The N-terminal peptide generated after activation of prophenoloxidase affects crayfish hematopoiesis

The circulating hemocytes of invertebrates are important mediators of immunity, and hemocyte homeostasis is of high importance for survival and health of crustaceans. The prophenoloxidase (proPO)-activating system is one of the most essential immune reactions, which can be activated by pattern recognition proteins from microorganisms. Activation of proPO by the proPO activating enzyme generates an N-terminal peptide, with cleavage site after Arg176, as well as the active enzyme phenoloxidase, which is the key enzyme for melanization. In the present study we demonstrate a role for the N-terminal proPO-peptide in hematopoiesis. Injection of this proPO-peptide increased the number of circulating hemocytes and especially granular hemocytes. We also show that the reactive oxygen species (ROS) production in the anterior proliferative center was enhanced after proPO peptide injection, which is a prerequisite for rapid hemocyte release from the hematopoietic tissue. Moreover, this peptide had an effect on ROS production in in vitro cultured hematopoietic cells and induced spreading of these cells within 72 h. Taken together, our findings show a role of the N-terminal proPO peptide in stimulation of hematopoiesis in crayfish, Pacifastacus leniusculus

Role of astakine1 in regulating transglutaminase activity

Transglutaminase (TGase) has been implicated in maintaining the undifferentiated stage of hematopoietic stem cells (HSC) in the crayfish Pacifastacus leniusculus. TGase activity has been reported to be regulated by astakine1, an essential crayfish cytokine for inducing new hemocyte synthesis in hematopoietic tissue (HPT). Here, the role of astakine1 in TGase activity regulation and clotting protein (CP) cross-linking was characterized. A reduction in TGase activity was observed by the addition of purified astakine1 in vitro for both endogenous crayfish TGase and a commercial purified guinea pig liver TGase. As a result, we observed that astakine1 inhibits TGase enzyme activity and acts as a non-competitive inhibitor for the TGase enzyme. Additionally, the clotting reaction was impaired in the presence of astakine1. A decrease in TGase-mediated crosslinking of ε(γ-glutamyl)-lysine bonds was also observed in the presence of astakine1. In conclusion, this study shows that astakine1 acts as an inhibitor of TGase activity and that it also affects CP cross-linking during crayfish hematopoiesis