Exocytosis and proteomic analysis of the vesicle content of granular hemocytes from a crayfish.

The circulating blood cells (hemocytes) of invertebrates are important in cellular immune reactions and to deliver immune factors synthesized in these cells to the external milieu. Previously, we have shown that release of vesicle contents is involved in a regulated exocytosis and here we show which proteins in the vesicles are most abundant and which are released by triggering of exocytosis using a calcium ionophore, lipopolysaccharides-peptidoglycan and peroxinectin, a cell adhesion and degranulation factor from the hemocytes. The ionophore caused release of nine proteins and six of them were characterized and found to be a masquerade-like protein, a masquerade-like serine proteinase, a mannose receptor protein, a vitelline membrane outer layer protein I, and two anti-microbial peptides. The released protein band with a mass of 76 kDa is more likely pro-phenoloxidase and/or peroxinectin. When peroxinectin was used as a trigger of exocytosis, seven proteins could be identified and for the lipopolysaccharides-peptidoglycan six proteins could be identified and all of them were also released by the ionophore treatment. Interestingly, several anti-microbial peptides were the most abundant proteins and were efficiently released by all treatments as were two masquerade-like proteins one of which is functioning as an opsonic protein.