The Anopheles gambiae salivary protein cE5 is a tight- and fast-binding thrombin inhibitor whose tissue-restricted expression is regulated at the post-transcriptional level

Mosquito saliva carries a large number of factors with anti-hemostatic, anti-inflammatory and immuno-modulatory activities. We have previously explored the salivary repertoire of the African malaria vector Anopheles gambiae and, among other components, we have identified a cDNA encoding the putative salivary protein cE5 (Arcà B. et al., 1999, Proc Natl Acad Sci U S A 96: 1516). The cE5 protein was then shown to share sequence similarity with anophelin, a thrombin inhibitor from the saliva of the New World mosquito Anopheles albimanus (Valenzuela J.G. et al., 1999, Biochemistry 38: 11209). AIM. The aim of this work was to carry out a detailed molecular and biochemical characterization of the An. gambiae cE5 protein. MATERIALS AND METHODS. The transcriptional profile of the cE5 gene was determined by RT-PCR and 3’-RACE in salivary glands and several other tissues/organs of adult male and/or female mosquitoes (midgut, ovaries, heads, malpighian tubules, hemolymph, hemocytes). The cE5 protein was expressed in recombinant form in E. coli, purified by affinity chromatography followed by ion-exchange chromatography and HPLC. An anti-cE5 polyclonal serum was obtained by immunization of BALB/c mice and employed for protein detection by Western blot analysis. The purified recombinant protein was used for biochemical assays to evaluate its anti-thrombin activity. RESULTS. The cE5 gene was found to encode mRNA isoforms carrying 3’UTRs of different length coexisting in several tissues of both male and female mosquitoes, a highly unusual profile for a gene involved in blood feeding and potentially encoding an anti-thrombin polypeptide. Interestingly, despite the widespread occurrence of cE5 transcripts in different mosquito tissues, the corresponding protein was only found in female salivary glands, where it undergoes post-translational modification. Expression of recombinant cE5 protein and assessment of its activity and inhibitory properties showed that it is a highly specific and tight-binding thrombin inhibitor, which differs from the An. albimanus orthologue for the fast-binding kinetics. CONCLUSIONS. Unusually, the tissue-specific restriction of An. gambiae cE5 is not achieved by transcriptional control, as common for mosquito salivary genes involved in hematophagy, but by post-trascriptional gene regulatory mechanism. Although An. gambiae and An. albimanus separated approximately 100 million years ago and the two proteins, anophelin and cE5, are quite divergent (43% identity, 57% similarity), their anti-thrombin function is fully preserved, since they were both found to be highly specific and tight-binding inhibitors of thrombin. On the contrary, they differ for the binding kinetics: the An. albimanus anophelin is a slow-binding thrombin inhibitor, whereas the An. gambiae cE5 protein behaves like a fast-binding inhibitor, a property that may confer a significant advantage to a blood feeder like a mosquito. Our observations provide a paradigm of post-transcriptional regulation as key determinant of tissue specificity for a protein from an important disease vector and point out that transcriptomic data should be interpreted with caution in the absence of concomitant proteomic support.