Identification and characterization of N-glycosylated and phosphorylated variants of proenkephalin A-derived peptides in bovine adrenal medulla, spinal cord and ileum

Recent studies have shown that during its biosynthesis in bovine adrenal medulla, the opioid precursor proenkephalin A, may be both N-glycosylated and phosphorylated. To investigate whether these chemical modifications were common to proenkephalin A processing in other tissues, we have sought to characterize enkephalin-containing peptides from bovine adrenal medulla, spinal cord and ileum. The peptides were identified using antiserum L189, specific for the C-terminus of Met-enkephalin Arg6Gly7Leu8 (MERGL), and L152, specific for the C-terminus of Met-enkephalin Arg6Phe7 (MERF). Glycosylated MERGL-immunoreactive peptides of 23, 20, 16 and 13 kDa were identified in adrenal medulla using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and concanavalin A-Sepharose affinity chromatography. Sephadex G50 gel filtration fractionated the glycosylated peptides into two immunoreactive peaks. Similar peaks of concanavalin A-binding MERGL immunoreactivity were detected in extracts of spinal cord and ileum, although there were differences in relative proportions of the two peaks. Antiserum L152 identified phosphorylated N-terminally extended variants of MERF when boiling water extracts of adrenal medulla, spinal cord and ileum were separated by anion exchange chromatography. In adrenal medulla these peptides were more than 99% phosphorylated, whereas in both ileum and spinal cord there was a relatively higher proportion of the unphosphorylated peptide. The results indicate that N-glycosylation and phosphorylation of proenkephalin A occurs in adrenal medulla, spinal cord and ileum, although there are tissue-specific differences in the relative proportions of the modified and unmodified peptides.     

Purification from brain of synenkephalin, the N-terminal fragment of proenkephalin

Abstract: The primary sequence of adrenal proenkephalin was recently deduced from the structure of the cloned cDNA that codes for this protein. Several enkephalin-containing proteins with molecular weights between 8,000 and 20,000 daltons were purified from the bovine adrenal medulla. These proteins appear to represent intermediates in the processing of proenkephalin into physiologically active opioid peptides. While the concentrations of these large processing intermediates in the adrenal medulla are quite high, similar proteins have not yet been shown to be present in brain, and there is some question as to whether the brain synthesizes an enkephalin precursor similar to adrenal proenkephalin. We report here the purification from bovine caudate nucleus of synenkephalin, the N-terminal fragment of adrenal proenkephalin. The amino acid composition of synenkephalin indicates that the protein represents residues 1–70 of adrenal proenkephalin. Thus the brain and adrenal glands appear to utilize a similar precursor for enkephalin biosynthesis.     

N-linked glycosylation of a proenkephalin A-derived peptide. Evidence for the glycosylation of an NH2-terminally extended Met-enkephalin Arg6-Gly7-Leu8 variant

To investigate the possibility that the opioid peptide precursor proenkephalin A was glycosylated, we utilized an antiserum raised against the COOH terminus of Met-enkephalin Arg6-Gly7-Leu8 (MERGL) to identify and characterize enkephalin-containing peptides from extracts of bovine adrenal medulla. Sephadex G-50 gel filtration separated two immunoreactive peaks which had apparent masses of 9 and 6 kDa. Anion-exchange chromatography and reverse-phase high pressure liquid chromatography (HPLC) revealed that the 9-kDa material was a heterogenous mixture of immunoreactive peptides, of which one (9K-MERGL Ia) was purified to homogeneity. The 6-kDa material separated into two major immunoreactive peaks (6K-MERGL I and 6K-MERGL II) on anion-exchange chromatography, and these were obtained in an homogenous form after reverse-phase HPLC. Amino acid sequencing, together with immunological characterization, indicated that the three peptides were identical in chain length, and corresponded to proenkephalin A 116-165. They contained the sequence Asn-Ser-Ser which is a potential N-glycosylation site. In 9K-MERGL Ia, but not the others, automated Edman amino acid sequencing was unable to detect the relevant asparagine residue, suggesting that this residue has been chemically modified. Further investigation of the 9K-MERGL material using lectin affinity chromatography provided direct evidence of glycosylation. Verification of this result was obtained using the specific enzyme glycopeptidase F (glycopeptide-N-glycosidase) which demonstrated that 9K-MERGL contained, in part, N-linked oligosaccharide chains. These results show that an NH2 terminally extended Met-enkephalin Arg6-Gly7-Leu8 variant was N-glycosylated, and hence indicate that the precursor polypeptide proenkephalin A can be glycosylated during translation in the rough endoplasmic reticulum.     

Novel Opioid Peptide Amidorphin: Characterization and Distribution of Amidorphin-Like Immunoreactivity in Bovine, Ovine, and Porcine Brain, Pituitary, and Adrenal Medulla

We have recently isolated from bovine adrenal medulla a novel C-terminally amidated opioid peptide, amidorphin, which derives from proenkephalin A. Amidorphin revealed a widespread distribution in bovine, ovine, and porcine tissue. Particularly high concentrations of amidorphin immunoreactivity were detected in adrenal medulla, posterior pituitary, and striatum, similar to the major gene products of proenkephalin A. In the adrenal medulla of each species, authentic amidorphin was the predominant immunoreactive form. Pituitary and brain, however, contained predominantly putative N-terminally shortened fragments of amidorphin of a slightly lower molecular weight and shorter retention times on HPLC. In addition, in ovine adrenal medulla, a putative high-molecular-weight form of amidorphin was detected. These findings are indicative of a tissue-specific processing of the proenkephalin A precursor, leading predominantly to authentic amidorphin in the adrenal medulla and further processing to smaller C-terminal fragments in the brain and pituitary.     

Characterisation of Met-enkephalin(Arg6,Phe7) immunoreactivity in human adrenal and human phaeochromocytoma

The molecular forms of opioid peptides in human adrenal have not been well characterised. These peptides are predominantly derived from the proenkephalin A precursor, which has the sequence of Met-enkephalin(Arg⁶,Phe⁷) as its carboxyl terminus. We have looked in the present study at the subcellular distribution and the molecular form of immunoreactivity to this sequence in post-mortem human adrenal medulla and in phaeochromocytoma. In the human adrenal homogenates, the immunoreactivity distributes on a sucrose gradient in a manner consistent with localisation in chromaffin granules. On chromatography, the immunoreactivity from adrenal medulla is predominantly in the heptapeptide form; the intermediate (3000–4000) molecular weight material is only a minor component of immunoreactivity, in contrast to bovine tissue extracts where this is the major form of immunoreactivity. In the phaeochromocytoma extracts, the heptapeptide sequence again predominates over a minor amount of intermediate sized material. The results are discussed in terms of post-mortem changes, precursor processing and the function of the adrenal medulla.     

Quantitation of proenkephalin A messenger RNA in bovine brain, pituitary and adrenal medulla: correlation between mRNA and peptide levels.

Concentrations of mRNA coding for the opioid peptide precursor proenkephalin A were measured in bovine brain areas, pituitary and adrenal medulla. In all tissues, a single hybridizable species of 1400 bases in size was found by Northern blot analysis using as a probe a single-stranded (ss) cDNA complementary to bovine proenkephalin A mRNA. In solution hybridization experiments the distribution of the mRNA was quantified. Considerable differences were found for the abundance of proenkephalin A mRNA in the various tissues: from 0.023% in the adrenal medulla to 0.00002% in the adenohypophysis. Relative abundance in the various brain areas varied greater than 20-fold, being highest in the caudate nucleus (0.0025%) and lowest in the thalamus and substantia nigra (0.0001%). Comparison with immunoreactive peptide concentrations in these tissues showed a close correlation between the levels of proenkephalin A mRNA and the immunoreactive peptides.     

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.     

The isolation and chemical characterization of phosphorylated enkephalin-containing peptides from bovine adrenal medulla

There is increasing evidence that the opioid peptide precursor, proenkephalin A, and its products undergo extensive post-translational modification, in addition to the cleavage at dibasic amino acid sites. We have used an antiserum directed toward the C terminus of Met-enkephalin Arg6-Phe7 in a radioimmunoassay to monitor the purification to homogeneity of four peptide B variants from bovine adrenal medulla, using gel filtration, anion exchange chromatography, and reverse phase high performance liquid chromatography. Amino acid sequence analysis, together with immunochemical data, confirmed that each comprised the primary sequence, proenkephalin A-(209-239). In addition, three of the four variants were shown to be phosphorylated by alkaline phosphatase digestion, microphosphate analysis, and ethanethiol derivatization coupled with amino acid sequence analysis; these variants were shown to have 1, 2, or 3 phosphate groups per peptide chain, which corresponded to their increasing acidic nature. The phosphorylation sites were clustered together at positions Ser7, Ser13, and Ser15 and were in close association with acidic residues. The clustering of phosphorylated residues is unique among regulatory peptide precursors. This region of proenkephalin A is well conserved, which suggests that it constitutes an important novel functional domain.     

Processing of Proenkephalin in Adrenal Chromaffin Cells

The processing of proenkephalin was studied using [35S]methionine pulse-chase techniques in primary cultures of bovine adrenal medullary chromaffin cells. Following radiolabeling, proenkephalin-derived peptides were extracted from the cells and separated by reverse-phase HPLC. Fractions containing proenkephalin fragments were digested with trypsin and carboxypeptidase B to liberate Met-enkephalin sequences and subjected to a second HPLC step to demonstrate association of radiolabel with Met-enkephalin. Processing of proenkephalin is complete within 2 h of synthesis, suggesting completion at or soon after incorporation into storage vesicles. Pretreatment of the cells with nicotine, histamine, or vasoactive intestinal peptide to enhance the rate of proenkephalin synthesis failed to alter the time course of processing and had minimal effects on the distribution of products formed. Addition of tetrabenazine, an inhibitor of catecholamine uptake into chromaffin vesicles, during radiolabeling and a 6-h chase period caused enhanced proenkephalin processing. These results suggest that the full range of proenkephalin fragments normally found in the adrenal medulla (up to 23.3 kDa) represents final processing products of the tissue and that termination of processing may depend on the co-storage of catecholamines.     

Bovine adrenal chromaffin granules are a site of synthesis of atrial natriuretic factor

We have previously reported the existence of a peptide factor in the adrenal medulla which inhibits aldosterone secretion in cultured bovine zona glomerulosa cells. The acid extracts of chromaffin granules from bovine adrenal medulla were purified by a four step high performance liquid chromatography procedure. Two active fractions exhibited sequence homology with bovine atrial natriuretic factor ANF (Ser99-Tyr126) and its polypeptide precursor (Asn1-Tyr126). The occurrence of both precursor and mature forms of ANF within chromaffin granules indicates the endogenous character of ANF in the adrenal medulla and suggests the potential usefulness of cultured adrenal chromaffin cells for investigating the synthesis, maturation and secretion of atrial peptides.     

Immunoreactive Met-enkephalin Arg6 in rat brain, and bovine brain, gut, and adrenal

Antibodies directed against the Met-enkephalin-related hexapeptide, Met-enk Arg6, have been used in radioimmunoassays in the characterization of material in rat brain, and bovine striatum, colon, and adrenal medulla. Met-enk Lys6 reacted 0.27 relative to Met-enk Arg6, but Leu-enk Arg6 and C-terminal extensions or deletions of Met-enk Arg6 showed less than 0.02 immunoreactivity. In rat brain, the concentration of Met-enk Arg6-like immunoreactivity was less than 20 pmol X g-1 in all regions, but after trypsinization of tissue extracts there were up to 80-fold increases in immunoreactivity as a result of cleavage of C-terminally extended forms. The tryptic product eluted as Met-enk Arg6 on gel filtration. In control extracts of rat brain there were at least three immunoreactive forms of Met-enk Arg6; one eluted in the position of the hexapeptide standard on gel filtration and HPLC while the others had properties of N-terminally extended forms. In bovine striatum and colon the hexapeptide-like material predominated; but in bovine adrenal extracts, there were relatively low concentrations of the hexapeptide and, instead, the dominant immunoreactive forms corresponded to two components that were probably N-terminally extended variants. Trypsin again produced marked increases in immunoreactivity. HPLC studies indicated that Met-enk Arg6Phe7- and Met-enk Arg6Gly7Leu8-like immunoreactive peptides were important substrates in bovine brain for the production of hexapeptide immunoreactivity after trypsin. The differences in the patterns of immunoreactive forms in bovine adrenal, colon, and brain are consistent with tissue variations in the pathways of posttranslational processing of the precursor molecules.     

Proenkephalin: A general pathway for enkephalin biosynthesis in animal tissues

Guinea pig adrenal, brain, and myenteric plexus have been shown to contain many polypeptides that yield free enkephalins on digestion with trypsin and carboxypeptidase B. The enkephalin-containing polypeptides (ECPs) range from 500 to >20,000 daltons and show similarities in their chromatographic behavior to the ECPs present in the chromaffin granules of the bovine adrenal medulla. Furthermore, the heptapeptide [Met]enkephalin-Arg⁶-Phe⁷, that is now known to represent the carboxyl terminal sequence of the proenkephalin found in bovine adrenal medulla (Gübler et al. (1982) Nature (London), in press), was identified in all three guinea pig tissues. It appears that processing of a proenkephalin similar to the one in adrenal medulla represents a general pathway for enkephalin biosynthesis in animal tissues.     

Specific polyclonal antibodies to the carboxyl terminus of [Met]enkephalin-Arg6-Gly7-Leu8

The octapeptide Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu was recently isolated from bovine adrenal chromaffin granules and serves as a marker for proenkephalin from which it is derived. Polyclonal antisera which are highly specific for the carboxyl terminus have been raised against the synthetic peptide. The only significant cross-reactivity was with the 18.2-k Da and 5.3-k Da enkephalin-containing peptides (EC peptides) which contain the octapeptide at their carboxyl termini and the [des-Tyr] and [des-Tyr-Gly] congeners of the octapeptide. Extracts of bovine adrenal medulla and rat spinal cord were shown to contain significant amounts of the octapeptide, the two larger EC peptides, and the two smaller congeners.     

Prohormone Thiol Protease and Enkephalin Precursor Processing: Cleavage at Dibasic and Monobasic Sites

Production of active enkephalin peptides requires proteolytic processing of proenkephalin at dibasic Lys-Arg, Arg-Arg, and Lys-Lys sites, as well as cleavage at a monobasic arginine site. A novel “prohormone thiol protease” (PTP) has been demonstrated to be involved in enkephalin precursor processing. To find if PTP is capable of cleaving all the putative cleavage sites needed for proenkephalin processing, its ability to cleave the dibasic and the monobasic sites within the enkephalin-containing peptides, peptide E and BAM-22P (bovine adrenal medulla docosapeptide), was examined in this study. Cleavage products were separated by HPLC and subjected to microsequencing to determine their identity. PTP cleaved BAM-22P at the Lys-Arg site between the two basic residues. The Arg-Arg site of both peptide E and BAM-22P was cleaved at the NH₂-terminal side of the paired basic residues to generate [Met]-enkephalin. Furthermore, the monobasic arginine site was cleaved at its NH₂-terminal side by PTP. These findings, together with previous results showing PTP cleavage at the Lys-Lys site of peptide F, demonstrate that PTP possesses the necessary specificity for all the dibasic and monobasic cleavage sites required for proenkephalin processing. In addition, the unique specificity of PTP for cleavage at the NH₂-terminal side of arginine at dibasic or monobasic sites distinguishes it from many other putative prohormone processing enzymes, providing further evidence that PTP appears to be a novel prohormone processing enzyme.     

Regulation of rat adrenal medullary enkephalins by glucocorticoids

Opioid peptides and their precursors of the proenkephalin family are found in the chromaffin cells of the rat adrenal medulla in low quantities. However, if the gland is denervated, there is a 10 to 20-fold increase in enkephalin-containing (EC) peptides consisting mostly of the precursor proenkephalin. The denervation-induced rise in medullary EC peptides is blocked by hypophysectomy, and partially reinstated by corticosterone, dexamethasone or ACTH treatment. In the intact rat, intermediate doses of corticosterone or dexamethasone reduce the denervation-induced increase in EC peptides, while a high dose of dexamethasone restores this response. These results indicate that glucocorticoids exert a permissive effect in vivo on the denervation-induced stimulation of EC peptide biosynthesis.     

Product inhibition of carboxypeptidase H

Carboxypeptidase H is one of several enzymes required for the processing of peptide hormone precursors. In this study, inhibition of carboxypeptidase H by its peptide products was investigated. Carboxypeptidase H activity in bovine adrenal medulla chromaffin granules and rat adrenal medulla homogenate was inhibited by the peptides Met- and Leu-enkephalin, vasopressin, oxytocin, luteinizing hormone-releasing hormone, substance P, and thyrotropin-releasing hormone, with oxytocin and ACTH 1-14 having the least effect, at concentrations of 2-20 mM. Inhibition by amidated peptide products (vasopressin, oxytocin, luteinizing hormone-releasing hormone, substance P, and thyrotropin-releasing hormone) show that the final products of the precursor processing pathway can regulate carboxypeptidase H. These levels of peptides are similar to known intragranular peptide concentrations indicating that product and feedback inhibition of carboxypeptidase H may play a role in the control of neuropeptide synthesis. The proenkephalin-derived peptides Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg6-Gly7-Leu8, and Met-enkephalin-Arg6-Phe7 competitively inhibited bovine and rat carboxypeptidase H with Ki values of 12.0, 6.5, 7.0, and 5.5 mM, respectively. The significantly greater Ki for Met-enkephalin may reflect the effects of higher intragranular concentration of Met-enkephalin, since one proenkephalin molecule contains four copies of Met-enkephalin and only one copy of each of the other enkephalin peptides. Thus, the products from one multivalent precursor molecule may equivalently inhibit carboxypeptidase H activity. Product inhibition of carboxypeptidase H and perhaps other processing enzymes may serve to limit the maximum peptide concentration within the secretory vesicle.     

Chromatographic Evidence for the Presence of Multiple Tachykinins in the Bovine Adrenal Medulla

Among the mammalian tachykinins, substance P (SP) has been shown to be the most potent at modulating the response due to nicotinic acetylcholine receptor stimulation of bovine adrenal chromaffin cells. SP-like immunoreactivity has been detected in nerve terminals innervating the adrenal medulla; however, little is known of the presence of other tachykinins in this tissue. In this study, reverse-phase HPLC was used to fractionate peptides in bovine adrenal medullary extracts, and the fractions were analyzed by radioimmunoassay using antisera to SP or neurokinin A (NKA). The results show that both NKA- and SP-like immunoreactivities are present in the adrenal medulla. The presence of neurokinin B is also indicated. The presence of multiple tachykinins in this tissue raises questions as to their functions in the adrenal medulla.     

Release in vivo of Met-enkephalin and encrypted forms of Met-enkephalin from brain and spinal cord of the anesthetized cat

Processing of the proenkephalin molecule will result in peptide fragments in which the pentapeptide YGGFM is included. We have employed a molecular sieve (2 kDa) separation, enzyme hydrolysis radioimmunoassay (RIA) treatment sequence which permits concurrent measurement of Met-enkephalin (Enk) and several enkephalin-encrypting (X-Enk) peptides in a single sample. Using this protocol, the release of Enk and X-Enk (total Enk - Enk) greater and less than 2 kDa from spinal cord and the mesencephalic aqueductal grey was assessed under resting conditions and during stimulation of the sciatic nerve in the chloralose-urethane anesthetized cat. Under resting conditions measurable levels of Enk (10.5±4.7; 9.1±2.1 pg/min) and X-Enk (47.8±19.7; 45.7±12.3 pg/min) are found in aqueductal and spinal superfusates, respectively. The X-Enk measured under resting and evoked conditions in aqueductal and spinal perfusates is associated almost exclusively (>90–95%) with fragments >2 kDa in size. These results, showing the relative absence of detectable levels of X-Enk forms <2 kDa, were confirmed by reverse phase chromatography. During sciatic nerve stimulation, the levels of both Enk and X-Enk were mildly elevated in spinal and ventricular perfusates. With the addition of thiorphan (10⁻⁵ M), though there was no effect on the resting release of either Enk or X-Enk, the levels of Enk measured under evoked conditions were significantly augmented in both ventricular and spinal perfusates.     

Affinity Purification of Synenkephalin-Containing Peptides Including a Novel 23.3-Kilodalton Species

Abstract: Affinity chromatography has been used for rapid and high-yield purification of synenkephalin (proenkephalin 1 -70) containing peptides present in bovine adrenal medulla (BAM) chromaffin granular lysate. A column of CN-Br-activated Sepharose 4B coupled to synenkephalin antiserum bound synenkephalin immunoreactivity which was eluted by a stepwise gradient of 50 mM ammonium acetate containing 20% (vol/vol) acetonitrile over the pH range 7–3. Synenkephalin immunoreactivity emerged as two peaks, eluting at pH 5.5 and 4.5. Characterization of the two peaks by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting indicated that the pH 5.5 peak contained principally low-molecular-weight proenkephalin species (8.6 and 12.6 kilodaltons), whereas the pH 4.5 peak contained, in addition, high-molecular-weight proenkephalin species (18.2 and 23.3 kilodaltons). The 8.6- and 12.6- kilodalton species were isolated from the pH 5.5 peak by TSK gel filtration HPLC, whereas the pH 4.5 peak was further purified by passage over successive affinity columns coupled to antiserum against BAM 22P (proenkephalin 182–203) and [Met⁵]-enkephalin-Arg⁶-Gly⁷-Leu⁸. The former column retains the 23.3-kilodalton species, whereas the latter column retains the 18.2-kilodalton species. The 23.3- kilodalton peptide represents a novel putative proenkephalin intermediate (proenkephalin-1–206), containing [Leu⁵]- enkephalin at the C-terminus.     

Characterization of the molecular forms of proenkephalin in bovine adrenal medulla and rat adrenal, brain, and spinal cord with a site-directed antiserum

An antiserum was generated against a synthetic peptide corresponding to amino acids 95-117 of bovine proenkephalin, and a sensitive radioimmunoassay was developed. Comparison of the reactivities of the synthetic peptide, its specific cleavage products, and other synthetic peptides showed that the important immunological determinant was contained within residues 101-109 of bovine proenkephalin (-Gly-Gly-Glu-Val-Leu-Gly-Lys-Arg-Tyr-). Radioimmunoassay of fractions after gel filtration of bovine adrenal medullary chromaffin granule lysate showed three pools of immunoreactivity: pool 1 (Mr 20,000-30,000), pool 2 (Mr 10,000-20,000), and pool 3 (Mr approximately 5,000). Further characterization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting showed that the antiserum recognized 27-, 20.5-, 16.5-, and 5.6-kilodalton enkephalin-containing proteins. The radioimmunoassay was also used to detect proenkephalin-like material in extracts of rat adrenal and regions of rat brain and spinal cord following gel filtration. Immunoreactivity from the rat adrenal chromatographed predominantly as high molecular weight material (Mr 31,500-43,500), whereas material in regions of rat brain showed a broader molecular weight distribution (Mr 4,000-43,500). This indicated differences in the processing of proenkephalin between rat adrenal and brain tissue. Differences were also seen in the molecular weight profile of immunoreactivity in different brain regions, most noticeable in the case of striatum and hypothalamus, suggesting regional differences in processing. Based on quantitation of higher molecular weight immunoreactive proenkephalin-like material and free Met-enkephalin immunoreactivity in different brain regions, it was apparent that extensive processing of proenkephalin occurs in brain. We concluded that antisera against proenkephalin-(95-117) recognize a wide range of intermediates in the processing of proenkephalin in both bovine adrenal medulla and rat adrenal, brain, and spinal cord, making it a useful tool for further studies concerned with the expression and post-translational processing of proenkephalin.