 people annually. High-quality, genome-enabled comprehensive characterization of toxin genes will facilitate development of effective humanized recombinant antivenom. We report a de novo near-chromosomal genome assembly of Naja naja, the Indian cobra, a highly venomous, medically important snake. Our assembly has a scaffold N50 of 223.35 Mb, with 19 scaffolds containing 95% of the genome. Of the 23,248 predicted protein-coding genes, 12,346 venom-gland-expressed genes constitute the \u27venom-ome\u27 and this included 139 genes from 33 toxin families. Among the 139 toxin genes were 19 \u27venom-ome-specific toxins\u27 (VSTs) that showed venom-gland-specific expression, and these probably encode the minimal core venom effector proteins. Synthetic venom reconstituted through recombinant VST expression will aid in the rapid development of safe and effective synthetic antivenom. Additionally, our genome could serve as a reference for snake genomes, support evolutionary studies and enable venom-driven drug discovery

Genomic view of the evolution of the complement system

The recent accumulation of genomic information of many representative animals has made it possible to trace the evolution of the complement system based on the presence or absence of each complement gene in the analyzed genomes. Genome information from a few mammals, chicken, clawed frog, a few bony fish, sea squirt, fruit fly, nematoda and sea anemone indicate that bony fish and higher vertebrates share practically the same set of complement genes. This suggests that most of the gene duplications that played an essential role in establishing the mammalian complement system had occurred by the time of the teleost/mammalian divergence around 500 million years ago (MYA). Members of most complement gene families are also present in ascidians, although they do not show a one-to-one correspondence to their counterparts in higher vertebrates, indicating that the gene duplications of each gene family occurred independently in vertebrates and ascidians. The C3 and factor B genes, but probably not the other complement genes, are present in the genome of the cnidaria and some protostomes, indicating that the origin of the central part of the complement system was established more than 1,000 MYA

Insights into complement convertase formation based on the structure of the factor B-cobra venom factor complex

Immune protection by the complement system critically depends on assembly of C3 convertases on the surface of pathogens and altered host cells. These short-lived protease complexes are formed through pro-convertases, which for the alternative pathway consist of the complement component C3b and the pro-enzyme factor B (FB). Here, we present the crystal structure at 2.2-Å resolution, small-angle X-ray scattering and electron microscopy (EM) data of the pro-convertase formed by human FB and cobra venom factor (CVF), a potent homologue of C3b that generates more stable convertases. FB is loaded onto CVF through its pro-peptide Ba segment by specific contacts, which explain the specificity for the homologous C3b over the native C3 and inactive products iC3b and C3c. The protease segment Bb binds the carboxy terminus of CVF through the metal-ion dependent adhesion site of the Von Willebrand factor A-type domain. A possible dynamic equilibrium between a ‘loading' and ‘activation' state of the pro-convertase may explain the observed difference between the crystal structure of CVFB and the EM structure of C3bB. These insights into formation of convertases provide a basis for further development of complement therapeutics

Flavoxobin, a serine protease from Trimeresurus flavoviridis (habu snake) venom, independently cleaves Arg726-Ser727 of human C3 and acts as a novel, heterologous C3 convertase

We have recently shown that crude Trimeresurus flavoviridis (habu snake) venom has a strong capability for activating the human alternative complement system. To identify the active component, the crude venom was fractionated and purified by serial chromatography using Sephadex G-100, CM-cellulose C-52, diethylaminoethyl-Toyopearl 650M, and Butyl-Toyopearl, and the active fractions were evaluated by the C3a-releasing and soluble membrane attack complex-forming activities. Two peak fractions with the highest activities were detected after gel filtration and ion exchange chromatography, and the first fraction was purified to homogeneity. The homogeneous protein was examined for its N-terminal amino acid sequence by Edman degradation. The determined sequence of 25 amino acids completely coincided with that of a previously reported serine protease with coagulant activity, flavoxobin, purified from the same snake venom. To elucidate the molecular mechanism of the complement activation, the reactive products of the mixture of the purified human C3 and flavoxobin were examined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The digesting pattern revealed that flavoxobin cleaves the α chain of the C3 molecule into two fragments. The N-terminal amino acid sequences for the remnant fragments of C3 disclosed that flavoxobin severs the human C3 at the Arg726-Ser727 site to form C3b and C3a the way C3bBb, the human alternative C3 convertase, does. In conclusion, flavoxobin acts as a novel, heterologous C3 convertase that independently cleaves human C3 and kick-starts the complement cascade