Toward a functional analysis of salivary proteins from the malaria vector Anopheles gambiae: a work in progress.

The mosquito salivary glands secrete a large number of compounds with anti-haemostatic, anti-inflammatory and immuno-modulatory activity that are of high adaptive value in relationship to haematophagy. Using a selective cloning strategy we previously identified twenty-two genes whose expression is either tissue-specific or highly enriched in the Anopheles gambiae salivary glands. Surprisingly, many of them do not show significant similarity to known proteins and, therefore, appear to represent “orphan” proteins in search for a function. For these reasons, as a preliminary step toward functional analysis, we started in vitro expression of “potentially” interesting salivary proteins. We initially focused our attention on two small putative proteins sharing similarity with anticoagulants from distantly related species: (a) gSG6 shows 24% identity and 65% similarity to AcAP6, an anti-coagulant from the haematophagous nematode Ancylostoma caninum that contains a TIL domain (Trypsin-Inhibitor-Like cystein-rich domain), typical of serine protease inhibitors; (b) gSG7 shares 19% identity and 45% similarity with a secreted phospholipase A2 from the venom of the cobra Naja naja atra that also possesses anticoagulant activity. The Pichia pastoris expression system was chosen because it combines advantages of prokaryotic and eukaryotic systems. The cDNAs encoding gSG6 and gSG7 were placed under control of the methanol-inducible alcohol oxidase promoter (AOX1) and a c-myc epitope and a six-histidine tag were included to facilitate detection and purification. Small scale expression trials in shake-flask were unsuccessful and, in the attempt to enhance the production, high-cell density cultures in a benchtop fermentor were performed. In this way it was possible to obtain and purify small amounts of the two proteins and use them for a few functional assays. Classical Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) tests indicated that recombinant gSG6 and gSG7 do not seem to affect either the extrinsic or the intrinsic pathway of the coagulation cascade. Since salivary secretions of haematophagous arthropods are known to contain also immuno-modulators we are presently testing the ability of these recombinant proteins to affect differentiation and maturation of dendritic cells.