Biochimie 93 (2011) 519e527
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Research paper
Characterisation of a mannose-binding C-type lectin from Oxyuranus scutellatus snake venomq Stephen T.H. Earl a, b, Jonathan Robson c, d, Manuela Trabi a, d, John de Jersey d, Paul P. Masci c, Martin F. Lavin a, b, * a
The Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Brisbane 4029, Australia The University of Queensland, UQ Centre for Clinical Research and School of Medicine, Central Clinical Division, PO Royal Brisbane Hospital, Brisbane 4029, Australia The University of Queensland, School of Medicine, Southern Clinical Division, Princess Alexandra Hospital, Ipswich Road, Woolloongabba 4102, Australia d The University of Queensland, School of Chemistry and Molecular Biociences, Brisbane 4072, Australia b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 August 2010 Accepted 17 November 2010 Available online 27 November 2010
C-type lectins are calcium-dependent sugar binding proteins and are distributed ubiquitously amongst vertebrate organisms. As part of a wider study on Australian snake venom components, we have identified and characterised a C-type lectin from the venom of Oxyuranus scutellatus (Australian coastal taipan) with mannose-binding activity. This protein exhibited a subunit molecular mass of 15 kDa and was found to bind mannose and also bind to and agglutinate erythrocytes in a Ca2þ-dependent manner. cDNA transcripts coding for C-lectin proteins were cloned and sequenced from six Australian elapid snake species and an antibody generated against the O. scutellatus mannose-binding C-lectin identified C-lectin proteins in the venom of 13 Australian elapid snakes by immunoblotting. Experimental evidence and molecular modelling also suggest that this protein exhibits a unique dimeric structure. This is the first confirmed example of a snake venom C-lectin with mannose-binding activity. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: C-type lectin Erythrocyte binding Mannose-binding Snake venom protein
1. Introduction Snake venoms are composed primarily of proteins and peptides. These include enzymatic proteins such as metalloproteinases, serine proteinases and phospholipase A2 enzymes, along with nonenzymatic proteins such as postsynaptic neurotoxins, nerve growth factors and lectins [1,2]. Together, these proteins exert potent effects, designed to disrupt prey homeostasis and not only bring about rapid prey death, but also initiate digestion [3]. A number of these snake venom components have been isolated and characterised in more detail, including lectins. Lectins are carbohydrate binding proteins that are found extensively throughout plant and animal species. Each lectin typically exhibits characteristic carbohydrate specificity [4]. Lectins bind carbohydrate groups through a combination of hydrogen
Abbreviations: CRD, carbohydrate recognition domain; MS, mass spectrometry. q The nucleotide sequence data reported have been submitted to the GenBankÒ database under the accession numbers EF914719, EF914724, EF914739, EF914741, EF914744 and EF914748. * Corresponding author. The Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Brisbane 4029, Australia. Tel.: þ617 3362 0341; fax: þ617 3362 0106. E-mail address:
[email protected] (M.F. Lavin). 0300-9084/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2010.11.006
bonding, van der Waals interactions and hydrophobic interactions [5]. Importantly, this binding is reversible and does not result in modification or hydrolysis of the saccharide group [6]. Carbohydrate binding is mediated through a carbohydrate recognition domain (CRD), which is variable between different lectin types. In animals, lectins are involved in an array of biological processes including cell trafficking, immune regulation, prevention of autoimmunity and defence against pathogens [7]. Several families of animal lectins have been defined based on structural and functional characteristics. These include the C, I, M, P, and R-types, galectins and calnexin [8e10]. C-type lectins are so named because they bind saccharides in a calcium-dependent manner. C-type lectins were first identified as a component of snake venom more than 20 years ago by Gartner et al. [11], when it was discovered that the haemagglutination property of the South American viper Bothrops atrox venom was caused by a lectin. Since then, C-lectins have been isolated from and characterised in multiple Viperidae venoms such as Crotalus atrox [12], Bitis arietans [13] and Agkistrodon piscivorus [14]. These proteins have been demonstrated to possess galactose-binding capabilities and also exhibit a wide variety of biological effects such as haemagglutination [11], mitogenic activity in lymphocytes [15], platelet aggregation [11], induction of paw oedema in mice [16], modulation of calcium release from skeletal muscle sarcoplasmic reticulum [17]
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and renal effects [18]. C-type lectin-like proteins that lack complete CRDs also exist in venoms but are not capable of binding sugars and demonstrate biological activities distinct from the true venom C-lectins such as anticoagulation by binding of factor IX/X [19] and inhibition of platelet aggregation [20]. While snake venom C-lectin-like proteins are heterodimers with an alpha and beta chain, the vast majority of true snake venom C-lectins characterised to date have been found to exist as homodimeric proteins, with the two subunits linked by a single disulfide bond [7]. These dimers can assemble into higher oligomeric structures, as is the case for the C-lectin from C. atrox venom which has been demonstrated to exist as a homodecamer (pentamer of dimers) [12,21]. The first snake venom C-lectin cDNA sequence reported was that from Trimeresurus stejnegeri [22]. To date, the primary sequences of more than 80 C-type lectins and C-type lectin-like proteins from snake venoms have been determined [8]. In contrast to the Viperidae family of snakes, lectins have only been identified and characterised at the protein level from the venom of one Elapidae species, Dendroaspis jamesoni. The D. jamesoni protein was found to be a lactose-binding lectin, with a molecular mass of approximately 26 kDa. Carbohydrate binding was shown to be unaffected by EDTA and Ca2þ, suggesting that the lectin may not be a C-type lectin. However, no sequence information was obtained from this protein to enable comparison to other lectin sequences [23]. Zha et al. [24], identified and sequenced venom gland cDNA transcripts for a predicted galactose-binding C-lectin from the elapid Bungarus multicinctus and one predicted galactose and one predicted mannose-binding C-lectin from the elapid Bungarus fasciatus. In addition, Ho et al. [25], identified and sequenced a venom gland cDNA transcript for a predicted mannose-binding C-lectin from the elapid Micrurus corallinus. Similarly, Fry et al. [26], have recently identified additional cDNA transcripts coding for predicted mannose-binding C-lectins from Enhydris polylepis and Thrasops jacksoni. These remain the only three published reports of a predicted mannose-binding C-lectin from a snake species. However, the presence of these proteins in the venom has not been investigated. As part of our comprehensive proteomic analysis of Australian elapid venoms, protein spots from 2-D gels of three species, Pseudonaja inframacula, Acanthophis antarcticus and Hoplocephalus stephensii, were matched by sequence similarity to C-lectins from the Asian elapid snakes B. multicinctus and B. fasciatus [1]. This represented the first identification of C-lectin proteins in the venom of Australian elapid snakes and only the second confirmed report of lectin proteins in the venom of Elapidae snake species. In order to address the lack of data on Elapidae venom C-lectins and investigate the Australian elapid C-lectins, further characterisation was undertaken, including cDNA cloning, generation of an antimannose-binding C-lectin antibody, molecular modelling and initial characterisation of the O. scutellatus venom C-lectin. As part of this work, we isolated and partially characterised the first confirmed example of a C-lectin with mannose-binding specificity from snake venom. 2. Materials and methods 2.1. Amplification of C-lectin transcripts from venom gland cDNA C-lectin transcripts were amplified from venom gland cDNA using PCR as previously described [27] with forward (50 -AGG GAA GGA AGG AAG ACC ATG-30 ) and reverse (50 -GCA GGT GAA GGA GCA ATT TGC-30 ) primers. These primers were designed by aligning C-lectin cDNA sequences from B. multicinctus (GenBank accession number AF354270) [24] and Bothrops insularis (GenBank accession number AY522720) [28] and choosing the area of conservation
immediately flanking the start and stop codons. Touchdown cycling conditions consisted of an initial denaturation at 95 C for 10 min, 14 cycles of 94 C for 30 s, 65 C for 1 min, with 1 C decrease per cycle down to 52 C and 72 C for 1 min, followed by a further 30 cycles of 94 C for 30 s, 55 C for 1 min and 72 C for 1 min with a final extension of 72 C for 7 min. This reaction was performed with O. scutellatus, H. stephensii, Pseudechis australis, Pseudechis porphyriacus, Tropidechis carinatus and Notechis scutatus cDNA and the appropriate no template control. Electrophoresis, band purification, cloning and sequencing were performed as previously described [27]. 2.2. Sequence analysis of C-lectin sequences BioEdit software (Isis Pharmaceuticals Inc., Carlsbad, CA, USA) was utilised to assemble and analyse sequence data and perform multiple alignments. For the prediction of signal peptide cleavage sites, the online SignalP 3.0 Server prediction tool was used [29]. 2.3. Recombinant expression of O. scutellatus mannose-binding C-lectin To produce recombinant protein for antibody generation, the full coding sequence from the O. scutellatus C-lectin was cloned into the bacterial expression plasmid pGEX-5X1 (GE Healthcare Biosciences, Uppsala, Sweden) utilising the EcoRI and NotI restriction sites. Restriction sites were incorporated into the cDNA at both ends using PCR with primers designed as follows; forward with EcoRI site (50 -CCG GAA TTC ATG GGG CGA TTC CTC TTG-30 ) and reverse with NotI site (50 -ATA GTT TAG CGG CCG CCT AGA ATC TGC ACT GGC AGA T-30 ). Recombinant protein expression with pGEX5X1 was then performed as per the manufacturer’s instruction. Cell lysis and GST-fusion protein purification was based on a previously described method by Frangioni and Neel [30]. 2.4. Generation of anti-mannose-binding C-lectin antibody Purified recombinant O. scutellatus mannose-binding C-lectinGST-fusion protein was divided into six 225 mg aliquots and stored at 70 C before being shipped to the Institute of Medical and Veterinary Science (Adelaide, Australia) for polyclonal antibody production in a rabbit. Aliquots were emulsified in Freund’s Complete Adjuvant for the primary inoculation and Freund’s Incomplete Adjuvant for subsequent inoculations into a rabbit. The immunisation schedule consisted of a primary inoculation followed by four subsequent inoculations three weeks apart. A pre-bleed was taken prior to immunisation, a test bleed two weeks after the third inoculation and the final bleed one week after the final inoculation. Serum was stored in aliquots at 70 C. 2.5. Purification of anti-mannose-binding C-lectin antibody The anti-mannose-binding C-lectin antibody was purified from the crude rabbit serum using a combination of ammonium sulfate precipitation and anti-GST affinity chromatography to remove GSTreactive antibodies. Ammonium sulfate was added to the serum to obtain 50% saturation in order to precipitate the Ig fraction from solution. The sample was centrifuged at 10,000g for 20 min, the supernatant removed and the pellet resuspended in 2 ml PBS. The solution was then dialysed overnight against PBS, before loading onto GST Sepharose column. This was prepared using 2.8 g of freeze dried cyanogen bromide (CNBr)-activated Sepharose (GE Healthcare Biosciences) and binding 50 mg of GST protein as per the manufacturer’s instructions. Unbound compounds from the GSTcolumn were washed off with PBS, monitored at 280 nm. When the
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base-line returned to zero, bound material was eluted by changing the buffer to 100 mM TriseHCl, 0.5 M NaCl, pH 2.7. 2.6. SDS-PAGE and western blotting with anti-C-lectin antibody A Bio-Rad Mini-Protean III apparatus was used to perform 1-D SDS-PAGE. SDS-PAGE gels and buffers were prepared using the Laemmli buffer system [31]. Australian elapid snake venoms were obtained in lyophilised form from Venom Supplies (Tanunda, Australia). Venoms were made up to a final concentration of 10 mg/ml in 50% saline, 50% glycerol and stored at 20 C. Protein samples consisting of 30 mg of crude venom were denatured by addition of 3 sample buffer (125 mM TriseHCl, pH 6.8, 6% SDS, 30% glycerol, 200 mM b-mercaptoethanol) to 1 in a 10 ml volume and heated for 10 min at 95 C. For non-denaturing electrophoresis, b-mercaptoethanol was omitted from the sample buffer and samples were not heated prior to SDS-PAGE. Proteins were resolved on separating gels of 15% acrylamide. Coomassie R-250 or G-250 [32] was used to detect protein. Immunoblotting was performed as previously described [1] with the rabbit polyclonal anti-C-lectin primary antibody diluted 1:1000 and sheep anti-rabbit horseradish peroxidase conjugated secondary antibody (Chemicon, Temecula, USA) diluted 1:4000.
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solution was then added to each well to fix bound erythrocytes. Following incubation at 4 C for 10 min, wells were washed twice more with saline before addition of 50 ml of milliQ H2O to lyse cells. The endogenous peroxidase activity of bound erythrocytes was measured by addition of 50 ml of 50% v/v 50 mM citric acid pH 4.0, 47.5% v/v milliQ H2O, 2% v/v 40 mM 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid), 0.5% v/v H2O2. After sufficient colour development, the reaction was stopped by addition of 250 mM HCl and the plate was read at 405 nm. 2.10. Erythrocyte agglutination assay A washed rabbit erythrocyte solution was prepared as described above, before being washed with agglutination buffer (0.15 M NaCl, 10 mM CaCl2). 50 ml aliquots of a 2% erythrocyte solution in agglutination buffer were incubated in a microfuge tube with 200 ml of 1 mg/ml fractionated venom samples with constant shaking for 30 min. Tubes were centrifuged at 3000g for 5 min, then vigorously shaken to see if erythrocytes became resuspended in solution. Samples positive for agglutination formed a solid aggregate and cells did not re-enter solution, while those returning a negative result were easily resuspended. 2.11. Molecular modelling of the O. scutellatus C-lectin
2.7. Purification of the C-lectin from O. scutellatus venom using mannose-affinity chromatography 200 mg of whole venom from O. scutellatus was incubated with 200 ml D-mannose agarose beads (SigmaeAldrich) in binding buffer (20 mM TriseHCl, pH 7.5, 100 mM NaCl, 500 mM CaCl2) for 1 h at 25 C. The sample was then centrifuged at 13,000g for 5 min, the supernatant removed and the beads washed twice with mannosebinding buffer. 50 ml of elution buffer (20 mM TriseHCl, pH 7.5, 10 mM EDTA) was then added to the beads and left to incubate for 1 h. Proteins eluted from the mannose agarose were analysed by SDS-PAGE. 2.8. Size exclusion chromatography of crude venom from O. scutellatus In order to partially purify larger quantities of the O. scutellatus venom C-lectin for characterisation, size exclusion chromatography was performed. A Sephacryl S-300 column was washed and equilibrated with column buffer (0.2 M ammonium acetate buffer, pH 6.8). Venom from O. scutellatus (1.090 g) was resuspended in 9.6 ml of column buffer before being loaded onto the column. The chromatography was carried out at 4 C with a flow rate of 48 ml/h. 10 ml fractions were collected and fractions constituting each elution peak were pooled together. 2.9. Erythrocyte binding assay 50 ml dilutions of fractionated O. scutellatus venom were combined with 50 ml of binding buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.0) in a Nunc Maxisorp 96 well EIA plate and incubated at 37 C for 1 h. The wells were then washed 3 times with PBS before being blocked with 200 ml of 2.5% BSA for 1 h at 37 C. The wells were washed 3 times with PBS before addition of 100 ml of a 2% washed rabbit erythrocyte solution. This was prepared by centrifuging whole blood at 3000g and resuspending the pellet in sterile saline (0.15 M NaCl). This process was repeated a further two times and erythrocytes diluted to a final concentration of 2% in saline solution containing 10 mM CaCl2. After incubation for 1 h at 37 C, the wells were washed with 100 ml of saline to remove unbound erythrocytes and 100 ml of a 2% formaldehyde in saline
A homology model of the O. scutellatus C-lectin was created using the SwissModel server [33] and PDB ID 1jzn [21], the X-ray structure of C. atrox C-lectin, as a template. Since the resulting model was lacking the terminal residues C1, C2 and F135, these residues were added in DeepView [34] and the model was subjected to 3 cycles of energy minimization (100 iterations each of steepest descent in the GROMOS force field). The coordinates of this O. scutellatus C-lectin model were subsequently submitted to molecular docking servers to find possible dimer orientations. For the following docking servers, the coordinates of the two monomers were submitted without definition of any contact or exclusion zones: PatchDock (with FireDock refinement) and SymmDock [35], ClusPro using ZDock or DOT [36,37] and GrammX [38]. The coordinates were again submitted to several molecular docking servers (FireDock, SymmDock, GrammX, ZDock), this time defining residues 74, 76, 77, 78, 86 as part of the binding site as per the C. atrox structure [21]. Disulfide bonded dimers of O. scutellatus C-lectin were created in Insight II (Accelrys Inc., San Diego, CA, USA) and subjected to 500 iterations of energy minimization followed by 1000 cycles of molecular dynamics simulation at 300 K. 3. Results As part of our thorough proteomic analysis of Australian elapid venoms, we have recently identified C-lectin proteins in Australian snake venoms for the first time [1]. Since this resulted in only partial sequence information, full-length venom gland cDNA was synthesised to further investigate and characterise C-lectins from the Australian elapids. 3.1. Amplification of C-lectin transcripts from venom gland cDNA C-lectin cDNA transcripts were amplified from O. scutellatus, N. scutatus, T. carinatus, H. stephensii, P. australis and P. porphyriacus using forward and reverse primers that were designed by aligning C-lectin cDNA sequences from B. multicinctus and B. insularis and selecting areas of conservation immediately flanking the coding sequence. Cloning and sequencing of these products confirmed these to be C-lectin cDNAs, highly homologous to the corresponding sequence from B. multicinctus. An alignment of the
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deduced amino acid sequences from the Australian elapid C-lectin cDNAs together with those from B. fasciatus, B. multicinctus and C. atrox is presented in Fig. 1. Comparison of Australian elapid sequences with the corresponding sequences from B. fasciatus and B. multicinctus revealed that three species (O. scutellatus, N. scutatus and T. carinatus) possessed a putative mannose-binding C-lectin, whereas the other three species (H. stephensii, P. australis and P. porphyriacus) had a putative galactose-binding C-lectin. This is evidenced by the presence of the consensus sequence EPN for mannose-binding or QPD for galactose-binding at positions 96 to 99 of the mature protein. A number of other residues, particularly towards the C-terminus, also appear to be conserved among the mannose and galactose-binding isoforms respectively. Overall, there is a high degree of identity between the Australian elapid and Bungarus species C-lectin sequences (approximately 65%), except for the signal peptide and associated signal peptide cleavage site which only exhibited 30% identity. Based on the predicted cleavage site for the signal peptide with SignalP software, the mature protein of the Bungarus species initiates with the residues YTC, whereas all the proteins from the Australian snakes begin with CCC. Other than the two additional cysteine residues observed at the N-terminus, all other cysteine residues throughout the rest of the proteins are conserved between the Bungarus and Australian elapid species. The cDNAs are predicted to code for mature C-lectin proteins with a molecular mass of approximately 16 kDa in monomeric form. 3.2. Generation and characterisation of an anti-mannose-binding C-lectin antibody In order to further characterise the C-lectin isoforms identified at the cDNA level, expression of one of these transcripts in E. coli was undertaken to produce a recombinant antigen for antibody generation. The mannose-binding C-lectin transcript from O. scutellatus was cloned into pGEX-5X1 and expressed as a GST-fusion protein. SDS-PAGE analysis confirmed the presence of the C-lectin GST-fusion protein at approximately 40 kDa in the cell lysate after induction with IPTG and this protein was subsequently purified using affinity chromatography with the GST moiety (Supplemental Fig. 1). Following elution from the beads, the GST-fusion protein
was tested for binding to mannose agarose and for binding to erythrocytes. No activity was detected and various refolding techniques also failed to produce an active recombinant C-lectin. 3.3. Detection of C-lectin proteins in different snake venoms The GST-fusion protein was successfully used as an antigen for polyclonal antibody production in a rabbit. Dot blot analysis confirmed that the antibody was specific for C-lectin protein (Supplemental Fig. 2). 1-D immunoblotting revealed the presence of reactive bands in 13 of the 20 venoms analysed at approximately 15 kDa (Fig. 2A), consistent with the predicted size of the mannosebinding C-lectin isoform. The most intense band was observed in Oxyuranus scutellatus (lane 17), with reactivity also observed in the Notechis (lanes 1e2), Pseudechis (lanes 11e14) and Austrelaps (lanes 9e10) species, together with Oxyuranus microlepidotus (lane 18), T. carinatus (lane 19), R. nigrescens (lane 20) and H. stephensii (lane 21). Faint bands at approximately 30 kDa also showed reactivity in the Pseudechis and Austrelaps species, consistent with the size of a C-lectin dimer, while larger molecular mass bands also showed reactivity in the Oxyuranus species and R. nigrescens. To investigate the quaternary structure of the C-lectin in the venom, the same immunoblot was performed under non-reducing and non-denaturing conditions (Fig. 2B). Under these conditions, both Oxyuranus species showed multiple reactive bands, with strongest reactivity at approximately 15 kDa (lanes 17e18). However, in the case of the Pseudechis species (lanes 12e14) reactive bands were observed at approximately 30 kDa and not at 15 kDa as occurred under reducing and denaturing conditions. 3.4. Partial purification and characterisation of the C-lectin from O. scutellatus venom Since it was not possible to express an active recombinant form of the C-lectin protein, mannose agarose chromatography was employed to purify and characterise the corresponding active venom form of O. scutellatus C-lectin. Analysis of the elution by denaturing and reducing SDS-PAGE showed the presence of a predominant protein band at approximately 15 kDa, consistent
Fig. 1. Deduced amino acid sequence alignment of C-lectin transcripts amplified from several Australian elapid snakes compared with C-lectin sequences from B. fasciatus, B. multicinctus and C. atrox. The Australian elapid C-lectin sequences show significant identity to the other snake venom C-lectin sequences. Those residues showing greater than 50% identity at each position are shaded in black and conserved cysteine residues are shaded in grey. Bungarus and Crotalus sequences are separated from the Australian elapid sequences by underlines. GenBank accession numbers are shown at the end of each sequence.
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Fig. 3. Analysis of eluted proteins from mannose agarose affinity chromatography. A) SDS-PAGE under reducing and denaturing conditions and B) Anti-mannose-binding Clectin immunoblot. A protein of approximately 15 kDa was eluted from the mannose beads and was subsequently confirmed to be the C-lectin by reactivity with the anti-Clectin antibody. Proteins were separated on a 15% gel and stained with Coomassie R250 or transferred for immunoblotting.
Fig. 2. Immunoblot against Australian elapid venoms with the anti-mannose-binding C-lectin antibody. A) Reducing and denaturing conditions. B) Non-reducing and nondenaturing conditions. Lane 1: molecular weight standards, Lane 2: N. scutatus, Lane 3: N. ater serventyi, Lane 4: N. ater niger, Lane 5: P. textilis, Lane 6: P. nuchalis, Lane 7: P. affinis, Lane 8: P. inframacula, Lane 9: A. superbus, Lane 10: A. ramsayi, Lane 11: P. porphyriacus, Lane 12: P. australis, Lane 13: P. guttatus, Lane 14: P. colletti, Lane 15: A. antarcticus, Lane 16: molecular weight standards, Lane 17: O. scutellatus, Lane 18: O. microlepidotus, Lane 19: T. carinatus, Lane 20: R. nigrescens, Lane 21: H. stephensii, Lane 22: D. vestigiata. Samples were separated on a 15% SDS-PAGE gel either stained with G250 or immunoblotted.
with the size of the C-lectin monomer (Fig. 3A). This band was subsequently found to be reactive with the C-lectin antibody (Fig. 3B), confirming its identity as the C-lectin protein. Thus, the C-lectin from O. scutellatus has mannose-binding activity as predicted from the mannose-binding consensus sequence EPN at positions 119e121. This result also confirms the O. scutellatus protein as the first example of a snake venom C-lectin with mannose-binding activity. Size exclusion chromatography was carried out on O. scutellatus venom with Sephacryl S-300 matrix as an alternative purification
step. Using this method, the venom was separated into seven major peaks (Fig. 4A). SDS-PAGE under reducing and denaturing conditions and immunoblot analysis of these fractions with the C-lectin antibody demonstrated the presence of the C-lectin protein in peaks three and four as judged by the reactive band at 15 kDa (Fig. 4B and C). Based upon calibration of the column, this elution volume for the C-lectin correlates to an approximate molecular mass of 25 kDa. This mass is consistent with a dimeric structure and provides evidence that the O. scutellatus C-lectin is present as a dimer in the venom. Since other C-lectins have been demonstrated to bind erythrocytes [14,39,40], this activity was investigated. Samples from each of the seven peak fractions were tested for the ability to bind rabbit erythrocytes. Erythrocyte binding activity was highest in peaks three and four, with no significant activity in the other peaks (Fig. 4D). Additionally, this erythrocyte binding was not observed in the absence of Ca2þ or when human erythrocytes were used (data not shown). Peak fractions were also examined for their ability to agglutinate rabbit erythrocytes. In this case, agglutination activity was also observed in peaks three and four (data not shown). Thus, erythrocyte binding and agglutination corresponds to the gel filtration peaks containing the C-lectin. These data provide evidence that the O. scutellatus venom C-lectin has activities in common with other C-lectin proteins. 3.5. Molecular modelling of the O. scutellatus C-lectin In order to investigate the molecular basis for mannose-binding activity and dimer formation in the Oxyuranus scutallatus C-lectin, homology-based molecular modelling was performed utilising the X-ray crystal structure of the C. atrox venom C-lectin. The O. scutellatus C-lectin monomer was modelled and various plausible dimer configurations were identified (Supplemental Figs. 3 and 4).
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Fig. 4. Further purification and characterisation of the O. scutellatus venom C-lectin. A) Chromatogram of venom from O. scutellatus separated by Sephacryl S-300 gel filtration in 0.2 M ammonium acetate, pH 6.8. The venom resolved into seven major peaks as labelled. B) SDS-PAGE and C) Immunoblot with the anti-mannose-binding C-lectin antibody of the seven peaks from Sephacryl S-300 gel filtration of O. scutellatus venom. Reactivity with the antibody confirmed that the C-lectin was present in peaks three and four. Lane 1: molecular weight standards, Lane 2: crude venom from O. scutellatus, Lane 3: Sephacryl S-300 peak 1, Lane 4: Sephacryl S-300 peak 2, Lane 5: Sephacryl S-300 peak 3, Lane 6: Sephacryl S-300 peak 4, Lane 7: Sephacryl S-300 peak 5, Lane 8: Sephacryl S-300 peak 6, Lane 9: Sephacryl S-300 peak 7. Proteins were separated on a 15% gel and stained with G250 or transferred for immunoblotting. D) Erythrocyte binding assay with Sephacryl S-300 fractionation peaks from the venom of O. scutellatus. Peaks three and four displayed concentration and Ca2þ-dependent binding of rabbit erythrocytes.
4. Discussion While C-lectins with various biological activities have been purified and characterised from several Viperidae snake venoms, only three previous publications have reported limited data on C-lectins from the Elapidae family. These include partial
characterisation of a lactose-binding lectin from D. jamesoni venom [23] and venom gland cDNA sequences for C-lectins from B. fasciatus and B. multicinctus [24] and M. corallinus [25]. As part of a comprehensive analysis of Australian elapid venoms, 2-D gels spots corresponding to C-lectins were identified in three species, P. inframacula, A. antarcticus and H. stephensii [1]. Close analysis of
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the MS data revealed that the matched peptides for all three species were unique for a galactose-binding C-lectin. Hence, it was suggested that Australian elapid C-lectins are galactose-binding proteins. However in this study, transcripts for both galactose and mannose-binding C-lectins were identified. Carbohydrate-binding specificity is predicted based upon the presence of the tripeptide carbohydrate binding consensus QPD for galactose or EPN for mannose as determined from previous studies [41]. These data represent only the fourth report of C-lectin sequences from the Elapidae family of snakes, together with those of Zha et al. [24], Ho et al. [25] and an unpublished mannose-binding C-lectin cDNA from B. multicinctus (GenBank accession number: DQ787090). Furthermore, all four of these reports provide evidence for the presence of mannose-binding C-lectins in Elapidae snake venoms when all viper venom C-lectins sequenced to date have been found to be galactose-binding proteins [8]. Indeed, when tested, the O. scutellatus venom C-lectin was found to possess mannose-binding activity which is consistent with the predicted carbohydrate binding specificity from the cDNA sequence. Given that all snake venom C-lectin proteins characterised to date at the protein level have been found to possess galactose-binding activity, the O. scutellatus protein is therefore the first confirmed example of a mannose-binding C-lectin isolated from snake venom. This discovery could prove to be yet another example of divergent evolution of snake venom toxins between the Viperidae and Elapidae families of venomous snakes. To date, only one 3-D structure of a venom C-lectin has been determined, namely that of a galactose-binding C-lectin from the venom of C. atrox [21]. This structure is consistent with earlier predictions as to which residues are important for carbohydrate recognition and binding and those involved in Ca2þ-binding [42]. From the crystallographic data, residues Q96, D98, E104, N119 and D120 were found to coordinate the Ca2þ ion, with Q96, D98, N119 and E104 also making direct hydrogen bonds to the bound galactose group [21]. Analysis of the molecular model of the O. scutellatus C-lectin monomer reveals that these same residues are predicted to coordinate carbohydrate and Ca2þ ion binding. However, instead of Q96 and D98 in C. atrox, the O. scutellatus C-lectin presents a glutamic acid in position 96 and an asparagine in position 98. As highlighted earlier, these differences are predicted to change carbohydrate-binding specificity from galactose to mannose. Indeed, in our model the 3-OH group of the mannose moiety interacts with the Ca2þ ion in approximately the same orientation as in the mannose-binding protein/mannose complex (PDB ID 2msb). Generally, it has been observed that in mannose specific C-lectins, the 3-OH group coordinates the Ca2þ ion, while in galactose-specific C-lectins, the 4-OH group of the carbohydrate takes its place in order to also be able to simultaneously form hydrogen bonds to E/Q96 and N/D98. Furthermore, it is interesting to note that all putative mannose-binding C-lectins identified here contain a serine residue at position 100, while all but one galactosebinding C-lectins contain an aromatic residue, F or Y, at this position (Fig. 1). It has been noted that in galactose-specific C-lectins, the aromatic ring of F/Y100 interacts with the D-galactose ring [21], while in mannose specific C-lectins, the 4-hydroxyl oxygen atom of the carbohydrate interacts with the hydroxyl bond of S100. It therefore stands to reason that residue 100 should be included in the definition of the carbohydrate specificity residues. The 3-D crystal structure of the C. atrox C-lectin also established that the protein exists as a decamer, composed of a series of five C-lectin homodimers. This result confirmed previous data suggesting that C. atrox C-lectin had a molecular mass of approximately 150 kDa [12]. The dimers were found to be linked by a disulfide bond between their C86 residues as well as hydrogen bonds [21]. In contrast, the majority of venom C-lectins isolated and characterised
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to date appear to be homodimers that do not form higher multimers [8]. A molecular modelling study of several venom C-lectin sequences has suggested that other Viperidae C-lectins also share the C86 interchain disulfide bond stabilising the homodimer [7]. Consistent with other snake venom C-lectins, the experimental evidence from this study suggests that the O. scutellatus protein exists as a dimer in solution. Indeed, the elution volume of the O. scutellatus C-lectin from the gel filtration column at approximately 25 kDa was consistent with a dimeric structure. Furthermore, the fact that the O. scutellatus C-lectin was able to agglutinate erythrocytes provides additional evidence that this protein exists as a dimer or higher oligomer in its native form in the venom. However, unlike the viperid C-lectin sequences, C86 is absent from the O. scutellatus and other Australian elapid C-lectin sequences. Apart from the eight highly conserved cysteine residues, shown in the C. atrox structure to form intrasubunit disulfide bonds, the only other cysteine residues from the Australian elapid C-lectin sequences are found at the first two residues of the mature protein, suggesting that dimer formation occurs by a different mechanism to Viperidae C-lectins. Molecular modelling with the O. scutellatus C-lectin identified at least six plausible mechanisms for dimer formation although it was difficult to single out one model as to the most likely to be correct as they all showed similar contacts. The immunoblot data provided evidence that other Australian elapid C-lectins also exhibit a dimeric structure. Under nonreducing and non-denaturing conditions, the predominant band detected in the Pseudechis and Austrelaps species was observed at 30 kDa, with no evidence of the 16 kDa band in the Pseudechis species. Furthermore, the immunoblot data suggested that the generated antibody was much more specific for the mannosebinding isoform than the galactose-binding isoform, despite significant sequence identity between the two. This is evidenced in that spots corresponding to the galactose-isoform were identified on 2-D maps of P. inframacula and A. antarcticus venom [1], but no bands were detected in these venoms by the mannose-binding Clectin antibody. It is of note that a mannose-binding C-lectin was identified in the venom of O. scutellatus in this study and the immunoblot with the mannose-binding C-lectin antibody suggests that the mannose-binding isoform may be present in several other Australian elapid venoms. However, no spots corresponding to the mannose-binding C-lectin were identified during our previous extensive proteomic analysis of 18 Australian elapid venoms [1]. As noted, the peptides from the spots of P. inframacula, A. antarcticus and H. Stephensii venoms matched to C-lectins were specific to the galactose-binding isoform. The failure to identify the mannosebinding isoform may be explained by a 2-D immunoblot of O. scutellatus venom with the mannose-binding C-lectin antibody. Despite a predicted pI of approximately eight, the C-lectin failed to resolve within the 3e10 isoelectric focussing strip, suggesting a pI of approximately 10 or higher (Supplemental Fig. 5). One possible explanation for this observation could be the presence of a posttranslational modification on the C-lectin protein conferring increased pI. Spots from this region of the gel were analysed but failed to identify any peptides from the mannose-binding C-lectin, possibly due to an abundance of phospholipase A2 peptides also in this region. Previous studies with snake venom C-lectins have confirmed that these proteins possess a wide variety of biological activities. One of the most common properties of snake venom C-lectins is the ability to agglutinate erythrocytes. This activity has been demonstrated with various snake venom C-lectins including Trimeresurus okinavensis, A. piscivorus and B. jararacussu [14,39,40] and is a result of the C-lectins binding cell surface carbohydrate groups on the erythrocyte surface. Consistent with this, the O. scutellatus C-lectin demonstrated the ability to bind and agglutinate rabbit erythrocytes.
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While it would be interesting to compare the relative potencies in agglutination between the different C-lectins, especially given their different sugar specificities, quantitative data on agglutination has not been reported in previous studies [14,39,40]. However, the galactose-binding C-lectins from T. okinavensis, A. piscivorus and B. jararacussu venoms have all demonstrated the ability to agglutinate human erythrocytes, whereas, the mannose-binding O. scutellatus C-lectin did not bind to human erythrocytes in this study. This result presumably can be attributed to differences in surface glycoproteins between human and rabbit erythrocytes. In all agglutination studies including with O. scutellatus C-lectin, the activity was found to be Ca2þ-dependent, consistent with the carbohydrate binding mechanism proposed for this class of proteins. Additionally, it is likely that C-lectin proteins from other Australian elapid venoms also possess erythrocyte binding and agglutination activity given the significant sequence identity. However, it remains to be seen whether the O. scutellatus and other Australian elapid venom C-lectins also possess some of the other known activities of venom C-lectins mentioned above. Similarly, the exact role C-lectins play in Australian elapid venoms also remains to be determined. It may be that these proteins function to exhibit toxic effects similar to those mentioned earlier, or have other new activities not previously associated with C-lectins that may have potential application as new human therapeutics. In conclusion, this study has identified and characterised a mannose-binding C-lectin from the venom of O. scutellatus, along with C-lectin proteins from other Australian elapid snake venoms. Significantly, this is the first confirmed report of a C-lectin protein from snake venom with mannose-binding activity. This C-lectin was demonstrated to bind and agglutinate rabbit erythrocytes in a Ca2þ-dependent manner. Acknowledgements This research was funded by The Australian Research Council and Venomics Pty Ltd. The authors thank Joe Sambono for the provision of venom glands and John Luff for collection of rabbit blood. We also acknowledge the support of the Australian NHMRC (Industry Fellowship, MT) Appendix. Supplementary data Supplementary data associated with this article can be found in the online version, at doi:10.1016/j.biochi.2010.11.006. References [1] G.W. Birrell, S.T. Earl, T.P. Wallis, P.P. Masci, J. de Jersey, J.J. Gorman, M.F. Lavin, The diversity of bioactive proteins in Australian snake venoms, Mol. Cell. Proteomics 7 (2007) 973e986. [2] B.G. Fry, From genome to “venome”: molecular origin and evolution of the snake venom proteome inferred from phylogenetic analysis of toxin sequences and related body proteins, Genome Res. 15 (2005) 403e420. [3] J. Nicholson, P. Mirtschin, F. Madaras, M. Venning, M. Kokkinn, Digestive properties of the venom of the Australian coastal Taipan, Oxyuranus scutellatus (Peters, 1867), Toxicon 48 (2006) 422e428. [4] J.F. Kennedy, P.M.G. Palva, M.T.S. Corella, M.S.M. Cavalcanti, L. Coelho, Lectins, versatile proteins of recognition e a review, Carbohydr. Polym. 26 (1995) 219e230. [5] R. Loris, T. Hamelryck, J. Bouckaert, L. Wyns, Legume lectin structure, Biochim. Biophys. Acta Protein Struct. Mol. Enzymol. 1383 (1998) 9e36. [6] H. Rudiger, H.J. Gabius, Plant lectins: occurrence, biochemistry, functions and applications, Glycoconj. J. 18 (2001) 589e613. [7] P.A. Abreu, M.G. Albuquerque, C.R. Rodrigues, H.C. Castro, Structureefunction inferences based on molecular modeling, sequence-based methods and biological data analysis of snake venom lectins, Toxicon 48 (2006) 690e701. [8] T. Ogawa, T. Chijiwa, N. Oda-Ueda, M. Ohno, Molecular diversity and accelerated evolution of C-type lectin-like proteins from snake venom, Toxicon 45 (2005) 1e14. [9] D.C. Kilpatrick, Animal lectins: a historical introduction and overview, Biochim. Biophys. Acta Gen. Subj. 1572 (2002) 187e197.
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