Novel function acquired by the Culex quinquefasciatus mosquito D7 salivary protein enhances blood feeding on mammals

Adult female mosquitoes require a vertebrate blood meal to develop eggs and continue their life cycle. During blood feeding, mosquito saliva is injected at the bite site to facilitate blood meal acquisition through anti-hemostatic compounds that counteract blood clotting, platelet aggregation, vasoconstriction and host immune responses. D7 proteins are among the most abundant components of the salivary glands of several blood feeding insects. They are members of a family of proteins that have evolved through gene duplication events to encode D7 proteins of several lengths. Here, we examine the ligand binding specificity and physiological relevance of two D7 long proteins, CxD7L1 and CxD7L2, from Culex quinquefasciatus mosquitoes, the vector of medical and veterinary diseases such as filariasis, avian malaria, and West Nile virus infections. CxD7L1 and CxD7L2 were assayed by microcalorimetry for binding of potential host ligands involved in hemostasis, including bioactive lipids, biogenic amines, and nucleotides/nucleosides. CxD7L2 binds serotonin, histamine, and epinephrine with high affinity as well as the thromboxane A2 analog U-46619 and several cysteinyl leukotrienes, as previously described for other D7 proteins. CxD7L1 does not bind any of the ligands that are bound by CxD7L2. Unexpectedly, CxD7L1 exhibited high affinity for adenine nucleotides and nucleosides, a binding capacity not reported in any D7 family member. We solved the crystal structure of CxD7L1 in complex with bound ADP to 1.97 Å resolution. The binding pocket for ADP is located between the two domains of CxD7L1, whereas all known D7s bind ligands either within the N-terminal or the C-terminal domains. We demonstrated that these two CxD7 long proteins inhibit human platelet aggregation in ex vivo experiments. CxD7L1 and CxD7L2 help blood feeding in mosquitoes by scavenging host molecules that promote vasoconstriction, 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 aggregation.

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 1. Introduction

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In previous studies 7, 8 , Culex quinquefasciatus salivary gland cDNA libraries were sequenced 81 resulting in the identification of 14 cDNA clusters with high sequence similarity to the 82 previously known two D7 long forms (D7clu1: AF420269 and D7clu12: AF420270) and a D7 83 short form (D7Clu32, AF420271). We compared the amino acid sequence of C. quinquefasciatus 84 D7 long proteins with other well characterized mosquito and sand fly D7 members, whose 85 function and structure have been solved. Exonic regions were conserved for all previously 86 studied mosquito proteins (Culex, Aedes and Anopheles) where the first exon corresponds to a 87 secretion signal peptide and the mature proteins are encoded by exons 2, 3, 4, and 5 ( Fig. 1).

Fig. 1. Multiple sequence alignment of C. quinquefasciatus D7 proteins and other related sequences.
Immunolocalization of CxD7L1 and CxD7L2 proteins in the salivary glands of C. quinquefasciatus. Salivary glands anti-CxD7L1 IgG was pre-adsorbed with CxD7L2 and anti-CxD7L2 IgG was pre-adsorbed with CxD7L1 ( Supplementary Fig. 1). Using preabsorbed antibodies allowed us to accurately localize 141 the Culex D7 long proteins within the female salivary glands. As shown in Figure 2d 146 Previous work demonstrated that members of the D7-related protein family can bind to biogenic 147 amines and eicosanoids 10,11,13,14 . Scavenging these proinflammatory and hemostatic mediators 148 may have conferred an evolutionary adaptation to blood-feeding in mosquitoes. While Culex D7 149 proteins were first described in 2003 21 and their transcripts were sequenced a year later 7 , their 150 biological activity remains unknown. The binding abilities of CxD7L1 were tested with a wide 151 panel of pro-hemostatic compounds including biogenic amines, nucleic acids, and 152 proinflammatory lipids using isothermal titration calorimetry (ITC). In contrast to its D7 153 orthologs in Aedes and Anopheles mosquitoes, CxD7L1 does not bind biogenic amines such as 154 serotonin, nor the pro-inflammatory lipids LTB4 and LTD4 or the stable analog of TXA2, U-155 46619 ( Supplementary Fig. 2). However, CxD7L1 has evolved to bind adenine-nucleosides and 156 nucleotides with high affinity (Table 1, Fig. 3), a novel function in a D7-related protein. 165 adenine (e). In panels j-f other purine and pyrimidine nucleotides and related substances showed no binding to adenosine monophosphate (i) and polyphosphate (j). The insets show the names and chemical formulas for these compounds.

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Our biochemical characterization shows that CxD7L1 specifically binds the purine nitrogenous 170 base adenine, its nucleoside (adenosine), and nucleotide derivates: AMP, ADP, and ATP, with 171 the highest affinity to ATP and ADP ( Fig. 3a-e). The binding is adenine-specific, as no binding 172 was observed with other purine or pyrimidine nucleotides such as GTP or TTP ( Fig. 3f-g).

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Although adenine is essential for binding, CxD7L1 did not bind to adenosine 3′-monophosphate

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(3'-AMP) or cyclic AMP ( Fig. 3h- between ATP and ADP and the lower affinity of AMP, which lacks the beta phosphate.

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We also investigated the anti-platelet aggregation activity of CxD7L1 and CxD7L2 using ADP

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We also used U-46619, the stable analog of TXA2 and widely accepted for platelet aggregation 313 studies 13,14,26,27 . When platelets are activated, TXA2 is synthesized from arachidonic acid 314 released from platelet membrane phospholipids. TXA2 is an unstable compound and cannot be 315 evaluated directly as a platelet aggregation agonist ex vivo. CxD7L2 inhibited U-46619-induced platelet aggregation in a dose-dependent manner. However, platelet shape change requires 317 minimal concentrations of TXA2, and it was not prevented by CxD7L2 (Fig. 8b). Shape change 318 was only abolished in the presence of 1 µM SQ29,548, a specific antagonist of the TXA2 319 receptor (Fig. 8b). This result is supported by our biochemical data showing that CxD7L2 binds 320 directly to U-46619 in vitro (Fig. 4h). However, we do not know whether this binding is retained 321 in vivo.

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To verify that this protein binds the biological active TXA2 ex vivo, we induced platelet 323 aggregation with its biosynthetic precursor, arachidonic acid, so that TXA2 would be released by 324 platelets. CxD7L2 inhibited platelet aggregation induced by arachidonic acid only at high doses 325 of protein (6 µM, Fig. 8b), most likely due to the low binding affinity observed for U-46619 and 326 arachidonic acid (Table 1). To further investigate whether this effect was a result of a direct 327 sequestering of TXA2 by CxD7L2, we pre-incubated platelets with indomethacin, a 328 cyclooxygenase-1 inhibitor, that prevents TXA2 biosynthesis. We observed almost no inhibition 329 of low dose collagen-induced platelet aggregation in the presence of CxD7L2 (Fig. 8b), 330 indicating that the anti-platelet aggregation activity of CxD7L2 is mediated by TXA2 binding.

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CxD7L1 does not bind U-46619 as shown by microcalorimetry ( Supplementary Fig. S2), but it 333 tightly binds ADP (Fig. 3b, Table 1). Platelet aggregation triggered by U-46619, arachidonic 334 acid, and low doses of collagen is highly dependent on ADP 28 . As a confirmation of this 335 dependence, CxD7L1 inhibits platelet aggregation stimulated by either U-46619 or arachidonic 336 acid as effectively as the antagonist of the TXA2 receptor SQ29,548. CxD7L1 also prevented 337 aggregation initiated by low dose of collagen in indomethacin-treated platelets (Fig. 8b).
Serotonin acts as a potentiator of platelet agonists such as ADP or collagen. Alone, serotonin can 339 initiate platelet aggregation, but in the absence of a more potent agonist, the platelets eventually 340 disaggregate (Fig. 9a). CxD7L2 tightly binds serotonin (Fig. 4a). Therefore, the initiation of 341 aggregation produced by serotonin was completely abolished in the presence of equimolar 342 concentrations of the recombinant protein (Fig. 9a). However, when a higher dose of serotonin 343 was used (10 μM), CxD7L2 was unable to sequester all the serotonin, resulting in no observed 344 inhibition of platelet aggregation (Fig. 9a). When serotonin and low doses of collagen were used 345 as aggregation agonists, CxD7L1 partially prevented aggregation, presumably due to its ADP 346 binding, while CxD7L2-serotonin binding resulted in full inhibition of platelet aggregation ( Fig.   347 9b). Serotonin also potentiated aggregation initiated by low doses of ADP (Fig. 9c). When 348 platelets were incubated with CxD7L2, the synergistic effect of serotonin and ADP in platelet 349 aggregation was abolished (Fig. 9c). CxD7L1, as a potent ADP-binder, completely abrogated 350 platelet aggregation initiated by serotonin and ADP combined. In addition, CxD7L2 partially 351 prevented aggregation initiated by serotonin and epinephrine (Fig. 9d).  3. Discussion

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An arthropod blood feeding event can be considered as a battle between the need of the 362 arthropod to acquire blood and the vertebrate host response to prevent blood loss. The outcome of this battle determines whether the arthropod can complete its life cycle, making a successful 364 blood feeding event a crucial process for the fate of the invertebrate. During a bite, arthropod 365 salivary proteins are injected into the host skin to counteract host hemostatic mediators. In this 366 work, we characterized the structure and function of the salivary D7 long proteins from C. 367 quinquefasciatus mosquitoes and described a novel mechanism of platelet aggregation inhibition 368 for a D7 salivary protein.

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CxD7L1 and CxD7L2 were found to be expressed in the distal-lateral and medial lobes of C.   C. quinquefasciatus mosquitoes that helped them to adapt to blood-feeding on mammals. Culex 497 tarsalis mosquitoes prefer to feed on birds but will readily feed on mammals in the absence of 498 their preferred host 49 . An alignment between CxD7L1 and C. tarsalis D7 long proteins showed 499 that most of the residues involved in ADP binding are conserved in C. tarsalis, suggesting that D7 proteins that bind ADP may be widespread in the genera Culex. More studies are necessary 501 to confirm this hypothesis.

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In conclusion, we determined the binding capabilities of the CxD7L1 and CxD7L2 proteins and 503 demonstrated their role in inhibiting human platelet aggregation through different mechanisms of 504 action. We identified a novel function of ADP-binding in the well-characterized D7 protein 505 family. Moreover, the structure of the complex CxD7L1-ADP was solved, showing a different 506 binding mechanism for a D7 with the binding pocket located between the N-terminal and C-507 terminal domains whereas most D7s bind ligands within one of these two respective domains.

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These proteins help blood feeding in mosquitoes by scavenging host molecules at the bite site 509 that promote vasoconstriction, platelet aggregation, itch, and pain. Accumulation of these 510 proteins in the salivary glands of females confers an evolutionary advantage for mosquito blood 511 feeding on mammals.  were centrifuged at 12,000 × g for 5 min and supernatants were kept at -80 °C until use.