Isolation of L-3-phenyllactyl-Leu-Arg-Asn-NH2 (Antho-RNamide), a sea anemone neuropeptide containing an unusual amino-terminal blocking group.

Abstract

Using a radioimmunoassay for the carboxylterminal sequence Arg-Asn-NH2, we have purified a peptide from acetic acid extracts of the sea anemone Anthopleura elegantissima. By classical amino acid analyses, mass spectrometry, and IH NMR spectroscopy, the structure of this peptide was determined as 3-phenyllactyl-Leu-Arg-Asn-NH2. By using reversed-phase HPLC and a chiral mobile phase, it was shown that the 3-phenyilactyl group had the L configuration. Immunocytochemical staining with antiserum against ArgAsn-NH2 showed that L-3-phenyflactyl-Leu-Arg-Asn-NH2 (Antho-RNamide) was localized in neurons of sea anemones. The L-3-phenyllactyl group has not been found earlier in neuropeptides of vertebrates or higher invertebrates. We propose that this residue renders Antho-RNamide resistant to nonspecific aminopeptidases, thereby increasing the stability of the peptide after neuronal release.

A comparative genomics study of neuropeptide genes in the cnidarian subclasses Hexacorallia and Ceriantharia

“…We have previously found that only neurons and no other cell types were stained with RNamide antibodies in the sea anemone C. parasitica, showing that the preprohormone is most likely expressed in neurons and that phelacLRNamide is probably a neuropeptide in all sea anemones listed in Table 3 [56].…”

“…Other neuropeptides had the C-terminal sequence RWamide, such as pQSLRWamide, or pQGLRWamide [ 9 – 11 , 54 , 55 ]. We also isolated and sequenced neuropeptides from sea anemones that had the unusual L-3-phenyllactyl residue as an N-terminal protecting group, such as L-3-phelac-LRNamide, L-3-phelac-YRIamide, or L-3-phelac-FKAamide [ 9 – 11 , 56 – 58 ]. For the last group of neuropeptides, the identification of their preprohormones would be especially rewarding, because we have not been able to clone these preprohormones using the degenerate primer PCR techniques of thirty years ago.…”

“…For the last group of neuropeptides, the identification of their preprohormones would be especially rewarding, because we have not been able to clone these preprohormones using the degenerate primer PCR techniques of thirty years ago. These preprohormones would also inform us on the biosynthesis of the N-terminal L-3-phenyllactyl group: Would this group originate from an N-terminal phenylalanine residue or would it be attached to the unprotected tripeptide by a, so far unknown, N-acyltranferase [ 9 , 10 , 56 ]?…”

Two-color double-labeling in situ hybridization of whole-mount Hydra using RNA probes for five different Hydra neuropeptide preprohormones: evidence for colocalization

“…This finding opened the possibility of staining the cnidarian neurons with neuropeptide antibodies, but it also showed that Hydra, for example, contained several neuronal populations that were neurochemically different (Grimmelikhuijzen et al 1982). Over the following years, we isolated numerous authentic neuropeptides from cnidarians and elucidated their structures (Grimmelikhuijzen and Graff 1986;Grimmelikhuijzen et al 1990Grimmelikhuijzen et al , 1996. This work also enabled us to develop better and more specific antisera to the various peptides and to visualize the cnidarian nervous system in an, at this time, unmatched quality and detail (Grimmelikhuijzen 1985;.…”

Structure of Neuropeptide Precursors in Cnidaria

“…From A. elegantissima , however, RNamides have been purified. 12 cDNAs for RNamides have not yet been cloned. It is remarkable that RNamide propeptide sequences are not contained in any of the so far cloned anthozoan precursors for antho‐RFamide.…”