Evolution of rhinocerase enzymes.

<p><i>A</i>. Amino acid sequence alignment of rhinocerases 2 to 5, <i>M. lebetina</i> α-fibrinogenase (ML-AF) and venom serine proteinase-like protein 2 (ML-P2), and <i>Bothrops atrox</i> batroxobin (BA-BT). The alignment was created using ClustalW. The amino acids in the batroxobin sequence are coloured according to the exons encoding them: amino acids encoded by exon 2 are coloured green, and residues encoded by exons 3 to 5 are coloured dark red, blue and orange respectively. The catalytic triad residues are coloured red in the <i>B.s gabonica</i> and <i>M. lebetina</i> sequences. Coloured shading is used to indicate the three surface segments within serine proteases identified by Doley et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021532#pone.0021532-LeBeau1" target="_blank">[26]</a> as undergoing accelerated change (ASSET). Residues in the first surface segment are shaded yellow or red depending on sequence similarity; residues in the second segment are shaded green or turquoise and the residues in the third segment are shaded pink.<i>B</i>. Schematic diagram of rhinocerases 2 to 5 indicating the different regions described in the text. The light shading represents the regions of each sequence corresponding to exon 2 and exons 3 to 5. Rhinocerases 2, 3 and 5 have similar N-terminal regions corresponding to exon 2 (pink), while rhinocerase 4 has a different sequence in this region (grey). In the C-terminal regions, corresponding to exons 3 to 5, rhinocerases 2 and 3 are similar (pale green) and rhinocerase 4 and 5 are similar to each other (pale blue). The brighter shading represents the three surface segments as shown in <i>A</i>. The first surface segment is identical in rhinocerases 2, 3 and 5 (yellow), and different in rhinocerase 4 (red). The second segment is identical in rhinocerases 3, 4 and 5 (green) and different in rhinocerase 2 (turquoise), and the third segment is very similar in all 3 sequences (pink).</p

Features of the nucleotide and protein sequences of rhinocerases 2 to 5.

<p>The predicted coding regions, molecular masses and isoelectric points were obtained from DNASTAR Lasergene version 7. The potential N-glycosylation sites were predicted by NetNGlyc.</p

Amino acid sequence alignment of rhinocerases with other viper venom serine protease homologues.

<p>The alignment was created using ClustalW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021532#pone.0021532-Larkin1" target="_blank">[12]</a> and the figure was generated using GeneDoc <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021532#pone.0021532-Nicholas1" target="_blank">[14]</a>. The sequence of bovine α-chymotrypsinogen (NCBI accession number: P00766) (BT-CHY) was included to allow conventional serine protease residue numbering to be assigned. The catalytic triad residues are coloured red, the primary specificity pocket residues are coloured blue and residue 193, involved in the oxyanion hole is coloured green. BG-SP1: AAR24534; BG-RHIN2: CBM40645; BG-RHIN3: CBM40646; EO-SP: ADE45141; ML-P2: Q9PT40; TF-SP2: O13057; TJ-SPH: B0ZT25; TG-SP2A: O13060; VS-KNH7: Q71Q10; VS-SPH1: QAY82; TJ-SP1: Q9DF68; VS-KNH4: Q71QJ4; BJu-SPH: Q7T229; BJa-HP3: Q5W958; ML-P3 and ML-P4 from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021532#pone.0021532-Siigur1" target="_blank">[5]</a>; Bas-SPL: Q072L6; BG-RHIN4: CBM40647; BG-RHIN5: CBM40648.</p

Phylogenetic tree showing relationship between serine protease homologues and serine proteases from the same snakes.

<p>65 amino acid sequences from 10 snakes were included together with bovine α-chymotrypsinogen (NCBI accession number: P00766) which was used as an outgroup. The alignment was generated using ClustalW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021532#pone.0021532-Larkin1" target="_blank">[12]</a> within MEGA 4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021532#pone.0021532-Kumar1" target="_blank">[16]</a> using a gap opening penalty of 10 and a gap extension penalty of 0.1 for the initial pairwise alignment, gap opening penalty of 3 and gap extension penalty of 1.8 for the multiple alignment and the Gonnet protein weight matrix. The phylogenetic tree was generated from this within MEGA 4 using the neighbour-joining method and the Jones-Taylor-Thornton substitution model. The bootstrap test was done using 2000 replications. In the diagram sequences are identified using a code which consists of up to 3 characters representing the snake name, (TG: <i>Trimeresurus gramineus</i>; VS: <i>Viridovipera stejnegeri</i>; TJ: <i>Trimeresurus jerdonii;</i> BJu: <i>Bothrops jararacussu</i> ; ML: <i>Macrovipera lebetina</i>; EO: <i>Echis ocellatus</i>; BG: <i>Bitis gabonica</i>; Bja: <i>Bothrops jararaca</i>; TF: <i>Trimeresurus flavoviridis;</i> BAs: <i>Bothrops asper</i>) followed by a dash and then up to 5 characters representing the protein name. Where possible NCBI accession numbers are also included. ML-P3 and ML-P4 sequences were obtained directly from the sequences named VLP3 and VLP4 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021532#pone.0021532-Siigur1" target="_blank">[5]</a>. Red circles indicate the sequences with mutations to the catalytic triad.</p

Identification of rhinocerases 1–5 in the venom of <i>B. g. rhinoceros</i>.

<p><i>A.</i> 1 mg of venom was mixed with non-reducing rotofor buffer containing ampholytes with pI 3–10 and separated under non-denaturing conditions using a micro rotofor. In total 10 fractions (indicated by the numbers at the top of the gel) were collected. 10 µl of each fraction were run in SDS-PAGE (10%) and stained with SimplyBlue™SafeStain. The numbers mentioned on the gel bands represent the bands which were excised and used for mass spectrometry. <i>B</i>. 20 µl of each rotofor fraction were used to measure serine protease activity using Arg-AMC fluorescent substrate. The data represent the mean±S.D. (<i>n</i> = 3). The hydrolytic activity measured for fraction 3 was taken as 100%. <i>C</i>. The sequences of rhinocerases 2–5 were aligned with the partial sequence of rhinocerase 1 obtained previously. Gel bands 1, 7, 8, 11 and 12 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021532#pone-0021532-g001" target="_blank">Fig. 1A</a>) were analysed by mass spectrometry and the corresponding peptide sequences are shown in different colours (grey: band 1; red: band 7; yellow: band 8; blue: band 11; green: band 12) in italics on rhinocerase 1, 2, 4, 3 and 5 respectively. The N-terminal sequences of serine proteases in the venom of <i>B. g. rhinoceros</i> identified by proteomic analysis previously are underlined. The symbols ⋆, : and . indicate conserved residues, biochemically related residues and biochemically less related residues respectively.</p

Structural models of rhinocerases.

<p>Structural models of rhinocerases 2 to 5 were created using the IntFOLD server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021532#pone.0021532-Roche1" target="_blank">[17]</a> using the structure of rat trypsin (PDB code: 1co9) as a template. <i>A.</i> Schematic diagram showing the overall similarities in structure between rhinocerase 2 (yellow) and rhinocerase 4 (red). The side chain atom positions for the catalytic triad residues are included. <i>B</i>. Detailed view of the amino acids corresponding to the catalytic triad residues in rhinocerase 2 (yellow), rhinocerase 4 (red) and chymotrypsin (PDB code: 1yph; cyan). Rhinocerase 2 has substitutions for the serine and histidine residues. <i>C</i>. Detailed view of the main constituents of the S1 specificity pocket in rhinocerase 2 (yellow), rhinocerase 4 (red), chymotrypsin (cyan) and trypsin (pdb code: 1co9; green). In chymotrypsin these residues are: S189 at the base of the specificity pocket, with G216 and G226 at the sides. In trypsin D189 is at the base of the pocket, with G216 and G226. All images were generated using PyMOL.</p

Characteristics of the sample population.

<p>Types I, II and III villages have <100, 100–250 and >250 houses respectively. The percentages in each case were calculated relative to the total population in each type of village. For the snakebite prevalence the percentages indicate the % of the male, female and total population in each village type who suffered snakebites and who died due to snakebite. This data was obtained from the 30 sampling villages.</p

Distribution of snake bites by age group.

<p>The red bars show the % of the total number of people which are in each age group identified in the study population. The blue bars show the % of the population of that age group who have been bitten by snakes.</p

Year-wise snakebites and death summary.

<p>From the study population, the information about the year of snakebite was obtained from the household members. The information obtained is presented accordingly for each type of study village.</p

Circumstances of snakebites and their socio-economic impacts.

<p>The circumstances of snakebite such as where and when the bite occurred, the activities of victims during bite and the place of bite on the body were obtained from the victims. In addition, the direct costs involved in the treatment of snakebites and their socio-economic impacts were also assessed. The information provided here was from 129 interviewed victims and percentages were calculated accordingly.</p