Four novel PYFs: members of NPY/PP peptide superfamily from the eyestalk of the giant tiger prawn Penaeus monodon.

An immunocytochemical method was used for localization of pancreatic polypeptide (PP) immunoreactive substances in the eyestalk of Penaeus monodon using anti-C-terminal hexapeptide of PP (anti-PP6) antiserum. Approximately 200 neuronal cell bodies were recognized in the ganglia between the medulla interna (MI) and medulla terminalis (MT) and surrounding MT in conjunction with the neuronal processes in medulla externa (ME), MI, MT and sinus gland. About half of the PP immunoreactive neurons were also recognized by a combination of three monoclonal antibodies raised against FMRFamide-like peptides. Isolation of the PP immunoreactive substances from the eyestalk was performed using 7500 eyestalks extracted in methanol/acetic acid/water (90/1/9) followed by five to six steps of RP-HPLC separation. Dot-ELISA with anti-PP6 antiserum was used to monitor PP-like substances in various fractions during the purification processes. Four new sequences of one hexapeptide; RARPRFamide, and three nonapeptides; YSQVSRPRFamide, YAIAGRPRFamide and YSLRARPRFamide were identified, and named as Pem-PYF1-4 due to their structural similarity to the PYF found in squid Loligo vulgaris. Each of the new peptides shares four to seven common residues with the C-terminus of the squid PYF and with the NPFs found in other invertebrates. The NPY/PP superfamily as well as the FMRFamide peptide family may be present throughout vertebrates and invertebrates.

Seven novel FMRFamide-like neuropeptide sequences from the eyestalk of the giant tiger prawn Penaeus monodon.

FMRFamide-like immunoreactivity (FLI) was localized in the eyestalk of Penaeus monodon by immunohistochemistry using a combination of three anti-FMRFamide-like peptide (FLPs) monoclonal antibodies. Approximately 3000 small neuronal cell bodies in the lamina ganglionalis; 100 medium to large size at the ganglion between the medulla interna and the medulla terminalis; and 250 medium size around the medulla terminalis were stained intensely. The neuronal processes in neuropils of the medulla externa, medulla interna, medulla terminalis, sinus gland and some nerve fibers in the optic nerve were also recognized. The small cell bodies, approximately 1500 cells, anterior to the medulla externa were stained inconsistently and the neuronal processes were not observed from these cells. Isolation of FLPs from 9000 eyestalks was performed using methanol/acetic/water (90:1:9) extraction. After the extract was partially purified using C18 cartridges, it was further purified by five to seven steps of RP-HPLC using three kinds of columns: C18; C8; and cyano, and three solvent systems: acetonitrile/trifluoro acetic acid; aceonitrile/heptafluoro butyric acid; and acetonitrile/triethyl ammonium acetate. Dot-ELISA using the combination of the same antibodies was used to monitor FLPs in the fractions during purification processes. Seven new sequences of FLPs were identified which can be divided into four subgroups according to the primary structure of the C-terminus: (1) GDRNFLRFamide; (2) AYSNLNYLRFamide; (3) AQPSMRLRFamide, SQPSMRLRFamide, SMPSLRLRFamide and DGRTPALRLRFamide; and (4) GYRKPPFNGSIFamide. These data indicate the high complexity of this peptide family in which multiple forms are usually exist.

Identification of two distinct molt-inhibiting hormone-related peptides from the giant tiger prawn Penaeus monodon.

Six peptides belonging to the crustacean hyperglycemic hormone (CHH) family were isolated from extracts of the sinus glands of the giant tiger prawn Penaeus monodon by reverse-phase high-performance liquid chromatography. These were designated Pem-SGP-A to Pem-SGA-F (Pem, Penaeus monodon; SGP, sinus gland peptide) and their amino-terminal amino acid sequences were analyzed. Five of the 6 peptides (Pem-SGP-A, -B, -D, -E, and -F) exhibited similar amino acid sequences to those of CHH peptides that had been characterized previously from the same prawn species by the other research groups, while 1 peptide (Pem-SGP-C) exhibited a novel sequence. Pem-SGP-C showed sequence similarity to known putative molt-inhibiting hormones (MIHs), particularly to an MIH from the kuruma prawn Penaeus japonicus, and less similarity to the CHHs determined thus far. Two similar complementary DNAs encoding Pem-SGP-C were cloned and found to encode very similar but distinct peptides, which were named Pem-SGP-C1 and Pem-SGA-C2. The open reading frame of each cDNA consisted only of a signal peptide and an MIH-like peptide. We also cloned 2 corresponding genes, both of which consisted of 3 exons and 2 introns. Analyses by reverse transcriptase polymerase chain reaction demonstrated that both Pem-SGP-C1 and Pem-SGP-C2 transcripts were detected only in cDNA synthesized using total RNA from the eyestalk but not in that from brain, thoracic ganglia, abdominal ganglia, abdominal muscle, hepatopancreas, or heart tissue of P. monodon.