Crustacean hyperglycemic hormone precursor transcripts in the hemocytes of the crayfish Procambarus clarkii: novel sequence characteristics relating to gene splicing pattern and transcript stability.

It was demonstrated in a previous study (Wu et al., 2012b) that crustacean hyperglycemic hormone (CHH) gene is expressed in the hemocyte of Procambarus clarkii. In the present study, 2 additional cDNAs (CHH2-L and tCHH2) from the hemocyte and a CHH gene (CHH2) from the abdominal muscle of the same species were cloned. Analyses of the cDNA and genomic sequences suggested that, similar to other previously reported CHH genes, 2 precursor transcripts (CHH2 and CHH2-L) would be derived from CHH2 gene through a process of RNA alternative splicing, and CHH2 and CHH2-L each encode a precursor containing a signal peptide, a CHH precursor-related peptide, and a mature peptide. Further, tCHH2 sequence consists of exon I, exon II, and a truncated segment of intron II of CHH2 gene, followed by a previously unknown 3'sequence. It is suggested that, because the truncation disrupts the highly conserved RNA splice acceptor site, the truncated segment is retained within tCHH2, resulting in encoding a precursor containing the typical precursor components except the mature peptide is truncated with only 40 residues. In addition, unlike 2 other previously identified transcripts (referred to as CHH1 and CHH1-L), CHH2-L, CHH2, tCHH2 contain in the 3'-UTRs 3-5 AU-rich elements (AREs). The data showed that multiple CHH genes are expressed in crayfish hemocytes. Novel sequence characteristics of the transcripts result in an RNA splicing pattern that yields a transcript (tCHH2) encoding a precursor with an atypical truncated mature peptide and possibly leads to a different expression dynamics of the precursors encoded by the ARE-containing transcripts.

Demonstration of expression of a neuropeptide-encoding gene in crustacean hemocytes.

Crustacean hyperglycemic hormone (CHH) was originally identified in a neuroendocrine system-the X-organ/sinus gland complex. In this study, a cDNA (Prc-CHH) encoding CHH precursor was cloned from the hemocyte of the crayfish Procambarus clarkii. Analysis of tissues by a CHH-specific enzyme-linked immunosorbent assay (ELISA) confirmed the presence of CHH in hemocytes, the levels of which were much lower than those in the sinus gland, but 2 to 10 times higher than those in the thoracic and cerebral ganglia. Total hemocytes were separated by density gradient centrifugation into layers of hyaline cell (HC), semi-granular cell (SGC), and granular cell (GC). Analysis of extracts of each layer using ELISA revealed that CHH is present in GCs (202.8±86.7 fmol/mg protein) and SGCs (497.8±49.4 fmol/mg protein), but not in HCs. Finally, CHH stimulated the membrane-bound guanylyl cyclase (GC) activity of hemocytes in a dose-dependent manner. These data for the first time confirm that a crustacean neuropeptide-encoding gene is expressed in cells essential for immunity and its expression in hemocytes is cell type-specific. Effect of CHH on the membrane-bound GC activity of hemocyte suggests that hemocyte is a target site of CHH. Possible functions of the hemocyte-derived CHH are discussed.