Identification Of New Proteins Involved In Feeding Process From Haementeria depressaLeech Salivary Complexes By Transcriptomic And Proteomic Analysis.

Faria, F¹; Ricci-Silva, ME¹; Junqueira-de-Azevedo, ILM²; Konno, K³; Fontes, W⁴; Ho, PL²; Yamane, T⁵; Chudzinski-Tavassi, AM¹

1 Laboratório de Bioquímica e Biofísica - Instituto Butantan; 2 Centro de Biotecnologia - Instituto Butantan; 3 Laboratório de Espectometria de Massa – CAT/CEPID; 4 Laboratório de Química de Proteínas - UnB; 5 Laboratório de Toxinologia Molecular – Instituto Butantan, São Paulo, Brasil

Saliva of hematophagous contains substances able to prevent blood clotting during ingestion and/or assimilation. Usually, in the studies of the salivary complex compounds from leeches, proteins involved in blood coagulation and fibrinolytic systems are identified and characterized. Only a factor Xa inhibitor, named lefaxin, and an aggregation platelet inhibitor nitridergic pathway with fibrino(geno)lytic properties (hementerin) were characterized in salivary complexes of the H. depressa by classical biochemistry methods. The aims of this study were identify new proteins involved in feeding process of the H.depressa leeches salivary complexes by transcriptomic and proteomic analysis. The salivary complexes composition 2D-eletrophoresis comparative analysis were carried out between the salivary complexes and muscular tissue to identify the exclusive spots of saliva. Parallelly all the EST sequences were assembled in clusters and searched against GenBank and dbEST NCBI databases via web network-client blast package using BlastX and BlastN algorithms, the first EST database from leeches was constructed. The EST sequences of transcripts involved in feeding were identified and analyzed individually. From 2D-gels, some spots exclusives of the salivary complexes were selected for Mass Spectrometry analysis. These spots were trypsin digested, sequenced by ESI Q-TOF MS, identified and confirmed in EST database deposited in GenBank. Six new groups of transcripts probably involved in feeding process were identified among the most abundants transcripts in new EST database (carbonic anhydrase, hemerythrin-like, platelet aggregation inhibitor, antistasin family inhibitor, factor XIIIa inhibitor and antimicrobial peptide), and three of them were also found in proteomic analysis (carbonic anhydrase, hemerytrin-like and platelet aggregation inhibitor). All these transcripts were completely sequenced (full-lenght clones), except the carbonic anhydrase clones, the primary structures were analyzed by alignment among their isoformes and the similar proteins from GenBank; the molecular masses, pIs and signal peptides were predicted. These proteins were described by the first time in H.depressa leeches.

Supported by FAPESP and CAPES.

Correspondence to: ferfaria@butantan.gov.br