THE PRIMARY STRUCTURE OF THE POL-RFAMIDE NEUROPEPTIDE PRECURSOR PROTEIN FROM THE HYDROMEDUSA POLYORCHIS-PENICILLATUS INDICATES A NOVEL PROCESSING PROTEINASE ACTIVITY

Neuropeptides containing the C-terminal sequence Arg-Phe-NH2 (RFamide) occur throughout the Animal Kingdom and are abundant in evolutionaril y 'old' nervous systems such as those of cnidarians. From the hydromed usa Polyorchis penicillatus we have previously isolated two neuropepti des, Pol-RFamide I (< Glu-Leu-Leu-Gly-Gly-Arg-Phe-NH2) and Pol-RFamide II (< Glu-Trp-Leu-Lys-Gly-Arg-Phe-NH2). Here we describe the cloning of a common precursor protein for these peptides from P. penicillatus. The precursor protein contains one copy of Pol-RFamide I, 11 copies o f Pol-RFamide II and one putative neuropeptide sequence. The Pol-RFami de I sequence is flanked by pairs of basic residues (Arg-Lys). At the C-tetmini of all Pol-RFamide II sequences, single basic residues (Arg) occur. Paired and single basic residues are established sites for pos ttranslational precursor cleavage. At the N-termini of the Pol-RFamide II sequences, however, basic residues are lacking and, instead, eithe r single Asp (in eight cases) or single Asn residues (in three cases) occur. This means that processing must take place at Asp and/or Asn re sidues. This is firm evidence for the presence of one or more unconven tional processing enzymes. The first type of processing enzyme could b e an endoproteinase or aminopeptidase hydrolysing at the C-terminal si de of Asp residues. Proteolytic cleavage at acidic amino acid residues has previously been inferred from other cnidarian neuropeptide precur sors. The second type of processing enzyme could be an endoproteinase or aminopeptidase hydrolysing at the C-terminal side of Asn residues.