RESUMO
Despite their physiological significance in immune and growth-controlling processes in plants and animals, no chitinolytic enzyme has been identified yet at the molecular level in Lophotrochozoa, one of the major clades of bilaterian animals. Here, we report the cloning and the characterization of a singular chitinase homologue from the bivalve mollusc Crassostrea gigas (Cg-Chit). This protein displays a modular structure including a conserved catalytic domain attached to a peritrophin-A type chitin-binding domain and an unconventional C-terminal hydrophobic sequence acting as a potential membrane anchor domain. Gene expression profiles monitored by quantitative RT-PCR in different adult tissues and during development support for the first time the involvement of such a protein in early embryonic development. Furthermore, Cg-Chit encoding gene was transcriptionally stimulated in haemocytes in response to either bacterial or LPS challenge. This suggests that Cg-Chit plays an important role as an immunity effector in molluscs.
Assuntos
Quitinases/genética , Crassostrea/embriologia , Crassostrea/genética , Crassostrea/imunologia , Sequência de Aminoácidos , Animais , Quitinases/química , Clonagem Molecular , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
A novel hypothalamic neuropeptide of the RFamide family, comprising 26 amino acids residues and thus termed 26RFa, has been recently characterized in human, and was found to be the endogenous ligand for the orphan G protein-coupled receptor GPR103. Intracerebroventricular injection of 26RFa provokes a robust increase in food intake in rodents. In the present study, we have investigated the solution conformation of 26RFa by using two-dimensional NMR spectroscopy in different media. In water, 26RFa exhibits mainly a random coil conformation although the presence of a nascent helix was detected between residues 6 and 15. In methanol, 26RFa adopts a well-defined conformation consisting of an amphipathic alpha-helical structure (Pro4-Arg17), flanked by two N- and C-terminal disordered regions. The strong conservation, from amphibians to mammals, of the amino acid sequence corresponding to the amphipathic helix and to the C-terminal flexible octapeptide of 26RFa, suggests that these two domains are crucial for the interaction of the peptide with its receptor.