Toward a functional analysis of salivary proteins from the African 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 have identified, in the last few years, more than twenty genes whose expression is either tissue-specific or highly enriched in the Anopheles gambiae adult female salivary glands (Arcà B et al, 1999 Proc Natl Acad Sci USA, 96: 1516-1521; Lanfrancotti A et al, 2002 FEBS Letters, 517: 67-71). Surprisingly, almost half of the genes identified so far either share only weak similarity to known proteins in the databases or do not show similarity at all; they represent therefore “orphan” proteins in search for a function. For these reasons we decided to proceed toward a functional analysis and, as a preliminary step in this direction, we started to express some of these proteins in vitro in a recombinant form. We have initially focused our attention on two small putative proteins sharing low level of 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 expression system based on the yeast Pichia pastoris was chosen because it seemed to combine advantages of prokaryotic and eukaryotic systems. The cDNAs encoding gSG6 and gSG7 were cloned downstream of the methanol-inducible alcohol oxidase promoter (AOX1); moreover, a c-myc epitope and a six-histidine tag were included to facilitate detection and purification of the recombinant proteins. The initial trials to express these two recombinant proteins in small scale shake-flask cultures were unsuccessful and, in the attempt to enhance the production of these proteins in P. pastoris, we performed high-cell density cultures in a 2 liters benchtop fermentor. Using this approach we could produce and purify by affinity chromatography on Ni-columns small amounts of the two proteins that could be used for a few functional assays. Classical Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) tests were performed to verify the ability of the recombinant proteins to affect either the extrinsic or the intrinsic pathway of the coagulation cascade. However, neither PT nor APTT showed any anti-coagulation property of the recombinant gSG6 or gSG7. 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 the immune system assaying their effect on differentiation and maturation of dendritic cells. Preliminary results seem compatible with immune-suppressive properties of the two proteins.