Abstract
Salivary gland homogenate (SGH) from the female mosquitoes Anopheles gambiae, An. stephensi, An. freeborni, An. dirus and An. albimanus were found to exhibit hemagglutinating (lectin) activity. Lectin activity was not found for male An. gambiae, or female Ae aegypti, Culex quinquefasciatus, Phlebotomus duboscqi, and Lutzomyia longipalpis. With respect to species-specificity, An. gambiae SGH agglutinates red blood cells (RBC) from humans, horse, sheep, goat, pig, and cow; it is less active for rats RBC, and not detectable for guinea-pigs or chicken RBC. Notably, lectin activity was inhibited by low concentrations of dextran sulfate 50–500 K, fucoidan, heparin, laminin, heparin sulfate proteoglycan, sialyl-containing glycans (e.g. 3′-sialyl Lewis X, and 6′-sialyl lactose), and gangliosides (e.g. GM3, GD1, GD1b, GTB1, GM1, GQ1B), but not by simple sugars. These results imply that molecule(s) in the salivary gland target sulfated glycans. SGH from An. gambiae was also found to promote agglutination of HL-60 cells which are rich in sialyl Lewis X, a glycan that decorates PSGL-1, the neutrophils receptor that interacts with endothelial cell P-selectin. Accordingly, SGH interferes with HL-60 cells adhesion to immobilized P-selectin. Because An. gambiae SGH expresses galectins, one member of this family (herein named Agalectin) was expressed in E. coli. Recombinant Agalectin behaves as a non-covalent homodimer. It does not display lectin activity, and does not interact with 500 candidates tested in a Glycan microarray. Gel-filtration chromatography of the SGH of An. gambiae identified a fraction with hemagglutinating activity, which was analyzed by 1D PAGE followed by in-gel tryptic digestion, and nano-LC MS/MS. This approach identified several genes which emerge as candidates for a lectin targeting sulfated glycans, the first with this selectivity to be reported in the SGH of a blood-sucking arthropod. The role of salivary molecules (sialogenins) with lectin activity is discussed in the context of inflammation, and parasite-vector-host interactions.
Highlights
Salivary glands of blood-sucking mosquitoes, sand flies, and ticks play a critical role in feeding [1]
Our results demonstrate that Agalectin (1 mM) did not induce agglutination in the absence or presence of 5 mM CaCl2 while it was observed with Salivary gland homogenate (SGH) of An.gambiae (Figure 3F)
Our results determined for the first time that An. gambiae hemagglutinating activity is specific for sulfated glycans
Summary
Salivary glands of blood-sucking mosquitoes, sand flies, and ticks play a critical role in feeding [1]. Blood is collected from hemorrhagic pools, as recently visualized by intravital video microscopy [2,3] During this process, saliva is released, and several salivary components counteract the host response triggered by the injury caused by bites [4,5]. Culex molestus and An. maculipennis promote hemagglutination of RBC from human, donkey, rabbit, and dog while Aedes angustivitatus agglutinates human O, A and B erythrocytes. Salivary lectins have not been molecularly characterized Identification of these components probably lays in the transcriptome and proteome (sialome) analysis of the glands, which characteristically contain several cDNAs coding for members of the C-type lectin and galectins super families [7,16]
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