Functional and structural similarities of D7 proteins in the independently-evolved salivary secretions of sand flies and mosquitoes

Abstract

The habit of blood feeding evolved independently in many insect orders of families. Sand flies and mosquitoes belong to separate lineages of blood-feeding Diptera and are thus considered to have evolved the trait independently. Because of this, sand fly salivary proteins differ structurally from those of mosquitoes, and orthologous groups are nearly impossible to define. An exception is the long-form D7-like proteins that show conservation with their mosquito counterparts of numerous residues associated with the N-terminal domain binding pocket. In mosquitoes, this pocket is responsible for the scavenging of proinflammatory cysteinyl leukotrienes and thromboxanes at the feeding site. Here we show that long-form D7 proteins AGE83092 and ABI15936 from the sand fly species, Phlebotomus papatasi and P . duboscqi , respectively, inhibit the activation of platelets by collagen and the thromboxane A 2 analog U46619. Using isothermal titration calorimetry, we also demonstrate direct binding of U46619 and cysteinyl leukotrienes C 4 , D 4 and E 4 to the P . papatasi protein. The crystal structure of P . duboscqi ABI15936 was determined and found to contain two domains oriented similarly to those of the mosquito proteins. The N-terminal domain contains an apparent eicosanoid binding pocket. The C-terminal domain is smaller in overall size than in the mosquito D7s and is missing some helical elements. Consequently, it does not contain an obvious internal binding pocket for small-molecule ligands that bind to many mosquito D7s. Structural similarities indicate that mosquito and sand fly D7 proteins have evolved from similar progenitors, but phylogenetics and differences in intron/exon structure suggest that they may have acquired the ability to bind vertebrate eicosanoids independently, indicating a convergent evolution scenario.

Sergentomyia schwetzi: Salivary gland transcriptome, proteome and enzymatic activities in two lineages adapted to different blood sources

“…gambiae, are able to bind biogenic amines and eicosanoids [85][86][87]. Recently, the sand flies' D7-related were identified as binders of cysteinyl leukotrienes and thromboxane A2 [88]. Despite these new findings, we are not able to connect the SschwD7_11 up-regulation to its specific function on reptile haemostasis or coagulation.…”

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

“…Both CxD7L1 and CxD7L2 proteins showed a potent inhibitory effect on platelet aggregation, explained by distinct mechanisms. CxD7L2 inhibits platelet aggregation in the classical mechanism observed in other eicosanoid-scavenging salivary proteins 13, 14, 26, 34, 35 . CxD7L2 inhibits low dose collagen-induced platelet aggregation in a dose dependent manner but did not affect aggregation induced by high doses of collagen or convulxin.…”

“…Previous work demonstrated that members of the D7-related protein family can bind to biogenic amines and eicosanoids 10, 11, 13, 14 . Scavenging these proinflammatory and hemostatic mediators may have conferred an evolutionary adaptation to blood-feeding in mosquitoes.…”

“…D7 proteins are widely distributed in the saliva of hematophagous Nematocera, including mosquitoes, black flies, biting midges, and sand flies 8 . D7 salivary proteins antagonize the hemostasis mediators through a non-enzymatic, non-receptor-based mechanism by binding and sequestering several host hemostasis mediators 8, 10, 11, 13, 14 . This mechanism of action requires a high concentration of salivary protein at the bite site.…”

“…Binding of biogenic amines by mosquito D7 proteins has been previously reported in the literature, highlighting the importance of removing these mediators at the bite site 10, 11, 30 . Binding affinities for the different amines vary, as D7 proteins have become highly specialized for specific ligands 10, 11, 13, 14 . CxD7L2 tightly binds serotonin and epinephrine in the same range as the short D7 proteins from An.…”