Identification of proNeuropeptide FF_A peptides processed in neuronal and non‐neuronal cells and in nervous tissue

Abstract

Peptides which should be generated from the neuropeptide FF (NPFF) precursor were identified in a neuronal (human neuroblastoma SH-SY5Y) cell line and in COS-7 cells after transient transfection of the human proNPFF A cDNA and were compared with those detected in the mouse spinal cord. After reverse-phase high performance liquid chromatography of soluble material, NPFF-related peptides were immunodetected with antisera raised against NPFF and identified by using on-line capillary liquid chromatography/ nanospray ion trap tandem mass spectrometry. Neuronal and non-neuronal cells generated different peptides from the same precursor. In addition to NPFF, SQA-NPFF (Ser-Gln-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) and NPAF were identified in the human neuroblastoma while only NPFF was clearly identified in COS-7 cells. In mouse, in addition to previously detected NPFF and NPSF, SPA-NPFF (Ser-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Pheamide), the homologous peptide of SQA-NPFF, were characterized. These data on intracellular processing of proNeuropeptide FFA are discussed in regard to the known enzymatic processing mechanisms. There is a large body of evidence that NPFF exhibits antiopioid properties; in rodents, morphine-induced analgesia decreased following administration of NPFF or NPFF analogues and increased, as stress-induced analgesia, in response to anti-NPFF antibody administration [6][7][8]. In contrast, intrathecal injections of NPFF analogues induced a long-lasting analgesia [9,10] by increasing opioid peptide release in the spinal cord through the functional blockade of presynaptic delta-opioid autoreceptors [11,12]. Recent data provided evidence that opioid and NPFF endogenous systems exert a tonic activity, NPFF counteracting tonic opioid analgesia under resting conditions [13]. NPFF is also implicated in morphine tolerance, morphine abstinence and also in several physiological processes, such as body thermoregulation, food intake and blood pressure regulation [7,[14][15][16][17][18][19][20][21].These pharmacological effects are mediated by two G-protein-coupled receptors, NPFF 1 and NPFF 2 , cloned in human and rat [22][23][24][25]. Pharmacological characterization of these receptors in recombinant cell lines showed a better selectivity of peptides deduced from proNPFF A sequence for NPFF 2 receptors binding, whereas proN-PFF B -derived peptides displayed a greater affinity for NPFF 1 receptors [26]. Autoradiographic studies performed on rat CNS with highly selective radioligands revealed the localization of both receptors in central nervous areas implicated in pain transmission [27]. The existence of two peptidergic systems of neurotransmission, mediated through NPFF 1 and NPFF 2 receptor stimulation by peptides generated by proNPFF B and proNPFF A processing, respectively, could explain the complex pharmacological effects of NPFF.The characterization of NPFF-related peptides generated by NPFF precursors processing is essential to identify peptides candidate to the role of neurotransmitter. This study f...

Modulatory role of neuropeptide FF system in nociception and opiate analgesia

“…In addition to NPFF, other NPFF-related peptides, as shown in Fig. 4, have also been identified in rat, mouse, and human tissues by HPLC followed by RIA or HPLC coupled with mass spectrometry (Bonnard et al, 2001;Bonnard et al, 2003;Burlet-Schiltz et al, 2002;Yang and Iadarola, 2006). We should first address the endogenous NPFF-like peptides that are derived from the NPFF precursor, because different NPFF-related peptides seem to differ in their bioactivities.…”

“…Rat NPA-NPFF (rNPA-NPFF) as predicted from the precursor was detected in a rat spinal cord extract and furthermore the quantity of rNPA-NPFF was determined by RIA to be much more abundant than that of NPFF (Bonnard et al, 2001). Mouse SPA-NPFF (mSPA-NPFF) predicted from the mouse NPFF precursor protein was identified in mouse spinal cord extract, but the quantity seemed to be much lower than that of NPFF (Bonnard et al, 2001;Bonnard et al, 2003). Processing of human proNPFF was studied by using human neuroblastoma and COS cells transfected with human proNPFF cDNA.…”

“…Processing of human proNPFF was studied by using human neuroblastoma and COS cells transfected with human proNPFF cDNA. All three NPFF-related peptides human SQA-NPFF (hSQA-NPFF), NPFF and human NPAF (hNPAF) were identified as the processed products (Bonnard et al, 2003) (Fig. 4).…”

“…The predicted peptides according to cleavage of consensus processing sites for rat and mouse were NPAFLFQPQRF-NH 2 (rNPA-NPFF) and SPA-FLFQPQRF-NH 2 (mSPA-NPFF), respectively. NPFF as well as the predicted peptides have all been identified in tissue extracts and the processing of NPAFLFQPQRF-NH 2 and SPAFLFQPQRF-NH 2 to NPFF has been speculated (Bonnard et al, 2003) but still remains unclear and to be determined. Sequence comparisons from recent whole genome sequencing efforts show that the NPFF precursor is highly conserved across many vertebrate species.…”

Role of LPXRFamide peptide in the neuroendocrine regulation of reproduction in fish

“…To date, five groups of the RFamide peptide family have been documented in vertebrates, as follows: (a) PQRFamide peptide group, e.g. neuropeptide FF (NPFF), neuropeptide AF (NPAF) and neuropeptide SF (NPSF) [4][5][6][7][8]; (b) LPXRFamide (X = L or Q) peptide group, e.g. gonadotropin-inhibitory hormone (GnIH), mammalian RFamide-related peptides (RFRPs), frog growth hormone-releasing peptide (fGRP) and goldfish LPXRFamide peptide (gfLPXRFa) [9][10][11][12][13][14][15][16][17][18][19][20]; (c) prolactin-releasing peptide (PrRP) group, e.g.…”