Autor(es): Alvarenga, Patricia H.; Francischetti, Ivo M. B.; Calvo, Eric; Sa-Nunes, Anderson; Ribeiro, Jose M. C.; Andersen, John F.

Resumo: A salivary protein from a malaria-transmitting mosquito uses a single domain to bind to thromboxane A2 and cysteinyl leukotrienes and prevent blood clotting and inflammation in the host on which it feeds. The highly expressed D7 protein family of mosquito saliva has previously been shown to act as an anti-inflammatory mediator by binding host biogenic amines and cysteinyl leukotrienes (CysLTs). In this study we demonstrate that AnSt-D7L1, a two-domain member of this group from Anopheles stephensi, retains the CysLT binding function seen in the homolog AeD7 from Aedes aegypti but has lost the ability to bind biogenic amines. Unlike any previously characterized members of the D7 family, AnSt-D7L1 has acquired the important function of binding thromboxane A2 (TXA2) and its analogs with high affinity. When administered to tissue preparations, AnSt-D7L1 abrogated Leukotriene C4 (LTC4)-induced contraction of guinea pig ileum and contraction of rat aorta by the TXA2 analog U46619. The protein also inhibited platelet aggregation induced by both collagen and U46619 when administered to stirred platelets. The crystal structure of AnSt-D7L1 contains two OBP-like domains and has a structure similar to AeD7. In AnSt-D7L1, the binding pocket of the C-terminal domain has been rearranged relative to AeD7, making the protein unable to bind biogenic amines. Structures of the ligand complexes show that CysLTs and TXA2 analogs both bind in the same hydrophobic pocket of the N-terminal domain. The TXA2 analog U46619 is stabilized by hydrogen bonding interactions of the omega -5 hydroxyl group with the phenolic hydroxyl group of Tyr 52. LTC4 and occupies a very similar position to LTE4 in the previously determined structure of its complex with AeD7. As yet, it is not known what, if any, new function has been acquired by the rearranged C-terminal domain. This article presents, to our knowledge, the first structural characterization of a protein from mosquito saliva that inhibits collagen mediated platelet activation. When feeding, a female mosquito must inhibit the blood clotting and inflammatory responses of the host. To do this, the insect produces salivary proteins that neutralize key host molecules participating in clotting and inflammation. Here, we describe a salivary protein AnSt-D7L1 that scavenges both thomboxane A2 and cysteinyl leukotrienes, two substances involved in blood vessel constriction, platelet aggregation, and inflammatory responses to an insect bite. We produced this protein in bacteria and showed that it tightly binds both these molecules, inhibiting the processes in which they are involved. We then determined its structure using X-ray crystallography and showed that there is a single binding site in one domain of the protein, accommodating both thromboxane A2 and cysteinyl leukotrienes, and that this site is responsible for the scavenging effect of the protein. These studies reveal the structural features of proteins needed to bind to key molecules of potential pharmacological importance and add to our understanding of the process of mosquito blood feeding, which is essential for transmission of the malaria parasite.

Palavras-Chave: Human diseases; Malaria; Collagen; Public health; Leukotrienes; Biogenic amines; Parasites; Platelet aggregation; Vectors; Protein families; Ileum; Thromboxane A2; Inflammation; Blood coagulation; Hydrogen bonding; Structure-function relationships; Crystal structure; Saliva; Aedes aegypti; Anopheles stephensi

Imprenta: Plos Biology, v. 8, n. 11, 2010.

Descritores: Aedes aegypti - Proteins ; Aedes aegypti - Infectious diseases ; Aedes aegypti - Inflammation ; Aedes aegypti - Transmission ; Aedes aegypti - Public health

Data de Publicação: 2010