Characterization of in vitro proteolytic processing of beta-endorphin by reversed-phase HPLC

In vitro, central and peripheral proteolytic processing of beta-endorphin by membrane-bound enzymes results in the formation of specific active fragments that have been recently shown to function in behavior, intestinal motility and in the central control of urinary bladder activity. A high resolution, reversed phase high performance liquid chromatography system capable of separating 28 beta-endorphin related fragments simultaneously was used to study the time-course processing of beta-endorphin by membrane associated peptidases in the brain and regions of the small intestine. The hypothesis we tested was that a homeostatic balance between alpha- and gamma-type endorphins exists in these tissues. The results of the study show that the rate and quantity of fragments produced between the mucosa and nerve-muscle regions of the small intestine are significantly different. Metabolic rates, pattern, and the ratio of alpha/gamma-type endorphins in the brain were very similar to the nerve-muscle region of the small intestine. This suggests that beta-endorphin processing to active fragments is occurring at the nerves of the small intestine and that a specific and similar balance of alpha/gamma-type endorphin exists in the brain and gastrointestinal system at neutral pH.     

Differential in vitro metabolism of β-endorphin in schizophrenia

Biologically active peptide fragments derived from the proteolytic cleavage of β-endorphin (βE) have been shown to be present in the brain. Based on clinical results using some of these fragments in neuropsychiatric disease studies we investigated the in vitro metabolism of βE by twice-washed membrane homogenates of postmortem putamen from sex and age matched controls versus subjects with a diagnosis of schizophrenia. The present study demonstrates that frozen (−80°C) postmortem human tissues are viable for these studies and that metabolism in control tissue proceeds similarly to fresh tissues. Furthermore, a significant increase in the formation of the putative neuroleptic-like peptide fragment desenkephalin-γ-endorphin in postmortem schizophrenic putamen versus controls was shown. A significant decrease in the formation of βE 6–21 was also reported. These data suggest that an approach using postmortem human brain is possible in studying β-endorphin catabolism and is therefore applicable to other neuropeptide systems.     

Proteolysis of β-endorphin in brain tissue

The processing of β-endorphin by brain enzymes into peptides related to the behaviorally active γ- and α-type endorphins and the sequence of proteolytic events in the conversion process are described. Multiple enzyme activities contribute to the generation of the peptides with neurotropic activity. It is proposed that the processing into γ- and α-type neuropeptides is a post-secretional event. The enzymes involved may have a key role in the nature and levels of neurotropic β-endorphin fragments in the brain.     

Characterization of β-endorphin- and α-MSH-related peptides in rat heart

POMC-derived peptides and mRNA have been identified in heart tissue, although POMC processing has not been fully characterized. In the present study, we found that β-lipotropin and ACTH were localized in rat heart, although they were almost entirely converted to β-endorphin- and α-MSH-related peptides. Ion exchange HPLC analysis revealed that β-endorphin(1–31) was further processed to α-N-acetyl-β-endorphin(1–31), which comprised 35.9 ± 0.1% of total immunoreactivity, and smaller amounts of β-endorphin(1–27), β-endorphin(1–26), and their α-N-acetylated derivatives. The predominant α-MSH immunoreactive peptides coeluted with α-MSH and N,O-diacetyl-α-MSH by reverse-phase HPLC, although small amounts of ACTH(1–13)-NH₂ were also present. Thus, multiple forms of β-endorphin and α-MSH are localized in rat heart. β-Endorphin(1–31) is a minor constituent, however, indicating that nonopioid β-endorphin peptides predominate.     

Evidence for a common precursor for αMSH and β-endorphin in the intermediate lobe of the pituitary of the reptile Anolis carolinensis

Immunohistochemical studies on the pituitary of Anolis carolinensis detected ACTH-like, β-endorphin-like, and 16K fragment-like immunoreactivity in distinct clusters of cells in the anterior lobe; ACTH-like, αMSH-like, β-endorphin-like, and 16K fragment-like immunoreactivity was detected in all the cells of the intermediate lobe. Crude acid extracts of both lobes, when alayzed by radioimmunoassay, gave displacement curves in ACTH and β-endorphin assays which were parallel to the appropriate synthetic standard. Only extracts of the intermediate lobe gave parallel displacement curves in an αMSH radioimmunoassay. Extracts of both lobes crossreacted with antiserum to 16K fragment, but the displacement curves were not parallel to that of mouse 16K fragment standard. The levels of immunoreactive ACTH and β-endorphin in the intermediate lobe were approximately 8-fold higher than in the anterior lobe. Fractionation of anterior lobe and intermediate lobe extracts by either gel filtration on Sephadex G-75 in 10% formic acid or sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed multiple forms of ACTH-related and β-endorphin-related substances in both lobes. In the anterior lobe the major forms of immunoreactivity were, respectively, ACTH-sized and β-endorphin-sized. In the intermediate lobe the major forms of immunoreactivity were αMSH-sized, CLIP-sized, and β-endorphin-sized. In both lobes, antisera directed against ACTH and β-endorphin detected high molecular weight material with an apparent molecular weight slightly less than that of mouse pro-ACTH/endorphin; this material probably represents the putative common precursor for ACTH and β-endorphin in this species.     

Naltrexone reduces weight gain, alters “β-endorphin”, and reduces insulin output from pancreatic islets of genetically obese mice

Naltrexone, an opiate antagonist, was administered to young obese (ob/ob) and lean mice for five weeks. Animals had continuous access to food and received 10 mg/kg SC twice daily with equivalent volumes of saline given to controls. The effects on body weight, and pituitary and plasma levels of β-endorphin-like material were measured. Naltrexone-injected obese animals gained weight more slowly over the first three weeks while the weight gain of lean animals was not affected by naltrexone. Plasma levels of β-endorphin were shown to be significantly higher in untreated ob/ob mice and this difference increased with age (4–20 weeks). With naltrexone treatment, plasma levels in +/? mice rose and exceeded those in ob/ob. Saline treatment appeared to be a stress, and pituitary β-endorphins rose 4–6 fold in ob/ob compared with +/?. While naltrexone reduced the levels in ob/ob pituitary towards normal, no effect on β-endorphin levels in pituitary of lean mice was obtained. In vitro studies of effects of the opiate antagonists, naloxone, on insulin secretion by isolated islets provided additional evidence of resistance of lean mice to naloxone relative to ob/ob. (IRI secretion fell only in naloxone treated ob/ob islets.) These observations support the contention that this form of genetic obesity is characterized by elevated endogenous opiate levels and an increased sensitivity to opiate antagonists such as naltrexone or naloxone.     

Proopiomelanocortin (POMC)-related peptides in the brain of the rainbow trout, Salmo gairdneri

We have investigated the presence of ACTH, -MSH and β-endorphin, three peptides which derive from the multifunctional precursor protein proopiomelanocortin (POMC) in the brain of the rainbow trout Salmo gairdneri. Using both the indirect immunofluorescence and peroxidase-antiperoxidase techniques, a discrete group of positive cells was identified in the hypothalamus, within the anterior part of the nucleus lateralis tuberis. -MSH-containing neurons represented the most abundant immunoreactive subpopulation. Coexistence of -MSH, ACTH and β-endorphin was observed in the lateral part of the nucleus. ACTH- and β-endorphin-containing cells were mainly distributed in the rostral and caudal regions of the nucleus. In the medial portion of the nucleus lateralis tuberis, numerous cells were only stained for -MSH. Moderate to dense plexuses of immunoreactive fibers were observed in the ventral thalamus and the floor of the hypothalamus. Some of these fibers projected towards the pituitary. The concentrations of ACTH, -MSH and β-endorphin-like immunoreactivities were measured in microdissected brain regions by means of specific radioimmunoassays. Diencephalon, mesencephalon and medulla oblongata extracts gave dilution curves which were parallel to standard curves. The highest concentrations of POMC-derived peptides were found in the diencephalon (-MSH: 4.28±0.43 ng/mg prot.; ACTH: 1.08±0.09 ng/mg prot.; β-endorphin: 1.02±0.1 ng/mg prot.), while lower concentrations were detected in the mesencephalon, medulla oblongata and telencephalon. The present results demonstrate that various peptides derived from POMC coexist within the same cell bodies of the fish hypothalamus. Taken together, these data suggest that expression and processing of POMC in the fish brain is similar to that occurring in pituitary melanotrophs.     

The posttranslational modification of β-endorphin in the intermediate pituitary of the toad, Bufo marinus, includes processing at a monobasic cleavage site

Fractionation of an acid extract of 15 B. marinus intermediate pituitaries by a combination of gel filtration chromatography and cation exchange chromatography revealed one major and five minor forms of β-endorphin in this tissue. Based on reversed-phase HPLC and immunological properties, as well as amino acid composition and primary sequence analysis, it was deduced that the sequence of the major form of B. marinus β-endorphin is N-acetyl-YGGFMTPE. Overall, the steady-state analyses of the minor forms of β-endorphin indicated that the posttranslational processing of β-endorphin in the toad intermediate pituitary includes endoproteolytic cleavage at both paired basic and monobasic cleavage sites.     

Reconstitution of the recombinant human γ-tubulin ring complex

Cryo-electron microscopy recently resolved the structure of the vertebrate γ-tubulin ring complex (γ-TuRC) purified from Xenopus laevis egg extract and human cells to near-atomic resolution. These studies clarified the arrangement and stoichiometry of γ-TuRC components and revealed that one molecule of actin and the small protein MZT1 are embedded into the complex. Based on this structural census of γ-TuRC core components, we developed a recombinant expression system for the reconstitution and purification of human γ-TuRC from insect cells. The recombinant γ-TuRC recapitulates the structure of purified native γ-TuRC and has similar functional properties in terms of microtubule nucleation and minus end capping. This recombinant system is a central step towards deciphering the activation mechanisms of the γ-TuRC and the function of individual γ-TuRC core components.     

Vitronectin and its fragments purified as serum inhibitors of Staphylococcus aureus γ-hemolysin and leukocidin, and their specific binding to the Hlg2 and the LukS components of the toxins

Staphylococcal γ-hemolysin and leukocidin are bi-component cytolysins, consisting of LukF (or Hlg1)/Hlg2 and LukF/LukS, respectively. Here, we purified serum inhibitors of γ-hemolysin and leukocidin from human plasma. Protein sequencing showed that the purified inhibitors of 62, 57, 50 and 38 kDa were the vitronectin fragments with truncation(s) of the C-terminal or both N- and C-terminal regions. The purified vitronectin fragments specifically bound to the Hlg2 component of γ-hemolysin and the LukS component of leukocidin to form high-molecular-weight complexes with them, leading to inhibition of the toxin-induced lysis of human erythrocytes and human polymorphonuclear leukocytes, respectively. Intact vitronectin also showed inhibitory activity to the toxins. The ability of γ-hemolysin and leukocidin to bind vitronectin and its fragments is a novel function of the pore-forming cytolysins.     

Cloning of a neoteleost (Oreochromis mossambicus) pro-opiomelanocortin (POMC) cDNA reveals a deletion of the γ-melanotropin region and most of the joining peptide region: implications for POMC processing

A signature feature of tetrapod pro-opiomelanocortin (POMC) is the presence of three melantropin (MSH) coding regions (α-MSH, β-MSH, γ-MSH). The MSH duplication events occurred early during the radiation of the jawed vertebrates well over 400 million years ago. However, in at least one order of modern bony fish (subdivision Teleostei; order Salmoniformes; i.e. salmon and trout) the γ-MSH sequence has been deleted from POMC. To determine whether the γ-MSH deletion has occurred in other teleost orders, a POMC cDNA was cloned from the pituitary of the neoteleost Oreochromis mossambicus (order Perciformes). In O. mossambicus POMC, the deletion is more extensive and includes the γ-MSH sequence and most of the joining peptide region. Because the salmoniform and perciform teleosts do not share a direct common ancestor, the γ-MSH deletion event must have occurred early in the evolution of the neoteleost fishes. The post-translational processing of O. mossambicus POMC occurs despite the fact that the proteolytic recognition sequence, (R/K)-X_n-(R/K) where n can be 0, 2, 4, or 6, a common feature in mammalian neuropeptide and polypeptide hormone precursors, is not present at several cleavage sites in O. mossambicus POMC. These observations would indicate that either the prohormone convertases in teleost fish use distinct recognition sequences or vertebrate prohormone convertases are capable of recognizing a greater number of primary sequence motifs around proteolytic cleavage sites.     

Dehydroepiandrosterone 7α-hydroxylation in human tissues: Possible interference with type 1 11β-hydroxysteroid dehydrogenase-mediated processes

Dehydroepiandrosterone (DHEA) is 7α-hydroxylated by the cytochome P450 7B1 (CYP7B1) in the human brain and liver. This produces 7α-hydroxy-DHEA that is a substrate for 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) which exists in the same tissues and carries out the inter-conversion of 7α- and 7β-hydroxy-DHEA through a 7-oxo-intermediary. Since the role of 11β-HSD1 is to transform the inactive cortisone into active cortisol, its competitive inhibition by 7α-hydroxy-DHEA may support the paradigm of native anti-glucocorticoid arising from DHEA. Therefore, our objective was to use human tissues to assess the presences of both CYP7B1 and 11β-HSD1. Human skin was selected then and used to test its ability to produce 7α-hydroxy-DHEA, and to test the interference of 7α- and 7β-hydroxy-DHEA and 7-oxo-DHEA with the 11β-HSD1-mediated oxidoreduction of cortisol and cortisone. Immuno-histochemical studies showed the presence of both CYP7B1 and 11β-HSD1 in the liver, skin and tonsils. DHEA was readily 7α-hydroxylated when incubated using skin slices. A S9 fraction of dermal homogenates containing the 11β-HSD1 carried out the oxidoreduction of cortisol and cortisone. Inhibition of the cortisol oxidation by 7α-hydroxy-DHEA and 7β-hydroxy-DHEA was competitive with a K_i at 1.85 ± 0.495 and 0.255 ± 0.005 μM, respectively. Inhibition of cortisone reduction by 7-oxo-DHEA was of a mixed type with a K_i at 1.13 ± 0.15 μM. These findings may support the previously proposed native anti-glucocorticoid paradigm and suggest that the 7α-hydroxy-DHEA production is a key for the fine tuning of glucocorticoid levels in tissues.     

Moieties of Complement iC3b Recognized by the I-domain of Integrin αXβ2

Complement fragment iC3b serves as a major opsonin for facilitating phagocytosis via its interaction with complement receptors CR3 and CR4, also known by their leukocyte integrin family names, αMβ2 and αXβ2, respectively. Although there is general agreement that iC3b binds to the αM and αX I-domains of the respective β2-integrins, much less is known regarding the regions of iC3b contributing to the αX I-domain binding. In this study, using recombinant αX I-domain, as well as recombinant fragments of iC3b as candidate binding partners, we have identified two distinct binding moieties of iC3b for the αX I-domain. They are the C3 convertase-generated N-terminal segment of the C3b α’-chain (α’NT) and the factor I cleavage-generated N-terminal segment in the CUBf region of α-chain. Additionally, we have found that the CUBf segment is a novel binding moiety of iC3b for the αM I-domain. The CUBf segment shows about a 2-fold higher binding activity than the α’NT for αX I-domain. We also have shown the involvement of crucial acidic residues on the iC3b side of the interface and basic residues on the I-domain side.     

Regional interactions of opioid peptides at μ and δ sites in rat brain

Opiate binding sites in five brain regions were labeled with the μ and δ markers, ³H-morphine and ³H-[D-Ala²,D-leu⁵]enkephalin, respectively. The highest densities of both ³H-morphine and ³H-DADLE labeled sites are found in striatum and frontal cortex. Hypothalamus and midbrain contain predominantly ³H-morphine labeled sites. The selectivity of the opioid peptides [D-Ala²,D-leu⁵]enkephalin, β-endorphin and dynorphin(1–13) for the two opiate sites was investigated by comparing the potency of these unlabeled compounds against the μ and δ markers in different brain regions. This determination has the effect of controlling for the breakdown of peptides within each region. While the enkephalin analogue shows a preference for the δ binding site and β-endorphin is more nearly equipotent towards the two binding sites, dynorphin(1–13) shows a high affinity and selective preference for the μ binding site over the δ site. The potency of the opioid peptides in displacing the μ and δ markers varies from region to region according to the relative densities of the two opiate binding site populations.     

Endorphins and the central inhibition of urinary bladder motility

The involvement of endogenous opioid mechanisms in the central neurogenic control of urinary bladder function has been examined in anesthetized rats. Intracerebroventricular (ICV) microinjection of β-endorphin (0.5–2.0 μg) produced powerful inhibition of rhythmic bladder contractions initiated by central reflex activity. The peptide fragments γ-endorphin and α-endorphin (4–16 μg), formed by the processing of β-endorphin by membrane homogenates of brain, were less active than the parent compound. The inhibitory effects of β-endorphin was reversed by ICV naloxone (1–2 μg) but higher doses were required to reverse γ- or α-endorphin effects. ICV naloxone administered alone increased intravesicular pressure and bladder contraction frequency. These observations support the hypothesis that the endorphins have a physiological role in the central regulation of urinary bladder activity.     

N-Acetylated endorphins in ovine anterior pituitary and neuro-intermediate lobe

We have used an antiserum for immunohistochemistry and RIA/RP-HPLC which recognizes all fragments of N-acetylated endorphin (NacEP). In the rat neurointermediate lobe (N-IL), in addition to the N-acetylated forms of immunoreactive-β-endorphin (ir-βEP) already reported, we have demonstrated NacβEP_1–17 as a minor component. In the sheep pituitary processing of βEP is markedly different. In the anterior pituitary (AP), staining was indistinguishable with βEP and NacEP antisera, in contrast with the rat where many fewer AP cells stained with the NacEP antiserum. Secondly, as in the rat, all N-IL cells stained with both antisera; on RP-HPLC, however, the major forms of NacEP in the sheep N-IL were NacβEP_1–17 (40%), NacβEP_1–27 (25%) and NacβEP_1–16 (20%), with NacβEP_1–31 (2%) as a minor component. A similar profile was seen on RP-HPLC of sheep AP. These data suggest that (1) patterns of processing in sheep AP are similar to those in N-IL, though the extent of acetylation is less and (2) in the sheep pituitary low molecular weight acetylated fragments predominate, in contrast with the rat.     

Endogenous C-terminal fragments of beta-amyloid precursor protein from Xenopus laevis skin exudate

Using a monoclonal antibody against the entire C-terminal end of human APP₆₉₅ (643–695 sequence) and a monoclonal antibody directed against human β[1–40] amyloid peptide (βA), we show the existence of endogenous peptides proteolytically derived from APP in skin exudate of the non transgenic Xenopus laevis frog. The majority of the immunoreactivity is found associated with a 30 kDa molecular species. Biochemical fractionation followed by mass spectrometry identification allowed us to assign this molecular species to C-terminal APP fragments containing all or part of βA. According to the nature of N- and C-terminal amino acids we identified endogenous β-, γ-, ε-secretase-like activities, caspase-like activity and numerous endogenous cleavage sites within the β-amyloid sequence at same sites as those observed in human βA sequence. All these homologies with human indicate that X. laevis skin exudate is a good natural model to study βA metabolism. In this way, interestingly, we identified endogenous cleavages at prohormone convertase-like sites not yet described at the same sites in human. Finally, all identified peptide fragments were stably associated with a 20.2 kDa protein. These new observed features suggest new research pathways concerning human βA metabolism and carriage of hydrophobic peptide fragments issued from APP processing.     

15β-hydroxysteroids (Part IV). Steroids of the human perinatal period: The synthesis of 3α,15β,17α-trihydroxy-5α-pregnan-20-one and its A/B-ring configurational isomers

In recent years several 15β-hydroxysteroids have emerged pathognomonic of adrenal disorders in human neonates of which 3α,15β,17α-trihydroxy-5β-pregnan-20-one (2) was the first to be identified in the urine of newborn infants affected with congenital adrenal hyperplasia. In this investigation we report the synthesis of the three remaining 3ξ,5ξ-isomers, namely 3α,15β,17α-trihydroxy-5α-pregnan-20-one (3), 3β,15β,17α-trihydroxy-5α-pregnan-20-one (7) and 3β,15β,17α-trihydroxy-5β-pregnan-20-one (8) for their definitive identification in pathological conditions in human neonates. 3β,15β-Diacetoxy-17α-hydroxy-5-pregnen-20-one (11), a product of chemical synthesis was converted to the isomeric 3 and 7, while conversion of 15β,17α-dihydroxy-4-pregnen-3,20-dione (4), a product of microbiological transformation, resulted in the preparation of 8. In brief, selective acetate hydrolysis of 11 gave 15β-acetoxy-3β,17α-dihydroxy-5-pregnen-20-one (12) which on catalytic hydrogenation gave 15β-acetoxy-3β,17α-dihydroxy-5α-pregnan-20-one (13) a common intermediate for the synthesis of the 3β(and α),5α-isomers. Hydrolysis of the 15β-acetate gave 7, whereas oxidation with pyridinium chlorochromate gave 15β-acetoxy-17α-hydroxy-5α-pregnan-3,20-dione (14) which on reduction with -Selectride and hydrolysis of the 15β-acetate gave 3. Finally, hydrogenation of 4 gave 15β,17α-dihydroxy-5β-pregnan-3,20-dione (10) which on reduction with -Selectride gave 8.     

Analysis of the α4β1 Integrin–Osteopontin Interaction

The integrin α4β1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via α4β1 using GST fusion proteins. We show that α4β1 expressed in J6 cells interacts with intact OPN when the integrin is in a high activation state, and by deletion mapping that the α4β1 binding region in OPN lies between amino acid residues 125 and 168 (aa125–168). This region contains the central RGD motif of OPN, which also interacts with integrins αvβ3, αvβ5, αvβ1, α8β1, and α5β1. Mutating the RGD motif to RAD had no effect on the interaction with α4β1. To define the binding site the region incorporating aa125–168 was divided into 5 overlapping peptides expressed as GST fusion proteins. Two peptides supported adhesion via α4β1, aa132–146, and aa153–168; of these only a synthetic peptide, SVVYGLR (aa162–168), derived from aa153–168 was able to inhibit α4β1 binding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhibitor of α4β1, and the primary α4β1 binding site within OPN.