Purification of rhinocerase from rotofor separated venom fractions.

<p><i>A</i>. Superdex 75 gel filtration chromatogram obtained during the elution of proteins from rotofor separated venom fractions 3 and 4. The peaks were numbered at the particular fractions which were used for analysis by SDS-PAGE and for analysis of the serine protease activity. <i>B</i>. 100 µl of the selected fractions shown in figure <i>A</i> were used to measure serine protease activity using Arg-AMC fluorescent substrate. Each bar shows the mean ± S.D. (<i>n</i> = 3). The hydrolytic activity measured for P2 was taken as 100%. <i>C</i>. 100 µl of the selected fractions indicated in figure <i>A</i> were analysed by SDS-PAGE (10%) and silver stained.</p

Glycosylation detection on rhinocerase.

<p>Deglycosylation was performed by mixing 100 µg of rhinocerase with 5 units of N glycosidase F in 50 µl of 0.02M Tris-HCl pH 7.4. 20 µg of glycosylated (lane1) and deglycosylated (lane2) rhinocerase were run in two separate SDS-PAGE (10%) gels and transferred to two PVDF membranes. <i>A</i>. One PVDF membrane was treated with rhinocerase-specific antibody. <i>B</i>. The second PVDF membrane was used to detect glycosylation using the ECL glycosylation detection module. Data are representative of three separate experiments.</p

Effect of rhinocerase on platelets.

<p>100 µg of rhinocerase in a 50 µl volume was added to 450 µl of washed human platelets or platelet rich plasma in a siliconised glass cuvette in an optical platelet aggregometer with continuous stirring (1200 g) at 37°C. As positive controls, 5 µg/ml concentration of CRP and 1 U/ml concentration of thrombin were used at same conditions. The traces show nil effect of rhinocerase and aggregation effects of CRP and thrombin on human washed platelets (<i>A</i>) and platelet rich plasma (<i>B</i>).</p

Sequence alignment of rhinocerase with other VVSPs.

<p>The rhinocerase sequence obtained by Edman degradation was aligned with BGSP, the only known serine protease sequence from <i>Bitis gabonica</i> (NCBI accession number: AAR24534) and two other VVSP sequences; bothrombin (NCBI accession number: P81661) and ancrod (NCBI accession number: AAA49195). The sequence identified by Q-TOF is underlined. ★ indicates conserved residues, : indicates biochemically more related residues and. indicates biochemically less related residues.</p

Fibrinogenolytic activity of rhinocerase.

<p>Coomassie brilliant blue stained SDS-PAGE gels containing samples taken at different time intervals during the incubation of 100 µg of rhinocerase with 400 µl (5 mg/ml) of plasminogen-free fibrinogen in 0.05M Tris-HCl; 0.1M NaCl, pH 7.8 or 400 µl of human plasma in the presence and absence of 20 mM EDTA. The numbers at the top of each lane represent the time (in minutes) when samples were taken during the digestion. <i>A</i>. The degradation profile of plasminogen-free fibrinogen by rhinocerase. α, β and γ represent the three chains of fibrinogen and A and B represent the degradation products. The position of rhinocerase has been labelled on the image. <i>B</i>. The degradation profile of fibrinogen present in human plasma after removal of albumin and IgG by Proteoprep immunoaffinity albumin and IgG depletion columns. The position of the α chain of fibrinogen is marked with an arrow. Data are representative of three separate experiments.</p

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