Hematopoiesis, Kazal Inhibitors and Crustins in a Crustacean

Abstract: Hemocytes are important as storage and producers of proteins of the innate immune defence, as well as actors of the cellular immune response. Therefore the hematopoietic process is critical for survival of most invertebrates. In order to search for molecules of importance for hemocyte development in crayfish we investigated proteins in crayfish plasma, which were increased after microbial challenge. As a result we were able to identify, purify and characterize a new invertebrate cytokine named astakine, and could clearly show that this protein is important for hematopoietic development in vivo as well as in an in vitro cell culture system. Astakine contains a prokineticin (PK) domain shown for the first time in an invertebrate, however, unlike the vertebrate PKs, astakine binds to a cell surface F1 ATP synthase ? subunit located on the hematopoietic tissue (hpt) cell membranes. Extracellular ATP synthases as receptors have earlier been reported in different vertebrate cells and here we show that extracellular ATP synthase ? subunit acts as a receptor for an invertebrate cytokine and is involved in hematopoiesis.We also found two other groups of proteins, which were increased in plasma after microbial challenge and they were further characterized. A great number of different Kazal type proteinase inhibitors were produced by the hemocytes and this type of proteinase inhibitors have variable reactive sites determining the specificity of their inhibition. In crayfish Kazal inhibitors with similar reactive sites were found as a response to specific microorganisms suggesting that the crayfish Kazal proteinase inhibitors may provide enough variability to participate in diverse innate immune reactions against different pathogens.Antimicrobial peptides were synthesized by the hemocytes and were likewise released in high amount upon microbial infection and we have characterized the main group of cystein-rich crustin-like antimicrobial peptides and investigated their tissue distribution and expression pattern.