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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
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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
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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
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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
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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
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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
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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
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I.7 Ai ms of thesis
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Part 1: Evolution of soft-tick anti-hemostatic factors of the BPTHike superfamily
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Chapter 2: Characterization of savignygrin, a platelet aggregation inhibitor from the
soft tick Ornithodoros savignyi
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2.1.1 Introduction: Integrins
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2. 1.2 allb~3 as model integrin
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2.1.3 Snake venom disi ntegrins as platelet aggregation inhibitors
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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
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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
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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?
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2.4 Discussion
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2.4.1 Savignygrin (+)/(-) isoforms: conformational and genetic origins
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2.4.2 Conformational and stability differences observed between the (+)/(-) forms 74
2.4.3 Electrophoretic behaviour of the savignygrins
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2.4.4 Biological activity of savignygrins
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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
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2.5 Summary
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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
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3.2 Materials and metIiods
3.2.1 Cloning, sequencing and sequence analysis of sav ignin
3.2.2 Molecu lar modeling of savignin
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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
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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
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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
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3.5 Summary
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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
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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
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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
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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
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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
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Part 2: Evolution of the tick lipocalin superfamily
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Chapter5: Localization of anti-hemostatic factors to specific salivary gland granules 130
5.1.1 Introduction: Tick salivary glands
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5.1.2 Agranular ac ini
5. 1.3 Granular ac ini
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5.1.4 Argasid granu lar acini structure
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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
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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
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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
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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
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5.5 Conclusions derived from this study
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5.6 Salivary glands and granule formation
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Chapter 6: Major salivary gland proteins from the tick O. savignyifunction as toxic and
granule biogenic proteins
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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
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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
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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
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6.4 Results
6.4.1 Identification of major salivary gland proteins
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6.4.2 Purification of dense core granules
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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
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6.4.5 Investigation into the possible neurotoxicity of SGE
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6.4.6 The effect of SGE on an isolated rat heart perfusion system
6.4.7 Purification of TSGPs and toxins
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6.4.8 Fractionation of toxic activities
6.4.9 The effect of different toxins on the cardiovascular system
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6.4.10 Desalting ofTSGPs
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6.4.11 Molecular mass analys is of TSGPs
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6.4.12 Peptide mass fingerprinting of TSGPs
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6.4.13 Amino acid analysis ofTSGPs
6.4.14 N-terminal amino acid analysis of the TSGPs
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6.4.15 Specificity of polyclonal anti-sera for TSGPs
6.4.16 Localization of TSGPs to sa livary gland granules
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6.4.17 Protein co ncentration and its influence on granule biogenesis
6.4.1 8 Molecular crowding in the cell
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6.4.19 TSGPs as densely packed spheres
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6.5 Discussion
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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
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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
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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
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7.3 Results
7.3.1 RACE of the TSGP's
7.3.2 Amino acid sequences of the TSGPs
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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?
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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
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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
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Chapter 8: Conclud ing discussion
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Summary
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Opsomming
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References
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IX
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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
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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
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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
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Fig. 3.16: Relationship between molecular mass and volume of various proteins
Fig. 3.17: A two-step mechanism for savignin binding to thrombin
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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
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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
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Fig. 5.3: Purification of apyrase and specificity of anti-sera
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Fig. 5.4: Scanning electron microscopy analysis of salivary glands from
O. savignyi
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Fig. 5.5: Sections though a salivary gland cell of an unfed tick
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Fig. 5.6: Salivary glands from ticks dissected 10 miilUtes after feeding to
engorgement on a rabbit
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Fig. 5.7: Salivary glands from ticks at different time periods after feeding
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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
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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
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192
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196
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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
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230
~~~
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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
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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