The structures of two salivary proteins from the West Nile vector Culex quinquefasciatus reveal a beta-trefoil fold with putative sugar binding properties

Abstract

Female mosquitoes require blood meals for egg development. The saliva of blood feeding arthropods contains biochemically active molecules, whose anti-hemostatic and anti-inflammatory properties facilitate blood feeding on vertebrate hosts. While transcriptomics has presented new opportunities to investigate the diversity of salivary proteins from hematophagous arthropods, many of these proteins remain functionally undescribed. Previous transcriptomic analysis of female salivary glands from Culex quinquefasciatus, an important vector of parasitic and viral infections, uncovered a 12-member family of putatively secreted proteins of unknown function, named the Cysteine and Tryptophan-Rich (CWRC) proteins. Here, we present advances in the characterization of two C. quinquefasciatus CWRC family members, CqDVP-2 and CqDVP-4, including their enrichment in female salivary glands, their specific localization within salivary gland tissues, evidence that these proteins are secreted into the saliva, and their native crystal structures, at 2.3 ​Å and 1.87 ​Å, respectively. The β-trefoil fold common to CqDVP-2 and CqDVP-4 is similar to carbohydrate-binding proteins, including the B subunit of the AB toxin, ricin, from the castor bean Ricinus communis . Further, we used a glycan array approach, which identifies carbohydrate ligands associated with inflammatory processes and signal transduction. Glycan array 300 testing identified 100 carbohydrate moieties with positive binding to CqDVP-2, and 77 glycans with positive binding to CqDVP-4. The glycan with the highest relative fluorescence intensities, which exhibited binding to both CqDVP-2 and CqDVP-4, was used for molecular docking experiments. We hypothesize that these proteins bind to carbohydrates on the surface of cells important to host immunology. Given that saliva is deposited into the skin during a mosquito bite, and acts as the vehicle for arbovirus inoculation, understanding the role of these proteins in pathogen transmission is of critical importance. This work presents the first solved crystal structures of C. quinquefasciatus salivary proteins with unknown function. These two molecules are the second and third structures reported from salivary proteins from C. quinquefasciatus , an important, yet understudied disease vector.

Aedes aegypti sialokinin facilitates mosquito blood feeding and modulates host immunity and vascular biology

“…Horizontal gene transfer has provided new tools for blood-feeding arthropods. The cysteine and tryptophan-rich protein family found in Culex (Kern et al, 2021;Ribeiro et al, 2004), Psorophora (Chagas et al, 2013), and Corethrella genuses (Ribeiro et al, 2014), and the vasodilator hormone vasopresin found in Ornithodoros ticks, have been acquired through horizontal gene transfer from prokaryotes (Kern et al, 2021;Ribeiro et al, 2004) and from the vertebrate host, respectively (Iwanaga et al, 2014). The Ae.…”

A deeper insight into the sialome of male and female Ochlerotatus triseriatus mosquitoes

“…Three biological replicates per male and per female were used for transcriptomic analysis. Saliva from starved female O. triseriatus mosquitoes was collected as described previously ( Kern et al, 2021 ). Briefly, 5- to 8-day old female O. triseriatus mosquitoes were starved for 12 h, transferred to Petri dishes, and sedated on ice for 10 min.…”

A deeper insight into the sialome of male and female Culex quinquefasciatus mosquitoes

“…Additional Western blot and immunofluorescence assays confirmed the presence of the native protein in the distal part of the lateral lobes of the salivary gland tissue, while mass spectrometry analysis of the mosquito saliva identified unique peptides from both proteins, confirming that they are indeed secreted into the host. The three-dimensional structure of both proteins was also solved by X-ray crystallography, confirming the presence of the β-trefoil fold and an initial functional screen suggests that they can bind to carbohydrates [ 11 ], however their biological function in blood feeding remains to be elucidated.…”