Department of Biochemistry, University of Pretoria
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Abstract |
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Introduction |
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Ticks (suborder Ixodida) are obligate hematophagous organisms that comprise three families, the Ixodidae (hard ticks), Argasidae (soft ticks), and Nuttalliellidae (monotypic; Hoogstraal 1956
). Their origins have been estimated to be in the Late Cretaceous,
120 MYA, which was the last time that Australia was part of Gondwanaland, indicating that this period played an important role in the origin of the Australian tick lineages, and by extension, the entire tick family (Klompen et al. 1996
, 2000
). Phylogenetic analysis indicates that the Ixodida are monophyletic (Black, Klompen, and Keirans 1997
), whereas the oldest tick fossil to date is an argasid (Carios jerseyi) found in New Jersey amber (9094 MYA; Klompen and Grimaldi 2001
). This indicates that ticks had already diverged by
92 MYA into the main tick families, as well as the argasid genus level. The Holothyrida, with the Ixodida and Mesostigmata, forms the superorder Parasitiformes, and it was indicated that the Holothyrida rather than Mesostigmata are a sister-group to ticks (Dobson and Barker 1999
). Holothyrida is a group of free-living scavenging mites, which mainly live on body fluids of dead arthropods. It has been suggested that ticks shared this trait before adaptation to a blood-feeding environment (Walter and Proctor 1998
). This scenario raises the following questions:
These questions can be addressed by studying the evolution of hemostatic factors secreted by ticks during feeding. The coagulation inhibitors and platelet aggregation inhibitors (PAIs) present in the saliva of hard and soft ticks have been reviewed extensively (Law, Ribeiro, and Wells 1992
; Sauer et al. 1995
; Bowman et al. 1997
; Nuttall et al. 2000
). Inhibitors for the central blood clotting enzymes, fXa (tick anticoagulant peptide [TAP] and fXa inhibitor [fXaI]) and thrombin (ornithodorin and savignin), have been described in Ornithodoros moubata and O. savignyi, respectively (Waxman et al. 1990
; Gaspar et al. 1996
; van de Locht et al. 1996
; Joubert et al. 1998
; Nienaber, Gaspar, and Neitz 1999
). They are slow tight-binding inhibitors of the Kunitz family. Canonical Kunitz inhibitors inhibit their respective serine proteases by presenting a substrate-binding loop (located around the second cysteine) to the active site of their enzymes, where it binds in an antiparallel ß-sheet fashion similar to that of natural serine protease substrates (Laskowski and Kato 1980
; Bode and Huber 1992
). The tick inhibitors, however, insert their N-terminal sequences into their enzymes' active site in a manner reminiscent of hirudin, where they bind in a parallel ß-sheet fashion (van de Locht et al. 1996
; Wei et al. 1998
). The tick fXaIs consist of a single BPTI-like domain (60 amino acids) that interacts with fXa via the N-terminal as well as secondary interactive sites in its C-terminal
-helix (Wei et al. 1998
). The tick thrombin inhibitors have two BPTI-like domains (
60 amino acids each with an eight-residue linker), of which the N-terminal domain (NTI) interacts with thrombins' active site via the N-terminal residues and secondary interaction sites, whereas the C-terminal domain (CTI) interacts via its C-terminal
-helix with the fibrinogen-binding exo-site of thrombin (van de Locht et al. 1996
; Mans, Louw, and Neitz 2002a
). PAIs from O. moubata (disagregin) and O. savignyi (savignygrin) have also been described (Karczewski, Endris, and Connolly 1994
; Mans, Louw, and Neitz 2002b
). Platelet aggregation is inhibited by targeting of the fibrinogen receptor
IIbß3, thereby blocking fibrinogen-binding and subsequent aggregation (Karczewski and Connolly 1997
; Mans, Louw, and Neitz 2002b
). It was shown that savignygrin presents RGD, an integrin recognition motif, on the substrate-binding loop of the canonical BPTI-fold (Mans, Louw, and Neitz 2002b
). As fXa, thrombin, and PAIs from O. savignyi all exhibit the BPTI-fold, the hypothesis that all share a common ancestor was advanced. This study describes a common origin for the tick BPTI-like inhibitors and discusses possible pathways by which these novel functions evolved.
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Materials and Methods |
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TAP_ORNMO: tick anticoagulant peptide (P17726). fXaI: fXa inhibitor (AAD09876). ORNT_ORNM1: ornithodorin domain 1 (P56409). Savignin1: savignin domain 1 (AF321524). DISG_ORNMO: disagregin (g544163), savignygrin (AF452885). ORNT_ORNM2: ornithodorin domain 2 (P56409). Savignin2: savignin second domain (AF321524). A4_RAT: Alzheimer's disease amyloid 4 (P08592). A4_MACFA: Alzheimer's disease amyloid 4 (P53601). A4_SAISC: Alzheimer's disease amyloid 4 (Q95241). A4_MOUSE: Alzheimer's disease amyloid 4 (P12023). APP2_RAT: amyloidlike protein 2 (P15943). APP2_HUMAN: amyloidlike protein 2 (Q06481). AMBP_BOV2: bovine alpha-1-microglobulin domain 2 (P00978). IATR_SHEEP2: sheep alpha-1-microglobulin domain 2 (P13371). AMBP_PIG2: pig alpha-1-microglobulin domain 2 (P04366). AMBP_HUMAN2: human alpha-1-microglobulin domain 2 (P02760). AMBP_RAT2: rat alpha-1-microglobulin domain 2 (Q64240). ITR4_RADMA: trypsin inhibitor Radiantus macrodactylus (P16344). ISH1_STOHE: serine protease inhibitor from Stichodactyla helianthus (P31713). BPTI1_BOVIN: bovine pancreatic trypsin inhibitor (P00974). BPTI2_BOVIN: spleen trypsin inhibitor (P04815). IBPS_BOVIN: serum basic protein (P00975). TFPI_HUMAN3: human tissue factor pathway inhibitor domain 3 (P10646). TFPI_RABIT3: rabbit tissue factor pathway inhibitor domain 3 (P19761). TFPI_RAT3: rat tissue factor pathway inhibitor domain 3 (Q02445). ISC1_BOMMO: silkworm chymotrypsin inhibitor (P10831). ISC2_BOMMO: silkworm chymotrypsin inhibitor (P10832). TIMTC3: silkworm chymotrypsin inhibitor (TIMTC3). SBPI_SARBU: grey flesh fly protease inhibitor (P26228). TIFHBP: flesh fly proteinase inhibitor (TIFHBP). CRPT_BOOMI: carrapatin Boophilus microplus (P81162). BMTI-2: B. microplus trypsin inhibitor second domain (Tanaka et al. 1999
). TFPI_HUMAN1: human tissue factor pathway inhibitor domain 3 (P10646). TFPI_RABIT1: rabbit tissue factor pathway inhibitor domain 3 (P19761). TFPI_RAT1: rat tissue factor pathway inhibitor domain 3 (Q02445). AMBP_BOV1: bovine alpha-1-microglobulin domain 1 (P00978). IATR_SHEEP1: sheep alpha-1-microglobulin domain 1 (P13371). AMBP_PIG1: pig alpha-1-microglobulin domain 1 (P04366). AMBP_HUMAN1: human alpha-1-microglobulin domain 1 (P02760). AMBP_RAT1: rat alpha-1-microglobulin domain 1 (Q64240). TIBOC: bovine colostrums inhibitor (TIBOC). TFPI_HUMAN2: human tissue factor pathway inhibitor domain 3 (P10646). TFPI_RABIT2: rabbit tissue factor pathway inhibitor domain 3 (P19761). TFPI_RAT2: rat tissue factor pathway inhibitor domain 3 (Q02445). AsKC1: kalicludine sea anemone toxin (AAB35413). AsKC2: kalicludine sea anemone toxin (AAB35414). IP52_ANESU: sea anemone protease inhibitor SA5 II (P10280). ISIK_HELPO: roman snail isoinhibitor K (P00994). IVBK_DENPO: dendrotoxin K (P00981). IVBI_DENAN: alpha dendrotoxin (P00980). IVBE_DENPO: dendrotoxin E (P00984). IVB1_VIPAA: venom trypsin inhibitor 1 (P00991). IVB3_VIPAA: venom basic protease inhibitor 3 (P00992). IVBT_ERIMA: venom trypsin inhibitor (P24541). IVB1_BUNFA: venom basic protease inhibitors IX and VIIIB (P25660). IVBT_NAJNA: venom trypsin inhibitor (P20229). IVB2_NAJNI: venom basic protease inhibitor II (P00986). IVB2_HEMHA: venom basic protease inhibitor II (P00985). IVB1_BUNMU: beta-1 bungarotoxin chain B (P00987). IVB3_BUNMU: beta-2 bungarotoxin chain B (P00989).
Multiple Sequence Alignment
Sequences were processed to give only a single BPTI core-domain by truncation of amino acid sequences, one amino acid before and one after the first and last cysteine of the BPTI-fold, respectively (Pritchard and Dufton 1999
). For BPTI-proteins that exist as multiple-domains, each domain was treated as a single BPTI-fold. Multiple sequence alignment was performed with ClustalX, using the PAM250 matrix and default gap penalty options (Jeanmougin et al. 1998
). Alignments were adjusted manually on the basis of conserved cysteine positions and secondary structure considerations (Antuch et al. 1994
).
Neighbor-Joining (NJ) Analysis of the BPTI-Family
Phylogenetic analysis of the total BPTI-family was conducted using MEGA version 2.0 (Kumar, Tamura, and Nei, 1994
). NJ 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 39 informative sites were used for analysis.
Maximum Parsimony (MP) Analysis of Tick-Derived BPTI-Inhibitors
NJ did not completely resolve the relationships within the soft tick BPTI-inhibitor clade. To obtain a more accurate description of the underlying relationships between tick BPTI-inhibitors, MP using the PHYLIP package, Version 3.2, were performed employing the PROTPARS method (Felsenstein 1989
). For comparative purposes, BPTI-inhibitors from hard ticks as well as insect hemolymph were included, whereas ß-bungarotoxin chain B, proposed to be an outlier of the whole BPTI-family, was used as outgroup (Dufton 1985
; Ikeo, Takahashi, and Gojobori 1992
).
Phylogeny of Soft Tick Inhibitors Based on Protein Structure
It has been indicated that structure comparison could resolve phylogenetic relationships (Johnson, Sutcliffe, and Blundell 1990
). Construction of a pairwise distance tree on the basis of root mean square deviation (RMSD) of the
-carbon backbone structure could assist in the estimation of distant homologies. As the X-ray diffraction structure of ornithodorin (PDB code: 1TOC; van de Locht et al. 1996
) and nuclear magnetic resonance structure of TAP (PDB code: 1TAP: Antuch et al. 1994
) are known, modeling of their orthologs (savignin and fXaI, respectively) was conducted using the SWISS-MODEL Automated Comparative Protein Modeling Server (Guex, Diemand, and Peitsch 1999
) and MODELLER (Sali et al. 1995
). The low identity observed among the PAIs and ornithodorin and TAP complicates their modeling using automated servers. Model structures could be obtained, however, using the MODELLER package (Mans, Louw, and Neitz 2002b
). The structure of BPTI (PDB code: 1BPI), considered to be the prototype BPTI-fold, was used as outgroup. RMSD values between structure pairs were determined by fitting of the backbone structures (using the same core structure of the BPTI-fold as was used for multiple alignment and phylogenetic analysis) using the McLachlan algorithm (McLaghlan 1982
), as implemented in the protein least squares fitting program, ProFit V1.8 (http://www.biochem.ucl.ac.uk/
martin/#profit). The phylogenetic tree was constructed using a pairwise distance matrix of RMSD values and the program NEIGHBOUR of the PHYLIP package. The quality of the modeled structures was assessed by construction of Ramachandran plots using Procheck (Laskowski et al. 1996
). Protein structures were obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Databank (Berman et al. 2000
; http://www.rcsb.org/pdb/).
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Results and Discussion |
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MP Analysis of the Tick-Derived BPTI-Inhibitors
MP analysis using the alignment obtained after gapped positions were removed grouped BPTI-inhibitors derived from insect hemolymph and hard ticks into a monophyletic clade, whereas the soft tick antihemostatic inhibitors were grouped into their own monophyletic clade (fig. 4A
). The same unresolved relationship between the soft tickderived inhibitors as with NJ was observed. In this case, fXaI and NTI are grouped together, whereas CTI is closer to PAIs. The gapped positions observed in the alignment, especially the insertions before the first ß-sheet and C-terminal -helix could be information-rich in terms of structural and functional constraints of these inhibitors. MP analysis using this information gave a more informative relationship between the tick-derived inhibitors (fig. 4B
). CTI is basal to this clade, followed by NTI, whereas fXaI and PAIs group as the terminal clade. This indicates that at least three separate paralogous gene duplication events had occurred, with the evolution of platelet aggregation inhibitory activity being one of the last events.
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Evolution of Tick BPTI-Proteins: A Paradox Resolved
It has been suggested that the only way by which new protein functions could evolve from duplicated genes is by way of gene sharing of a single-domain protein or from an existing bifunctional multidomain protein, where duplication of each domain leads to acquisition of individual functions (Hughes 1994
). In the light of this observation, a probable evolutionary scenario based on considerations of thrombin's structure and function is shown in figure 6
.
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The BPTI-Fold as Evolutionary Unit
It is not surprising that soft ticks have used the BPTI-like fold to evolve new protein functions. The BPTI-fold per se is not novel to serine protease inhibitors. A rather large group of these proteins is found in snake venoms, where they act as toxins by blocking ion channels of the cardiac and nervous systems (Pritchard and Dufton 1999
). Furthermore, the substrate-presenting loop of the BPTI-like fold is the ideal place for the presentation of recognition motifs, as evidenced by the RGD-containing proteins. What is perhaps surprising is the absence of an RGD-motif in the snake venom BPTI-like proteins, where the disintegrin protein family exploits this motif (Huang 1998
). This might imply that in the case of snake venoms, the disintegrins were already present when the BPTI-like proteins acquired their respective functions. In ticks, such a disintegrinlike protein might have been absent, forcing the tick to rely on protein families already present in its repertoire, to generate functional diversity. The loss of a restricted canonical substrate-binding loop conformation probably further allowed utilization of this loop for a novel presentation mechanism, although the utilization of the fibrinogen-mimicking binding site of CTI could have been important for targeting to the relevant binding site.
Independent Evolution of Antihemostatic Inhibitors in Hard and Soft Ticks
It is interesting that BPTI-inhibitors from the hard tick B. microplus are not grouped with the antihemostatic factors of soft ticks, but rather with hemolymph derived protease inhibitors that inhibit their respective enzymes by the classical BPTI mechanism. fXa and thrombin inhibitors from hard ticks have also been described with molecular masses (1765 kDa) that differ significantly from that of the BPTI-fold (Bowman et al. 1997
). Furthermore, variabilin, a PAI from the hard tick Dermacentor variabilis, does not resemble the PAIs from soft ticks (Wang et al. 1996
). Its cysteine pattern as well as the localization of its RGD-motif differs completely from the observed BPTI-fold and the motif found in savignygrin. This suggests that soft tickderived BPTI-inhibitors only acquired their specific mechanisms of action after the divergence of hard and soft ticks, indicating independent adaptation to a blood-feeding environment by hard and soft ticks. This is of interest because it would have been expected that ticks, being monophyletic, would have adapted to a blood-feeding environment before divergence. It also raises the question whether antihemostatic functions observed in the genus Ornithodoros are represented in other soft tick families. Other signs of independent adaptation of hard and soft ticks to a blood-feeding environment include different salivary gland morphologies, feeding behavior, and reproductive strategies that are all intimately linked with blood-feeding (Sonenshine 1991
). But although this study suggests independent acquisition of novel antihemostatic components by the two main tick families, the presence of apyrase in both families has been indicated (Law, Ribeiro, and Wells 1992
). Apyrase, (ATP-diphosphohydrolase; EC 3.6.1.5) inhibits platelet aggregation induced by ADP and is also able to disaggregate platelets aggregated by ADP (Mans et al. 1998
, 2000
). It has been identified in all the hematophagous arthropod families investigated so far (Ribeiro 1995
), which suggests that this enzyme might have been present in the ancestral nonhematophagous tick. The absence of apyrase in the saliva of the tick Amblyomma americanum (Bowman et al. 1997
) is worth noting.
The Driving Force Behind Tick Divergence
Independent adaptation to a blood-feeding environment indicates a rapid divergence soon after the origin of the Ixodida (120 MYA), with ticks being adapted to a blood-feeding life by 92 MYA. The question that is raised is, What could have triggered such a rapid diversification into different tick families? A current controversy exists around the divergence of early birds and placental mammals, where the fossil records argue for divergence around the Cretaceous-Tertiary (K-T) boundary (65 MYA), whereas molecular phylogeny evidence suggests a much earlier divergence in the Early Cretaceous (12080 MYA; Benton 1999
; Easteal 1999
; Madsen et al. 2001
; Murphy et al. 2001
). A rapid divergence and independent acquisition of hematophagous mechanisms in ticks from about 120 to 90 MYA fits with the molecular phylogeny hypothesis. Radiation of birds as well as placental mammals would have provided ample opportunity for ticks to find novel niches in which they could excel as blood-feeding arthropods. It could thus be argued that the emergence of hematophagy in ticks was triggered by the divergence of early birds and mammals. This provides an interesting counterpoint to suggestions that evolution of ticks was not so much influenced by host-specificity as by ecological factors (Klompen et al. 1996
). Although this might be the case for adaptation to the environment, independent evolution of antihemostatic strategies would suggest that host diversity could have influenced the adaptation of ticks to the vertebrate hemostatic system. Positive Darwinian selection indicates that the hemostatic system of the host played a decisive role in the evolution of hematophagy in ticks.
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Conclusions |
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Acknowledgements |
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Footnotes |
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Keywords: anticoagulant
evolution
platelet aggregation inhibitor
tick
Address for correspondence and reprints: Albert W. H. Neitz, Department of Biochemistry, University of Pretoria, Pretoria 0002, South Africa. albert.neitz{at}bioagric.up.ac.za
.
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