Studies on the WFDC-domain-containing protein, carcinin
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The whey acidic four-disulphide core (WFDC) domain-containing proteins are a diverse group of antimicrobials and/or anti-proteases, widespread throughout animal taxa. Carcinin was the first WFDC-domain containing protein to be isolated from a marine invertebrate on the basis of its antibacterial properties, and is the prototypic member of a large WFDC family, designated as the crustins. The previous research conducted on carcinin did not sufficiently reveal its vital role, despite its high abundance in haemolymph. In addition, the vast majority of reported crustins were investigated by studying gene transcripts and/or producing the recombinant peptide via simple bacterial expression systems. These approaches did not give the required knowledge regarding the biological role of these peptides that were assumed to be multifunctional. The present study aimed to optimise a purification method to increase the yield of the isolated pure carcinin. A specific polyclonal antibody was raised against the native peptide, being the first specific antibody against crustin to be employed to investigate the immune staining in various tissues and organs. In addition, the native purified carcinin was employed in functional studies to investigate its involvement in host defence reactions. Carcinin was detected mainly in the haemocytes, and was secreted from these cells to support the immunological functions of various tissues. A unique deposition of carcinin was detected in the gonads and the eyestalk, showing a distinct presence through different maturation stages and its importance for the regenerating tissues. Carcinin was found to affect the viability of bacterial cells by inducing pore formation. Furthermore, carcinin was shown to support phagocytosis as an opsonin, and to aid the elimination of unwanted particles including bacteria and defective cells. This study succeeded in revealing the multifunctionality of carcinin, and emphasized the importance of utilising the native protein in the characterization of novel antimicrobial peptides.