Novel opioid peptides derived from casein (beta-casomorphins). II. Structure of active components from bovine casein peptone

Material with opioid activity had been isolated from an enzymatic casein digest. It was shown to contain a pure heptapeptide with the sequence Tyr-Pro-Phe-Pro-Gly-Pro-Ile. The identity between the opioid principle and the peptide was proven by the fact that chemical reagents or enzymes effecting one would effect the other. After carboxypeptidase Y digestion a pentapeptide, Tyr-Pro-Phe-Pro-Gly, could be isolated; this peptide showed a higher opioid activity than the heptapeptide. The opioid peptides were highly resistant towards proteolysis, even by pronase. The sequence of the hepatapeptide identified it as a fragment of bovine beta-casein. Therefore it was named beta-casomorphin.     

一种新阿片肽的分离纯化

报道了一种新阿片肽OP1的分离纯化。将重组毕赤酵母经适宜的生长和表达培养后,所得的发酵液经离心得无细胞上清液,上清液经超滤后过Sephadex G-10柱。将经Sephadex G-10柱所得具有阿片活性的粗组分用HPLC-MS分析,根据阿片肽N-端均有一个酪氨酸残基,且在肽链的第三或第四位上有一个芳香族氨基酸残基这一性质,依据分子量确定活性组分中可能存在的所有阿片肽,然后根据这些阿片肽的等电点,利用AKTA Purifier 100快速纯化系统的DEAE-阴离子交换纤维素柱将其进一步分离,活性组分再用Sephasil peptide C18反相高压液相柱分离得到活性组分OP1肽,鉴定纯度后测定其氨基酸组成。最后确定该肽的一级序列为YPFPGPIRYG,该阿片肽序列目前尚未见报道。     

Isolation and Characterization of Opioid Peptides from Rabbit Cerebellum

The rabbit cerebellum has been shown to contain significant quantities of opioid receptors consisting of both mu- and kappa-subtypes. To determine the nature of the endogenous opioid ligands in this tissue, extracts from rabbit cerebellum were separated by various chromatography techniques and fractions were assayed initially for opioid peptides with a radioimmunoassay capable of detecting all peptides with an amino-terminal Tyr-Gly-Gly-Phe sequence. This sequence is common to all mammalian opioid peptides and is critical for recognition by all known opioid receptors. Each of the three immunoreactive opioid peptide peaks detected was purified to homogeneity and subjected to amino acid composition and sequence analysis. One peak was analyzed further by mass spectrometry. This identified the major opioid peptides in the cerebellum as [Met5]enkephalin, [Leu5]enkephalin, and heptapeptide [Met5]enkephalyl-Arg6-Phe7. The comprehensiveness of this initial detection scheme in identifying biologically active opioid peptides was substantiated through subsequent analysis. Using specific radioimmunoassays for representative opioid peptides of the three opioid systems currently known, no other peptides of either the proenkephalin, proopiomelanocortin, or prodynorphin series were detected in any appreciable amounts. Collectively, these results are consistent with the position that rabbit cerebellar opioids are derived from proenkephalin. However, given that no appreciable quantities of either [Met5]enkephalyl-Arg6-Arg7-Val8-NH2 (metorphamide) or [Met5]enkephalyl-Arg6-Gly7-Leu8 were detected suggests that rabbit proenkephalin may have a slightly altered sequence and/or is differentially processed relative to other mammalian species studied.     

Isolation and sequence of a non-opioid peptide derived from proenkephalin

A non-opioid peptide derived from adrenal proenkephalin has been isolated and sequenced. The sequence of this peptide is Ser-Pro-His-Leu-Glu-Asp-Glu-Thr-Lys-Glu-Leu-Gln (Proenkephalin 168-180). This sequence represents the portion of Peptide I that is cleaved to yield Peptide E. This peptide is processed in a similar manner to the opioid peptides and is present at approximately the same level as Peptide E.     

Inhibition of nicotine-induced secretion from bovine chromaffin cells by the amidated C-terminal sequence of the opioid peptide amidorphin

The nicotine-induced release of catecholamines and opioid peptides from bovine chromaffin cells is inhibited by the amidated opioid peptide amidorphin. The active site of this inhibitory activity is located at the peptide's C-Terminus, which is, in contrast to the N-terminal sequence TYR-GLY-GLY-PHE, not responsible for the opioidergic activity of opioid peptides. The noradrenaline-secretion induced by histamine, a non-cholinergic secretagogue, has not been inhibited by amidorphin.     

Hemorphin and hemorphin-like peptides isolated from dog pancreas and sheep brain are able to potentiate bradykinin activity in vivo

Hemorphins are biologically active peptides, derived from hemoglobin, which presents a number of physiological activities. Proteolytic generation of these peptides is not fully understood; however, among their roles, is to provoke reduction on blood pressure. In this work, this particular biological effect was chosen as the monitor for the selection of mammalian vasoactive peptides. By combining high-performance liquid chromatography and mass spectrometry, including 'de novo' sequencing, several hemorphin-like peptides were identified presenting bradykinin potentiating activity. Moreover, taking LVV-hemorphin-7 as model compound, we evaluated its biological effect on blood pressure of anaesthetized rats. By summarizing all the results, it is possible to present the hemorphins as a family of proteolytically generated peptides that are able to potentiate bradykinin activity in vivo.     

Ligands for kappa-opioid and ORL1 receptors identified from a conformationally constrained peptide combinatorial library.

We have screened a synthetic peptide combinatorial library composed of 2 x 10(7) beta-turn-constrained peptides in binding assays on four structurally related receptors, the human opioid receptors mu, delta, and kappa and the opioid receptor-like ORL1. Sixty-six individual peptides were synthesized from the primary screening and tested in the four receptor binding assays. Three peptides composed essentially of unnatural amino acids were found to show high affinity for human kappa-opioid receptor. Investigation of their activity in agonist-promoted stimulation of [(35)S]guanosine 5'-3-O-(thio)triphosphate binding assay revealed that we have identified the first inverse agonist as well as peptidic antagonists for kappa-receptors. To fine-tune the potency and selectivity of these kappa-peptides we replaced their turn-forming template by other turn mimetic molecules. This "turn-scan" process allowed the discovery of compounds with modified selectivity and activity profiles. One peptide displayed comparable affinity and partial agonist activity toward all four receptors. Interestingly, another peptide showed selectivity for the ORL1 receptor and displayed antagonist activity at ORL1 and agonist activity at opioid receptors. In conclusion, we have identified peptides that represent an entirely new class of ligands for opioid and ORL1 receptors and exhibit novel pharmacological activity. This study demonstrates that conformationally constrained peptide combinatorial libraries are a rich source of ligands that are more suitable for the design of nonpeptidal drugs.     

BmK-YA, an enkephalin-like peptide in scorpion venom

By screening extracts of venom from the Asian scorpion Buthus martensii Karsch (BmK) for their abilities to activate opioid receptors, we have identified BmK-YA, an amidated peptide containing an enkephalin-like sequence. BmK-YA is encoded by a precursor that displays a signal sequence and contains four copies of BmK-YA sequences and four of His(4)-BmK-YA, all flanked by single amino acid residues. BmK-YA and His(4)-BmK-YA are amidated and thus fulfill the characteristics expected of bioactive peptides. BmK-YA can activate mammalian opioid receptors with selectivity for the δ subtype while His(4)-BmK-YA is inactive at opioid receptors. The discovery of BmK-YA suggests that scorpion venom may represent a novel source of bioactive molecules targeting G protein-coupled receptors (GPCRs) and reveal additional insights on the evolution of the opioid precursors.     

Conformational properties of deltorphin: new features of the delta-opioid receptor

Deltorphin is an opioid peptide with the sequence H-Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, recently isolated from the skin of Phyllomedusa sauvagei. Its enormous selectivity towards the delta-opioid receptor and the similarity of the N-terminal part of the sequence with that of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), a mu selective peptide isolated from the same natural source, prompted a comparative conformational study. A 1H-NMR study in two different solvent systems showed that the conformational preferences of the N-terminal sequences of the two peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects. Besides a general trend consistent with the role of the membrane in the preselection of the peptides, the present study demonstrates the crucial role played by charged residues in the interaction inside the receptors.     

Inhibition of recombinant dipeptidyl peptidase III by synthetic hemorphin-like peptides

In order to find the most effective antagonist for dipeptidyl peptidase III degrading enkephalin, we synthesized hemorphin-like pentapeptides with aliphatic or aromatic amino acids at the N-termini, such as VVYPW, LVYPW, IVYPW, YVYPW, FVYPW and WVYPW. Among those pentapeptides, IVYPW and WVYPW showed the strongest inhibitory activity toward rDPP III. The K(i) values of IVYPW and WVYPW were 0.100+/-0.011 and 0.126+/-0.015 microM (mean+/-S.E.), respectively. The order of K(i) values was Ile> or =Trp>Phe> or =Tyr>Leu>Ala>Val>Ser>Gly. rDPP III activity is inhibited in a non-competitive manner by these peptides. The peptide VYPW did not inhibit rDPP III activity, but the sequence is essential for the expression of inhibitory activity.     

Search of the human proteome for endomorphin-1 and endomorphin-2 precursor proteins

Based on the promising opioid pharmacological profile of the peptide, Tyr-Pro-Trp-Gly-NH(2) (Tyr-W-MIF), Zadina et al. [Zadina, J.E., Hackler, L., Ge, L.-J., Kastin, A.J., 1997. A potent and selective endogenous agonist for the mu-opiate receptor. Nature 386, 499-5502] synthesized and screened other Gly(4)-substituted peptides, culminating in the synthesis of Tyr-Pro-Trp-Phe-NH(2) (endomorphin-1), which displayed high affinity and selectivity for the mu-opioid receptor. The amidated peptide was then isolated from bovine brain frontal cortex, as was a related peptide, Tyr-Pro-Phe-Phe-NH(2) (endomorphin-2), that displayed similar high affinity and selectivity for the mu-opioid receptor. The biosynthesis of the endomorphins in the brain remains obscure, since the putative precursor proteins for the peptides have not been identified. With the completion of the human genome sequencing project, we hypothesized that we should uncover the biological precursors of the peptides using a bioinformatic approach to search the current human proteome for proteins that contained the endomorphin peptide sequences followed by Gly-Lys/Arg, the consensus sequence for peptide alpha-amidation and precursor cleavage. Twelve proteins were identified that contained the endomorphin-1 Tyr-Pro-Trp-Phe sequence, however none contained the Tyr-Pro-Trp-Phe-Gly sequence necessary for alpha-amidation. Twenty-two distinct proteins contained the endomorphin-2 tetrapeptide sequence, and two of those contained the sequence, Tyr-Pro-Phe-Phe-Gly, however, none contained the requisite peptide-Gly-Lys/Arg sequence. Western blot analysis using an endomorphin-2 antibody detected 4 prominent proteins in mouse brain, necessitating reinterpretation of previous immunocytolocalization studies in the brain. Screening of the current human proteome yielded no evidence for endomorphin precursor proteins based on accepted biochemical criteria.     

Cloning and characterization of Xen-dorphin prohormone from Xenopus laevis: a new opioid-like prohormone distinct from proenkephalin and prodynorphin

Opioid-like peptides mediate analgesia and induce behavioral effects such as tolerance and dependence by ligand-receptor-mediated mechanisms. The classical opioid prohormones can generate several bioactive peptides, and these divergent families of prohormones share a common well conserved ancestral opioid motif (Tyr-Gly-Gly-Phe). Evidence from pharmacological and molecular cloning studies indicates the presence of multiple isoforms of opioid ligands and receptors that are as yet uncharacterized. To identify potential new members we used the opioid motif as an anchor sequence and isolated two distinct isoforms (Xen-dorphins A and B) of an opioid prohormone from Xenopus laevis brain cDNA library. Xen-dorphin prohormones can generate multiple novel opioid ligands distinct from the known members of this family. Both isoforms are present in a wide variety of tissues including the brain. Two potential bioactive peptides, Xen-dorphin-1A and -1B, that were chemically synthesized showed opioid agonist activity in frog and rat brain membranes using a [35S]GTPgammaS assay. Initial radioligand binding experiments demonstrated that Xen-dorphin-1B binds with high affinity to opioid receptor(s) and with potential preference to the kappa-opioid receptor subtype. Cloning of the Xen-dorphin prohormone provides new evidence for the potential presence of other members in the opioid peptide superfamily.     

Measurement of Total Opioid Peptides in Rat Brain and Pituitary by Radioimmunoassay Directed at the α-N-Acetyl Derivative

A sensitive assay, which cross-reacts with and is specific for diverse opioid peptides, is described. This is based on the prior acetylation of samples and subsequent radioimmunoassay with an antiserum highly specific for the acetylated NH₂ terminus of opioid peptides. The result is a procedure that can be used to investigate multiple forms of opioid peptides in extracts of biological material. The sensitivity of the assay is ?15 fmol of β-endorphin per incubation tube, i.e., ? 100-fold greater sensitivity than the radioreceptor assay used in our laboratory. The peptide concentration required for 50% displacement of trace ranged from 0.65 nM (β-endorphin) to 1.6 nM (Met-enkephalin). The assay apparently shows an absolute requirement for a free (or acetylated) NH₂ terminus corresponding to either a Leu- or Met-enkephalin sequence. Use of the assay with and without prior acetylation of sample provides a method for estimation of the ratio of acetylated:nonacetylated opioid peptides in crude or fractionated extracts. The procedure is used to investigate the forms of opioid peptide found in rat brain and pituitary.     

The Relationship Between Structure and Activity Among Opioid Peptides

Summary Since the discovery and isolation of the endogenous opioid peptides Leu- and Met-enkephalin, structural studies have been focused on deducing the bioactive conformation of the peptide ligands. Theoretically, linear peptides can have many different backbone conformations, yet early, X-ray studies on enkephalin and its analogues showed only two different backbone conformations: extended and single β-bend. More recent reports include a third conformation for Leu-enkephalin and constrained opioid peptides from two ‘new’ classes (i.e. cyclic and ‘allaromatic’ peptides). In this report the relationship between solid-state X-ray structure and opioid peptide activity is examined. The N-terminal amine nitrogen and the two aromatic rings have previously been identified as structural features important to the biological activity of opioid peptides. From X-ray studies we find that the distances between the centroids of the aromatic rings, and between the N-terminal amine nitrogen and the centroid of the phenylalanine ring, vary over a large range. There is a discernible relationship, however, between the separation of the two rings and their orientation that correlates with activity.     

New features of the delta opioid receptor: conformational properties of deltorphin I analogues

Deltorphin I is an opioid peptide of sequence H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2, recently isolated from the skin of Phyllomedusa bicolor. Its enormous selectivity towards the delta opioid receptor and the similarity of the conformation of the N-terminal part of the sequence with that of dermorphin (H-Tyr-D-Ala-he-Gly-Tyr-Pro-Ser-NH2), a mu selective peptide, prompted the synthesis, biological evaluation and comparative conformational study of four analogs. A 1H-NMR study showed that the conformational preferences of the N-terminal sequences of all peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects in the interaction with the membrane and at the receptor site and of hydrophobicity of the C-terminal part, when structured in a folded conformation.     

Solid Phase Synthesis and Application of Labeled Peptide Derivatives: Probes of Receptor-Opioid Peptide Interactions

Solid phase synthetic methodology has been developed in our laboratory to incorporate an affinity label (a reactive functionality such as isothiocyanate or bromoacetamide) into peptides (Leelasvatanakij and Aldrich J Peptide Res 56, 80, 2000), and we have used this synthetic strategy to prepare affinity label derivatives of a variety of opioid peptides. To date side reactions have been detected only in two cases, both involving intramolecular cyclization. We have identified several peptide-based affinity labels for δ opioid receptors that exhibit wash-resistant inhibition of binding to these receptors and are valuable pharmacological tools to study opioid receptors. Even in cases where the peptide derivatives do not bind covalently to their target receptor, studying their binding has revealed subtle differences in receptor interactions with particular opioid peptide residues, especially Phe residues in the N-terminal “message” sequences. Solid phase synthetic methodology for the incorporation of other labels (e.g. biotin) into the C-terminus of peptides has also been developed in our laboratory (Kumar and Aldrich Org Lett 5, 613, 2003). These two synthetic approaches have been combined to prepare peptides containing multiple labels that can be used as tools to study peptide ligand-receptor interactions. These solid phase synthetic methodologies are versatile strategies that are applicable to the preparation of labeled peptides for a variety of targets in addition to opioid receptors.     

Purification and sequence of a non-opioid peptide derived from ovine proenkephalin: implications for possible species specific processing

A non-enkephalin containing pentadeca peptide derived from ovine adrenal proenkephalin has been purified and sequenced. The sequence of the peptide is: Phe-Ala-Glu-Pro-Leu-Pro-Ser-Glu-Glu-Glu-Gly-Glu-Ser-Tyr-Ser (preproenkephalin 237-251) representing the amino portion of peptide B (preproenkephalin 237-268). The sequence is identical to bovine preproenkephalin 237-251, differing from the corresponding human sequence at positions 240 and 244. This peptide can be generated by a processing event common to other opioid peptides and is present in chromaffin granules in significant amounts. The presence of this peptide in substantial quantities suggests a possible difference in proenkephalin processing between the bovine and ovine adrenal medulla.     

Opioid peptides in adrenal gland

Enkephalin-like immunoreactive peptides have been observed in adrenal glands of all species studied with the highest contents found in dogs and cows, and the lowest in rats. These peptides are located both in gland cells and in afferent nerve terminals. Bovine adrenal glands contain opioid peptides in many molecular forms. The peptides include a group of low molecular weight forms (M.W. <1000) which are capable of binding to the opiate receptor, and a group of high molecular weight forms (M.W. >1000) which contain enkephalin within their peptide sequence, but are devoid of opioid activity unless treated with trypsin. The physiological role(s) of the adrenal enkephalin-like material is not clear at present. However, it has been observed that nicotine-stimulated release of catecholamine from isolated chromaffin cells can be reduced by opiate agonists, suggesting that enkephalin-like peptide in nerve terminals may act on chromaffin cells. Several lines of evidence suggest that enkephalin-like peptides in gland cells can be released into the bloodstream.     

Properties and functions of human placental opioid system.

Human placental villus tissue contains opioid receptors and peptides. Kappa opioid receptors (the only type present in this tissue) were purified with retention of their binding properties. The purified kappa receptor is a glycoprotein with an apparent molecular weight of 63,000. Two opioid receptor mediated functions were identified in trophoblast tissue, namely regulation of acetylcholine and hormonal (human chorionic gonadotrophin and human placental lactogen) release. Placental content of kappa receptors increases with gestational age. Term placental content of kappa receptors correlates with route of delivery (higher in those abdominally obtained). Opioid use and/or abuse during pregnancy affects placental receptor content at delivery, as well as its mediated functions. Opioid peptides identified in placental extracts were beta-endorphin, methionine enkephalin, leucine enkephalin and dynorphins 1-8 and 1-13. Dynorphin 1-8 seem to be the predominant opioid peptide present in placental villus tissue.     

Opioid peptide biosynthesis: enzymatic selectivity and regulatory mechanisms

Certain general principles determine the biosynthesis of most biologically active peptides, including the opioid peptides, from large protein precursors. In almost all instances, the active peptide is embedded in the precursor flanked on both sides by pairs of basic amino acids. The first step in processing involves a trypsinlike enzyme, cleaving to the carboxyl terminus of basic amino acids, and leaving the active peptide with a basic amino acid on the carboxyl terminus. A carboxy-peptidase peptidase B-like enzyme then removes the remaining basic amino acid. It has been unclear whether any endopeptidases with trypsinlike activity are selective for one or another basic amino acid. Recently a soluble endopeptidase has been identified that can cleave to both the carboxyl and amino termini of basic amino acids. Enkephalin convertase (carboxypeptidase E, H) (EC 3.4.17.10) has considerable selectivity, and appears to be physiologically associated with the biosynthesis of enkephalin as well as a limited number of other neuropeptides. The turnover of opioid peptides and other neuropeptides is most effectively ascertained by measuring levels of mRNA either biochemically or by in situ hybridization. Striking dynamic alterations include a pronounced increase in levels of proenkephalin mRNA in the corpus striatum after blockade of dopamine receptors, but changes in opioid peptide mRNA after opiate addiction are less clear.