A long form of shrimp astakine transcript: Molecular cloning, characterization and functional elucidation in promoting hematopoiesis

Hemocytes play important roles in crustacean immune responses. Generation of new hemocytes (hematopoiesis) is thus necessary to maintain homeostasis which is vital to crustaceans. In vertebrates, certain cytokines have been demonstrated to regulate hematopoiesis and immune responses. In invertebrates, however, little is known about cytokines related to hematopoiesis. In the present study, we cloned an astakine molecule from hemocytic cDNA of tiger shrimp (Penaeus monodon) which was 1509 bp in length with a 5′-UTR of 143 bp, a coding region of 375 bp and a 3′-UTR of 991 bp. The present clone (GenBank accession no. EU980444) showed to be a longer form of astakine transcript with an extra insert of 671 bp in the 3′-UTR than the NCBI-recorded shrimp astakine cDNA sequence (GenBank accession no. AY787657). The deduced protein had 124 amino acid residues, including a signal peptide and one prokineticin domain. The calculated molecular weight (MW) of the mature peptide was 11,295 Da and pI was 5.2. Phylogenetically, this molecule is most similar to astakine-related molecules of arthropod including tiger shrimp astakine, crayfish astakines 1, 2a and 2b, aphid astakine-like molecule and parasitic wasp astakine-like molecule. Nested RT-PCR showed that astakine mRNA is expressed in many tissues and organs of the shrimp such as eyestalk, subcuticular epithelium, gills, heart, hepatopancreas, lymphoid organ, intestine, muscle, nerve and hemocytes. Real-time PCR further revealed that astakine mRNA is expressed mainly in the hemocytes. The astakine transcript is not inducible in the hemocytes until 24 h post LPS injection of shrimp. The recombinant protein of shrimp astakine (rPmAst) was synthesized using insect cell-baculovirus expression system. The authenticity of rPmAst protein was examined by MALDI-MS/MS spectrometry. Using ESI-MS it was determined that the MW of C-terminally histidine-tagged recombinant protein is 12,107 Da. It is 10 Da less than the computer-predicted MW (12,117 Da), allowing the formation of five pairs of disulfide bonds. Using BrdU incorporation assay it was demonstrated that the injection of rPmAst to the shrimp promoted cell proliferation in hematopoietic tissues. Therefore, we conclude that shrimp astakine functions as a cytokine that influences cell proliferation in the hematopoietic tissues.