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1.
PLoS Negl Trop Dis ; 10(4): e0004581, 2016 Apr.
Article in English | MEDLINE | ID: mdl-27129103

ABSTRACT

BACKGROUND: Triatomines are hematophagous insects that act as vectors of Chagas disease. Rhodnius neglectus is one of these kissing bugs found, contributing to the transmission of this American trypanosomiasis. The saliva of hematophagous arthropods contains bioactive molecules responsible for counteracting host haemostatic, inflammatory, and immune responses. METHODS/PRINCIPAL FINDINGS: Next generation sequencing and mass spectrometry-based protein identification were performed to investigate the content of triatomine R. neglectus saliva. We deposited 4,230 coding DNA sequences (CDS) in GenBank. A set of 636 CDS of proteins of putative secretory nature was extracted from the assembled reads, 73 of them confirmed by proteomic analysis. The sialome of R. neglectus was characterized and serine protease transcripts detected. The presence of ubiquitous protein families was revealed, including lipocalins, serine protease inhibitors, and antigen-5. Metalloproteases, disintegrins, and odorant binding protein families were less abundant. CONCLUSIONS/SIGNIFICANCE: The data presented improve our understanding of hematophagous arthropod sialomes, and aid in understanding hematophagy and the complex interplay among vectors and their vertebrate hosts.


Subject(s)
Insect Vectors , Peptide Hydrolases/analysis , Peptide Hydrolases/genetics , Rhodnius/physiology , Saliva/chemistry , Salivary Proteins and Peptides/analysis , Salivary Proteins and Peptides/genetics , Animals , Genomics , Mass Spectrometry , Molecular Sequence Data , Proteomics , Sequence Analysis, DNA
2.
PLoS One ; 9(2): e87140, 2014.
Article in English | MEDLINE | ID: mdl-24586264

ABSTRACT

BACKGROUND: The role of intracellular radical oxygen species (ROS) in pathogenesis of cerebral malaria (CM) remains incompletely understood. METHODS AND FINDINGS: We undertook testing Tempol--a superoxide dismutase (SOD) mimetic and pleiotropic intracellular antioxidant--in cells relevant to malaria pathogenesis in the context of coagulation and inflammation. Tempol was also tested in a murine model of CM induced by Plasmodium berghei Anka infection. Tempol was found to prevent transcription and functional expression of procoagulant tissue factor in endothelial cells (ECs) stimulated by lipopolysaccharide (LPS). This effect was accompanied by inhibition of IL-6, IL-8, and monocyte chemoattractant protein (MCP-1) production. Tempol also attenuated platelet aggregation and human promyelocytic leukemia HL60 cells oxidative burst. In dendritic cells, Tempol inhibited LPS-induced production of TNF-α, IL-6, and IL-12p70, downregulated expression of co-stimulatory molecules, and prevented antigen-dependent lymphocyte proliferation. Notably, Tempol (20 mg/kg) partially increased the survival of mice with CM. Mechanistically, treated mice had lowered plasma levels of MCP-1, suggesting that Tempol downmodulates EC function and vascular inflammation. Tempol also diminished blood brain barrier permeability associated with CM when started at day 4 post infection but not at day 1, suggesting that ROS production is tightly regulated. Other antioxidants-such as α-phenyl N-tertiary-butyl nitrone (PBN; a spin trap), MnTe-2-PyP and MnTBAP (Mn-phorphyrin), Mitoquinone (MitoQ) and Mitotempo (mitochondrial antioxidants), M30 (an iron chelator), and epigallocatechin gallate (EGCG; polyphenol from green tea) did not improve survival. By contrast, these compounds (except PBN) inhibited Plasmodium falciparum growth in culture with different IC50s. Knockout mice for SOD1 or phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (gp91(phox-/-)) or mice treated with inhibitors of SOD (diethyldithiocarbamate) or NADPH oxidase (diphenyleneiodonium) did not show protection or exacerbation for CM. CONCLUSION: Results with Tempol suggest that intracellular ROS contribute, in part, to CM pathogenesis. Therapeutic targeting of intracellular ROS in CM is discussed.


Subject(s)
Antioxidants/pharmacology , Cyclic N-Oxides/pharmacology , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Malaria, Cerebral/drug therapy , Thromboplastin/metabolism , Animals , Antioxidants/therapeutic use , Cells, Cultured , Chemokine CCL2/metabolism , Cyclic N-Oxides/therapeutic use , Enzyme-Linked Immunosorbent Assay , Humans , Interleukin-6/metabolism , Interleukin-8/metabolism , Malaria, Cerebral/metabolism , Mice , Reactive Oxygen Species/metabolism , Real-Time Polymerase Chain Reaction , Spin Labels
3.
Blood ; 122(25): 4094-106, 2013 Dec 12.
Article in English | MEDLINE | ID: mdl-24159172

ABSTRACT

The identity of vampire bat saliva anticoagulant remained elusive for almost a century. Sequencing the salivary gland genes from the vampire bat Desmodus rotundus identified Desmolaris as a novel 21.5-kDa naturally deleted (Kunitz 1-domainless) form of tissue factor pathway inhibitor. Recombinant Desmolaris was expressed in HEK293 cells and characterized as a slow, tight, and noncompetitive inhibitor of factor (F) XIa by a mechanism modulated by heparin. Desmolaris also inhibits FXa with lower affinity, independently of protein S. In addition, Desmolaris binds kallikrein and reduces bradykinin generation in plasma activated with kaolin. Truncated and mutated forms of Desmolaris determined that Arg32 in the Kunitz-1 domain is critical for protease inhibition. Moreover, Kunitz-2 and the carboxyl-terminus domains mediate interaction of Desmolaris with heparin and are required for optimal inhibition of FXIa and FXa. Notably, Desmolaris (100 µg/kg) inhibited FeCl3-induced carotid artery thrombus without impairing hemostasis. These results imply that FXIa is the primary in vivo target for Desmolaris at antithrombotic concentrations. Desmolaris also reduces the polyphosphate-induced increase in vascular permeability and collagen- and epinephrine-mediated thromboembolism in mice. Desmolaris emerges as a novel anticoagulant targeting FXIa under conditions in which the coagulation activation, particularly the contact pathway, plays a major pathological role.


Subject(s)
Anticoagulants/chemistry , Anticoagulants/pharmacology , Chiroptera , Factor Xa Inhibitors , Salivary Proteins and Peptides/chemistry , Salivary Proteins and Peptides/pharmacology , Thrombosis/drug therapy , Animals , Bradykinin/chemistry , Bradykinin/genetics , Bradykinin/metabolism , Chlorides/adverse effects , Chlorides/pharmacology , Disease Models, Animal , Factor Xa/chemistry , Factor Xa/genetics , Factor Xa/metabolism , Ferric Compounds/adverse effects , Ferric Compounds/pharmacology , HEK293 Cells , Humans , Inflammation/drug therapy , Inflammation/genetics , Inflammation/metabolism , Kallikreins/chemistry , Kallikreins/genetics , Kallikreins/metabolism , Mice , Noxae/adverse effects , Noxae/pharmacology , Protein Structure, Tertiary , Salivary Proteins and Peptides/genetics , Thrombosis/chemically induced , Thrombosis/genetics , Thrombosis/metabolism
4.
J Biol Chem ; 288(20): 14341-14361, 2013 May 17.
Article in English | MEDLINE | ID: mdl-23564450

ABSTRACT

The function of the antigen-5/CAP family of proteins found in the salivary gland of bloodsucking animals has remained elusive for decades. Antigen-5 members from the hematophagous insects Dipetalogaster maxima (DMAV) and Triatoma infestans (TIAV) were expressed and discovered to attenuate platelet aggregation, ATP secretion, and thromboxane A2 generation by low doses of collagen (<1 µg/ml) but no other agonists. DMAV did not interact with collagen, glycoprotein VI, or integrin α2ß1. This inhibitory profile resembles the effects of antioxidants Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in platelet function. Accordingly, DMAV was found to inhibit cytochrome c reduction by O2[Symbol: see text] generated by the xanthine/xanthine oxidase, implying that it exhibits antioxidant activity. Moreover, our results demonstrate that DMAV blunts the luminescence signal of O2[Symbol: see text] generated by phorbol 12-myristate 13-acetate-stimulated neutrophils. Mechanistically, inductively coupled plasma mass spectrometry and fluorescence spectroscopy revealed that DMAV, like Cu,Zn-SOD, interacts with Cu(2+), which provides redox potential for catalytic removal of O2[Symbol: see text]. Notably, surface plasmon resonance experiments (BIAcore) determined that DMAV binds sulfated glycosaminoglycans (e.g. heparin, KD ~100 nmol/liter), as reported for extracellular SOD. Finally, fractions of the salivary gland of D. maxima with native DMAV contain Cu(2+) and display metal-dependent antioxidant properties. Antigen-5/CAP emerges as novel family of Cu(2+)-dependent antioxidant enzymes that inhibit neutrophil oxidative burst and negatively modulate platelet aggregation by a unique salivary mechanism.


Subject(s)
Copper/metabolism , Free Radical Scavengers/metabolism , Neutrophils/metabolism , Platelet Aggregation , Respiratory Burst , Triatoma/enzymology , Amino Acid Sequence , Animals , Antioxidants/metabolism , Cattle , Collagen/metabolism , Glycosaminoglycans/metabolism , Horses , Humans , Hydrogen Peroxide/analysis , Molecular Sequence Data , Oxygen/metabolism , Phylogeny , Platelet Adhesiveness , Salivary Glands/enzymology , Sequence Alignment , Sharks , Sulfur/chemistry , Surface Plasmon Resonance , Swine
5.
J Proteomics ; 82: 288-319, 2013 Apr 26.
Article in English | MEDLINE | ID: mdl-23411029

ABSTRACT

Vampire bats are notorious for being the sole mammals that strictly feed on fresh blood for their survival. While their saliva has been historically associated with anticoagulants, only one antihemostatic (plasminogen activator) has been molecularly and functionally characterized. Here, RNAs from both principal and accessory submaxillary (submandibular) salivary glands of Desmodus rotundus were extracted, and ~200 million reads were sequenced by Illumina. The principal gland was enriched with plasminogen activators with fibrinolytic properties, members of lipocalin and secretoglobin families, which bind prohemostatic prostaglandins, and endonucleases, which cleave neutrophil-derived procoagulant NETs. Anticoagulant (tissue factor pathway inhibitor, TFPI), vasodilators (PACAP and C-natriuretic peptide), and metalloproteases (ADAMTS-1) were also abundantly expressed. Members of the TSG-6 (anti-inflammatory), antigen 5/CRISP, and CCL28-like (antimicrobial) protein families were also sequenced. Apyrases (which remove platelet agonist ADP), phosphatases (which degrade procoagulant polyphosphates), and sphingomyelinase were found at lower transcriptional levels. Accessory glands were enriched with antimicrobials (lysozyme, defensin, lactotransferrin) and protease inhibitors (TIL-domain, cystatin, Kazal). Mucins, heme-oxygenase, and IgG chains were present in both glands. Proteome analysis by nano LC-MS/MS confirmed that several transcripts are expressed in the glands. The database presented herein is accessible online at http://exon.niaid.nih.gov/transcriptome/D_rotundus/Supplemental-web.xlsx. These results reveal that bat saliva emerges as a novel source of modulators of vascular biology. BIOLOGICAL SIGNIFICANCE: Vampire bat saliva emerges as a novel source of antihemostatics which modulate several aspects of vascular biology.


Subject(s)
Chiroptera/metabolism , Disease Vectors , Proteome/biosynthesis , Rabies , Salivary Proteins and Peptides/biosynthesis , Submandibular Gland/metabolism , Transcriptome , Animals , Chiroptera/virology , Female , Humans , Proteomics/methods , Submandibular Gland/virology
6.
PLoS One ; 7(9): e44612, 2012.
Article in English | MEDLINE | ID: mdl-23049752

ABSTRACT

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.


Subject(s)
Ctenocephalides/genetics , Saliva/metabolism , Salivary Glands/metabolism , Salivary Proteins and Peptides/genetics , Transcriptome/genetics , Amino Acid Sequence , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Blood Coagulation/drug effects , Cats , Coagulants/chemistry , Coagulants/pharmacology , Gene Expression Profiling , Gene Library , Molecular Sequence Data , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/pharmacology , Phylogeny , Rats , Saliva/chemistry , Salivary Glands/chemistry , Salivary Proteins and Peptides/pharmacology , Sequence Alignment , Sequence Homology, Amino Acid , Xenopsylla/genetics
7.
Arterioscler Thromb Vasc Biol ; 32(9): 2185-98, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22796577

ABSTRACT

OBJECTIVE: Blood-sucking arthropods' salivary glands contain a remarkable diversity of antihemostatics. The aim of the present study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. METHODS AND RESULTS: Several L. longipalpis salivary proteins were expressed in human embryonic kidney 293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant ≈3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl(3)-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. Finally, salivary gland of sand flies was found to inhibit FXa and to interact with the enzyme. CONCLUSIONS: Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and anti-inflammatory activities. It is a useful tool to understand FXa structural features and its role in prohemostatic and proinflammatory events.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Factor Xa Inhibitors , Fibrinolytic Agents/pharmacology , Inflammation/prevention & control , Insect Proteins/pharmacology , Psychodidae/chemistry , Receptor, PAR-2/antagonists & inhibitors , Salivary Glands/chemistry , Thrombosis/prevention & control , Amino Acid Sequence , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/isolation & purification , Blood Coagulation/drug effects , Calorimetry , Cell Line, Tumor , Chlorides , Cloning, Molecular , Disease Models, Animal , Dose-Response Relationship, Drug , Factor Xa/metabolism , Female , Ferric Compounds , Fibrinolytic Agents/chemistry , Fibrinolytic Agents/isolation & purification , HEK293 Cells , Humans , Inflammation/blood , Inflammation/metabolism , Insect Proteins/chemistry , Insect Proteins/isolation & purification , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Molecular Weight , Partial Thromboplastin Time , Protein Binding , Prothrombin Time , Rats , Receptor, PAR-2/metabolism , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , Surface Plasmon Resonance , Thromboplastin/antagonists & inhibitors , Thromboplastin/metabolism , Thrombosis/blood , Thrombosis/chemically induced , Thrombosis/metabolism , Time Factors
8.
Toxins (Basel) ; 4(5): 296-322, 2012 05.
Article in English | MEDLINE | ID: mdl-22778902

ABSTRACT

Bloodsucking arthropods are a rich source of salivary molecules (sialogenins) which inhibit platelet aggregation, neutrophil function and angiogenesis. Here we review the literature on salivary disintegrins and their targets. Disintegrins were first discovered in snake venoms, and were instrumental in our understanding of integrin function and also for the development of anti-thrombotic drugs. In hematophagous animals, most disintegrins described so far have been discovered in the salivary gland of ticks and leeches. A limited number have also been found in hookworms and horseflies, and none identified in mosquitoes or sand flies. The vast majority of salivary disintegrins reported display a RGD motif and were described as platelet aggregation inhibitors, and few others as negative modulator of neutrophil or endothelial cell functions. This notably low number of reported disintegrins is certainly an underestimation of the actual complexity of this family of proteins in hematophagous secretions. Therefore an algorithm was created in order to identify the tripeptide motifs RGD, KGD, VGD, MLD, KTS, RTS, WGD, or RED (flanked by cysteines) in sialogenins deposited in GenBank database. The search included sequences from various blood-sucking animals such as ticks (e.g., Ixodes sp., Argas sp., Rhipicephalus sp., Amblyommasp.), tabanids (e.g., Tabanus sp.), bugs (e.g., Triatoma sp., Rhodnius prolixus), mosquitoes (e.g., Anopheles sp., Aedes sp., Culex sp.), sand flies (e.g., Lutzomyia sp., Phlebotomus sp.), leeches (e.g., Macrobdella sp., Placobdella sp.) and worms (e.g., Ancylostoma sp.). This approach allowed the identification of a remarkably high number of novel putative sialogenins with tripeptide motifs typical of disintegrins (>450 sequences) whose biological activity remains to be verified. This database is accessible online as a hyperlinked worksheet and displays biochemical, taxonomic, and gene ontology aspects for each putative disintegrin. It is also freely available for download (right click with the mouse) at links http://exon.niaid.nih.gov/transcriptome/RGD/RGD-Peps-WEB.xlsx (web version) and http://exon.niaid.nih.gov/transcriptome/RGD/RGD-sialogenins.zip (stand alone version).


Subject(s)
Disintegrins/chemistry , Amino Acid Motifs , Animals , Disintegrins/metabolism , Oligopeptides , Salivary Glands/metabolism
9.
Am J Trop Med Hyg ; 86(6): 1005-14, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22665609

ABSTRACT

Triatoma matogrossensis is a Hemiptera that belongs to the oliveirai complex, a vector of Chagas' disease that feeds on vertebrate blood in all life stages. Hematophagous insects' salivary glands (SGs) produce potent pharmacologic compounds that counteract host hemostasis, including anticlotting, antiplatelet, and vasodilatory molecules. Exposure to T. matogrossensis was also found to be a risk factor associated with the endemic form of the autoimmune skin disease pemphigus foliaceus, which is described in the same regions where Chagas' disease is observed in Brazil. To obtain a further insight into the salivary biochemical and pharmacologic diversity of this kissing bug and to identify possible allergens that might be associated with this autoimmune disease, a cDNA library from its SGs was randomly sequenced. We present the analysis of a set of 2,230 (SG) cDNA sequences, 1,182 of which coded for proteins of a putative secretory nature.


Subject(s)
Autoimmune Diseases/epidemiology , Pemphigus/epidemiology , Transcriptome , Triatoma/genetics , Amino Acid Sequence , Animals , Autoimmune Diseases/parasitology , Autoimmune Diseases/pathology , Brazil , Computational Biology , Gene Library , Insect Proteins/genetics , Insect Proteins/metabolism , Insect Vectors/genetics , Insect Vectors/metabolism , Molecular Sequence Data , Multigene Family , Pemphigus/parasitology , Pemphigus/pathology , Salivary Glands/metabolism , Sequence Analysis, DNA , Triatoma/classification
10.
J Med Entomol ; 49(3): 563-72, 2012 May.
Article in English | MEDLINE | ID: mdl-22679863

ABSTRACT

The kissing bug Triatoma rubida (Uhler, 1894) is found in southwestern United States and parts of Mexico where it is found infected with Trypanosoma cruzi, invades human dwellings and causes allergies from their bites. Although the protein salivary composition of several triatomine species is known, not a single salivary protein sequence is known from T. rubida. Furthermore, the salivary diversity of related hematophagous arthropods is very large probably because of the immune pressure from their hosts. Here we report the sialotranscriptome analysis of T. rubida based on the assembly of 1,820 high-quality expressed sequence tags, 51% of which code for putative secreted peptides, including lipocalins, members of the antigen five family, apyrase, hemolysin, and trialysin families. Interestingly, T. rubida lipocalins are at best 40% identical in primary sequence to those of T. protracta, a kissing bug that overlaps its range with T. rubida, indicating the diversity of the salivary lipocalins among species of the same hematophagous genus. We additionally found several expressed sequence tags coding for proteins of clear Trypanosoma spp. origin. This work contributes to the future development of markers of human and pet exposure to T. rubida and to the possible development of desensitization therapies. Supp. Data 1 and 2 (online only) of the transcriptome and deducted protein sequences can be obtained from http://exon.niaid.nih.gov/transcriptome/Trubida/Triru-S1-web.xlsx and http://exon.niaid.nih.gov/transcriptome/Trubida/Triru-S2-web.xlsx.


Subject(s)
Transcriptome , Triatoma/metabolism , Animals , Antigens/genetics , Antigens/isolation & purification , Antigens/metabolism , Chagas Disease , Expressed Sequence Tags , Insect Vectors/genetics , Insect Vectors/immunology , Insect Vectors/metabolism , Saliva/chemistry , Salivary Glands/metabolism , Triatoma/genetics , Triatoma/immunology
11.
Thromb Haemost ; 107(1): 111-23, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22159626

ABSTRACT

Salivary glands from haematophagous 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-haemostatic prostaglandins. Our results demonstrate that triplatin inhibits platelet aggregation induced by low doses of collagen, thromboxane A2 (TXA(2)) 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, cTXA(2), TXB(2), U46619 or prostaglandin (PG)H(2) mimetic (U51605). Consistent with its ligand specificity, triplatin induces relaxation of rat aorta contracted with U46619. Triplatin also interacts with PGF(2α) and PGJ(2), 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 high-density lipoprotein, ApoD was tested as a putative TXA(2)-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 elucidated without ambiguity as a novel TXA(2)- and PGF(2α)- binding protein. It conceivably blocks platelet aggregation and vasoconstriction, thus contributing to successful blood feeding at the vector-host interface.


Subject(s)
Platelet Aggregation Inhibitors/pharmacology , Platelet Membrane Glycoproteins/chemistry , Salivary Glands/metabolism , Salivary Proteins and Peptides/therapeutic use , Thromboxane A2/metabolism , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology , Amines/chemistry , Animals , Arachidonic Acid/metabolism , Horses , Humans , Ligands , Models, Biological , Platelet Aggregation/drug effects , Prostaglandins H/pharmacology , Protein Binding , Snakes
12.
J Proteomics ; 74(9): 1693-700, 2011 Aug 24.
Article in English | MEDLINE | ID: mdl-21601023

ABSTRACT

Panstrongylus megistus, a vector for the Chagas disease parasite Trypanosoma cruzi, is a hematophagous bug widely distributed in South America. This ubiquitous triatomine is known to colonize different wild life habitats. Additionally, P. megistus synanthropy, preying upon mammals, birds, reptiles, and eventually being predators upon insect's hemolymph probably increases its ability to survive after prolonged fasting. It was suspected that the P. megistus mechanisms of adaptation to survival might include a salivary gland complex tool-box with a diversity of pharmacologically active proteins for obtaining blood meals. Herein we describe comprehensive proteome and transcriptome of the P. megistus salivary gland. The proteomic analysis led to the identification of 159 proteins, and the transcriptome revealed 47 complete cDNAs. A diversity of protein functions associated to blood feeding was identified. The most prevalent proteins were related to blood clotting, anti-platelet aggregation and anti-vasoconstriction activities, which correlate with the insect's ability to obtain meals from different sources. Moreover, a gene of resistance to insecticides was identified. These features augments the comprehension towards P. megistus enormous capacity to survive in adverse wild life-changing habitats.


Subject(s)
Feeding Behavior , Insect Proteins/analysis , Panstrongylus/chemistry , Salivary Proteins and Peptides/physiology , Animals , Anticoagulants , Hemolymph , Insect Proteins/physiology , Insect Vectors , Insecticide Resistance , Panstrongylus/parasitology , Panstrongylus/physiology , Platelet Aggregation Inhibitors , Salivary Glands/chemistry , Salivary Glands/parasitology , Triatoma , Trypanosoma cruzi , Vasoconstriction/drug effects
13.
J Proteome Res ; 10(2): 669-79, 2011 Feb 04.
Article in English | MEDLINE | ID: mdl-21058630

ABSTRACT

Dipetalogaster maxima is a blood-sucking Hemiptera that inhabits sylvatic areas in Mexico. It usually takes its blood meal from lizards, but following human population growth, it invaded suburban areas, feeding also on humans and domestic animals. Hematophagous insect salivary glands produce potent pharmacologic compounds that counteract host hemostasis, including anticlotting, antiplatelet, and vasodilatory molecules. To obtain further insight into the salivary biochemical and pharmacologic complexity of this insect, a cDNA library from its salivary glands was randomly sequenced. Salivary proteins were also submitted to one- and two-dimensional gel electrophoresis (1DE and 2DE) followed by mass spectrometry analysis. We present the analysis of a set of 2728 cDNA sequences, 1375 of which coded for proteins of a putative secretory nature. The saliva 2DE proteome displayed approximately 150 spots. The mass spectrometry analysis revealed mainly lipocalins, pallidipins, antigen 5-like proteins, and apyrases. The redundancy of sequence identification of saliva-secreted proteins suggests that proteins are present in multiple isoforms or derive from gene duplications.


Subject(s)
Insect Proteins/analysis , Proteome/analysis , Triatominae/metabolism , Amino Acid Sequence , Animals , Cluster Analysis , Electrophoresis, Polyacrylamide Gel , Gene Expression Profiling , Gene Library , Insect Proteins/classification , Insect Proteins/metabolism , Mass Spectrometry , Molecular Sequence Data , Peptide Mapping , Proteome/metabolism , RNA, Messenger/chemistry , RNA, Messenger/isolation & purification , Salivary Glands/chemistry , Salivary Glands/metabolism , Sequence Alignment
14.
J Biol Chem ; 285(50): 39001-12, 2010 Dec 10.
Article in English | MEDLINE | ID: mdl-20889972

ABSTRACT

Dipetalodipin (DPTL) is an 18 kDa protein cloned from salivary glands of the triatomine Dipetalogaster maxima. DPTL belongs to the lipocalin superfamily and has strong sequence similarity to pallidipin, a salivary inhibitor of collagen-induced platelet aggregation. DPTL expressed in Escherichia coli was found to inhibit platelet aggregation by collagen, U-46619, or arachidonic acid without affecting aggregation induced by ADP, convulxin, PMA, and ristocetin. An assay based on incubation of DPTL with small molecules (e.g. prostanoids, leukotrienes, lipids, biogenic amines) followed by chromatography, mass spectrometry, and isothermal titration calorimetry showed that DPTL binds with high affinity to carbocyclic TXA(2), TXA(2) mimetic (U-46619), TXB(2), PGH(2) mimetic (U-51605), PGD(2,) PGJ(2), and PGF(2α). It also interacts with 15(S)-HETE, being the first lipocalin described to date to bind to a derivative of 15-lipoxygenase. Binding was not observed to other prostaglandins (e.g. PGE(1), PGE(2), 8-iso-PGF(2α), prostacyclin), leukotrienes (e.g. LTB(4), LTC(4), LTD(4), LTE(4)), HETEs (e.g. 5(S)-HETE, 12(S)-HETE, 20-HETE), lipids (e.g. arachidonic acid, PAF), and biogenic amines (e.g. ADP, serotonin, epinephrine, norepinephrine, histamine). Consistent with its binding specificity, DPTL prevents contraction of rat uterus stimulated by PGF(2α) and induces relaxation of aorta previously contracted with U-46619. Moreover, it inhibits angiogenesis mediated by 15(S)-HETE and did not enhance inhibition of collagen-induced platelet aggregation by SQ29548 (TXA(2) antagonist) and indomethacin. A 3-D model for DPTL and pallidipin is presented that indicates the presence of a conserved Arg(39) and Gln(135) in the binding pocket of both lipocalins. Results suggest that DPTL blocks platelet aggregation, vasoconstriction, and angiogenesis through binding to distinct eicosanoids involved in inflammation.


Subject(s)
Dinoprost/metabolism , Hydroxyeicosatetraenoic Acids/chemistry , Insect Proteins/chemistry , Lipocalins/metabolism , Neovascularization, Pathologic , Platelet Aggregation/drug effects , Saliva/metabolism , Thromboxane A2/metabolism , Triatominae/metabolism , Vasoconstriction , Animals , Aorta/drug effects , Aorta/metabolism , Female , Horses , Lipocalins/chemistry , Rats , Rats, Wistar , Salivary Glands/metabolism , Uterus/drug effects
15.
Insect Biochem Mol Biol ; 38(2): 213-32, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18207082

ABSTRACT

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 anticlotting, antiplatelet, and vasodilatory molecules. To obtain a further insight into the salivary biochemical and pharmacological complexity of this insect, a cDNA library from its SG was randomly sequenced. Also, salivary proteins were submitted to two-dimensional gel (2D-gel) electrophoresis followed by MS analysis. We present the analysis of a set of 1534 (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.


Subject(s)
Lipocalins/metabolism , Saliva/metabolism , Salivary Proteins and Peptides/metabolism , Triatoma/metabolism , Amino Acid Sequence , Animals , Apyrase/metabolism , Capsid Proteins/analysis , Chagas Disease/transmission , DNA Transposable Elements , Defensins/metabolism , Gene Expression Profiling , Gene Library , Host-Parasite Interactions/physiology , Inositol Phosphates/metabolism , Molecular Sequence Data , Nymph/metabolism , Proteomics , Receptors, Odorant/metabolism , Saliva/chemistry , Salivary Glands/metabolism , Sequence Analysis, DNA , Serpins/metabolism , Triatoma/immunology
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