Caracterização molecular e correlações estruturais com adiponectina humana de uma proteína purificada do látex de Cryptostegia grandiflora r. BR.

Abstract

Several plant species produce a fluid with milky aspect called latex, which is synthesized and stored under pressure in a system of channels formed by specialized cells termed laticifers. Latex is an emulsion of many kind of substances suspended in aqueous media. Many studies have suggested that latex compounds are involved in plant defense. Here, we describe the purification, cDNA cloning and molecular modeling of an osmotin (CgOsm) from Crytostegia grandiflora latex. Osmotin of C. grandiflora, named CgOsm, was purified by using a simple protocol consisting cation exchange chromatography on MonoS column coupled to FPLC system. The protein purity was confirmed by mass spectrometry. Total RNA was isolated from young leaves of C. grandiflora by Concert (Invitrogen) method. The yield and purity of RNA was determined spectrophotometrically and the quality of RNA was tested on 1% agarose gel. The first strand of cDNA was synthesized by the process of reverse transcription standard. The second strand of cDNA and then subsequent amplification of CgOsm cDNA was performed using forward primer (based on first seven N-terminal amino acid of CgOsm) and oligo dT as reverse primer. The PCR products were subcloned into pGEM-T vector and then introduced into Escherichia coli cloning host TOP10F'. The recombinant plasmids were purified from transformed bacterial cell and submitted to DNA sequencing. The 3D model of CgOsm was constructed using SWISS-MODEL program by homology modeling using X-ray crystal structure of act d 2 (PDB ID: 4BCT). Model quality was evaluated by MolProbity server. The computational analysis of the deduced sequence of CgOsm shown that it is an protein of apparent molecular weight about 21.966 Da and isoelectric point of 8.1. The complete structure of CgOsm is composed of three domains: domain I consists of 11 beta-strands, arranged in the shape of a beta-sandwich, domain II consists of several loops extending from domain I and stabilized by four disulfide bonds and domain III consists of a small loop with two disulfide bonds. MolProbity analysis revealed that 96.5% residues were present in most favored region. In silico analysis revealed that the structural segments of CgOsm show similarity with folding adiponectin, a human protein hormone synthesized by adipocytes whose action has implications in obesity, type II diabetes, cardiovascular disease and atherosclerosis. Tests have demonstrated that modeling CgOsm could interact with the adiponectin receptor, fact that leads to the hypothesis that CgOsm could mimic activities of human adiponectin. Thus, the results described in this study show a perspective to be explored about the pharmacological potential of this protein