A cluster of D7-related genes is expressed in the salivary glands of the African malaria vector Anopheles gambiae.

The salivary glands of haematophagous arthropods secrete a wide array of factors whose pharmacological activities are of essential importance for their ability to feed on blood. A variety of anti-coagulants, vasodilators and inhibitors of platelet aggregation are produced in the glands as result of the intense tissue-specific gene expression taking place in this organ (James AA, 1994, Bull Inst Pasteur, 92: 133-150; Ribeiro JMC, 1995, Infect Agents Dis, 4: 143-152; Arcà B et al., 1999, Parassitologia, 41: 483-487). We have recently started a systematic analysis of the salivary glands of the malaria vector Anopheles gambiae with the aim to identify both salivary functions connected to haematophagy and receptors potentially involved in the recognition and invasion by Plasmodium sporozoites. Using the Signal Sequence Trap (SST) technique we were able to identify several cDNAs coding for secreted proteins expressed in the salivary glands of An. gambiae (Arcà B et al., 1999, Proc Natl Acad Sci USA, 96: 1516-1521). Three of these cDNA fragments showed a different degree of similarity to the D7 protein of the yellow fever mosquito Aedes aegypti and we named the corresponding genes as D7-related (D7r1, D7r2 and D7r3). The D7 protein is abundantly and exclusively expressed in the salivary glands of adult Ae. aegypti females and its function is still unknown (James AA et al., 1991, Mol Biochem Parasitol, 44:245-254). The aim of the present study is to analyze the structure and the expression of the An. gambiae D7-related genes and try to shed some light on their possible functions. Using a PCR-based method we first screened a salivary gland-enriched cDNA library and obtained the full-length cDNAs encoded by D7r1, D7r2 and D7r3. The putative polypeptides encoded by these cDNAs are respectively 165, 168 and 169 aminoacids in length, considerably shorter than the 321 aminoacids long Ae. Aegypti D7. The three D7-related proteins can be easily aligned for their entire length to the carboxy-terminal portion of D7 suggesting that this region, corresponding to exon 4 and exon 5, may be of crucial functional importance. The D7r1 putative protein is the most closely related to the Ae. Aegypti D7 (36% identity, 44% similarity) whereas D7r2 and D7r3 are the most similar to each other (62% identity, 65% similarity) and are likely the result of a more recent duplication event. Developmental expression analysis revealed that the D7-related genes are exclusively transcribed in adult females where, as previously shown by RNA in situ hybridization, they are mainly or exclusively expressed in the distal lateral lobes of the salivary glands. Because of our interest in salivary gland-specific promoters we have also isolated the corresponding genes. We show here that these three D7-related genes are closely clustered together in a 5 kb region located on the right arm of the third chromosome and that they have a very similar structure consisting of three exons separated by two small introns. Preliminary cross-hybridization analysis under moderate stringency conditions showed the presence of homologous sequences in other mosquito species suggesting that members of this protein family may be widely spread both in Anophelinae and in Culicinae. This observation, joined to the redundancy found in An. gambiae, suggests that the D7 family of proteins must play some essential role, most likely in connection to blood-feeding. In vitro expression of active recombinant protein(s) and their biochemical analysis may help to address the question concerning the functions of members of this intriguing protein family