An additional round of Signal Sequence Trap discloses novel salivary gland cDNAs from the malaria vector Anopheles gambiae.

Mosquito salivary glands are an interesting organ not only for the crucial role they play in the malaria parasite sporogonic cycle but also because they produce a wide array of secreted compounds that are delivered with the saliva at the vertebrate injured site. Indeed, anti-hemostatic activities (platelet inhibitors, vasodilators and anticoagulants) are the better-known components of the hematophagous arthropods saliva and they help blood-feeding by affecting the host hemostatic response. Since a few years we have been undertaking a detailed molecular analysis of the Anopheles gambiae salivary glands with the aim of identifying genes which are specifically expressed in this organ and that encode secreted proteins and/or potential sporozoite receptors. To this purpose we have successfully employed the Signal Sequence Trap (SST) method, a strategy that allows the entrapment of cDNAs coding for proteins that contain a signal peptide, a distinguished feature of secreted and membrane-anchored molecules (Arcà et al, 1999 Proc Natl Acad Sci USA, 96: 1516-1521). Briefly, a salivary cDNA expression library is screened by transfection of COS-7 cells. cDNAs coding for signal peptide-containing proteins, such as receptors and secreted factors, can be expressed as recombinant fusion proteins on the surface of transfected COS-7 cells and can be easily detected by immunostaining with a FITC-conjugated monoclonal antibody. We report here the final outcome of a second round of SST screening that was performed after the initial subtraction by colony hybridization of the previously identified clones. 15 novel cDNAs coding for putative secreted proteins were identified. According to tissue and developmental RT-PCR expression analysis these newly identified genes could be grouped in four main categories: (i) five genes exhibited an expression profile restricted to female salivary glands and are therefore presumably implicated in blood-feeding; (ii) four genes were expressed both in female salivary glands and in adult males and likely represent genes involved in sugar feeding or in more general functions of the glands; (iii) one gene showed an enriched expression in the female glands and, finally, (iv) four additional genes were ubiquitously expressed. Full-length cDNAs corresponding to the most interesting clones were isolated and the deduced proteins were used to search databases revealing some interesting similarity. Two polypeptides (gSG6 and gSG7) showed a weak but potentially meaningful similarity to anticoagulants from the hematophagous nematode Ancylostoma caninum (AcAPs) and to a phospholipase A2 (NnPLA2) from the venom of the Chinese cobra Naja naja atra, respectively. Strikingly, both AcAps and NnPLA2 exert a FXa-directed anticoagulant activity. Two putative proteins with similarity to the previously isolated gSG1 were also identified. Therefore, the An. gambiae genome encodes five gSG1-like proteins whose functions are not understood but may be connected to blood-feeding. The overall conservation suggests that they are members of a novel protein family involved in salivary gland functions and for this reason we named these proteins as glandins. A fourth member of the D7 family of protein was also identified and its properties are described elsewhere (see abstract by Arcà B et al.). Finally, the putative protein encoded by Ag9 was similar to a family of polypeptides widely conserved among evolutionarily distant organisms from Drosophila to humans. Intriguingly, this group of proteins included also two insect proteins that act as platelet aggregation inhibitors, the salivary apyrases of Phlebotomus papatasi and Cimex lectularius. We would like to stress that several others polypeptides failed to show any similarity to known proteins suggesting that they represent previously unidentified functions. Taken together these data emphasize the complexity of the salivary secretions of hematophagous insects suggesting that several pharmacological activities still remain to be identified. It is expected that purification from salivary extracts and/or in vitro expression of recombinant proteins will allow further biochemical analyses that will certainly lead to a better understanding of the physiological functions of mosquito salivary glands.