Functional analyses yield detailed insight into the mechanism of thrombin inhibition by the antihemostatic salivary protein cE5 from Anopheles gambiae

Abstract

Saliva of blood-feeding arthropods carries several antihemostatic compounds whose physiological role is to facilitate successful acquisition of blood. The identification of novel natural anticoagulants and the understanding of their mechanism of action may offer opportunities for designing new antithrombotics disrupting blood clotting. We report here an in-depth structural and functional analysis of the anophelin family member cE5, a salivary protein from the major African malaria vector that specifically, tightly, and quickly binds and inhibits thrombin. Using calorimetry, functional assays, and complementary structural techniques, we show that the central region of the protein, encompassing amino acids Asp-31-Arg-62, is the region mainly responsible for α-thrombin binding and inhibition. As previously reported for the orthologue anophelin, cE5 binds both thrombin exosite I with segment Glu-35-Asp-47 and the catalytic site with the region Pro-49-Arg-56, which includes the highly conserved DPGR tetrapeptide. Moreover, the N-terminal Ala-1-Ser-30 region of cE5 (which includes an RGD tripeptide) and the additional C-terminal serine-rich Asn-63-Glu-82 region (absent in orthologues from anophelines of the New World species and) also played some functionally relevant role. Indeed, we observed decreased thrombin binding and inhibitory properties even when using the central cE5 fragment (Asp-31-Arg-62) alone. In summary, these results shed additional light on the mechanism of thrombin binding and inhibition by this family of salivary anticoagulants from anopheline mosquitoes.

A selective αvβ5 integrin antagonist hidden into the anophelin family protein cE5 from the malaria vector Anopheles gambiae

“…Such explorative experiments showed no effect (data no shown) letting hypothesize no interaction between peptide and α 5 β 1 . Altogether these results indicate that the peptide APQ30 has a significant anti‐adhesion activity against α v β 3 and α v β 5 despite the previously reported lack of activity on αIIbβ3 integrin …”

“…APQ16 surprisingly exhibited high activity and, interestingly, strong selectivity against α v β 5 integrin, letting hypothesize a potential and relevant use as theranostic agent. Under a different point of view, the ability of the cE5 protein to bind α v β 3 and α v β 5 integrins (although, as previously reported, it does not significantly interact with integrin αIIbβ3) opens up a question concerning a potential physiological role of the N‐terminal RGD. In this scenario cE5 could play not only a role as thrombin inhibitor, but perhaps also some additional functions through integrin binding (either as full protein or as its cleavage product), for example interfering with the inflammatory and/or immune response of the vertebrate host.…”

“…To analyze in greater detail the sequence requirements for integrin binding we designed and synthesized a new shorter version of APQ30, the APQ16 peptide (Figure ) encompassing the first 16 amino acid residues and fully conserved in all cE5/anophelin family members from A. gambiae species complex carrying the RGD. The length of the peptide was reduced taking care to preserve the acidic region DEED present in all members of the anophelin family . The length reduction has the advantage to facilitate the synthesis and it is an added value also for the reduction of production times and costs.…”

“…A structural and functional characterization of the salivary thrombin inhibitor cE5, a member of the anophelin family of proteins from the major African malaria vector Anopheles gambiae , was recently carried out revealing that cE5, as its orthologue from Anopheles albimanus , is an intrinsically disordered protein. To evaluate the possible role of different cE5 regions in thrombin binding and inhibition, three peptides encompassing the entire cE5 sequence were designed and their interaction with thrombin assessed: P1 (cE5A1‐S30, here named APQ30), P2 (cE5D31‐R62), and P3 (cE5N63‐E82) (Figure ) . Binding studies and thrombin inhibition assays indicated the central region corresponding to P2 (cE5D31‐R62) as the main segment responsible for both activities.…”

“…The disintegrins RGD motif is typically positioned in the loop of peptide hairpins stabilized by disulfide bonds, which is not the case for the cE5 RGD. Actually, cE5 and APQ30 were tested in in vitro assays but only little effect on platelet aggregation was observed, ruling out the functional interaction of cE5 RGD with integrin αIIbβ3 . However, the question of the possible in vivo binding of the N‐terminal cE5 RGD motif to other integrin family members remained open .…”