About 24 intrinsic neurosecretory neurons within the pericardial organs (POs) of the crab Carcinus maenas produce a novel crustacean hyperglycaemic hormone (CHH)-like peptide (PO-CHH) and two CHH-precursor-related peptides (PO-CPRP I and II) as identified immunochemically and by peptide chemistry. Edman sequencing and MS revealed PO-CHH as a 73 amino acid peptide (8630Da) with a free C-terminus. PO-CHH and sinus gland CHH (SG-CHH) share an identical N-terminal sequence, positions 1–40, but the remaining sequence, positions 41–73 or 41–72, differs considerably. PO-CHH may have different precursors, as cDNA cloning of PO-derived mRNAs has revealed several similar forms, one exactly encoding the peptide. All PO-CHH cDNAs contain a nucleotide stretch coding for the SG-CHH41–76 sequence in the 3′-untranslated region (UTR). Cloning of crab testis genomic DNA revealed at least four CHH genes, the structure of which suggest that PO-CHH and SG-CHH arise by alternative splicing of precursors and possibly post-transcriptional modification of PO-CHH. The genes encode four exons, separated by three variable introns, encoding part of a signal peptide (exon I), the remaining signal peptide residues, a CPRP, the PO-CHH1–40/SG-CHH1–40 sequences (exon II), the remaining PO-CHH residues (exon III) and the remaining SG-CHH residues and a 3′-UTR (exon IV). Precursor and gene structures are more closely related to those encoding related insect ion-transport peptides than to penaeid shrimp CHH genes. PO-CHH neither exhibits hyperglycaemic activity in vivo, nor does it inhibit Y-organ ecdysteroid synthesis in vitro. From the morphology of the neurons it seems likely that novel functions remain to be discovered.
Abbreviations used: CHH, crustacean hyperglycaemic hormone; CPRP, CHH-precursor-related peptide; MALDI-TOF MS, matrix-assisted laser desorption ionization–time-of-flight MS; MT, medulla terminalis; TFA, trifluoroacetic acid; SG, sinus gland; ITP, ion-transport peptide; PO, pericardial organ; Spe-, S-pyridylethylated; EP-AspN, endoproteinase AspN; CID, collision-induced dissociation; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcriptase PCR; MIH, moult-inhibiting hormone; RP-HPLC, reversed-phase HPLC; UTR, untranslated region.