Department of Biochemistry, University of Pretoria, Pretoria, South Africa
Correspondence: E-mail: albert.neitz{at}bioagric.up.ac.za.
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Abstract |
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Key Words: evolution gene duplication lipocalins tick toxicoses
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Introduction |
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Scorpion toxins share a common protein fold, gene, and intron organization, which suggests that they were all derived from a common ancestor (Froy et al. 1999). Spider toxins however, differ completely from those of scorpions in terms of structure, with many sharing the cystine knot motif (Escoubas, Diochot, and Corzo 2000). This difference suggests that toxins evolved independently within the toxic arachnida. The Acari (mites and ticks) are closest related to the Ricinulei or the "hooded-tickspiders," a group of nontoxic arachnids (Cooke 1967; Evans 1992). Within the Acari, ticks group closest to the holothyrida, a group of scavengers (Dobson and Barker 1999). As such, homology of tick toxins with either scorpion or spider toxins would seem to be a remote possibility. A common origin for tick toxins would thus have to derive from within the tick lineage itself.
In terms of toxicoses, the soft tick, Ornithodoros savignyi, is of special importance because it causes livestock losses in areas where it occurs. Mortality has been considered to be due to exsanguination, but evidence indicates that toxins secreted by the tick during feeding are the causal agents of pathogenesis (Neitz, Howell, and Potgieter 1969; Howell, Neitz, and Potgieter 1975; Neitz et al. 1983; Mans et al. 2002). Recently, four highly abundant tick salivary gland proteins (TSGPs) proposed to function in tick salivary gland granule biogenesis have been described in this species (Mans et al. 2001). In addition, TSGP2 and TSGP4 were identified as toxins that affect the cardiovascular system of the host (Mans et al. 2002). It was shown that the pathogenic and biochemical properties of these toxins differ significantly from those of tick paralysis toxins, and it was therefore suggested that tick toxicoses evolved independently over time (Mans et al. 2002). The present study shows that the TSGPs form part of the tick lipocalin family. This is the first study to identify a specific protein family involved in tick toxicoses and also the first to identify toxic lipocalins. Evidence is provided that toxicoses caused by O. savignyi are due to recent gene duplication events. The results presented suggest that tick toxicoses do not have a common origin within the tick lineage.
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Materials and Methods |
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Data Retrieval and Sequence Preparation
Sequences used during this study were all retrieved from the National Center for Biotechnology Information GenBank database (release 132.0). All sequences used in this analysis contained signal peptides as predicted by SignalP (von Heijne 1990; Nielsen et al. 1997). Each peptide was removed before sequence analysis to give the mature full-length proteins. All sequences are described by their abbreviation used in this study, followed by a description and, in parentheses, a GenBank accession number. Sequences from the soft tick O. savignyi are these: TSGP1, tick salivary gland protein 1 (gi|259913871|); TSGP2, tick salivary gland protein 2 (gi|25991389|); TSGP3, tick salivary gland protein 3 (gi|25991391|); TSGP4, tick salivary gland protein 4 (gi|25991438|). From O. moubata, we obtained the inhibitor specific for collagen-induced platelet aggregation (Keller et al. 1993), MOUB: moubatin (gi|159945|); from the hard tick Ripicephalus appendiculates, three histamine-binding proteins (Paesen et al. 1999): HBP1, female-specific histamine-binding protein 1 (gi|3452085|); HBP2, female-specific histamine-binding protein 2 (gi|3452089|); and HBP3, male-specific histamine-binding protein 3 (gi|3452093|). From the hard tick Dermacentor reticulates, we obtained the serotonin- and histamine-binding protein (Sangamnatdej et al. 2002) SHBP, serotonin- and histamine-binding protein (gi|18032205|). A variety of potential lipocalins derived from the sequencing of a salivary gland cDNA library from partially engorged female Ixodes scapularis hard ticks (Valenzuela et al. 2002): PSP1, potential secretory protein (gi|22164318|); PSP2, potential secretory protein 2 (gi|22164276|); IPHBP1, Ixodes potential histamine-binding protein 1 (gi|22164270|); IPHBP2, Ixodes potential histamine-binding protein 2 (gi|15428292|); IPHBP3, Ixodes potential histamine-binding protein 3 (gi|22164268|); PSP3, potential secretory protein 3 (gi|22164278|); PSP4, potential secretory protein 4 (gi|22164320|). A number of proteins identified as immunodominant antigens secreted during feeding of the hard tick I. scapularis (Das et al. 2001) were also obtained: Salp25A, salivary protein 25 kDa (gi|15428310|); Salp25B, salivary protein 25 kDa B (gi|15428306|); Salp26B, salivary protein 26 kDa B (gi|15428306|); Salp17, salivary protein 17 kDa (gi|15428298|).
Homology Detection for the TSGPs
The amino acid sequences of TSGP1-4 were analyzed using the EMBL advanced WU-Blast 2.08 server with the nonredundant database (nrdb95), using the default settings (expect = 100) (Yuan et al. 1998). A database search (GenBank release 132.0) of the TSGPs was also performed using the Smith-Waterman algorithm as employed by the SSEARCH3 program (Pearson 1991). To evaluate distant relationships between tick lipocalin sequences, the statistical significance by random shuffling (PRSS) was determined using 1,000 random generated sequences and a window size of 20. The PRSS program (version 3.4t20, Blosum 50 matrix, gap and extension penalties of -10 and -2, respectively) compares a query sequence to shuffled sequences using the Smith-Waterman algorithm (Pearson 1996).
Multiple Alignment and Phylogenetic Analysis
For phylogenetic analysis, multiple alignments were performed with ClustalX using the PAM250 matrix (Jeanmougin et al. 1998). A profile based on the secondary structure obtained from the crystal structure of HBP2 (PDB code: 1QVT) was constructed and used during the alignments to set the local gap penalties. Phylogenetic analysis was performed with Mega version 2.0 (Kumar, Tamura, and Nei 1994). Neighbor-Joining analysis was performed using the number of amino acid differences per site to construct a distance matrix between sequences. Positions that contained gaps were completely deleted so that 79 informative sites were used for analysis. The percentage confidence of the obtained unrooted tree was assessed using 10,000 bootstraps.
Molecular Modeling of Soft Tick Lipocalins
The only tick lipocalin for which structures are presently available is the female histamine-binding protein 2 (HBP2) (Paesen et al. 1999). Crystal structures were determined for the native protein (PDB code: 1QFT) and the apoprotein (PDB code: 1QVT). In both cases an asymmetric unit composed of two crystallographically distinct molecules (A and B) was obtained, and the two units differ, with a root mean square deviation (RMSD) value of 0.6. For modeling purposes, all four models were used (1QFTA, 1QFTB, 1QFVA, 1QFVB) as templates. The sequences of the TSGPs were manually aligned and threaded onto the HBP2 structures to obtain optimal threading energies calculated with the Swiss-PdbViewer program (v3.7b2). The mean force potential energies (Sippl 1990) were all negative (TSGP1, -4.3; TSGP2, -7.9; TSGP3, -6.7; and TSGP4, -14.1). After threading, sequences were submitted to the SWISS-MODEL Automated Comparative Protein Modeling Server with the default settings (Guex and Peitsch 1997). The modeling method used was ProMod II, and the structure obtained was minimized using steepest descent (200 cycles) with GROMOS96 and validated with WHATIF (Vriend 1990). For TSGP1-3, the N-terminal portions were rotated around Thr 9 (TSGP1) or Thr 8 (TSGP2 and TSGP3) to allow the formation of disulfide bonds, after which the proteins were put through an energy minimization cycle using steepest descent (200 cycles) with the GROMOS 43B1 force field of the Swiss Pdb-Viewer program (Guex and Peitsch 1997).
Generation of Polyclonal Antisera Against the TSGPs and Western Blot Analysis
The TSGPs were purified as described and further fractionated by tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue staining (Mans et al. 2001). Bands (10 µg) were excised, homogenized, and suspended in 400 µl of 0.1 M phosphate buffered saline, pH 7.2 (PBS) and emulsified as described using Freund's complete adjuvant (Vulliet 1996). New Zealand White rabbits were first bled to obtain naïve sera and then immunized with antigen emulsified in Freund's complete adjuvant. After 6 weeks the rabbits were boosted with antigen prepared in Freund's incomplete adjuvant, and that schedule was repeated until a specific response could be seen at x1,000 dilution (
18 weeks) with Western blotting. To gauge the immune response, rabbits were bled 10 days after each immunization. Western blot analysis of pooled salivary gland extracts (20 glands) from either O. moubata or O. savignyi was performed by standard procedures.
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Results and Discussion |
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WU-Blast Analysis of the TSGPs
An advanced WU-Blast analysis indicated sequence similarity to moubatin (swiss|Q04669|) for TSGP1 (P(N)-value: 0.0016), TSGP2 (P(N) value: 1.8e-36), and TSGP3 (P(N)-value: 5.2e-37). Moubatin is an inhibitor specific for collagen-induced platelet aggregation (Keller et al. 1993; Waxman and Connolly 1993). It was recently suggested that moubatin might be related to the hard tick histamine-binding proteins based on sequence similarity (Paesen et al. 1999). WU-Blast analysis of TSGP4 showed significant similarity to the serotonin- and histamine-binding protein (SHBP; sptrembl|Q8WSK7|; P(N)-value: 0.0007) from the hard tick D. reticulates. Serotonin- and histamine-binding protein is homologous to the other hard tick histamine-binding proteins, all of which exhibit the lipocalin protein fold (Sangamnatdej et al. 2002). Other hits were a 22.5 kDa putative secreted protein (PSP1,sptrembl|Q8MV98|) from the hard tick I. scapularis (P(N)-value: 0.033) described recently (Valenzuela et al. 2002) and moubatin (P(N)-value: 0.94). A WU-Blast analysis of moubatin identified TSGP3 (P(N)-value: 5.5e-37), TSGP2 (P(N)-value: 1.9e-36), TSGP1 (P(N)-value: 0.0012) and TSGP4 (P(N)-value: 0.95) as the first four hits.
SSEARCH of the TSGPs
Database searches with the TSGPs sequences were also performed using SSEARCH, a program that employs the more robust, but slower Smith-Waterman algorithm (Pearson 1991). The only significant hit for TSGP1-3 was moubatin, with E values of 0.0004, 1.1e-33, and 2.3e-29, respectively (table 1). In the case of TSGP4 several tick proteins were obtained, listed in order of appearance: SHBP (gi|18032205|, E value: 0.0034); the 22.5 kDa protein from I. scapularis (PSP1: gi|22164318|, E value: 0.012); Salp26B (gi|15428306|, E value: 0.035); an immunodominant protein from the salivary gland of I. scapularis (Das et al. 2001), HBP2 (gi|3452089|, E value: 0.18); a putative 25 kDa secretory protein (PSP2: gi|22164276|, E value: 0.48) from I. scapularis (Valenzuela et al. 2002); Salp25A (gi|15428310|, E value: 0.72), another immunodominant protein from the salivary glands of I. scapularis (Das et al. 2001); Salp25B (gi|15428302|, E value: 0.72), a related immunodominant protein from I. scapularis (Das et al. 2001); and moubatin (E value: 1.1). It is clear that the only sequences homologous to the TSGPs are other tick proteins now present in the databases.
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Homology Assessment of Putative Tick Lipocalins
WU-Blast as well as SSEARCH analysis suggested that the TSGPs are homologous to one another via similarity to moubatin and to the hard tick lipocalins, even though their relationship might be quite distant. To investigate the possibility that the TSGPs are related to the tick lipocalins, the statistical significance of their pairwise similarities was evaluated using the PRSS program with a library of 1,000 shuffled sequences and a window size of 20 (fig. 4). With these parameters, an E value below 0.2 is considered to be a clear indication of homology (Pearson 1996). Expected values obtained in this way indicate that the tick lipocalins can be divided into three distinct groups: TSGP1-3, which are clearly related to moubatin; the hard tick histamine-binding proteins, which are related to one another and some sequences from I. scapularis (fig. 4); and a third group not clearly related to the HBPs or TSGPs, proteins from I. scapularis. Although, no distinct relationships exist among these groups, some proteins like TSGP4 are related to members of the other groups. It is clear that TSGP4 is related to HBP2 and SHBP and that it is distantly related to HBP1 and PSP1 from the hard tick lipocalins and to TSGP2, TSGP3 and moubatin from the soft tick proteins. Based on the principle that proteins that share a common homolog must themselves be homologous, even if they share no significant sequence similarity (Pearson 1996), it can be concluded that all the described tick proteins are homologous and probably share the same lipocalin fold. Both identities as well as similarities are rather low between sequence pairs, except for a few that show identities above 30% (fig. 4). Most fall within the twilight zone (20%30% identity) or the midnight zone (average around 8% identity). Similarities are however, significantly higher than identities (>50% in most cases using the PAM250 matrix) for all sequence pairs, and similarity is a feature generally associated with homologous sequences (Rost 1999). This observation is in fact quite important, as it was found that similarity was higher than identity in only 10% of structurally similar pairs (Rost 1999). The low sequence identity observed between the tick lipocalins is consistent with the general trend of the lipocalin family where paralogous members show less than 20% identity (Flower 1996; Flower, North, and Sansom 2000).
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Phylogenetic Analysis of the Tick Lipocalins
Neighbor-Joining grouped the tick lipocalins into three main branches, the soft tick lipocalins, the histamine-binding proteins, and lipocalins derived from I. scapularis (fig. 6). The fact that database analysis of the tick lipocalins detects only other tick lipocalins suggests that these proteins evolved exclusively within the tick lineage. Phylogenetic analysis also suggests that most tick lipocalins evolved after the divergence of the main tick families, and in most cases even up to genus level. This would suggest extensive gene duplication events of the tick lipocalins as ticks diversified, and it also suggests a selective pressure on ticks to evolve new protein functions. In general this happens when organisms adapt to novel environments, and in this case we propose that it occurred during the adaptation of ticks to a blood-feeding environment.
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Independent Evolution of Tick Toxicoses
If TSGP2 and TSGP3 are not orthologs of moubatin, it follows that they must be recent gene duplicates (paralogs) that have lost their ability to inhibit collagen-induced platelet aggregation. This raises the possibility that these gene duplication events occurred after the divergence of O. moubata and O. savignyi. If so, then it could be expected that TSGP2-3 will be absent from salivary gland extracts (SGE) of O. moubata. Western blot analysis indicates that TSGP2-3 is indeed absent in SGE of O. moubata (fig. 7), supporting this hypothesis. The presence of a cross-reactive band similar to TSGP1 and another cross-reactive band similar to TSGP4 correlates with the fact that both group basal to the rest of the soft tick lipocalins, suggesting that they were present before the divergence of O. savignyi and O. moubata. N-terminal sequence similarity to the highly abundant antigenic protein 20A1 of O. moubata was also observed for TSGP1 (Mans et al. 2001). However, caution should be exercised in the case of TSGP4, where the main antigenic band detected with antiserum against TSGP4 is not present in O. moubata. This suggests that this toxic form is absent in O. moubata salivary glands. The most parsimonious explanation for this is that this gene was lost in the O. moubata lineage. However, as these are paralogous genes and nothing is known about their last common ancestor, it could be that TSGP4 duplicated after divergence of the two tick species from a shared but as yet unidentified nontoxic ancestral gene. Toxicosis associated with O. savignyi differs significantly from other forms of toxicosis such as tick paralysis (Mans et al. 2002). The absence of toxins in O. moubata and the possibility that toxic proteins evolved after divergence of these tick species correlate with this being a novel form of toxicosis. Gain of toxic function after divergence of O. moubata and O. savignyi has important implications for the origins of toxicity in ticks. Recent aquisition of genes coding for toxins through gene duplication discounts a common ancient origin for all tick toxins and suggests the probability of multiple unrelated origins for tick toxins. Independent evolution of tick toxicosis is not really so surprising, as recent evidence suggests that the main tick families independently evolved a hematophagous life-style (Mans, Louw, and Neitz 2002b).
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Acknowledgements |
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Footnotes |
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