Molecular, mass spectral, and physiological analyses of orcokinins and orcokinin precursor-related peptides in the lobster Homarus americanus and the crayfish Procambarus clarkii

Abstract

Recently, cDNAs encoding prepro-orcokinins were cloned from the crayfish Procambarus clarkii; these cDNAs encode multiple copies of four orcokinin isoforms as well as several other peptides. Using the translated open reading frames of the P. clarkii transcripts as queries, five ESTs encoding American lobster Homarus americanus orthologs were identified via BLAST analysis. From these clones, three cDNAs, each encoding one of two distinct prepro-hormones, were characterized. Predicted processing of the deduced prepro-hormones would generate 13 peptides, 12 of which are conserved between the 2 precursors: the orcokinins NFDEIDRSGFGFN (3 copies), NFDEIDRSGFGFH (2 copies) and NFDEIDRSGFGFV (2 copies), FDAFTTGFGHN (an orcomyotropin-related peptide), SSEDMDRLGFGFN, GDY (SO3)DVYPE, VYGPRDIANLY and SAE. Additionally, one of two longer peptides (GPIKVRFLSAIFIPIAAPARSSPQQDAAAGYTDGAPV or APARSSPQQDAAAGYTDGAPV) is predicted from each prepro-hormone. MALDI-FTMS analyses confirmed the presence of all predicted orcokinins, the orcomyotropin-related peptide, and three precursor-related peptides, SSEDMDRLGFGFN, GDYDVYPE (unsulfated) and VYGPRDIANLY, in H. americanus neural tissues. SAE and the longer, unshared peptides were not detected. Similar complements of peptides are predicted from P. clarkii transcripts; the majority of these were detected in its neural tissues with mass spectrometry. Truncated orcokinins not predicted from any precursor were also detected in both species. Consistent with previous studies in the crayfish Orconectes limosus, NFDEIDRSGFGFN increased mid-/hindgut motility in P. clarkii. Surprisingly, the same peptide, although native to H. americanus, did not affect gut motility in this species. Together, our results provide the framework for future investigations of the regulation and physiological function of orcokinins/orcokinin precursor-related peptides in astacideans.