Mohamed Amiche

The dermaseptin precursors: a protein family with a common preproregion and a variable C-terminal antimicrobial domain

Preprodermaseptins are a group of antimicrobial peptide precursors found in the skin of a variety of frog species. Precursors of this family have very similar N‐terminal preprosequences followed by markedly different C‐terminal domains that correspond to mature antimicrobial peptides. Some of these peptides are 24–34 amino acids long and form well‐behaved amphipathic α‐helices, others are disulfide‐linked peptides of 20–46 residues, still others, highly hydrophobic, are the smallest antimicrobial peptides known so far being only 10–13 residues in length. All these peptides are broad‐spectrum microbicides that kill many bacteria, protozoa, yeasts and fungi by destroying or permeating the microbial membrane. In frogs belonging to the genus Phyllomedusinae, preprodermaseptins encoded peptides also include dermorphins and deltorphins, D‐amino acid‐containing heptapeptides which are very potent and specific agonists of the μ‐ or δ‐opioid receptors. The remarkable similarity between preproregions of precursors that give rise to peptides with very different primary structures, conformations and activities suggests that the corresponding genes originate from a common ancestor. The high conservation of the precursor prepropart indicates that this region must have an important function.

Isolation of dermenkephalin from amphibian skin, a high-affinity (δ-selective opioid heptapeptide containing a D-amino acid residue

The predicted amino acid sequence of the biosynthetic precursor of dermorphin, a highly potent and nearly specific μ‐opioid peptide from amphibian skin, contains four repeats of the dermorphin progenitor sequence and one single copy of a different heptapeptide sequence. We have developed a specific enzyme immunoassay and used synthetic peptides to detect and purify the new predicted heptapeptide 2.4 μg/g dry skin) from the skin of the Phyllomedusa sauvagei frog from which dermorphin was originally isolated. The identity of the novel pro‐dermorphin related peptide, Tyr‐D‐Met‐Phe‐His‐Leu‐Met‐Asp‐NH2, was established by co‐chromatography with synthetic peptides on reverse‐phase HPLC, immunological analysis, gas‐phase sequencing, mass spectrometry and by pharmacological assays. Opioid‐binding assays in vitro demonstrated that both the natural and synthetic heptapeptides displayed exceptionally high selectivity and affinity towards the δ‐opioid receptors. Because of its origin and its δ‐opioid (enkephalin) activity and specificity, this novel D‐amino acid containing peptide is named dermenkephalin.

Enter a new post-translational modification: D-amino acids in gene-encoded peptides.

The post-translational processing of peptides plays a key role in conferring biological activity on those peptides. Recently, ribosomally made peptides that contain D-amino acids at specific positions have been discovered in microorganisms as well as in vertebrates and invertebrates. This points to yet another strategy of circumventing stereochemical limitations imposed by the genetic code and conveying biological activity to otherwise inert molecules.

Characterisation and visualisation of [3H]dermorphin binding to μ opioid receptors in the rat brain

Dermorphin, Tyr-DAla-Phe-Gly-Tyr-Pro-Ser-NH2, a potent opioid peptide isolated from amphibian skin, is endowed with outstanding structural and biological features. It has no common structure with mammalian opioid peptides and is a unique example of a peptide, synthesized by an animal cell, which contains a D-amino acid in its native sequence. We have undertaken a complete evaluation of the receptor selectivity of dermorphin, together with the binding characteristics and receptor distribution of [3H]dermorphin in the rat brain. 1. Dermorphin was tested for its relative affinity to mu-, delta- and chi-opioid receptors by determining its potency in displacing the selective mu-receptor ligand [3H]Tyr-DAla-Gly-MePhe-Gly-ol (where Gly-ol = glycinol), the prototypic delta-receptor ligand [3H]Tyr-DPen-Gly-Phe-DPen (where DPen = beta, beta-dimethylcysteine) and the chi ligand [3H]ethylketocyclazocine from rat brain and/or guinea pig cerebellum membrane preparations. Inhibitory constant (Ki) values of dermorphin were 0.7 nM, 62 nM and greater than 5000 nM respectively for mu, delta and chi sites, indicating a selectivity ratio Ki(delta)/Ki(mu) = 88. Under similar conditions, Tyr-DAla-Gly-MePhe-Gly-ol, which is regarded as one of the most selective high-affinity mu-agonist available, exhibited a selectivity ratio of 84. 2. Specific binding properties of tritium-labeled dermorphin (52 Ci/mmol) were characterized in the rat brain. Equilibrium measurements performed over a large range of concentrations revealed a single homogeneous population of high-affinity binding sites (Kd = 0.46 nM; Bmax = 92 fmol/mg membrane protein). 3. Profound differences were observed in the potencies displayed by various selective opiates and opioids ligands in inhibiting the specific binding of [3H]dermorphin. The rank order of potency was in good agreement with that obtained with other mu-selective radiolabeled ligands. 4. Receptor autoradiography in vitro was used to visualize the distribution of [3H]dermorphin binding sites in rat brain. The labeling pattern paralleled that observed using other mu probes. Binding parameters and selectivity profile of [3H]dermorphin on slide-mounted sections were similar to those obtained with membrane homogenates. 5. Finally, intracerebroventricular administration of synthetic dermorphin into mice showed that this peptide is the most potent analgesic known to date, being up to 5 and 670 times more active than beta-endorphin and morphine, respectively. Higher doses induced catalepsy. The overall data collected demonstrate that dermorphin is the first among the naturally occurring peptides to be highly potent and nearly specific super-agonist towards the morphine (mu) receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular and cellular specificity of post-translational aminoacyl isomerization in the crustacean hyperglycaemic hormone family.

d‐aminoacyl residues have been detected in various animal peptides from several taxa, especially vertebrates and arthropods. This unusual polymorphism was shown to occur in isoforms of the crustacean hyperglycaemic hormone (CHH) of the American lobster because a d‐phenylalanyl residue was found in position 3 of the sequence (CHH and d‐Phe3 CHH). In the present study, we report the detailed strategy used to characterize, in the lobster neuroendocrine system, isomers of another member of the CHH family, vitellogenesis inhibiting hormone (VIH). We have demonstrated that the fourth residue is either an l‐ or a d‐ tryptophanyl residue (VIH and d‐Trp4 VIH). Furthermore, use of antisera specifically recognizing the epimers of CHH and VIH reveals that aminoacyl isomerization occurs in specialized cells of the X organ–sinus gland neurosecretory system and that the d‐forms of the two neuropeptides are not only present in the same cells, but, importantly, also are co‐packaged within the same secretory vesicles.

Specific opioid binding sites for dermorphin in rat brain: a radioreceptor assay using the tritiated hormone as primary ligand

Dermorphin, a heptapeptide amide isolated from amphibian skin, is the most potent of the naturally occurring opioid peptides. (3H)-dermorphin (52 Ci/mmol, 1294 GBq/mmol) was prepared by catalytic tritiation of the synthetic (2,5-iodotyrosyl 1,5)-dermorphin precursor. High affinity specific binding sites for dermorphin were labeled in rat brain membranes using tritiated dermorphin as primary ligand. The binding was saturable and time-dependent. Scatchard analysis revealed a single population of non-interacting high affinity sites (Kd = 0.86 nM). Dermorphin and the specific opiate antagonist naloxone inhibited specific (3H)-dermorphin binding in a concentration dependent manner. The displacement curves could be fit to a simple competitive model assuming only one population of binding sites, with IC 50 of 1.6 nM and 3.4 nM for dermorphin and naloxone, respectively. The use of tritiated dermorphin will be helpful to ascertain unequivocally the selectivity of dermorphin for the different opioid receptor subtypes in the central nervous system.

The aspartic acid in deltorphin I and dermenkephalin promotes targeting to δ-opioid receptor independently of receptor binding

Recent studies on the highly potent and selective delta-opioid agonists demenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) suggested that key structural features necessary for specific targetting to the delta-opioid receptor are located within the C-terminal halves of these naturally occurring heptapeptides. To investigate the contribution of aspartic acid 4 residue in deltorphin I and aspartic acid 7 residue in dermenkephalin to the delta-addressing ability of the C-terminal ends, fourteen analogs were synthesized and assessed for their ability to bind to mu and delta-opioid receptors in rat brain membrane homogenates. Results showed that i/ although the tetrapeptide C-terminus of dermenkephalin and deltorphin I differ in amino acid composition, they play a similar role in specifying correct addressing of these peptides to the delta-receptor, ii/ the negatively charged side chain of aspartic acid 4 residue in deltorphin I and aspartic acid 7 residue in dermenkephalin is not involved in binding contact at the delta-receptor site, nor in maintaining a delta-bioactive folding of the peptides, iii/ these side chains are, in contrast, functionally or structurally required to confer high delta-selectivity by preventing mu-site recognition and/or binding.

Opioid activity of dermenkephalin analogues in the guinea-pig myenteric plexus and the hamster vas deferens

1 To elucidate the structural features required for selective and potent action of dermenkephalin at the δ‐opioid receptor, a series of analogues of dermenkephalin and dermorphin were tested for their effectiveness in depressing electrically‐evoked contractions of the vas deferens of the hamster (δ‐opioid receptors) and the guinea‐pig myenteric plexus‐longitudinal muscle preparation (μ‐ and κ‐opioid receptors). 2 Dermenkephalin was more selective and more potent at δ‐receptors than the δ‐ligand [d‐Pen2, d‐Pen5]‐enkephalin. The responses to dermenkephalin in the hamster vas deferens were increased by addition of peptidase inhibitors; the maximum effect was obtained with 3 μm thiorphan. 3 [l‐Met2]‐dermenkephalin had 0.2% and [l‐Ala2]‐dermorphin 0.01% of the agonist activity of the corresponding endogenous peptides which have d‐amino acids in position 2. The pharmacological activity of these analogues was unaffected by inhibition of peptidases. This emphasizes the role that the d‐configuration plays in determining the bioactive folding of these highly active peptides. 4 Dermenkephalin‐(1–6)‐NH2 was more potent at δ‐receptors than at μ‐receptors whereas, dermenkephalin‐(1–4)‐NH2 is a selective μ‐agonist, having no activity at δ‐receptors. 5 Substitution of the C‐terminal tripeptide of dermorphin with the C‐terminal tripeptide of dermenkephalin abolished the μ‐receptor preference of dermorphin. The resulting hybrid peptide, Tyr‐d‐Ala‐Phe‐Gly‐Leu‐Met‐Asp‐NH2 was as potent as dermenkephalin at δ‐receptors. A shift towards a preference for δ‐receptors was obtained when the C‐terminal tetrapeptide of dermorphin was replaced by the C‐terminal tetrapeptide of dermenkephalin. 6 Substitution of Asp by Asn in position 7 of dermenkephalin caused an increase in μ‐receptor potency and a decrease in δ‐receptor potency, resulting in a 20 fold decrease in μ‐receptor selectivity. Dermenkephalin‐(1–6)‐NH2 and [Asn7]‐dermenkephalin have almost identical δ‐receptor agonist potencies and ratios of IC50 in the myenteric plexus to IC50 in the hamster vas deferens. 7 The results obtained emphasise the importance of a negative charge at the C‐terminus of dermenkephalin for selectivity at the δ‐opioid receptor. Furthermore, the hydrophobic residues Leu5and Met6may be critical in ensuring tight binding to the receptor which results in high agonist potency.