Simplagrin, a Platelet Aggregation Inhibitor from Simulium nigrimanum Salivary Glands Specifically Binds to the Von Willebrand Factor Receptor in Collagen and Inhibits Carotid Thrombus Formation In Vivo

Chagas, Andrezza Campos; McPhie, Peter; San, Hong; Narum, David L.; Reiter, Karine; Tokomasu, Fuyuki; Brayner, Fábio André; Alves, Luíz Carlos; Ribeiro, José M. C.; Calvo, Eric

doi:10.1371/journal.pntd.0002947

Cited by 13 publications

(10 citation statements)

References 31 publications

(42 reference statements)

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“…To investigate the biochemical and biological activities of these proteins, CxD7L1 and CxD7L2 mature cDNA sequences were codon-optimized for a eukaryotic cell expression system and engineered to contain a 6x-histidine tag in the C-terminal end followed by a stop codon. Both genes were subcloned into a VR2001-TOPO DNA cloning plasmid (Vical Inc.) as described by Chagas et al 22 . Recombinant CxD7L1 and CxD7L2 proteins were expressed in human embryonic kidney (HEK293) cells and purified by affinity and size exclusion chromatography (Fig.…”

Section: Resultsmentioning

confidence: 99%

ADP binding by the Culex quinquefasciatus mosquito D7 salivary protein enhances blood feeding on mammals

et al. 2020

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During blood-feeding, mosquito saliva is injected into the skin to facilitate blood meal acquisition. D7 proteins are among the most abundant components of the mosquito saliva. Here we report the ligand binding specificity and physiological relevance of two D7 long proteins from Culex quinquefasciatus mosquito, the vector of filaria parasites or West Nile viruses. CxD7L2 binds biogenic amines and eicosanoids. CxD7L1 exhibits high affinity for ADP and ATP, a binding capacity not reported in any D7. We solve the crystal structure of CxD7L1 in complex with ADP to 1.97 Å resolution. The binding pocket lies between the two protein domains, whereas all known D7s bind ligands either within the Nor the C-terminal domains. We demonstrate that these proteins inhibit hemostasis in ex vivo and in vivo experiments. Our results suggest that the ADP-binding function acquired by CxD7L1 evolved to enhance blood-feeding in mammals, where ADP plays a key role in platelet aggregation.

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Section: Resultsmentioning

confidence: 99%

ADP binding by the Culex quinquefasciatus mosquito D7 salivary protein enhances blood feeding on mammals

et al. 2020

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“…Proteins of this family have a more complex amino terminus and a less complex glycine rich, acidic, carboxy terminus. A protein named Simplagrin was found in salivary transcriptomes of black flies; very divergent from mosquito aegyptins, but conserving the general structure of a complex followed by a less complex amino acid sequence and was also able to inhibit collagen-induced platelet aggregation [65], indicating this protein family was present~150 million years ago in the blood-feeding precursors of black flies and mosquitoes. This protein family is unique to Culicoidea.…”

Section: Kda Antigen/aegyptin Familymentioning

confidence: 99%

An insight into the sialotranscriptome and virome of Amazonian anophelines

et al. 2019

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Background: Saliva of mosquitoes contains anti-platelet, anti-clotting, vasodilatory, anti-complement and antiinflammatory substances that help the blood feeding process. The salivary polypeptides are at a fast pace of evolution possibly due to their relative lack of structural constraint and possibly also by positive selection on their genes leading to evasion of host immune pressure. Results: In this study, we used deep mRNA sequence to uncover for the first time the sialomes of four Amazonian anophelines species (Anopheles braziliensis, A. marajorara, A. nuneztovari and A. triannulatus) and extend the knowledge of the A. darlingi sialome. Two libraries were generated from A. darlingi mosquitoes, sampled from two localities separated~1100 km apart. A total of 60,016 sequences were submitted to GenBank, which will help discovery of novel pharmacologically active polypeptides and the design of specific immunological markers of mosquito exposure. Additionally, in these analyses we identified and characterized novel phasmaviruses and anpheviruses associated to the sialomes of A. triannulatus, A. marajorara and A. darlingi species. Conclusions: Besides their pharmacological properties, which may be exploited for the development of new drugs (e.g. anti-thrombotics), salivary proteins of blood feeding arthropods may be turned into tools to prevent and/or better control vector borne diseases; for example, through the development of vaccines or biomarkers to evaluate human exposure to vector bites. The sialotranscriptome study reported here provided novel data on four New World anopheline species and allowed to extend our knowledge on the salivary repertoire of A. darlingi. Additionally, we discovered novel viruses following analysis of the transcriptomes, a procedure that should become standard within future RNAseq studies.

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“…To investigate the biochemical and biological activities of these proteins, CxD7L1 and CxD7L2 mature cDNA sequences were codon optimized for a eukaryotic cell expression system and engineered to contain a 6x-histidine tag in the C-terminal end followed by a stop codon. Both genes were subcloned into a VR2001-TOPO DNA cloning plasmid (Vical Inc) as described in Chagas et al 22 . Recombinant CxD7L1 and CxD7L2 proteins were expressed in human embryonic kidney (HEK293) cells and purified by affinity and size exclusion chromatography (Fig.…”

Section: Resultsmentioning

confidence: 99%

Novel function acquired by theCulex quinquefasciatusmosquito D7 salivary protein enhances blood feeding on mammals

Martín-Martín

Paige

Valenzuela-León

et al. 2020

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9Adult female mosquitoes require a vertebrate blood meal to develop eggs and continue their life 10 cycle. During blood feeding, mosquito saliva is injected at the bite site to facilitate blood meal 11 acquisition through anti-hemostatic compounds that counteract blood clotting, platelet 12 aggregation, vasoconstriction and host immune responses. D7 proteins are among the most 13 abundant components of the salivary glands of several blood feeding insects. They are members 14 of a family of proteins that have evolved through gene duplication events to encode D7 proteins 15 of several lengths. Here, we examine the ligand binding specificity and physiological relevance 16 of two D7 long proteins, CxD7L1 and CxD7L2, from Culex quinquefasciatus mosquitoes, the 17 vector of medical and veterinary diseases such as filariasis, avian malaria, and West Nile virus 18 infections. CxD7L1 and CxD7L2 were assayed by microcalorimetry for binding of potential host 19 ligands involved in hemostasis, including bioactive lipids, biogenic amines, and 20 nucleotides/nucleosides. CxD7L2 binds serotonin, histamine, and epinephrine with high affinity 21 as well as the thromboxane A2 analog U-46619 and several cysteinyl leukotrienes, as previously 22 described for other D7 proteins. CxD7L1 does not bind any of the ligands that are bound by 23 CxD7L2. Unexpectedly, CxD7L1 exhibited high affinity for adenine nucleotides and 24 nucleosides, a binding capacity not reported in any D7 family member. We solved the crystal 25 structure of CxD7L1 in complex with bound ADP to 1.97 Å resolution. The binding pocket for 26 ADP is located between the two domains of CxD7L1, whereas all known D7s bind ligands either 27 within the N-terminal or the C-terminal domains. We demonstrated that these two CxD7 long 28 proteins inhibit human platelet aggregation in ex vivo experiments. CxD7L1 and CxD7L2 help 29 blood feeding in mosquitoes by scavenging host molecules that promote vasoconstriction, 30 platelet aggregation, itch, and pain at the bite site. The novel ADP-binding function acquired by CxD7L1 evolved to enhance blood feeding in mammals where ADP plays a key role in platelet 32 aggregation. 33 Culex quinquefasciatus (Diptera: Culicidae) commonly known as the southern house mosquito, 35 is a vector of medical and veterinary importance of filaria parasites, including Wuchereria 36 bancrofti and Dirofilaria immitis 1, 2 and avian malaria parasites (Plasmodium relictum) 3 . They 37 also can transmit several arboviruses including Rift Valley fever, West Nile, St. Louis or 38 Western equine encephalitis viruses 4, 5 . Adult female mosquitoes need to acquire vertebrate 39 blood for egg development. During blood feeding, mosquito saliva is injected at the bite site and 40 facilitates blood meal acquisition through anti-hemostatic compounds that prevent blood clotting, 41 platelet aggregation and vasoconstriction as well as host immune responses 6 . 42D7 proteins are among the most abundant components in the salivary glands of several blood 43 feeding ar...

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Simplagrin, a Platelet Aggregation Inhibitor from Simulium nigrimanum Salivary Glands Specifically Binds to the Von Willebrand Factor Receptor in Collagen and Inhibits Carotid Thrombus Formation In Vivo

Cited by 13 publications

References 31 publications

ADP binding by the Culex quinquefasciatus mosquito D7 salivary protein enhances blood feeding on mammals

ADP binding by the Culex quinquefasciatus mosquito D7 salivary protein enhances blood feeding on mammals

An insight into the sialotranscriptome and virome of Amazonian anophelines

Novel function acquired by theCulex quinquefasciatusmosquito D7 salivary protein enhances blood feeding on mammals

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