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r Functional perspectives on the evolution of argasid tick salivary gland protein superfamilies Functional perspectives on the evolution of argasid tick salivary gland protein superfamilies by Barend Johannes Mans Submitted in fulfillment of the degree Philosophiae Dor;tor Department of Biochemistry Faculty of Natural and Agricultural Sciences U ni versity of Pretoria June 2002 ©University of Pretoria CONTENTS Chapter1: Evolution of hematophagy in ticks 1.1.1 Ticks as a relevant problem 1.1.2 Tick control 1. 1.3 Concealed antigens as vaccine targets 1.1 .4 Exposed antigens as vaccine targets 1 2 3 4 1.2.1 Evolution of hematophagy 1.2 .2 The Ixodida or ticks 1.2. 3 The origin of ticks 1.2.4 Tick fossil records 1.2.5 Speciation of ticks 1.2.6 Ticks were free-living scavengers 1.2.7 Questions regarding tick evolution 8 8 9 1.3.1 Tick-host interactions lJ.2 Platelet aggregation and its role in hemostasis 1.3.3 Co llagen and its role in hemostasis 1.3.4 Platelet acti vation 1.3.5 Inhibition of adeny late cyclase 1.3.6 The cyclo-oxygenase pathway 1.3.7 The phospholipase C pathway 1. 3.8 Secretion of granul e components 1.3.9 Blood coagulation 9 10 10 1I II 11 12 13. l3 1.4.1 Anti-hemostatic components produced by ticks 1.4.2 Immunomodul atory components in tick sa li va 1.4.3 Blood clotting inhibitors from ti cks 1.4.4 Platelet aggregation inhibitors from ticks 14 15 15 16 1.5.1 Argasidae as models of a primitive ancestor 1.5.2 Ornithodoros savignyi as a model of argasid biology 1.5.3 The relationship between O. savignyi and 0. moubma 1.5.4 Distribution of O. savignyi 1.5.5 Co ntrol of O. savignyi 1.5.6 Bio-active compo nents from O. savignyi 17 18 1.6.1 Molecular evolution as mechanism of adaptation to a blood-feeding environment 1.6.2 Gene duplicati on and the acquis ition of no vel protein function 1.6.3 Orthol ogous genes 1.6.4 Paralogous genes 1.6.5 Alleles 5 6 7 8 19 20 20 20 21 22 23 24 24 1.6.6 Homology and analogy 1.6.7 Detecting homology 1.6.8 Detecting homology by immunological means 1.6.9 Detecting homology by database search 1.6.10 Detecting homology with multiple sequence alignment and ph ylogenetic analysis 1.6.1 1 S tructure as means of detecting homology 25 25 26 26 27 27 I.7 Ai ms of thesis 28 Part 1: Evolution of soft-tick anti-hemostatic factors of the BPTHike superfamily 29 Chapter 2: Characterization of savignygrin, a platelet aggregation inhibitor from the soft tick Ornithodoros savignyi 30 2.1.1 Introduction: Integrins 30 2. 1.2 allb~3 as model integrin 30 2.1.3 Snake venom disi ntegrins as platelet aggregation inhibitors 33 34 ' 2.1.4 Tick-derived allb~3 antagoni sts 2.2 Materials and methqds 2.2.1 Chemicals used 2.2.2 Collection of ticks and preparation of tick sali vary gland extract 2.2.3 Measuring platel et aggregation using an aggregometer 2.2.4 Disaggregation of aggregated platelets by sav ignygrin 2.2.5 Electron microscopical analysis of platelet disaggregation by savignygrin 2.;2.6 Platelet aggregation assay usi ng a mJcro-plate fTlethod 2.2.7 Inhibition of binding between monoclonal antibody P2 (a-CD41 ) and platelets by savignygrin 2.2.8 Inhibition of allb~3 binding to immobilized fibrinogen 2.2 .9 Inhibition of osteosarcoma ce ll adhes ion by sav ignygrin 2.2.10 Inhibition of platelet adhesion to fibrinogen 2.2.1 1 Temperature stabi lity of the sav ignyg rins 2.2.12 High performance liquid chro matography 2.2.13 Size exclusion HPLC 2.2.14 Anion exchange HPLC 2.2. 15 Reversed phase HPLC 2.2. 16 Quantitation of proteins 2.2.17 Amino acid analysis 2.2.18 Total cysteine determination by performic acid oxidation 2.2.19 Alkylation of cysteines with 4-vinyl pyridine 2.2.20 Tryptophan determination by methane sulfonic acid (MSA) hydrolysis 2.2.21 N-termin al sequence analysis 2.2.22 Tricine sodium dodecyl su lphate pol yacrylamide ge l electrophores is 2.2.23 Staini ng of SDS-PAGE gels 2.2.24 Tricine SDS-PAGE in the presence of urea 2.2.25 Electrospray mass spectrometry 11 34 34 35 35 36 36 36 37 37 38 38 39 39 39 40 40 41 41 42 42 42 43 43 43 44 44 2.2.26 Peptide mapping using MALDI-TOF-MS 2.2.27 Origins of (+) and (-) forms: alleles or gene duplicates 2.2.28 Total RNA purification 2.2.29 cDNA synthesis from total RNA 2.2.30 Rapid amplification of 3' cDNA ends (3'RACE) 2.2.31 Rapid amplification of 5' cDNA ends (5'RACE) 2.2.32 Cloning of low-molecular mass sav ignygrin inhibitor 2.2.33 Analysis of PCR products 2.2.34 Quantitation of DNA 2.2.35 Elution of RACE products from agarose gels 2.2.36 Purification of PCR products 2.2.37 AfT cloning of RACE products into pOEM T Easy vector 2.2.38 Preparation of competent cells 2.2.39 Transformation of SURE E. coli cells 2.2.40 Screening for recombinant clones 2.2.41 Miniprep of plasmids 2.2.42 High pure plasmid isolation 2.2.43 Sequencing of recombinant pOEM T Easy plasmids 2.2.44 Primers used during study 44 45 45 45 46 47 48 49 49 49 49 50 50 50 51 51 52 52 · 54 2.3 Results 2.3.1 Purification of the savignygrins 2.3.2 Tricine SDS-PAOE analysis of the savignygrins 2.3.3 Electrospray mass spectrometry of the savignygrins 2.3.4 Peptide mass fingerprinting of the savignygrins 2.3.5 Amino acid analysis of the savignygrins 2.3.6 Determination of disulphide content of the savignygrins 2.3.7 Disulphide bonds and their relevance for activity 2.3.8 Biological activity of the savignygrins 2.3.9 The influence of savignygrin on platelet shape change 2.3.10 Targeting of allb~3 by savignygrin 2.3.1 1 Integrin specificity of savignygrin 2.3.12 N-terminal amino acid sequence determination of the savignygrins 2.3.13 Isolation of total RNA 2.3.14 Cloning and sequencing of savignygrin 2.3.15 Analysis of the recombinant amino acid sequence of savignygrin 2.3.16 Comparison of data obtained from the deduced amino acid sequence and data from native savignygrin 2.3.17 Identity of savignygrin with disagregin 2.3.18 Origins of (+)/(-) forms : alleles or gene duplication? 55 55 57 59 59 60 61 62 62 63 64 67 67 68 69 69 71 72 72 2.4 Discussion 74 2.4.1 Savignygrin (+)/(-) isoforms: conformational and genetic origins 74 2.4.2 Conformational and stability differences observed between the (+)/(-) forms 74 2.4.3 Electrophoretic behaviour of the savignygrins 75 2.4.4 Biological activity of savignygrins 75 III 2.4.5 The RGD motif of the savignygrins 2.4.6 Secretion of savignygrin during feeding 2.4. 7 Independent adaptation of hard and soft ticks to a blood feeding environment 76 77 77 2.5 Summary 78 Chapter 3: Homology modeling of savignin, a thrombin inhibitor from the tick' Ornithodoros savignyi 3.1.1 Introduction: The mechanism of serine protease activity 3.1.2 Specificity of serine proteases 3.1.3 Seri ne protease zymogens and inhibitors 3. 1.4 The BPTI IKunitz family of serine protease inhibitors 3.1.5 Tick-derived serine protease inhibitors of fXa and thrombin 3.1.6 The TAP-fXa complex 3.1.7 The ornithodOlin-thrombin complex 79 79 80 81 82 82 · 83 84 3.2 Materials and metIiods 3.2.1 Cloning, sequencing and sequence analysis of sav ignin 3.2.2 Molecu lar modeling of savignin 86 · 86 86 3.3 Results 3.3.1 RACE and sequencing of savignin 3.3.2 Analysis of the recombinant amino acid sequence of savignin 3.3.3 Comparison of the recombinant sequence data with data from native savlgrun . 3.3.4 Sequence alignment of savignin with omithodorin 3.3.5 Homology modeling of savignin 3.3.6 Interaction of savignin with thrombin 3.3.7 Quality assessment of the modeled structure of savignin 3.3.8 Unusual confonnation of savignin and thrombin 87 87 87 3.4 Discussion 3.4. 1 Interaction of savignin with the thrombin active-site 3.4.2 Interaction of savignin with thrombin's fibrinogen recognitio n site 3.4.3 Unusual confonnation of complexed savignin and thrombin 3.4.4 Kinetic mechanism of thrombin inhibition 98 98 99 99 100 3.5 Summary 101 Chapter 4: Evolution of soft tick KunitzlBPTI anti-hemostatic components 4.1.1 Introduction: The KunitzlBPTI protein family 4.1.2 ~-b u ngarotoxins 4.1.3 Inter alpha-trypsin inhibitor 4.1.4 Sea anemo ne Kunitz inhibitors 4.1.5 Alzheimer's ameloid Kunitz domain 102 102 102 102 104 104 IV 88 89 90 92 95 96 4.1.6 Tissue factor pathway inhibitor 4.1.7 BPTI 4. 1. 8 Colostrum BPTI-inhibitors 4.1.9 Snake venom Kunitz inhibitors 4. 1.10 Arthropod-derived KUllitz inhibitors 4.1.11 Tick-derived Kunitz inhibitors 104 104 104 104 105 105 4.2 Materials and methods 4.2.1 Protein fo ld prediction of platelet aggregation inh ibitors 4.2.2 Retrieval of BPT sequences 4.2.3 Mu lti ple sequence alignment 4.2.4 Neighbor-Joining Analysis of the BPTI-Family 4.2.5 Maximum Parsimony Analysis of Tick derived BPTI-Inhibitors 4.2.6 Phylogeny of Soft Tick Inhibitors based on Protein Structure 4.2.7 Assay for serine protease inhibitory activity 105 105 105 107 107 107 108 109 4.3 Results 4.3.1 Protein Fold Prediction for the Platelet Aggregation Inhibitors 4.3.2 Multiple alignment of savignygrin with BPTI inhibitors 4.3.3 Identity and similarity within ortholog groups of the BPTI-family 4.3.4 Ne ighbor-joini ng analysis of the BPTI-family 4.3.5 Maximum parsimony analys is of the tick-derived BPTI-inhibitors 4.3.6 Structu ral comparison of the soft tick-derived BPTI-like inhibitors 4.3.7 Homology modeling of savignygrin 4.3.8 Analysis of modeled structure 4.3.9 Structu ral impl icatio ns for savignygrin 4.3.10 Serine protease inhibitory activity 109 109 110 112 11 3 115 116 117 118 120 121 4.4 Discussion 4.4.1 A novel BPTI-platelet aggregation inhibitor 4.4.2 Implications of the BPTI-fold fo r the structure of savignygrin 4.4.3 Sav ignin is a highly conserved protein: imp lications for structu re 4.4.4 Soft tick BPTI pro teins: a functiona l paradox 4.4.5 Evolu tion of tick BPTI-proteins: a paradox reso lved 4.4.6 The BPTI-Fold as Evolutionary Unit 4.4.7 Independent evolution of anti-hemostatic inhjb itors in hard and soft ticks 4.4.8 T he driv ing fo rce behi nd tick divergence 4.4.9 Implications for pharmacological and vaccine development 122 122 122 122 123 123 125 127 128 128 Part 2: Evolution of the tick lipocalin superfamily 129 Chapter5: Localization of anti-hemostatic factors to specific salivary gland granules 130 5.1.1 Introduction: Tick salivary glands 130 13 1 5.1.2 Agranular ac ini 5. 1.3 Granular ac ini 13 1 131 5.1.4 Argasid granu lar acini structure v 5.1.5 Biological significance of sal ivary gland proteins during feeding 5.1.6 Evolutionary significance of tick salivary glands 5.1.7 Salivary glands of other arthropods 5.1.8 Localization of components to granule types 132 133 133 134 5.2 Materials and methods 5.2.1 Assay for apyrase activity 5.2.2 Purification of apyrase by HPLC 5.2.3 Preparation of anti-sera 5.2.4 Western-blot analysis of SGE 5.2.5 Secretion of sali vary gland components by various stimulants 5.2.6 Preparation of tick sa li vary glands for immuno-Iocali zatio n 5.2.7 Immuno-cytochemicallabeling of thin sections 5.2.8 Immuno-fluorescent localization of savignygrin, using confocal rrucroscopy 5.2.9 Immuno-fluorescent localization of microtubules using confocal rrucroscopy 5.2.10 Carbohydrate and membrane staining of salivary glands using the Thiery test 5.2.11 Scanning electr!? n microscopy of the salivary gland membrane system 5.2.12 Sub-cellular fractionation of salivary gland granules using co Llagenase 134 134 135 135 135 136 137 137 5.3 Results 5.3.1 Purification of apyrase and generation of spec ific polyclonal anti-sera 5.3.2 Morphologi cal studies on the salivary glands of O. savignyi 5.3.3 Ultrastructural studies of the salivary glands 5.3.4 Morphology of salivary glands from fed ticks 5.3.5 Morphology of salivary glands several days after feeding 5.3.6 Secretion of salivary gland components induced by various stimulants 5.3.7 Secretion efficiency of salivary glands during feeding 5.3.8 Immunolocalizalion of apyrase and savignygrin on light microscope level 5.3.9 Co-localization of apyrase and savignygrin at the ultrastructural le vel 5.3.10 Immuno-fluorescent localization of savignygrin to sali vary gland granules 5.3.11 Absence of membrane vesic les involved in granu le forma tion in mature cells 5.3.12 SEM investigation into membrane systems of salivary glands 5.3.13 Sub-cellular fractionation of salivary gland granules after collagenase treatment 5.3.14 Immuno-I ocali zation of Cl.-tubulin to tick salivary glands 5.4 Discussion 5.4.1 Importance of the salivary glands in secretion 5.4.2 Secretory mechanisms of argasid tick salivary glands 5.4.3 Localization of apyrase and sav ignygrin 5.4.4 Paradoxes between light, TEM and confocal microscopy 5.4.5 Thiery staining of salivary glands VI 138 139 139 139 140 141 141 142 143 144 145 146 147 148 ISO J 5'? 153 154 155 156 15 8 158 158 159 160 160 5.4.6 ER and Golgi ap paratu s in the sali vary glands 5.4.7 Relationship between ER, Golgi apparatus and secretory granules 161 16 1 5.5 Conclusions derived from this study 162 5.6 Salivary glands and granule formation 162 Chapter 6: Major salivary gland proteins from the tick O. savignyifunction as toxic and granule biogenic proteins 164 6.1 Part 1: the secretory pathway 6.1.1 Targetin g of secretory proteins to the secretory pathway 6. 1.2 Targetin g to the ER and Golgi-apparatus 6. 1.3 The constitutive and regulated secrelion palhway 6. 1.4 "Sorting fo r entry" or "sorting by retenti on" 6.1.5 Aggregation as means of sorting 6. 1.6 Granule biogenes is in tick salivary glands 6. 1.7 Granule biogenesis and tick toxicoses 164 164 164 165 166 166 166 167 6.2 Part 2: Tick toxi ns 6.2. 1 Tick toxicoses 6.2.2 Toxicoses from an evo lutionary perspective 6.2.3 Tick paralysis 6.2.4 Pathogenic mecha nisms oftick paralysis toxins 6.2.5 Dermacentor andersoni 6.2.6 Ixodes holocyclus 6.2 .7 Ripicephalus evertsi evertsi 6.2.8 Argas walke rae 6.2.9 Common ancestors for paralysis toxins 6.2. 10 Other fo rms of toxicoses considered as non-paralytic 6.2. 11 Toxins from tick eggs 6.2. 12 Sweating sickness caused by Hyalomma truncatum 6.2.13 Sand tampan toxicoses 6.2. 14 Pathogenesis of sand tampan toxicoses 167 167 168 168 169 169 170 6.3 Materials and methods 6.3.1 Two-di mensional polyacrylamide gel electrophoresis 6.3.2 Isolation of salivary gland granu les after dispase treatment 6.3.3 Electron microscopy and X-ray microanalysis of granules 6.3.4 Reconstituting the conditions present in the rrans-Golgi network (TGN) 6.3.5 Purification of tick sali vary gland proteins (TSGPs) 6.3.6 Characterization of TSGPs 6.3.7 The effect of SGE on an isolated rat heart perfusion system 6.3 .8 The effect of toxic fractions on mouse ECG patterns 176 176 176 176 177 177 178 178 178 6.4 Results 6.4.1 Identification of major salivary gland proteins 179 179 Vll l70 171 172 172 173 173 174 174 6.4.2 Purification of dense core granules 180 6.4.3 Aggregation of proteins in acidic medium with high calcium concentration 181 6.4.4 A comparison of the toxicity of SGS and SGE 182 6.4.5 Investigation into the possible neurotoxicity of SGE 183 184 6.4.6 The effect of SGE on an isolated rat heart perfusion system 6.4.7 Purification of TSGPs and toxins 185 185 6.4.8 Fractionation of toxic activities 6.4.9 The effect of different toxins on the cardiovascular system 188 6.4.10 Desalting ofTSGPs 191 6.4.11 Molecular mass analys is of TSGPs 192 6.4.12 Peptide mass fingerprinting of TSGPs 194 195 6.4.13 Amino acid analysis ofTSGPs 6.4.14 N-terminal amino acid analysis of the TSGPs 196 197 6.4.15 Specificity of polyclonal anti-sera for TSGPs 6.4.16 Localization of TSGPs to sa livary gland granules 197 199 6.4.17 Protein co ncentration and its influence on granule biogenesis 6.4.1 8 Molecular crowding in the cell 199 6.4.19 TSGPs as densely packed spheres 200 6.5 Discussion , 6.5.1 Granule biogenesis in tick sa livary glands 6.5.2 Evidence ofTSGPs being granule biogenic protein's 6.5.3 Toxins from 0. savignyi 6.5.4 Non-paralytic nature of sand tampan toxicoses 6.5.5 Cardio-pathogenic properties of sand tampan toxin 6.5.6 Toxicity of TSGP2 versus the non-toxicity ofTSGP3 6.5.7 Biological functionality of toxins 203 203 203 204 205 206 207 207 Chapter 7: The major tick salivary gland proteins are part of the tick lipocalin family 7.1.1 Introduction: The lipocalin protein superfamily 7.1.2 Lipocalin function 7.1.3 Lipocalin three-dimensional structure 7.1.4 Conserved motifs of the lipocalins 7.1.5 Evolution of the lipocal ins 7.1.6 Lipocalins from hematophagous organisms 209 209 209 209 21 1 212 213 7.2 Materials and methods 7.2.1 Cloning and sequencing of TSGPs 7.2.2 Multiple alignment of lipocalins 7.2.3 Phylogenetic analysis of lipocalins 7.2.4 Molecular modeling of lipocalins 7.2.5 Partial purification of savignygen 214 214 215 216 216 216 7.3 Results 7.3.1 RACE of the TSGP's 7.3.2 Amino acid sequences of the TSGPs 217 217 217 Vlll 7.3 .3 Comparison of data from native toxins and deduced amino acid sequences 7.3.4 Multiple alignment of tick lipocalins 7.3.5 Phylogenetic analysis of tick derived lipocalins in relation to the lipocalin family 7.3.6 Absence of TSGP2-4 in SGE from O. moubata 7.3 .7 Molecular modeling of the TSGPs 7.3.8 Analysis of the TSGP structures 7.4.9 Implications of the TSGP structures 7.4.10 Are TSGP2 and TSGP3 platelet aggregation inhibitors? 224 231 233 234 236 237 7.5 Discussion 7.5.1 Evolution of tick lipocalins 7.5.2 TSGP 1 7.5.3 TSGP2 and TSGP3 7.5.4 Toxicity vs. non-toxicity: TSGP2 and TSGP3 7.5.5 TSGP4 7.5.6 Evolution of tick lipocalins and its implication for tick toxicoses 7.5.7 Tick Jipocalins as toxins . 7.5.8 Structure of TSGPs 7.5.9 Implications of IT}olecular crowding for the evolution of tick lipocalins 7.5.10 The tick lipocalin family 239 239 239 240 240 240 241 241 241 243 244 Chapter 8: Conclud ing discussion 245 Summary 256 Opsomming 258 References 260 \ IX 222 223 List of Figures Fig. 1.1: Hierarch y of the Ixodida Fig. 1.2: Platelet activation mechanisms Fig. 1.3: The blood coagulation cascade Fig. 1.4: External anatomy of a female O. savignyi 7 12 14 19 Fig. 2.1: Activation dependent confonnational change of aUb~3 Fig. 2.2: Three-dimensional structures of aIlb~3 antagonists Fig. 2.3: Amino acid sequences of different a llb~3a ntagoni s ts Fig. 2.4: Cloning strategy to obtain ORF and 3'UTR Fig. 2.5: Cloning strategy to obtain S'UTR and ORF Fig. 2.6: Purification of savignygrin Fig. 2.7: Electrophoretic analysis of the savignygrin s Fig. 2.8: Electrospray mass spectrometry analysis of the savignygrins Fig. 2.9: Peptide mass fingerprint analysis of thesavignygrins Fig. 2.10: Amino acid analysis of savignygrins Fig. 2.11: Determination of the disu lphide content of the savignygrins Fig. 2.12: The effect of disulphide bonds on activity Fig. 2.13: Inhibition of platelet aggregation by savignygrin Fig. 2.14: The disaggregation effect of savignygrin on aggregated platelets Fig. 2.15: Targeting of aIlb~3 by savignygrin Fig. 2.16: Integrin specificity of savignygrins Fig. 2.17: N-terminal sequence analysis of savignygrin Fig. 2.18: Electrophoretic analysis of total RNA Fig. 2.19: RACE of the high and low mass forms of savignygri n Fig. 2.20: Full-length cDNA sequence for savignygcin Fig. 2.21: Comparison of native savignygrin and the deduced sequence Fig. 2.22: Alignment of sav ignygrin with disagregin Fig. 2.23: Analysis of savignygrin isofonns in twenty individuals Fig. 2.24: The relatio nship between the different isoforms of sav ignygrin 31 32 33 47 48 56 58 59 60 61 61. 62 63 6S 66 67 68 68 69 70 71 72 73 78 Fig. 3.1: Mechanism of action of serine proteases Fig. 3.2: The catalytic triad of the serine proteases Fig. 3.3: Schechter-Berger notation for binding sites in the binding pocket Fig. 3.4: BPT! -Kunitz inhibitor structure Fig. 3.5: The fXa-TAP complex Fig. 3.6: Alignment of fXa! and TAP Fig. 3.7: The ornithodorin-thrombin complex Fig. 3.8: RACE of savignin Fig. 3.9: cDNA sequence and deduced protein sequence of savign in Fig. 3.10: Amino acid composition of native and recombinant savignin Fig. 3.11: cDNA sequence alignment of sav ignin with ornithodorin Fig. 3.12: Protein sequence alignment of savignin with ornithodorin Fig. 3.13: Modeled structure of savignin Fig. 3.14: Interaction of savignin with thrombin Fig. 3.15 : Ramachandran plot of modeled structure of savignin 79 80 81 82 84 84 85 87 88 88 90 90 91 93 95 x Fig. 3.16: Relationship between molecular mass and volume of various proteins Fig. 3.17: A two-step mechanism for savignin binding to thrombin 97 Fig. 4.1: UPGMA dendrogram from 74 KunitzlBPTI-homologous sequences Fig. 4.2: Multiple sequence alignment of BPTI inhibitors with the sequences of disagregin and savignygrin included Fig. 4.3: Percent identity and similarity observed between orthologs of the different functional classes from the BPTI-family Fig. 4.4: A Neighbor-Joining dendrogram of 62 BPTI sequences Fig. 4.5 : Maximum parsimony analysis of tick BP'r1 inhibitors Fig. 4.6: A phylogenetic tree based on pairwise distances of root mean square deviation values (RMSD) obtained from comparison of different structural models Fig. 4.7: Comparison of the PAl structural models with the fXa inhibitors Fig. 4.8: Ramachandran plot of the modeled structure of savignygrin Fig. 4.9: The structure of savignygrin Fig. 4.10: Evolutionary mechanisms for the acquisition of new anti-hemostatic functions in soft ticks . 103 to 1 III 112 114 115 117 118 119 121 126 130 Fig. 5.1: Salivary glands from the soft tick 0. savignyi Fig. 5.2: A schematic representation of the granular acini of the argasid tick, Argas perslcus 132 Fig. 5.3: Purification of apyrase and specificity of anti-sera 142 Fig. 5.4: Scanning electron microscopy analysis of salivary glands from O. savignyi 143 Fig. 5.5: Sections though a salivary gland cell of an unfed tick 144 Fig. 5.6: Salivary glands from ticks dissected 10 miilUtes after feeding to engorgement on a rabbit 145 Fig. 5.7: Salivary glands from ticks at different time periods after feeding 146 147 Fig. 5.8: Secretion of apyrase from exposed acini induced by dopamine Fig. 5.9: A comparison between salivary glands of unfed and fed ticks 148 149 Fig. 5.10: lmmunolocalization of apyrase and savignygrin Fig. 5.11: lmmunolocalization at a higher magnification ISO Fig. 5.12: Co- localization of apyrase and savignygrin using immuno-gold labeling IS I Fig. 5.13: Granular cells that did not show significant labeling 152 Fig. 5.14: Localization of savignygrin to a specific granule cell type with immunofluorescence confocal microscopy 153 Fig. 5.15: Thiery stain of salivary glands 154 Fig. 5.16: SEM analysis of membrane systems of the salivary glands after osmium maceration ISS Fig. 5.17: Sub-cellular fractionation of salivary gland granules using velocity sedimentatio n after collagenase treatment 156 F ig. 5.18: Localization of u-tubulin to salivary gland granules 157 Xl Fig. 6.1: The proteome of tick salivary gland extract Fig. 6.2: Purification of dense core granules Fig. 6.3: Reconstitution of in vitro conditions present in the TGN Fig. 6.4: Analysis of temperature inactivated SGE Fig. 6.5: The effect of SGE on a perfused rat heart system Fig. 6.6: Ion exchange HPLC of tick salivary gland extract Fig. 6.7: Fractionation of toxic fractions from AEHPLC Fig. 6.8. HIHPLC of acidic TSGPs Fig. 6.9: The effect of TSGP4 on the mouse ECG patterns Fig. 6.10: The effect of TSGP2 on the mouse ECG patterns Fig. 6.11: RPHPLC ofTSGPs Fig. 6.12. Reducing tricine SDS-PAGE analysis of purified TSGPs Fig. 6.13. MALDI-TOF-MS of TSGPs Fig. 6.14: ESMS spectra obtained for the TSGPs Fig. 6.15: Peptide mass fingerprints of TSGPs Fig. 6.16: Amino acid analysis of the TSGPs Fig. 6.17: N-terminal sequence analysis of the TSGPs Fig. 6.18: Specificity of polyclonal anti-sera raised against different TSGPs Fig. 6.19: Localization of the TSGPs to different salivary gland granules Fig. 6.20: The principly of volume exclusion Fig. 6.21: Precipitation of the TSGPs in the presence of increasing concentrations of dextran 179 180 182 184 184 186 187 188 189 191 192 193 193 194 195 196 196 197 198 200 202 Fig. 7.1: The structure of the lipocalin fold Fig. 7.2: An unwound view of the lipocalin structure Fig. 7.3: A phylogenetic tree of the lipocalins Fig. 7.4: RACE of the TSGPs. Fig. 7.5: Full-length sequence ofTSGPI Fig. 7.6: Full-length sequence of TSGP2 and TSGP3 Fig. 7.7: Full-length sequence of TSGP4 Fig. 7.8: Multiple sequence alignment of the tick lipocalins Fig. 7.9: Alignment of the lipocalin family used for phylogenetic analysis Fig. 7.10: Phylogenetic analysis of the lipocalins family Fig. 7.11: A comparison of the SGE proteomes from O. savignyi and O. moubata Fig. 7.12: Western blot analysis of TSGPs in SGE of O. moubata and O. savignyi Fig. 7.13: Structural models obtained for the TSGPs fitted onto the structure 210 211 213 217 219 220 221 224 225 230 ~~~ 234 235 236 Fig. 7.14: Ramachandran plots for the TSGPs Fig. 7.15: The structures of the TSGPs and their intact disulphide bonds Fig. 7.16: Identification of savignygen, an inhibitor specific for collagen-induced platelet aggregation Fig. 7.17: The proposed disulphide bond pattern of the tick toxins Xll 231 233 238 243 List of Tabl es Table 1.1: Arthropods that evolved hematophagy independently Table 1.2: Biologically active compounds secreted by activated platelets Table 1.3: Anticoagulants from ticks Table 1.4: Platelet aggregation inhibitors from ticks Table Table Table Table 2.1: Flow conditions used during AEHPLC 2.2: Flow conditions used during RPHPLC 2.3: HPLC gradient conditions for amino acid analysis 2.4: Primers used in study 6 13 16 17 40 40 42 54 Table 3.1: Experimental parameters for native savignin compared with the values calculated from the recombinant sequence 89 Table 3.2: Residues of omithodorin and savignin that interacts with thrombin as predicted by Ligplot 94 Table 3.3: Statistics of ramachandran plots of sav ignin and omithodorin 96 Table 3.4: Proteins used for determination of the relationship between molecular mass and volume . 97 . Table 4.1: Ramachandran analysis values for fXaI and savignygrin Table 4.2: Inhibition of se rine protease activity 11 9 121 Table 6.1 : The effect of dilution on toxicity of SGE and SGS 183 Table. 7.1: Comparison of the amino acid composition from the TSGPs and their deduced amino acid seq uences 222 Table 7 .2: A comparison of a theoretical tryptic digest of the TSGPs with peptide mass fingerprints previously obtained by MALDl-TOF-MS 223 XUl Abbreviations AA arac hidonic acid ADP adenosine diphosphate ADS an tibody dilution solutio n AEHPLC anion exchange HPLC ATP adenosine triphosphate BAPNA N - 0.- benzoy 1-L-argi n i ne-4-n itra n iii de BLAST basic local alignment searc h too l BMTI Boophilus microplus tryps in inhi bitor BPTI bovinefbasic pancreatic trypsin inhibitor BSA bov ine serum albumi n CEC cation exchange chromatogrqphy CI collagen specific inhi bitor COX cyclo oxygenase CTI C-terminal domain of the soft tick thrombin inhi bitors DAG diacyl glycerol DAB 3,3' diaminobenzidine DMEM Dulbecco's modified Eagle's med iu m DMSO dimethyl sulfox ide DTT dithiothreitol DEPC diethyl pyrocarbonate EDTA ethylene diamine tetra-acetic acid EGTA ethylene-bis(oxyethylene nitrilo) tetra-acetic acid ELISA enzyme linked immunosorbent assay ER endop lasmic reticulum ESMS electrospray mass spectrometry FAP foca l adhesion poi nt FCS fetal calf serum FXaI fXa inhibitors GdCI guanidinium chloride GSP ge ne specific primer HlHPLC hydrophobic interaction HPLC HPLC high performance liquid chro matography IP, inositol triphosphate IPTG isopropyl ~-D-thiogalactopyranoside ISG immature secretory granule XIV MALDI-TOF-MS matrix assisted laser desorption ionization time of flight mass spectrometry MOPS 3-(N-morpholino)propane sui phonic acid MSA methanesulfonic acid NJ neighbor joining 4-NPGB p-nitrophenyl-p' -guanidinobenzoate NTI N-terminal domain of the soft tick thrombin inhibitors OmGD Ornatin-glycine-aspartic acid . PAF platelet activating factor PAGE poly-acrylamide gel electrophoresis PAl platelet aggregation inhibito rs PBS phosphate buffered saline POB protein databank PG prostaglandin PGO, prostaglandin D2 PGI, prostglandin 12 PKC protein kinase C PLC phospholipase C RGO arginine-glycine-aspartic acid RMSD root mean sq uare deviation RNAse ribonucleic acid hydrolase RPHPLC reversed phase HPLC SCR structural conserved region SCOP structural classification of proteins SO standard deviation SOS sodium dodecyl sulphate SEHPLC size exclusion HPLC SEM scanning electron microscopy SGE salivary gland extract SGS salivary gland secretion TAE Tris-acetate-EDTA TAl tick adhesion inhibitor TCH thiocarbohydrazide TEM transmission electron microscopy TEMED N,N,N' ,N' -tetramethyl-ethylenediamine TFPI tissue factor pathway inhibitor xv TGN trans-Golgi Network TRAP thrombin receptor activating peptide ' Tris tris(hydroxymeth yl) aminomethane Tricine N-[Tris(hydroy methyl) methyl) glycine TSGP tick salivary gland proteins TXA2 thromboxane A2 4-VP 4-vinyl pyridine vWf vo n Willebrandt's factor UPGMA UTR unweighted pair group method with arithmetic mean X-gal 5-bromo-4-chloro-3-indolyl ~- D-galacto-pyranoside untranslated region XV I Acknowledgements Professor Albert Neitz, who started tick research in this department. Without the research that he has done himself or has supervised over several decades, this thesis would not have been possible. Thank you for your never-ending support, the encouragement and freedom you gave me . to pursue whatever avenue I thought worthwhile. For this I will be forever, grateful. Professor Abraham Louw, you were always there to talk to about research and life. Your criticism and ideas improved the quality of my research. Professor Lewis Coons, who took a complete stranger' into his house and showed me the true spirit of America. Your insight into tick biology was truly a revelation and a fountain for the growth 01 new ideas. The use of your EM equipment and confocal microscope was truly appreciated. Prof. Wolf Brandt, for the use of his N-terminal sequencing et1uipment and MALDI-TOF-MS. Prof. Chris Steinmann, for his assistance with electro-cardiology and heart perfusion experiments. Thank you for the keen interest you took in our project. • Dr. Marthinus van der Merwe from the electrospray mass spectrometry unit at the University of Stellenbosch who performed the ESMS analysis. Dr. Fourie Joubert, who introduced me to bio-informatical methods. Mr. Nic Taljaard who performed many, many amino acid analyses during the course of this study, without ever complaining. Without your help, this study would have been much more difficult. Mrs. Sandra van Wyngaardt who have always listened to me and assisted in numerous experiments over the years. XVII Mr. Jacobus Venter, my friend as well as colleague, without whom I would not have been able to perform most of the experiment's that required live animals. . Mrs. Renate Filter, for the superb nucleotide sequencing facility that is maintained at the University of Pretoria. The Laboratory for Microscopy and Microanalysis at the University of Pretoria, for the use of their equipment and their professional expertise in the use of electron microscopical methodologies. To my wife Janet, for your love and understanding, when I was more at the lab than at home. Your support and interest have truly made a difference during this time. My family who were always supportive during this time of study. Thank you for enduring me during this period. The Lord GOD, who created this world through his WORD. For the mysteries that we can investigate, for those precious few moments when we discover HIS divine grace in the molecules we study, for the opportunity to contemplate briefly the wonderful life locked up in nature. XVUl Jumrruu y SUMMARY Thesis title: Functional perspectives on the evolution of argas id lic k sal ivary gland protein superfamilies Student: B.J. Mans Promoters: Prof. A.W.H. Neitz, Prof. A.I. Louw and Dr. A.R.M. Gaspar Department: Biochemistry Degree: Philosophia Doc tor Ticks evolved an obligate, hematophagous lifestyle approximately 120 million years ago while the vertebrate hemostatic system has existed for at least 400 milli on years. Thi s implies that ticks adapted to an established and efficient hemostatic system . Adaptation to a new environment at a molec ular level implies the gain of, new protein. function s. Mechanisms for the acqui sition of new protein functions include gene dupli cati on and subseq uent gain/loss qf protein function. This predicts the presence of multi-gene or protein famili es. The present study in vestigated the adaptation of ticks to a blood-feedi ng environment through the use of such multi-gene families present in the sa li vary gland pro teins of the soft tick Ornithodoros savignyi. In this study, a fami ly of platelet aggregation inhi bitors named savignygrins was characteri zed. These savignygrins fo r which gene duplication was indi cated inhibit platelet aggregation induced by various agonists, disaggregate aggregated plate lets and inhibit the binding of the monoclonal antibody P2 to integrin Cl.Ub~3 and CI.(lb~3 to fibrinogen. This indicates that the savignygrin s target the fibrinogen receptor, wh ich was confirmed by sequence identity to disagregin, a fibrinogen receptor antagonist from the closely related ti ck specie Ornithodoros moubata. Savignygrin, however, differs from disagregin due to the presence of the integrin recognition motif RGD . The thrombin inhibitor savlgmn was cloned and sequenced. Savignin cons ists of two BPTI-Kunitz domains. Homology modeling using the structure of ornilhodorin, a thrombin inhibitor from 0. moubata, shows similar mec hani sms of inhibition. This 256 Summary includes targeting of thrombin 's active site with its N-tenninal BPTI-domain and thrombin's fibrinogen recognition exosite with its C-tenninal domain. Protein fo ld prediction as well as phylogenetic anal ys is indicated that the sav ignygrins share the BPTI-fold with thrombin and fXa inhibitors previpusly described for the Ornithodoros genus. A model of protein evolution for the tick BPTI-inhibitors is proposed that indicates a sequential evo lution of inhibition of the substrate recognition capability of thrombin (targeting of the fibrinogen binding exosite), its catalytic capabi lity (targeting of the active site), the catalytic capability of fXa (similar to that of thrombin) and platelet aggregation. This model acco unts for the different inhibitory mechanisms of the tick anti-coagulants relative to that of the canonical BPTI-family. The unique presentation mode of the RGD motif on the BPTI substrate-presenting loo p of the platelet aggregation inhibitors is also explained. Four highly abundant proteins (TSGPs) of the lipoca lin family were characteri zed. It was proposed that these protein s function during sali vary gland granule biogenesis. TSGP2 and TSGP4 were also identified as toxins that affect the cardi ac system. [n co ntrast to savignygrin and apyrase, which localizes to two specific sali vary gra nule types, the TSGPs localize to all the different granule types identifi ed in the sali vary glands . Localization studies also indi cate that instead of the previously described three granul ar cell types in soft ti ck salivary glands, there are fi ve. Phyl ogenetic analysis of the ti ck lipocalins indica tes a series of gene duplication eve nts and subsequen t gain/loss of protein function. The absence of the toxins in the salivary glands of O. moubata sugges ts that the toxin s as well as the non-toxic TSGP3 might be recent gene dup li cations that occurred after the di vergence of these two tick speci es. 257 Opsomming OPSOMMING Titel van tesis: Functional perspectives on the evolution of argasid tick salivary gland protein superfamilies Promoters: Prof. A.W.H. Neitz, Prof. A.I. Louw en Dr. A.R .M. Gaspar Departement: Biochernie Graad: Phi losophia Doctor Bos luise het 'n verpligte bloedvoedende lewenstyl ongeveer 120 miljoen jaar gelede ontwikkel. Werweldiere se hemostatiese sisteem bestaan al ongeveer 400 miljoen jaar. Bosluise moes dus by 'n bestaande en hoogs ontwikkelde hemostatiese sisteem aanpas. Aanpassing op molekulere vlak impliseer die oiltwikkeling van nuwe proteYen funksie. Meganismes vir die ontwikkeling van nuwe proteien funksie sl uit geen duplikasie en die daaropvlogende wins/verlies van prote"ien funksie in. Dit voorspel die bestaan van multigeen of protei"en families. Die huidige stud ie ondersoek die aanpassing van bosluise by 'n bloedvoedende lewenstyl deur gebruik te maak van multi-geen families leenwoordig in die speekselkl iere van die sagte bosluis Ornithodoros savignyi. 'n Familie van bloedplaatjie aggregasie inhibilore is gekaJakteriseer. Geen duplikasie kon aangetoon word. Bloedplaaljie aggregasie deur verskeie agoniste sowel as binding van die monoklonale teenliggaam P2 aan die integrien allb~) en allb~) aan fibrinogeen is deur savignygrin voorkom. Dit idenlifiseer die fibrinogeen reseptor as teiken van savignygrin. Identiteit aan die fibrinogeen reseptor antagonis disagregien bevestig die hipotese. Savignygrin verskil egter van disagregin a.g.v. die teenwoordigheid van die integrien herkennings motief, RGD. Die trombien inhibitor, savignin se geen volgorde is bepaal deur klonering. Savignin bestaan uit twee Kunitz-BPTI domeine. 'n Struktuur model gegrond op omithodorin , 'n trombien inhibitor van O. moubata, voorspel soortgelyke inhibitor meganismes. Dit sluit die teikening van trombien se aktiewe setel deur die N-terminale BPTI-domein en die fibr inogeen herkennings setel deur die C-terminale BPTI-domein in. 258 Opsomming ProteIen-vou voorspelling en filogenetiese analise dui aan dat savignygrin die BPTI-vou dee I saam met die trombien en fXa inhibitore geidentifiseer in the genus Ornithodoros. 'n Model vir die opeenvolgende onwikkeling van die boslui s BPTI-inhibitor funksie word voorgestel. Dit sluit in die teiken van trombien se substraat herkennings setel, trombien se aktiewe sentrum, fXa se aktiewe setel en bloedplaatjie aggregasie. Die model verklaar die uiteenlopende inhibisie meganismes van the bosluis BPTI-inhibitore ten opsigte van die kanonikale BPTI-inhibitore. Die unieke presentering van die ROD motief op die BPTIsubstraat herkennings Ius word ook verklaar. Vier van die mees volopste speekselklier proteIene (TSOPs) wat deel is van die lipokalien familie is gekarakteriseer. 'n Funhie in die biogenese van speekselkl ier 'granules is voorgestel. TSOP2 en TSOP4 is ook as toksiene aang.edui wat die hart aaotas. In teenstelling met savignyrin en ap irase wat in slegs twee granule tipes gevind word, word die TSOPs in al ,die granule sel tipes gevind. In plaas van die oorspronklike drie granule sel tipes van sagte bosluise, is vyf aangedui. Filogenetiese analise van die lipokaliene dui 'n reeks geen duplikasies aan met dienooreenkomstige wins/verlies van funksie. Die afwesigheid van die toksiene in die speekselklier ekstrakte van O. moubata dui ook aan dat die toksiene sowel as die nie-toksiese TSOP3 geen duplikasies mag wees wat plaasgevind het na die spesiasie van die twee bosluis spesies. 259