The Major Tick Salivary Gland Proteins and Toxins from the Soft Tick, Ornithodoros savignyi, Are Part of the Tick Lipocalin Family: Implications for the Origins of Tick Toxicoses

Ben J. Mans, Abraham I. Louw and Albert W. H. Neitz

Department of Biochemistry, University of Pretoria, Pretoria, South Africa

Correspondence: E-mail: albert.neitz{at}bioagric.up.ac.za.


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
The origins of tick toxicoses remain a subject of controversy because no molecular data are yet available to study the evolution of tick-derived toxins. In this study we describe the molecular structure of toxins from the soft tick, Ornithodoros savignyi. The tick salivary gland proteins (TSGPs) are four highly abundant proteins proposed to play a role in salivary gland granule biogenesis of the soft tick O. savignyi, of which the toxins TSGP2 and TSGP4 are a part. They were assigned to the lipocalin family based on sequence similarity to known tick lipocalins. Several other tick lipocalins were also identified using Smith-Waterman database searches, bringing the tick lipocalin family up to 20. Phylogenetic analysis showed that most tick lipocalins group within genus-specific clades, suggesting that gene duplication and divergence of tick lipocalin function occurred after tick speciation, most probably during the evolution of a hematophagous lifestyle. TSGP2 and TSGP3 show high sequence identity and group terminal to moubatin, an inhibitor of collagen-induced platelet aggregation from the tick, O. moubata. However, no platelet aggregation inhibitory activity is associated with the TSGPs using ADP or collagen as agonists, suggesting that TSGP2 and TSGP3 duplicated after divergence of O. savignyi and O. moubata. This timing is supported by the absence of TSGP2-4 in the salivary gland extracts of O. moubata. The absence of TSGP2 and TSGP4 in salivary gland extracts from O. moubata correlates with the nontoxicity of this tick species. The implications of this study are that the various forms of tick toxicoses do not have a common origin, but must have evolved independently in those tick species that cause pathogenesis.

Key Words: evolution • gene duplication • lipocalins • tick toxicoses


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
The Arachnida are notorious for the venomous organisms that form part of this class. The best known are scorpions and spiders, although toxic reactions have also been described in response to the bites of mites and ticks (Wikel 1984, pp. 371–396). Spiders and scorpions evolved toxins for defense as well as for predatory purposes (Froy et al. 1999; Escoubas, Diochot, and Corzo 2000). The advantages of being venomous are less clear for ticks, which are obligate, hematophagous parasites that depend on their host for a blood meal. It has been suggested that tick paralysis, a major form of tick toxicoses, may be a vestigial function retained in ticks when they evolved a parasitic lifestyle (Stone et al. 1989). However, only ~81 of ~869 tick species have been implicated in tick toxicoses (Gothe 1999). This would suggest that if tick toxicosis was a remnant of a previous predatory lifestyle, evolution of parasitism favored the demise of toxicoses. Research into the origins of tick toxicoses is thus of importance as it might elucidate character traits associated with the ancestral tick species. This knowledge could illuminate important processes that occurred during the adaptation of ticks to a blood-feeding lifestyle.

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.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
Rapid Amplification of cDNA Ends (RACE) to Obtain the Full-Length Genes
The cloning strategy used for savignin was followed (Mans, Louw, and Neitz 2002a). To obtain the coding gene and 3' untranslated region, degenerate primers were designed from the obtained N-terminal amino acid sequences. For TSGP1 a primer (20 kD: GGI CCI GAY GGI TGY GT) was designed using the first 6 amino acids (GPDGCV), for TSGP2 the primer (TOE: TTY CCI ACI GAR GCN TA) was designed from amino acids 6–11 (FPTEAY), and for TSGP3 the primer (TOC: TTY CCI ACI GAY GCN TA) was also designed from amino acids 6–11 (FPTDAY). Finally, the primer for TSGP4 (Toks1: GCN AAY GAY GTI TGG AAY GT) was designed from the first 7 amino acids (ANDVWNV). To obtain the full-length genes, 3' RACE studies were performed on single-stranded cDNA synthesized from total RNA. For 5' RACE, primers used were TSGP1 (20KDC1: GTG TAG GGG ATG GGG CCA), TSGP2 (C1T2: CTA GCA GTC CTT GTC TT), TSGP3 (NTC1: GTT CCA ACA TCC ACA TG), and TSGP4 (C1T1: CTA CGG AAC TCT GCA GCC TT). The primer 20KDC1 is complementary to a region in the 3' untranslated region of TSGP1; C1T2 is complementary to the last five amino acids and stop codon of TSGP2 (QDKDC-); NTC1 is complementary to an internal sequence (DMWMLE) of TSGP3 that differs from that of TSGP2 (EMWMLE) at the last position in the codon of aspartic acid; and C1T1 is complementary to the last five amino acids and stop codon of TSGP4 (EGCRVP-). 5' RACE reactions were performed using double-stranded cDNA synthesized from total RNA. The products of at least three RACE reactions were cloned and at least three different clones of each product were sequenced from both the upstream end and the downstream end (Mans, Louw, and Neitz 2002a).

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.


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
Amino Acid Sequences of the TSGPs
The full-length gene sequences of the TSGPs all include a stop codon, a polyadenylation site (TSGP1/TSGP4: AATAAA and TSGP2/TSGP3: AGTAAA), and a poly-A tail (figs. 1–3GoGo). The AGTAAA site was previously also identified in savignin, a thrombin inhibitor from this tick species (Mans, Louw, and Neitz 2002a). The translated amino acid sequences of the immature proteins contain a signal peptide and consist of 190 (TSGP1), 163 (TSGP2), 163 (TSGP3), and 176 (TSGP4) amino acids (figs. 1–3GoGo). Signal P predicted the presence of the signal peptide and the correct cleavage site in all cases (von Heijne 1990; Nielsen et al. 1997). The mature proteins consist of 171 (TSGP1—18,613 Da), 144 (TSGP2—15,872 Da), 144 (TSGP3—15,950 Da), and 156 (TSGP4—1,7161 Da) amino acids and include the previously determined N-terminal sequences (Mans et al. 2001). Of interest is E16 in the mature TSGP2/TSGP3 sequences, which showed up as a modified amino acid during N-terminal sequencing. The elution profile of this residue during N-terminal sequencing (personal observation) suggests carboxyl methylation, which could be involved in salivary gland granule packaging and exocytotic secretion (van Waarde 1987).



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FIG. 1. Full-length sequence of TSGP1. The 5' adapter, 3' gene-specific, and 3' anchor primers are shown in bold. The stop codon (tag), polyadenylation site (AATAAA) and poly-A tail are boxed. The N-terminal amino acid sequence previously obtained with N-terminal Edman degradation is underlined, and the N-terminal sequence used for degenerate primer design is shown in bold. The signal sequence is underlined with a dashed line. Fragments and their corresponding molecular masses that correspond to peptide mass fingerprints for TSGP1 are indicated in bracketed lines. The GenBank accession code for TSGP1 is gi|25991387|

 


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FIG. 2. Full-length sequences of TSGP2 and TSGP3. Synonymous nucleotide differences are boxed, and nonsynonymous nucleotide differences are boxed in gray. The 5' adapter, 3' gene-specific, and 3' anchor primers are shown in bold. The stop codon (TAG), polyadenylation site (AGTAAA) and poly-A tail are indicated by black boxes. For the deduced amino acid sequences, the differences are indicated by a slash (e.g., TSGP2/TSGP3). The N-terminal amino acid sequence previously obtained with N-terminal Edman degradation is underlined, and the N-terminal sequence used for degenerate primer design is shown in bold. The signal sequence is underlined with a dashed-line. Fragments and their corresponding molecular masses that correspond to peptide mass fingerprints for TSGP2 and TSGP3 are indicated in bracketed lines. The GenBank accession codes for TSGP2 and TSGP3 are gi|25991389| and gi|25991391|, respectively

 


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FIG. 3. Full-length sequence of TSGP4. The 5' adapter, 3' gene-specific, and 3' anchor primers are shown in bold. A stop codon (tag), polyadenylation site (AATAAA) and poly-A tail are boxed. The N-terminal amino acid sequence previously obtained with N-terminal Edman degradation is underlined, and the N-terminal sequence used for degenerate primer design is shown in bold. The signal sequence is underlined with a dashed-line. Fragments and their molecular masses that correspond to peptide mass fingerprints for TSGP4 are indicated in bracketed lines. The GenBank accession code for TSGP4 is gi|25991438|

 
Comparison of Data from Native TSGPs and Their Deduced Amino Acid Sequences
Amino acid compositions of the deduced amino acid sequences compare favorably with data from the native proteins (results not shown). The calculated molecular masses also correlate well with those obtained from electrospray mass spectrometry analysis (Mans et al. 2002). In contrast to previous reports, these data clearly show that the TSGPs are not glycosylated and that there is no post-translational proteolytic processing of the mature proteins (Neitz et al. 1983). Theoretical mapping with trypsin corresponds well with peptide mass fingerprints previously obtained (figs. 1–3GoGo; Mans et al. 2001). It also shows that the peptide fragments correspond with fragments across the full length of the TSGP sequences. These results indicate that the correct sequences have been obtained. In the case of TSGP1 there were, however, a few peptides that could not be matched to the sequence. Reversed-phase high performance liquid chromatography (HPLC) did, however, indicate that TSGP1 eluted as a broad tailing peak that could indicate microheterogeneity on the sequence level (Mans et al. 2001).

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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Table 1 SSEARCH 3 of the GenBank Database Using Various Tick Proteins. Specific Hits Are Indicated with Their Accession Numbers and E Values.

 
Identification of Other Potential Tick Lipocalin Homologs
Previous analysis of Salp25B indicated similarity to the tick histamine-binding proteins (Das et al. 2001). Salp25A and Salp26B did not, however, show any similarities to other sequences in GenBank. It would thus seem that numerous new tick-derived lipocalins are present in the databases. To assess whether other unidentified tick lipocalins are present in the database, SSEARCH was also performed with each new tick sequence identified using TSGP4 and other potential sequences that could be identified as potential lipocalins. The highest hits in all cases are other tick lipocalins (table 1), whereas other nontick protein hits show insignificant E values. New lipocalins identified are potential secretory proteins from I. scapularis (PSP1, PSP2, PSP3, and PSP4) and proteins previously identified as potential histamine-binding proteins (IPHBP1, IPHBP2, and IPHBP3) (Valenzuela et al. 2002). A 17 kDa immunodominant antigen (Salp17) was also identified (Das et al. 2001).

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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FIG. 4. Statistical analysis by random shuffling of tick lipocalin sequences. Expected values (E values) obtained for a pairwise analysis of each sequence pair using the Smith-Waterman algorithm, estimated against a library of 1,000 shuffled sequences obtained from one of the sequences in the pair are indicated in the upper right quadrant. Those boxes shaded in black indicate clearly related sequences (E values < 1e-3); boxes in gray indicate distantly related sequences (E values < 0.2) and unshaded boxes indicate unrelated sequences. The lower left quadrant shows the percentage identity/similarity as obtained from the multiple alignments used for phylogenetic analysis. Similarities are based on the PAM250 matrix and identities above 25% are boxed in black

 
Multiple Alignment of the Tick Lipocalins
Alignment of TSGP1–4 with moubatin and the histamine-binding proteins shows that there are areas of similarity, although identities are very low between sequences (fig. 5A). The highest conserved regions of the tick lipocalins correspond to those of the secondary structure previously obtained for HBP2 and consist of two N-terminal {alpha}-helices, an 8-stranded antiparallel ß-barrel with a (+1)7 topology and a C-terminal {alpha}-helix, characteristic of the lipocalin fold (Paesen et al. 1999). Residues involved in the low- and high-affinity binding sites of the HBPs are not conserved in the TSGPs and correlate with the inability of the TSGPs to bind to histamine (results not shown) (fig. 5A). The tick lipocalins do not contain any of the structural conserved regions (SCR) used to classify lipocalins as kernel (those that possess all three SCRs) or outlier (those that possess two or fewer SCRs) and are thus outlier lipocalins (Flower 1996; Flower, North, and Sansom 2000). Although HBP2 possess the TDYD sequence that is conserved in SCR2 and is one of the regions involved in immunoglobin E binding of lipocalin allergens (Mäntyjärvi, Rautiainen, and Virtanen 2000), this sequence is absent in the TSGPs.



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FIG. 5. Multiple sequence alignment of the tick lipocalins. (A) Alignment of TSGPs with the HBPs from the hard tick, R. appendiculatus, and moubatin, from the soft tick, O. moubata, the inhibitor specific for collagen-induced platelet aggregation. Secondary structure based on that of HBP2 is boxed and designated as {alpha}-helixes or ß-strands. Solid lines indicate conserved cysteines and their corresponding disulfide bonds, as deduced from the structure of Ra-HBP2. Dotted lines indicate hypothetical disulfide bonds of the remaining cysteines for moubatin, TSGP1-3, and TSGP4. Residues of Ra-HBP2 involved in the interaction with histamine are indicated for its high-affinity (black rectangles) and low-affinity (white rectangles) binding sites. (B) Structure models of the TSGPs. Models were fitted to the structure of HBP2 using Pdb-Viewer. Indicated are the RMSD values and the position of the intact disulfide bonds

 
Disulfide Bonds of the TSGPs
It was previously shown that all cysteines of the TSGPs are involved in disulfide bonds (Mans et al. 2001). Four cysteines conserved in all tick lipocalins are involved in disulfide bonds in the HBPs and serve to pin the C-terminal {alpha}-helix to the side of the barrel (Paesen et al. 1999). Cys 119–Cys 148 pins the start of the C-terminal {alpha}-helix to the start of strand ß-G, whereas Cys 48–Cys 168 links the C-terminal ß-I strand with the base of the {Omega}-loop. Assuming that this disulfide bond pattern is consistent for other tick lipocalins, the remaining disulfide bonds present in the TSGPs can be inferred from the sequence alignment (fig. 5). In the case of TSGP1-3 and moubatin, the extended N-terminal helix is disulfide bonded to the C-terminal {alpha}-helix (Cys 2–Cys 122 for TSGP2-3) and contrasts with HBP3, which lacks the N-terminal cysteine and forms a disulfide-linked dimer (Paesen et al. 1999). In the case of TSGP4, Cys 117–Cys 141 also pins the end of the C-terminal {alpha}-helix to the start of the ß-H sheet. In all cases the disulfide bonds do not stabilize the whole structure, but are all localized to one side of the protein fold, where it is involved in pinning the {alpha}-helix to the ß-barrel. This could explain the observed lability of the toxins during purification procedures (Mans et al. 2002). A free cysteine residue is necessary for prostaglandin D synthase activity (Nagata et al. 1991). As all cysteines could be assigned to disulfide bond partners in the TSGPs, they probably do not exhibit this activity. It would seem as if pinning of the C-terminal {alpha}-helix to the side of the ß-barrel is an important conserved feature of the lipocalin fold, probably to ensure a stable, tight packing of the helix against the barrel. This is particularly striking in TSGP4, where a shift in the disulfide bond structure led to an even more stable double-pinning of the C-terminal {alpha}-helix to the barrel.

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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FIG. 6. Phylogenetic analysis of the tick lipocalin family. Indicated is an unrooted tree constructed by Neighbor-Joining analysis, based on amino acid differences per site. The percentage confidence is indicated for 10,000 bootstraps

 
The Absence of Antihemostatic Activities Associated with the TSGPs
The tick salivary gland proteins TSGP2 and TSGP3 group below moubatin, while high sequence similarity was also observed between moubatin and TSGP2-3 (73%). This suggests that TSGP2 and/or TSGP3 might be the orthologs of moubatin. Moreover, lipocalins, which have been identified in the salivary gland secretions from various hematophagous organisms, play a key role in the control of their host's hemostatic systems (Montford, Weichsel, and Andersen 2000). This raises the question of whether the TSGPs are involved in the regulation of the haemostatic system. Inhibition of the blood coagulation cascade by the TSGPs may be excluded, as it was found that no inhibitory effects of the extrinsic or intrinsic pathways of the blood-coagulation cascade or specifically fXa or thrombin were associated with the TSGPs (Gaspar, Crause, and Neitz 1995; Gaspar et al. 1996; Nienaber, Gaspar, and Neitz 1999). Inhibition of ADP-induced platelet aggregation was not observed for the TSGPs (Mans, Louw, and Neitz 2002b). In the present study, no inhibition of collagen-induced platelet aggregation could be indicated for the TSGPs (results not shown). Instead, both ADP-induced and collagen-induced platelet aggregation inhibitory activities were observed after reversed-phase HPLC in a distinct peak, the first part of which (29–33 min) corresponds to savignygrin, an {alpha}IIBß3 integrin antagonist that inhibits both ADP-induced and collagen-induced platelet aggregation (Mans, Louw, and Neitz 2002b). The last part of the peak (38–40 min) corresponded to a 17 kDa protein (savignygen), which purified with the collagen-specific activity and is probably moubatin's ortholog, as it also showed cross-reactivity with anti-TSGP2 serum (results not shown). Further support for this is that savignygen and moubatin elute at ~20%-25% acetonitrile, whereas the TSGPs elute at 40%-45% acetonitrile (Waxman and Connolly 1993).

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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FIG. 7. Western blot analysis of TSGPs in salivary gland extracts of O. moubata and O. savignyi.Indicated are molecular mass markers (MM), Coomassie brilliant blue staining of salivary gland extracts of O. savignyi (S) and O. moubata (M), and the corresponding Western blots for TSGP1-4

 
Conclusion
This study is the first to assign tick toxins to a specific protein family. It also provides conclusive evidence that toxicosis induced by the tick O. savignyi is a very recent event that probably occurred after the divergence of O. savignyi and O. moubata. The TSGPs are part of what is becoming an extended family of tick lipocalins. It is clear that these different paralogs evolved by gene duplication events, and it is foreseen that more lipocalins with diverse functions will be found in different tick species. Although lipocalins have a conserved fold, there are several significant differences between those from ticks and the rest of the lipocalin family. Anti-tick feeding vaccines targeted at these specific features may thus be possible, and it is feasible that a wide range of cross-protection toward a wide range of tick species may be accomplished with these proteins.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
The authors thank the National Research Foundation and University of Pretoria for financial support, Mr. N. J. Taljaard for performing amino acid composition determination, and Mr. Pieter Vrey for the part he played in the elucidation of the initial partial sequence of TSGP1.


    Footnotes
 
Peer Bork, Associate Editor Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 

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Accepted for publication March 24, 2003.