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1.  Novel Family of Insect Salivary Inhibitors Blocks Contact Pathway Activation by Binding to Polyphosphate, Heparin, and Dextran Sulfate 
Objective
Polyphosphate and heparin are anionic polymers released by activated mast cells and platelets that are known to stimulate the contact pathway of coagulation. These polymers promote both the autoactivation of factor XII and the assembly of complexes containing factor XI, prekallikrein, and high-molecular-weight kininogen. We are searching for salivary proteins from blood-feeding insects that counteract the effect of procoagulant and proinflammatory factors in the host, including elements of the contact pathway.
Approach and Results
Here, we evaluate the ability of the sand fly salivary proteins, PdSP15a and PdSP15b, to inhibit the contact pathway by disrupting binding of its components to anionic polymers. We attempt to demonstrate binding of the proteins to polyphosphate, heparin, and dextran sulfate. We also evaluate the effect of this binding on contact pathway reactions. We also set out to determine the x-ray crystal structure of PdSP15b and examine the determinants of relevant molecular interactions. Both proteins bind polyphosphate, heparin, and dextran sulfate with high affinity. Through this mechanism they inhibit the autoactivation of factor XII and factor XI, the reciprocal activation of factor XII and prekallikrein, the activation of factor XI by thrombin and factor XIIa, the cleavage of high-molecular-weight kininogen in plasma, and plasma extravasation induced by polyphosphate. The crystal structure of PdSP15b contains an amphipathic helix studded with basic side chains that forms the likely interaction surface.
Conclusions
The results of these studies indicate that the binding of anionic polymers by salivary proteins is used by blood feeders as an antihemostatic/anti-inflammatory mechanism.
doi:10.1161/ATVBAHA.113.302482
PMCID: PMC4191670  PMID: 24092749
blood coagulation factor inhibitors; bradykinin; factor XI; factor XII; inflammation; kallikreins; leishmania
2.  Characterization of anti-hemostatic factors in the argasid, Argas monolakensis: Implications for the evolution of blood-feeding in the soft tick family 
To date, the only anti-hemostatic factors characterized for softs ticks are for Ornithodoros moubata and O. savignyi, ticks that feeds mainly on mammals. This includes thrombin (ornithodorin and savignin), fXa (TAP and fXaI) and platelet aggregation (disagegin and savignygrin) inhibitors that belong to the BPTI-Kunitz protein family. This raises the question on how well anti-hemostatic factors will be conserved in other soft tick genera that feeds on other vertebrates such as birds. We characterized the anti-hemostatic factors from Argas monolakensis, a soft tick that feeds mainly on Californian gulls. The main anti-clotting factor (monobin) is an ortholog of ornithodorin and savignin and shows similar slow tight-binding kinetics. The main anti-platelet activities are apyrase and fibrinogen receptor antagonists (monogrins). The monogrins are orthologs of disagregin and savignygrin and like savignygrin presents the RGD integrin-recognition motif on the BPTI substrate-binding presenting loop. This implies that the anti-hemostatic factors evolved in the ancestral soft tick lineage and has been maintained in soft tick species from two distinct genera with different host preferences. The Argas derived anti-hemostatic factors bind to mammalian targets with affinities similar to that observed for their orthologs in the Ornithodoros genus. This cross-reactivity could have facilitated the switching of soft ticks from avian to mammalian hosts and can explain in part the ability of Argas ticks, to feed on humans, thereby remaining a possible health risk.
doi:10.1016/j.ibmb.2007.09.002
PMCID: PMC4274796  PMID: 18070663
Argas monolakensis; blood-feeding; blood-clotting; evolution; platelet aggregation; ticks
3.  The Tick Salivary Protein Sialostatin L2 Inhibits Caspase-1-Mediated Inflammation during Anaplasma phagocytophilum Infection 
Infection and Immunity  2014;82(6):2553-2564.
Saliva from arthropod vectors facilitates blood feeding by altering host inflammation. Whether arthropod saliva counters inflammasome signaling, a protein scaffold that regulates the activity of caspase-1 and cleavage of interleukin-1β (IL-1β) and IL-18 into mature molecules, remains elusive. In this study, we provide evidence that a tick salivary protein, sialostatin L2, inhibits inflammasome formation during pathogen infection. We show that sialostatin L2 targets caspase-1 activity during host stimulation with the rickettsial agent Anaplasma phagocytophilum. A. phagocytophilum causes macrophage activation and hemophagocytic syndrome features. The effect of sialostatin L2 in macrophages was not due to direct caspase-1 enzymatic inhibition, and it did not rely on nuclear factor κB or cathepsin L signaling. Reactive oxygen species from NADPH oxidase and the Loop2 domain of sialostatin L2 were important for the regulatory process. Altogether, our data expand the knowledge of immunoregulatory pathways of tick salivary proteins and unveil an important finding in inflammasome biology.
doi:10.1128/IAI.01679-14
PMCID: PMC4019176  PMID: 24686067
4.  Evidence for a Lectin Specific for Sulfated Glycans in the Salivary Gland of the Malaria Vector, Anopheles gambiae 
PLoS ONE  2014;9(9):e107295.
Salivary gland homogenate (SGH) from the female mosquitoes Anopheles gambiae, An. stephensi, An. freeborni, An. dirus and An. albimanus were found to exhibit hemagglutinating (lectin) activity. Lectin activity was not found for male An. gambiae, or female Ae aegypti, Culex quinquefasciatus, Phlebotomus duboscqi, and Lutzomyia longipalpis. With respect to species-specificity, An. gambiae SGH agglutinates red blood cells (RBC) from humans, horse, sheep, goat, pig, and cow; it is less active for rats RBC, and not detectable for guinea-pigs or chicken RBC. Notably, lectin activity was inhibited by low concentrations of dextran sulfate 50–500 K, fucoidan, heparin, laminin, heparin sulfate proteoglycan, sialyl-containing glycans (e.g. 3′-sialyl Lewis X, and 6′-sialyl lactose), and gangliosides (e.g. GM3, GD1, GD1b, GTB1, GM1, GQ1B), but not by simple sugars. These results imply that molecule(s) in the salivary gland target sulfated glycans. SGH from An. gambiae was also found to promote agglutination of HL-60 cells which are rich in sialyl Lewis X, a glycan that decorates PSGL-1, the neutrophils receptor that interacts with endothelial cell P-selectin. Accordingly, SGH interferes with HL-60 cells adhesion to immobilized P-selectin. Because An. gambiae SGH expresses galectins, one member of this family (herein named Agalectin) was expressed in E. coli. Recombinant Agalectin behaves as a non-covalent homodimer. It does not display lectin activity, and does not interact with 500 candidates tested in a Glycan microarray. Gel-filtration chromatography of the SGH of An. gambiae identified a fraction with hemagglutinating activity, which was analyzed by 1D PAGE followed by in-gel tryptic digestion, and nano-LC MS/MS. This approach identified several genes which emerge as candidates for a lectin targeting sulfated glycans, the first with this selectivity to be reported in the SGH of a blood-sucking arthropod. The role of salivary molecules (sialogenins) with lectin activity is discussed in the context of inflammation, and parasite-vector-host interactions.
doi:10.1371/journal.pone.0107295
PMCID: PMC4160252  PMID: 25207644
5.  Examination of the Ligand-Binding and Enzymatic Properties of a Bilin-Binding Protein from the Poisonous Caterpillar Lonomia obliqua 
PLoS ONE  2014;9(6):e95424.
The bilin-binding proteins (BBP) from lepidopteran insects are members of the lipocalin family of proteins and play a special role in pigmentation through the binding of biliverdin IXγ. Lopap, a BBP-like protein from the venom of the toxic caterpillar Lonomia obliqua has been reported to act as a serine protease that activates the coagulation proenzyme prothrombin. Here we show that BBPLo, a variant of lopap from the same organism binds biliverdin IXγ, forming a complex that is spectrally identical with previously described BBP proteins. Although BBPLo is nearly identical in sequence to lopap, no prothrombinase activity was detected in our recombinant preparations using reconstituted systems containing coagulation factors Xa and Va, as well as anionic phospholipids. In addition to biliverdin, BBPLo was found to form a 1∶1 complex with heme prompting us to examine whether the unusual biliverdin IXγ ligand of BBPs forms as a result of oxidation of bound heme in situ rather than by a conventional heme oxygenase. Using ascorbate or a NADPH+-ferredoxin reductase-ferredoxin system as a source of reducing equivalents, spectral changes are seen that suggest an initial reduction of heme to the Fe(II) state and formation of an oxyferrous complex. The complex then disappears and a product identified as a 5-coordinate carbonyl complex of verdoheme, an intermediate in the biosynthesis of biliverdin, is formed. However, further reaction to form biliverdin was not observed, making it unlikely that biliverdin IXγ is formed by this pathway.
doi:10.1371/journal.pone.0095424
PMCID: PMC4074040  PMID: 24972000
6.  Structure and ligand-binding properties of the biogenic amine-binding protein from the saliva of a blood-feeding insect vector of Trypanosoma cruzi  
Biogenic amine-binding proteins mediate the anti-inflammatory and antihemostatic activities of blood-feeding insect saliva. The structure of the amine-binding protein from R. prolixus reveals the interaction of biogenic amine ligands with the protein.
Proteins that bind small-molecule mediators of inflammation and hemostasis are essential for blood-feeding by arthropod vectors of infectious disease. In ticks and triatomine insects, the lipocalin protein family is greatly expanded and members have been shown to bind biogenic amines, eicosanoids and ADP. These compounds are potent mediators of platelet activation, inflammation and vascular tone. In this paper, the structure of the amine-binding protein (ABP) from Rhodnius prolixus, a vector of the trypanosome that causes Chagas disease, is described. ABP binds the biogenic amines serotonin and norepinephrine with high affinity. A complex with tryptamine shows the presence of a binding site for a single ligand molecule in the central cavity of the β-barrel structure. The cavity contains significant additional volume, suggesting that this protein may have evolved from the related nitrophorin proteins, which bind a much larger heme ligand in the central cavity.
doi:10.1107/S0907444912043326
PMCID: PMC3532134  PMID: 23275168
lipocalins; Rhodnius prolixus; Triatominae; serotonin; norepinephrine; tryptamine; nitrophorin
7.  Structure and mechanism in salivary proteins from blood-feeding arthropods 
The saliva of blood-feeding arthropods contains rich mixtures of ligand binding proteins targeted at inhibiting hemostasis and inflammation in the host. Since blood feeding has evolved many times, different taxonomic groups utilize completely different families of proteins to perform similar tasks. Structural studies performed on a number of these proteins have revealed biologically novel and sophisticated mechanisms used to perform their functions. Here, the results of these structural and mechanistic studies are reviewed.
doi:10.1016/j.toxicon.2009.11.002
PMCID: PMC2889010  PMID: 19925819
Lipocalin; odorant-binding protein; D7; nitrophorin; tick; mosquito; Diptera; X-ray crystallography
8.  The crystal structures of two salivary cystatins from the tick Ixodes scapularis and the effect of these inhibitors on the establishment of Borrelia burgdorferi infection in a murine model 
Molecular microbiology  2010;77(2):456-470.
Summary
We have previously demonstrated that two salivary cysteine protease inhibitors from the Borrelia burgdorferi (Lyme disease) vector Ixodes scapularis-namely sialostatins L and L2-play an important role in tick biology, as demonstrated by the fact that silencing of both sialostatins in tandem results in severe feeding defects. Here we show that sialostatin L2 -but not sialostatin L- facilitates the growth of Borrelia burgdorferi in murine skin. To examine the structural basis underlying these differential effects of the two sialostatins, we have determined the crystal structures of both sialostatin L and L2. This is the first structural analysis of cystatins from an invertebrate source. Sialostatin L2 crystallizes as a monomer with an ‘unusual’ conformation of the N-terminus, while sialostatin L crystallizes as a domain-swapped dimer with an N-terminal conformation similar to other cystatins. Deletion of the ‘unusual’ N-terminal five residues of sialostatin L2 results in marked changes in its selectivity, suggesting that this region is a particularly important determinant of the biochemical activity of sialostatin L2. Collectively, our results reveal the structure of two tick salivary components that facilitate vector blood feeding and that one of them also supports pathogen transmission to the vertebrate host.
doi:10.1111/j.1365-2958.2010.07220.x
PMCID: PMC2909360  PMID: 20545851
9.  The tick salivary protein sialostatin L inhibits the Th9-derived production of the asthma-promoting cytokine interleukin-9 and is effective in the prevention of experimental asthma1 
Ticks developed a multitude of different immune evasion strategies in order to obtain a blood meal. Sialostatin L is an immunosuppressive cysteine protease inhibitor present in the saliva of the hard tick Ixodes scapularis. Herein we demonstrate that sialostatin L strongly inhibits the production of IL-9 by Th9 cells. Since we could show recently that Th9-derived IL-9 is essentially involved in the induction of asthma symptoms, sialostatin L was used for the treatment of experimental asthma. Application of sialostatin L in a model of experimental asthma almost completely abrogated airway hyperresponsiveness and eosinophilia. Our data suggest that sialostatin L can prevent experimental asthma, most likely by inhibiting the IL-9 production of Th9 cells. Thus, alternative to IL-9 neutralization sialostatin L provides the basis for the development of innovative therapeutic strategies to treat asthma.
doi:10.4049/jimmunol.1100529
PMCID: PMC3523721  PMID: 22327077
Th9 cells; Lung; Allergy; Parasites; Rodents
10.  Recognition of Anionic Phospholipid Membranes by an Antihemostatic Protein from a Blood-Feeding Insect 
Biochemistry  2004;43(22):6987-6994.
The saliva of blood-feeding insects contains a variety of molecules having antihemostatic activity. Here we describe nitrophorin 7 (NP7), a salivary protein that binds with high affinity to anionic phospholipid membranes. The protein is apparently targeted to the negatively charged surfaces of activated platelets and other cells where it can serve as a vasodilator, antihistamine, platelet aggregation inhibitor, and anticoagulant. As with other members of the nitrophorin group, NP7 reversibly binds a molecule of NO and binds histamine with high affinity. The protein differs from other nitrophorins in that it binds to membranes containing phosphatidylserine. Sedimentation and surface plasmon resonance experiments, revealed two classes of phospholipid binding site having Kd values of 4.8 and 755 nM. NP7 inhibits prothrombin activation by blocking phospholipid binding sites for the prothrombinase complex on the surfaces of vesicles and activated platelets. As a NO complex, NP7 inhibits collagen and ADP-induced platelet aggregation and induces disaggregation of ADP-stimulated platelets by an NO-mediated mechanism. Molecular modeling of NP7 revealed a putative, positively charged membrane interaction surface comprised mainly of a helix lying outside of the lipocalin β-barrel structure.
doi:10.1021/bi049655t
PMCID: PMC2915585  PMID: 15170336
Nitric oxide; histamine; prothrombinase; phosphatidyserine; Rhodnius prolixus
11.  The crystal structure and functional characterization of an immunomodulatory salivary cystatin from the soft tick Ornithodoros moubata 
The Biochemical journal  2010;429(1):103-112.
Synopsis
The saliva of blood-feeding parasites is a rich source of peptidase inhibitors that help overcome the host’s defense during host-parasite interactions. Using proteomic analysis, the cystatin OmC2 was demonstrated in the saliva of the soft tick Ornithodoros moubata, an important disease-vector transmitting African swine fever virus and the spirochaete Borrelia duttoni. A structural, biochemical and biological characterization of this peptidase inhibitor was undertaken. Recombinant OmC2 was screened against a panel of physiologically relevant peptidases and found to be an effective broad-specificity inhibitor of cysteine cathepsins, including endopeptidases (cathepsins L and S) and exopeptidases (cathepsins B, C and H). The crystal structure of OmC2 was determined at a resolution of 2.45 Å and used to describe the structure-inhibitory activity relationship. The biological impact of OmC2 was demonstrated both in vitro and in vivo. OmC2 affected the function of antigen-presenting mouse dendritic cells by reducing the production of the proinflammatory cytokines TNF-α and IL-12, and proliferation of antigen-specific CD4+ T cells. This suggests that OmC2 may suppress the host’s adaptive immune response. Immunization of mice with OmC2 significantly suppressed the survival of O. moubata in infestation experiments. We conclude that OmC2 is a promising target for the development of a novel anti-tick vaccine to control O. moubata populations and combat the spread of associated diseases.
doi:10.1042/BJ20100280
PMCID: PMC3523712  PMID: 20545626
cathepsin; cystatin; immune cells; structure-activity relationship; parasite; peptidase inhibitor
12.  A novel family of RGD-containing disintegrin (Tablysin-15) from the salivary gland of the horsefly Tabanus yao targets integrins αIIbβ3 and αVβ3 and inhibits platelet aggregation and angiogenesis 
Thrombosis and haemostasis  2011;105(6):1032-1045.
A novel family of RGD-containing molecule (Tablysin-15) has been molecularly characterized from the salivary gland of the hematophagous horsefly Tabanus yao. Tablysin-15 does not share primary sequence homology to any disintegrin discovered so far, and displays an RGD motif in the N-terminus of the molecule. It is also distinct from disintegrins from Viperidae since its mature form is not released from a metalloproteinase precursor. Tablysin-15 exhibits high affinity for platelet αIIbβ3 and endothelial cell αvβ3 integrins, but not for α5β1 or α2β1. Accordingly, it blocks endothelial cell adhesion to vitronectin (IC50 ~ 1 nM) and marginally to fibronectin (IC50 ~ 1 µM), but not to collagen. It also inhibits FGF-induced endothelial cell proliferation, and attenuates tube formation in vitro. In platelets, Tablysin-15 inhibits aggregation induced by collagen, ADP and convulxin, and prevents static platelet adhesion to immobilized fibrinogen. In addition, solid-phase assays and flow cytometry demonstrates that αIIbβ3 binds to Tablysin-15. Moreover, immobilized Tablysin-15 supports platelet adhesion by a mechanism which was blocked by anti-integrin αIIbβ3 monoclonal antibody (e.g. abciximab) or by EDTA. Furthermore, Tablysin-15 dose-dependently attenuates thrombus formation to collagen under flow, without affecting platelet adhesion to collagen fibrils. Consistent with these findings, Tablysin-15 displays antithrombotic properties in vivo suggesting that it is a useful tool to block αIIbβ3, or as a prototype to develop antithrombotics. The RGD motif in the unique sequence of Tablysin-15 represents a novel template for studying the structure-function relationship of the disintegrin family of inhibitors.
doi:10.1160/TH11-01-0029
PMCID: PMC3499621  PMID: 21475772
hematophagy; blood-sucking; disintegrin; thrombosis; sialogenins
13.  An Insight into the Sialotranscriptome of the Cat Flea, Ctenocephalides felis 
PLoS ONE  2012;7(9):e44612.
Background
Saliva of hematophagous arthropods contains a diverse mixture of compounds that counteracts host hemostasis. Immunomodulatory and antiinflammatory components are also found in these organisms' saliva. Blood feeding evolved at least ten times within arthropods, providing a scenario of convergent evolution for the solution of the salivary potion. Perhaps because of immune pressure from hosts, the salivary proteins of related organisms have considerable divergence, and new protein families are often found within different genera of the same family or even among subgenera. Fleas radiated with their vertebrate hosts, including within the mammal expansion initiated 65 million years ago. Currently, only one flea species–the rat flea Xenopsylla cheopis–has been investigated by means of salivary transcriptome analysis to reveal salivary constituents, or sialome. We present the analysis of the sialome of cat flea Ctenocephaides felis.
Methodology and Critical Findings
A salivary gland cDNA library from adult fleas was randomly sequenced, assembled, and annotated. Sialomes of cat and rat fleas have in common the enzyme families of phosphatases (inactive), CD-39-type apyrase, adenosine deaminases, and esterases. Antigen-5 members are also common to both sialomes, as are defensins. FS-I/Cys7 and the 8-Cys families of peptides are also shared by both fleas and are unique to these organisms. The Gly-His-rich peptide similar to holotricin was found only in the cat flea, as were the abundantly expressed Cys-less peptide and a novel short peptide family.
Conclusions/Significance
Fleas, in contrast to bloodsucking Nematocera (mosquitoes, sand flies, and black flies), appear to concentrate a good portion of their sialome in small polypeptides, none of which have a known function but could act as inhibitors of hemostasis or inflammation. They are also unique in expansion of a phosphatase family that appears to be deficient of enzyme activity and has an unknown function.
doi:10.1371/journal.pone.0044612
PMCID: PMC3458046  PMID: 23049752
14.  Inhibition of tissue factor by ixolaris reduces primary tumor growth and experimental metastasis in a murine model of melanoma 
Thrombosis research  2012;130(3):e163-e170.
Melanoma is a highly metastatic cancer and there is strong evidence that the clotting initiator protein, tissue factor (TF), contributes to its aggressive pattern. TF inhibitors may attenuate primary tumor growth and metastasis. In this study, we evaluated the effect of ixolaris, a TF inhibitor, on a murine model of melanoma B16F10 cells. Enzymatic assays performed with B16F10 and human U87-MG tumor cells as the TF source showed that ixolaris inhibits the generation of FX in either murine, human or hybrid FVIIa/TF complexes. The effect of ixolaris on the metastatic potential was further estimated by intravenous injection of B16F10 cells in C57/BL6 mice. Ixolaris (250 μg/kg) dramatically decreased the number of pulmonary tumor nodules (4 ± 1 compared to 47 ± 10 in the control group). Furthermore, a significant decrease in tumor weights was observed in primary tumor growth assays in animals treated with ixolaris (250 μg/kg) from days 3 to 18 after a subcutaneous inoculation of melanoma cells. Remarkably, immunohistochemical analyses showed that inhibition of melanoma growth by ixolaris is accompanied by a significant downregulation of both vascular endothelial growth factor (VEGF) expression and microvascular density in the tumor mass. Our data demonstrate that ixolaris targets B16F10 cell-derived TF, resulting in the reduction of both the primary tumor growth and the metastatic potential of melanoma, as well as the inhibition of tumor angiogenesis. Therefore TF may be a potential target for the treatment of this aggressive malignancy.
doi:10.1016/j.thromres.2012.05.021
PMCID: PMC3424357  PMID: 22683021
Tissue factor; melanoma; ixolaris; angiogenesis; metastasis; anticoagulant therapy
15.  An insight into the sialome of the blood-sucking bug Triatoma infestans, a vector of Chagas' disease 
Triatoma infestans is a hemiptera, vector of Chagas’ disease, that feeds exclusively on vertebrate blood in all life stages. Hematophagous insects’ salivary glands (SG) produce potent pharmacological compounds that counteract host hemostasis, including anti-clotting, anti-platelet, and vasodilatory molecules. To obtain a further insight into the salivary biochemical and pharmacological complexity of this insect, a cDNA library from its salivary glands was randomly sequenced. Also, salivary proteins were submitted to two dimentional gel (2D-gel) electrophoresis followed by MS analysis. We present the analysis of a set of 1,534 (SG) cDNA sequences, 645 of which coded for proteins of a putative secretory nature. Most salivary proteins described as lipocalins matched peptide sequences obtained from proteomic results.
doi:10.1016/j.ibmb.2007.11.001
PMCID: PMC2262853  PMID: 18207082
Hematophagy; Saliva; Transcriptome; Triatoma infestans; Feeding; Sialome
16.  Triplatin, a platelet aggregation inhibitor from the salivary gland of the triatomine vector of Chagas Disease, binds to TXA2 but does not interact with GPVI 
Thrombosis and haemostasis  2011;107(1):111-123.
Salivary glands from hematophagous animals express a notable diversity of negative modulators of platelet function. Triplatin is an inhibitor of collagen-induced platelet aggregation which has been described as an antagonist of glycoprotein VI (GPVI). Because triplatin displays sequence homology to members of the lipocalin family of proteins, we investigated whether triplatin mechanism of action could be explained by interaction with pro-hemostatic prostaglandins. Our results demonstrate that triplatin inhibits platelet aggregation induced by low doses of collagen, thromboxane A2 (TXA2) mimetic (U46619), and arachidonic acid (AA). On the other hand, it does not inhibit platelet aggregation by convulxin, PMA, or low-dose ADP. Isothermal titration calorimetry (ITC) revealed that triplatin binds AA, cTXA2, TXB2, U46619 or PGH2 mimetic (U51605). Consistent with its ligand specificity, triplatin induces relaxation of rat aorta contracted with U46619. Triplatin also interacts with PGF2α and PGJ2, but not with leukotrienes, AA or biogenic amines. Surface plasmon resonance experiments failed to demonstrate interaction of triplatin with GPVI; it also did to inhibit platelet adhesion to fibrillar or soluble collagen. Because triplatin displays sequence similarity to apolipoprotein D (ApoD)—a lipocalin associated with HDL, it was tested as a putative TXA2-binding molecule. ITC failed to demonstrate binding of ApoD to all prostanoids described above, or to AA. Furthermore, ApoD was devoid of inhibitory properties towards platelets activation by AA, collagen, or U46619. In conclusion, Triplatin mechanism of action has been elucidate without ambiguity as a novel TXA2- and PGF2α- binding protein. It conceivably blocks platelet aggregation and vasoconstriction, thus contributing to successful blood feeding at the vector-host interface.
doi:10.1160/TH11-10-0685
PMCID: PMC3408606  PMID: 22159626
17.  Mitochondrial Reactive Oxygen Species Modulate Mosquito Susceptibility to Plasmodium Infection 
PLoS ONE  2012;7(7):e41083.
Background
Mitochondria perform multiple roles in cell biology, acting as the site of aerobic energy-transducing pathways and as an important source of reactive oxygen species (ROS) that modulate redox metabolism.
Methodology/Principal Findings
We demonstrate that a novel member of the mitochondrial transporter protein family, Anopheles gambiae mitochondrial carrier 1 (AgMC1), is required to maintain mitochondrial membrane potential in mosquito midgut cells and modulates epithelial responses to Plasmodium infection. AgMC1 silencing reduces mitochondrial membrane potential, resulting in increased proton-leak and uncoupling of oxidative phosphorylation. These metabolic changes reduce midgut ROS generation and increase A. gambiae susceptibility to Plasmodium infection.
Conclusion
We provide direct experimental evidence indicating that ROS derived from mitochondria can modulate mosquito epithelial responses to Plasmodium infection.
doi:10.1371/journal.pone.0041083
PMCID: PMC3399787  PMID: 22815925
18.  An insight into the sialome of the bed bug, Cimex lectularius 
Journal of proteome research  2010;9(8):3820-3831.
The evolution of insects to a blood diet leads to the development of a saliva that antagonizes their hosts' hemostasis and inflammation. Hemostasis and inflammation are redundant processes, and thus a complex salivary potion comprised of dozens or near one hundred different polypeptides is commonly found by transcriptome or proteome analysis of these organisms. Several insect orders or families evolved independently to hematophagy creating unique salivary potions in the form of novel pharmacological use of endogenous substances, and in the form of unique proteins not matching other known proteins, these probably arriving by fast evolution of salivary proteins as they evade their hosts' immune response. In this work we present a preliminary description of the sialome (from the Greek Sialo = saliva) of the common bed bug Cimex lectularius, the first such work from a member of the Cimicidae family. This manuscript is a guide for the supplemental database files http://exon.niaid.nih.gov/transcriptome/C_lectularius/S1/Cimex-S1.zip and http://exon.niaid.nih.gov/transcriptome/C_lectularius/S2/Cimex-S2.xls
doi:10.1021/pr1000169
PMCID: PMC2917537  PMID: 20441151
Bedbug; saliva; salivary transcriptome; salivary proteome
19.  NITROPHORIN 2, A FIX(a)-DIRECTED ANTICOAGULANT, INHIBITS ARTERIAL THROMBOSIS WITHOUT IMPAIRING HEMOSTASIS 
Thrombosis and haemostasis  2010;104(6):1116-1123.
Summary
Nitrophorin 2 (NP2) is a 20 kDa lipocalin identified in the salivary gland of the blood sucking insect, Rhodnius prolixus. It functions as a potent inhibitor of the intrinsic pathway of coagulation upon binding to factor IX (FIX) or FIXa. Herein we have investigated the in vivo antithrombotic properties of NP2. Surface plasmon resonance assays demonstrated that NP2 binds to rat FIX and FIXa with high affinities (KD = 43 and 47 nM, respectively), and prolongs the aPTT without affecting the PT. In order to evaluate NP2 antithrombotic effects in vivo two distinct models of thrombosis in rats were carried out. In the rose Bengal/laser induced injury model of arterial thrombosis, NP2 increased the carotid artery occlusion time by ≈35 and ≈155%, at doses of 8 and 80 µg/kg, respectively. NP2 also inhibited thrombus formation in an arterio-venous shunt model, showing ≈60% reduction at 400 µg/kg (i.v. administration). The antithrombotic effect lasted for up to 48 h after a single i.v. dose. Notably, effective doses of NP2 did not increase the blood loss as evaluated by tail-transection model. In conclusion, NP2 is a potent and long-lasting inhibitor of arterial thrombosis with minor effects on hemostasis. It might be regarded as a potential agent for the treatment of human cardiovascular diseases.
doi:10.1160/TH10.1160/TH10-03-0186
PMCID: PMC2996477  PMID: 20838739
Animal models; Arterial thrombosis; Coagulation inhibitors; Haemostasis; Nitrophorin
20.  The Function and Three-Dimensional Structure of a Thromboxane A2/Cysteinyl Leukotriene-Binding Protein from the Saliva of a Mosquito Vector of the Malaria Parasite 
PLoS Biology  2010;8(11):e1000547.
A salivary protein from a malaria-transmitting mosquito uses a single domain to bind to thromboxane A2 and cysteinyl leukotrienes and prevent blood clotting and inflammation in the host on which it feeds.
The highly expressed D7 protein family of mosquito saliva has previously been shown to act as an anti-inflammatory mediator by binding host biogenic amines and cysteinyl leukotrienes (CysLTs). In this study we demonstrate that AnSt-D7L1, a two-domain member of this group from Anopheles stephensi, retains the CysLT binding function seen in the homolog AeD7 from Aedes aegypti but has lost the ability to bind biogenic amines. Unlike any previously characterized members of the D7 family, AnSt-D7L1 has acquired the important function of binding thromboxane A2 (TXA2) and its analogs with high affinity. When administered to tissue preparations, AnSt-D7L1 abrogated Leukotriene C4 (LTC4)-induced contraction of guinea pig ileum and contraction of rat aorta by the TXA2 analog U46619. The protein also inhibited platelet aggregation induced by both collagen and U46619 when administered to stirred platelets. The crystal structure of AnSt-D7L1 contains two OBP-like domains and has a structure similar to AeD7. In AnSt-D7L1, the binding pocket of the C-terminal domain has been rearranged relative to AeD7, making the protein unable to bind biogenic amines. Structures of the ligand complexes show that CysLTs and TXA2 analogs both bind in the same hydrophobic pocket of the N-terminal domain. The TXA2 analog U46619 is stabilized by hydrogen bonding interactions of the ω-5 hydroxyl group with the phenolic hydroxyl group of Tyr 52. LTC4 and occupies a very similar position to LTE4 in the previously determined structure of its complex with AeD7. As yet, it is not known what, if any, new function has been acquired by the rearranged C-terminal domain. This article presents, to our knowledge, the first structural characterization of a protein from mosquito saliva that inhibits collagen mediated platelet activation.
Author Summary
When feeding, a female mosquito must inhibit the blood clotting and inflammatory responses of the host. To do this, the insect produces salivary proteins that neutralize key host molecules participating in clotting and inflammation. Here, we describe a salivary protein AnSt-D7L1 that scavenges both thomboxane A2 and cysteinyl leukotrienes, two substances involved in blood vessel constriction, platelet aggregation, and inflammatory responses to an insect bite. We produced this protein in bacteria and showed that it tightly binds both these molecules, inhibiting the processes in which they are involved. We then determined its structure using X-ray crystallography and showed that there is a single binding site in one domain of the protein, accommodating both thromboxane A2 and cysteinyl leukotrienes, and that this site is responsible for the scavenging effect of the protein. These studies reveal the structural features of proteins needed to bind to key molecules of potential pharmacological importance and add to our understanding of the process of mosquito blood feeding, which is essential for transmission of the malaria parasite.
doi:10.1371/journal.pbio.1000547
PMCID: PMC2994686  PMID: 21152418
21.  The role of salivary lipocalins in blood feeding by Rhodnius prolixus 
In order to overcome host mechanisms that prevent blood loss, the bloodsucking bug Rhodnius prolixus has evolved a complex salivary secretion containing dozens of different proteins. A number of these have been characterized and found to have roles in inhibiting various hemostatic or inflammatory systems. Interestingly, many of these biologically active salivary proteins belong to the lipocalin protein family. A proliferation of lipocalin genes has occurred via gene duplication and subsequent divergence. Functional genomic, proteomic and functional studies have been performed to probe the role of salivary lipocalins in blood feeding. In the course of these investigations, anticoagulant, antiplatelet, antiinflammatory and vasodilatory molecules have been described.
doi:10.1002/arch.20032
PMCID: PMC2915583  PMID: 15660358
22.  The transcriptome of the salivary glands of the female western black-legged tick Ixodes pacificus (Acari: Ixodidae) 
Summary
Sequencing of an Ixodes pacificus salivary gland cDNA library yielded 1068 sequences with an average undetermined nucleotide of 1.9% and an average length of 487 base pairs. Assembly of the expressed sequence tags yielded 557 contigs, 138 of which appear to code for secreted peptides or proteins based on translation of a putative signal peptide. Based on the BLASTX similarity of these contigs to 66 matches of Ixodes scapularis peptide sequences, only 58% sequence identity was found, indicating a rapid divergence of salivary proteins as observed previously for mosquito and triatomine bug salivary proteins. Here we report 106 mostly full-length sequences that clustered in 16 different families: Basic-tail proteins rich in lysine in the carboxy-terminal, Kunitz-containing proteins (monolaris, ixolaris and penthalaris families), proline-rich peptides, 5-kDa.-, 9.4 kDa.-, and 18.7 kDa.-proteins of unknown functions, in addition to metalloproteases (class PIII-like) similar to reprolysins. We also have found a family of disintegrins, named ixodegrins that display homology to variabilin, a GPIIb/IIIa antagonist from the tick Dermacentor variabilis. In addition, we describe peptides (here named ixostatins) that display remarkable similarities to the cysteine-rich domain of ADAMST-4 (aggrecanase). Many molecules were assigned in the lipocalin family (histamine-binding proteins); others appear to be involved in oxidant metabolism, and still others were similar to ixodid proteins such as the anticomplement ISAC. We also identified for the first time a neuropeptide-like protein (nlp-31) with GGY repeats that may have antimicrobial activity. In addition, 16 novel proteins without significant similarities to other tick proteins and 37 housekeeping proteins that may be useful for phylogenetic studies are described. Some of these proteins may be useful for studying vascular biology or the immune system, for vaccine development, or as immunoreagents to detect prior exposure to ticks. Electronic version of the manuscript can be found at http://www.ncbi.nlm.nih.gov/projects/omes/.
doi:10.1016/j.ibmb.2005.05.007
PMCID: PMC2887698  PMID: 16102420
Ixodes pacificus; sialome; tick; blood-feeding; Kunitz inhibitor; Lyme disease; vascular biology; Ixolaris; vector biology; transcriptome; proteome
23.  The immunomodulatory action of sialostatin L on dendritic cells reveals its potential to interfere with autoimmunity1 
Sialostatin L (SialoL) is a secreted cysteine protease inhibitor identified in the salivary glands of the Lyme disease vector Ixodes scapularis. Here, we reveal the mechanisms of SialoL immunomodulatory actions on the vertebrate host. LPS-induced maturation of dendritic cells from C57BL/6 mice was significantly reduced in the presence of SialoL. Although OVA degradation was not affected by the presence of SialoL in dendritic cell cultures, cathepsin S activity was partially inhibited, leading to an accumulation of 10 KDa invariant chain intermediate (Ii-p10) in these cells. As a consequence, in vitro antigen-specific CD4+ T cell proliferation was inhibited in a time-dependent manner by SialoL and further studies engaging cathepsin S−/− or cathepsin L−/− dendritic cells confirmed that the immunomodulatory actions SialoL are mediated by inhibition of cathepsin S. Moreover, mice treated with SialoL displayed decreased early T cell expansion and recall response upon antigenic stimulation. Finally, SialoL administration during the immunization phase of experimental autoimmune encephalomyelitis in mice significantly prevented disease symptoms, which was associated with impaired IFN-γ and IL-17 production and specific T cell proliferation. These results illuminate the dual mechanism by which a human disease vector protein modulates vertebrate host immunity and reveals its potential in prevention of an autoimmune disease.
doi:10.4049/jimmunol.0900075
PMCID: PMC2694955  PMID: 19494265
Dendritic cells; T cells; Autoimmunity; Antigen Presentation/Processing; Cell Proliferation
24.  An Insight into the Sialome of the Black Fly, Simulium vittatum 
Journal of proteome research  2009;8(3):1474-1488.
Adaptation to vertebrate blood feeding includes development of a salivary ‘magic potion’ that can disarm host hemostasis and inflammatory reactions. Within the lower Diptera, a vertebrate blood-sucking mode evolved in the Psychodidae (sand flies), Culicidae (mosquitoes), Ceratopogonidae (biting midges), Simuliidae (black flies), and in the frog-feeding Corethrellidae. Sialotranscriptome analyses from several species of mosquitoes and sand flies and from one biting midge indicate divergence in the evolution of the blood-sucking salivary potion, manifested in the finding of many unique proteins within each insect family, and even genus. Gene duplication and divergence events are highly prevalent, possibly driven by vertebrate host immune pressure. Within this framework, we describe the sialome (from Greek sialo, saliva) of the black fly Simulium vittatum and discuss the findings within the context of the protein families found in other blood-sucking Diptera. Sequences and results of Blast searches against several protein family databases are given in Supplemental Tables S1 and S2, which can be obtained from http://exon.niaid.nih.gov/transcriptome/S_vittatum/T1/SV-tb1.zip and http://exon.niaid.nih.gov/transcriptome/S_vittatum/T2/SV-tb2.zip.
doi:10.1021/pr8008429
PMCID: PMC2778207  PMID: 19166301
Simulium vittatum; black fly; sialotranscriptomes; salivary gland transcriptome; sialome; proteome; hematophagy; onchocerciasis
25.  Cyr61/CCN1 Displays High-Affinity Binding to the Somatomedin B 1–44 Domain of Vitronectin 
PLoS ONE  2010;5(2):e9356.
Background
Cyr61 is a member of the CCN (Cyr61, connective tissue growth, NOV) family of extracellular-associated (matricellular) proteins that present four distinct functional modules, namely insulin-like growth factor binding protein (IGFBP), von Willebrand factor type C (vWF), thrombospondin type 1 (TSP), and C-terminal growth factor cysteine knot (CT) domain. While heparin sulphate proteoglycans reportedly mediate the interaction of Cyr61 with the matrix and cell surface, the role of other extracellular associated proteins has not been revealed.
Methods and Findings
In this report, surface plasmon resonance (SPR) experiments and solid-phase binding assays demonstrate that recombinant Cyr61 interacts with immobilized monomeric or multimeric vitronectin (VTNC) with KD in the nanomolar range. Notably, the binding site for Cyr61 was identified as the somatomedin B domain (SMTB 1–44) of VTNC, which mediates its interaction with PAI-1, uPAR, and integrin αvβ3. Accordingly, PAI-1 outcompetes Cyr61 for binding to immobilized SMTB 1–44, and Cyr61 attenuates uPAR-mediated U937 adhesion to VTNC. In contrast, isothermal titration calorimetry shows that Cyr61 does not display high-affinity binding for SMTB 1-44 in solution. Nevertheless, competitive ELISA revealed that multimeric VTNC, heat-modified monomeric VTNC, or SMTB 1–44 at high concentrations attenuate Cyr61 binding to immobilized VTNC, while monomeric VTNC was ineffective. Therefore, immobilization of VTNC exposes cryptic epitopes that recognize Cyr61 with high affinity, as reported for a number of antibodies, β-endorphin, and other molecules.
Conclusions
The finding that Cyr61 interacts with the SMTB 1–44 domain suggests that VTNC represent a point of anchorage for CCN family members to the matrix. Results are discussed in the context of the role of CCN and VTNC in matrix biology and angiogenesis.
doi:10.1371/journal.pone.0009356
PMCID: PMC2829074  PMID: 20195466

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