Pathway infiammatorio del Sistema del Complemento nelle ascidie: sequenziamento e caratterizzazione funzionale del recettore dell’anafilatossina C3a di Ciona intestinalis
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Melillo, Daniela
Tota, Bruno
Panno, Maria Luisa
Pinto, Maria Rosaria
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Dottorato di Ricerca in Biologia Animale, XIX Ciclo,a.a.2005-2006; In mammals, the bioactive fragment C3a, released from C3 during
complement activation, is a potent mediator of inflammatory reactions and
exerts its functional activity through the specific binding to cell surface G
protein-coupled seven-transmembrane receptors. Recently, a C3a-mediated
chemotaxis of hemocytes has been demonstrated in the deuterostome
invertebrate Ciona intestinalis and an important role for this molecule in
inflammatory processes has been suggested.
In this study, we have cloned and characterized the CiC3aR molecule
involved in the CiC3a-mediated chemotaxis and studied its expression
profile. The sequence of CiC3aR, encoding a 95,394 Da seventransmembrane
domain protein, shows the highest sequence homology with
mammalian C3aRs. Northern blot analysis revealed that the CiC3aR is
expressed abundantly in the heart and neural complex and to a lesser extent,
in the ovaries, hemocytes, and larvae. Three polyclonal antibodies raised
against peptides corresponding to CiC3aR regions of the first and second
extracellular loop and of the third intracellular loop, react specifically in
Western blotting with a single band of 98-102 kDa in hemocyte protein
extracts. Immunostaining performed on circulating hemocytes with the three
specific antibodies revealed that CiC3aR is constitutively expressed only in
hyaline and granular amoebocytes. In chemotaxis experiments, the antibodies
against the first and second extracellular loop inhibited directional migration
of hemocytes toward the synthetic peptide reproducing the CiC3a C-terminal
sequence, thus providing the compelling evidence that C. intestinalis.expresses a functional C3aR homologous to the mammalian receptor. These
findings further elucidate the evolutionary origin of the vertebrate
complement-mediated proinflammatory process; Università della CalabriaSoggetto
Biologia animale; Vertebrat
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BIO/09;