Isolation and characterization of authentic Phe‐Met‐Arg‐Phe‐NH₂ and the novel Phe‐Thr‐Arg‐Phe‐NH₂ peptide from Nereis diversicolor

Abstract

Immunocytochemical studies have shown that peptides like Phe-Met-Arg-Phe-NH2 (FMRFamide) are widely distributed throughout the nervous system of three Nereidae. In Nereis diversicolor we have isolated these peptides from an extract of total worms by affinity chromatography and two steps of reversed-phase high-performance liquid chromatography. The sequences of the purified peptides have been determined by amino acid sequencing and on the basis of their reactivity with an anti-FMRFamide serum specific for the determinant Arg-Phe-NH,. Two primary structures have been established: Phe-Thr-Arg-Phe-NH2 (FTRFamide) and Phe-Met-Arg-Phe-NH2 (FMRFamide). Furthermore a methionine sulfoxide derivative of the FMRFamide has been identified. We have synthesized the FTRFamide peptide and in all cases, the native peptides were indistinguishable from the synthetic counterparts. The structure of the two native peptides and of the methionyl sulfoxide derivate have been confirmed by fast-atom-bombardment and tandem mass spectrometry. [6, 71, crustaceans [8-lo], insects [ll-131, and vertebrates [ l l , 141. FMRFamide immunoreactivities have been observed in various tissues: central and peripheral nervous systems, gastrointestinal tract and reproductive system. These observations suggest that a family of FMRFamide-like peptides exists within the animal kingdom. Several of the peptides responsible for these immunoreactivities have been isolated and sequenced in molluscan as well as in non-molluscan taxa. It appears that generally the FMRFamide-like invertebrates peptides are characterized by the carboxy-terminal tetrapeptide Phe-Xaa-Arg-Phe-NH2 in which Xaa is Met, Leu or Ile and they can differ in having various extended aminoterminal sequences. It was demonstrated that FMRFamide and related peptides are encoded in multiple copies by a single Correspondence to B. Baratte, Laboratoire d'Endocrinologie des Invertebres,

Immunofluorescence analysis of the internal brain anatomy of Nereis diversicolor (Polychaeta, Annelida)

“…The presence of Phe-Met-Arg-Phe-NH2 (FMRFamide) in the central nervous system of N. diversicolor was reported by Porchet and Dhainaut-Courtois (1988). In a subsequent biochemical study, Baratte et al (1991) isolated and characterized RF-amides by means of affinity chromatography in the same species. Based on ontogenetic and regeneration studies, Orrhage and co-workers provided a detailed analysis of the number and position of commissures that create an initial scaffold for the developing brain in a wide variety of polychaete representatives including N. diversicolor (summarized by Orrhage and Müller 2005).…”

“…Our observations have shown that each of the neuroactive substances tested occurs only in a limited number of perikarya and neuropil fibers, FMRFamide immunoreactivity being more widely distributed in the brain than immunoreactivity for either serotonin or histamine. This abundance of FMRF-amidergic neurons is possibly the result of cross-reactivity of the antiserum to structurally related FR-amides, e.g., FTRF-amide or a methionine sulfoxide derivative of FMRF-amide (Baratte et al 1991).…”

A review of FMRFamide- and RFamide-like peptides in metazoa

“…In addition to FMRFa, a methionyl sulphoxide derivative has been identified in both polychaetes and leeches, e.g., Nereis diversicolor and Hirudo medicinalis. Unusually, there is another tetrapeptide where T replaces M or L, FTRFamide, Table 6, (Baratte et al 1991). There are also two tetrapeptides where tyrosine replaces phenylalanine at position 1, YMRFa and YLRFa, both found in H. medicinalis (Evans et al 1991).…”

“…Compared with other phyla relatively few FLPs have been identified in annelids but these include both FMRFa and FLRFa, Table 6 ( Baratte et al 1991;Evans et al 1991). In addition to FMRFa, a methionyl sulphoxide derivative has been identified in both polychaetes and leeches, e.g., Nereis diversicolor and Hirudo medicinalis.…”

Immunoaffinity-based mass spectrometric characterization of the FMRFamide-related peptide family in the pericardial organ of Cancer borealis

“…Since then, numerous peptides with the C-terminal motif Arg-Phe-NH 2 have been discovered and characterized in a wide variety of invertebrate and vertebrate species [4-10]. Physiological studies of FMRFamide-related peptides (FaRPs) in invertebrates has revealed their highly varied biological functions including cardioexcitatory activities [1, 11-13], control of muscle contraction [13-16], neuromodulation [17, 18], and control of locomotor activity [13, 19].…”