"Joining peptide" of pro-opiomelanocortin. II. Interspecies heterogeneity of the joining peptide fragment

The use of an antiserum raised against the joining peptide sequence -23 to -14 of bovine pro-opiomelanocortin (POMC) enabled the detection of related immunoreactive sequences of peptides in bovine, porcine, mouse and guinea-pig pituitaries, as well as in mouse brain and cerebral cortex, guinea-pig cerebral cortex, and bovine hypothalamus. Gel chromatography of pituitary extracts (Sephadex G-75 and Bio-Gel P-4) indicated the presence of several immunoreactive joining peptide fragments ranging in the molecular weight range (Mr) of 1,500 to 2,300. Furthermore, high molecular weight (Mr greater than 22,500) immunoreactive-precursor from bovine anterior pituitary was readily digested with trypsin into an immunoreactive fragment of approximately Mr 1,500. Analyses of these immunoreactive peptides by reverse-phase high-performance liquid chromatography (HPLC) led to their resolution into six distinct peptides. The only apparent correspondence in the elution profiles of immunoreactive peptide profiles between different mammalian species was the identification of a similar fragment (Mr 2,000) from bovine and guinea-pig pituitaries. Thus, we conclude that immunoreactivity to the joining peptide region of POMC from various mammalian species exhibits a degree of heterogeneity in its composition. The relatively low levels of immunoreactivity in comparison to that of ACTH also suggest that the joining peptide domain may be further processed. The hormonal status of the joining peptide region remains to be determined.     

Characterization of sulfated forms of the pro-opiomelanocortin amino-terminal glycopeptide in rat intermediate lobe cells

Explants of rat neurointermediate lobes were incubated in the presence of radioactive amino acids, sugars or sulfate and the labeled proteins were separated by two-dimensional gel electrophoresis. A double series of acidic peptides (Mr = 16,000-21,500) were identified as variant forms of the amino-terminal glycopeptide of pro-opiomelanocortin (N-POMC). The series of peptides with the higher molecular weights (Mr = 18,000-21,500) contain a tryptic fragment (tentatively identified as the tryptic peptide of the "joining peptide": sequence 77 to 93 of rat POMC) which is absent from the forms of the lower molecular weight series (Mr = 16,000 to 18,000). Pulse-chase studies further showed that the high molecular weight forms of N-POMC could be post-translationally cleaved albeit slowly into the species of Mr = 16,000-18,000 which constitute, at least in part, the final maturation products of the N-terminal region of the precursor molecule. All the variant forms of the N-POMC glycopeptide could be labeled with [35S]sulfate. Our results strongly suggest that most of the sulfate groups are attached to N-linked oligosaccharide side chains of N-POMC. We therefore propose that one of the final maturation products of the N-terminal portion of POMC in rat intermediate lobes is a sulfated glycopeptide (Mr = 16,000-18,000) composed of the 1-74 sequence of rat POMC.     

Pro-opiomelanocortin-derived peptides in the pig pituitary: alpha- and gamma 1-melanocyte-stimulating hormones and their glycine-extended forms

Pro-opiomelanocortin (POMC)-related peptides in extracts of anterior and neurointermediate pituitary lobes from pigs were characterized by gel chromatography, reversed-phase chromatography and radioimmunoassays. The peptide content was ca. 3-fold greater in the anterior lobe compared to the neurointermediate lobe (19.8 nmol POMC/anterior lobe vs 7.0 nmol/neurointermediate lobe). In the neurointermediate lobe 93% of POMC was processed to alpha-melanocyte-stimulating hormone (alpha-MSH) and analogs exclusively of low molecular weight. Most of the remaining adrenocorticotropic hormone (ACTH)-related material consisted of the glycine-extended intermediate ACTH-(1-14) and analogs. In contrast only one fourth to one third of the N-terminal part of POMC (N-POMC) was processed to amidated gamma-MSH and its C-terminal glycine-extended precursor. The relative amount of amidated gamma-MSH was the same as alpha-MSH and analogs (94%). However, more than 95% of these peptides were of high molecular weight. In the anterior lobe 2.3% of N-POMC was processed and 94% was amidated gamma-MSH of only high molecular weight. These results show that gamma-MSH and alpha-MSH are amidated to the same extent and that gamma 1-MSH and gamma 2-MSH immunoreactivity are present in both the anterior lobe and the neurointermediate lobe. The results suggest that the production of amidated peptides is not regulated by the amidation process itself but at an earlier step (e.g. at the proteolytic cleavage).     

Investigation of the structural requirements for peptide precursor processing in AtT-20 cells using site-directed mutagenesis of proadrenocorticotropin/endorphin.

Mouse pro-ACTH/endorphin (or POMC) contains in its sequence each of the four possible pairs of basic amino acids recognized as potential cleavage sites in the production of bioactive peptides from higher mol wt precursors: KR (lysine-arginine), RR, RK, and KK. To examine the structural requirements for processing and routing in one region of pro-ACTH/endorphin, a reporter mutation was introduced into the mouse pro-ACTH/endorphin cDNA; a methionine residue was mutated to an isoleucine residue to allow biosynthetic double labeling with [3H]Ile and [35S]Met. Analysis of stable cell lines expressing the reporter cDNA indicated that this mutation did not affect processing or secretion. Therefore, additional mutations were introduced on the reporter background to investigate important structural features of the precursor. First, the tripeptide signal for N-linked glycosylation in the N-terminal glycopeptide (Asn65,Ser66,Ser67) was disrupted by the conservative substitution of asparagine65 with a glutamine residue. Secondly, O-glycosylation was prevented by substitution of threonine45 with an alanine residue. Finally, lysine50 was mutated to an arginine residue, transforming the RK doublet preceding the gamma 3MSH sequence into an RR doublet. The results show that the enzymatic machinery of AtT-20 cells fails to cleave efficiently at the Arg-Lys (RK) site even after elimination of any possible structural hindrance by carbohydrate side-chains. Elimination of O-linked oligosaccharides to the N-terminal side of gamma 3MSH did not allow cleavage at the RK site, and elimination of N-linked oligosaccharides did not alter the processing and routing of pro-ACTH/endorphin in AtT-20 cells. However, mutation of the RK sequence to RR allowed extensive cleavage regardless of the occurrence of O- or N-glycosylation.     

Alpha-amidated peptides derived from pro-opiomelanocortin in normal human pituitary.

Normal human pituitaries were extracted in boiling water and acetic acid, and the alpha-amidated peptide products of pro-opiomelanocortin (POMC), alpha-melanocyte-stimulating hormone (alpha MSH), gamma-melanocyte-stimulating hormone (gamma 1MSH), and amidated hinge peptide (HP-N), as well as their glycine-extended precursors, were characterized by sequence-specific radioimmunoassays, gel-chromatography, h.p.l.c. and amino acid sequencing. alpha MSH and gamma 1MSH constituted 0.27-1.32% and 0.10-5.10%, respectively, of the POMC-derived products [calculated as the sum of adrenocorticotropic hormone (ACTH)-(1-39), ACTH-(1-14) and alpha MSH immunoreactivity]. alpha MSH and ACTH-(1-14) were only present in non- or mono-acetylated forms. Only large forms of gamma 1MSH and gamma 2MSH were present in partly glycosylated states. The hinge peptides were amidated to an extent two to three orders of magnitude greater than alpha MSH and gamma 1MSH. Most (99%) of the HP-N was of low molecular mass and consisted mainly of HP-N-30. The remaining part was high-molecular-mass HP-N, probably HP-N-108, although the presence of HP-N-44 could not be completely excluded. These results show that all the possible amidated POMC-related peptides are present in normal human pituitary. It also shows that cleavage in vivo at all dibasic amino acids but one, takes place at the N-terminal POMC region; the exception is at the POMC-(49-50) N-terminal of the gamma MSH sequence. The pattern of peptides produced suggests that the generation of amidated peptides is mainly regulated at the endopeptidase level.     

Detection of a novel sequence change in the major form of alpha-MSH isolated from the intermediate pituitary of the reptile, Anolis carolinensis.

Intermediate pituitaries of the reptile, Anolis carolinensis, were separately pulse labeled with [3H]Trp and [3H]Tyr. The major form of alpha-MSH was purified by immunoprecipitation and isolated by reverse phase HPLC. Tryptic peptide analysis indicated that the [3H]Trp-labeled C-terminal fragment of Anolis alpha-MSH had the same retention time as mammalian ACTH(9-13) amide; however, the [3H]Tyr-labeled N-terminal fragment did not coelute with either mammalian ACTH(1-8) or N-acetyl-ACTH(1-8). Purification of alpha-MSH from 76 Anolis intermediate pituitaries confirmed that a sequence change had occurred in the N-terminal region of Anolis alpha-MSH. The tissues were acid extracted and purified by Sephadex G-25 chromatography and reverse phase HPLC to yield 4.5 micrograms of purified Anolis alpha-MSH for amino acid composition analysis and automated Edman degradation sequence analysis. The major form of Anolis alpha-MSH is nonacetylated and has the following novel primary sequence: Ser-Tyr-Ala-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro(Val-amide). The presence of Val-amide was verified by immunological analysis, tryptic peptide analysis and amino acid composition analysis.     

Production of monoclonal antibodies against the N-terminal glycopeptide of porcine pro-opiomelanocortin: their use for solid-phase radioimmunoassay, western blotting, and immunogold cytochemistry.

We have prepared two monoclonal antibodies for the N-terminal glycopeptide of pro-opiomelanocortin 1-77 (N-POMC(1-77)) purified from porcine pituitaries. Antibody 1-244 recognizes an epitope located within the gamma 3-melanotropin (gamma 3-MSH or POMC(51-77)) sequence, whereas antibody 2-197 binds specifically to a determinant in the 1-49 region of N-POMC. These monoclonal antibodies were used to construct a two-site solid-phase radioimmunoassay that can detect as little as 50 pg of N-POMC(1-77). The assay is linear between 0.5 and 5 ng of porcine peptide and recognizes equally well the homologous peptides purified from human and bovine pituitaries. The assay has been used to analyze reversed-phase high pressure liquid chromatography fractions of crude bovine pituitary extracts and detected a peptide with chromatographic properties identical to those of N-POMC(1-77). When used to stain immunoblots of bovine intermediate pituitary extracts, both the 2-197 and 1-244 antibodies could recognize a major peptide comigrating with purified N-POMC(1-77). In addition, antibody 2-197 also detected a peptide with a mobility similar to that of standard N-POMC(1-49). When used in conjunction with a second anti-mouse antibody coupled to colloidal gold particles, antibody 2-197 stained N-POMC immunoreactive material located in granules in thin sections of pituitary.     

Prohormone-converting enzymes: regulation and evaluation of function using antisense RNA.

Several putative peptide-processing endoproteases have been identified by homology to the yeast Kex2 endoprotease, including furin, PC2, and PC1. However, the question is still open as to which might be involved in peptide posttranslational processing. To enable detailed comparisons of physiological changes in peptide processing with biochemical and molecular biological studies, we cloned rat pituitary cDNAs for PC1 and PC2. The amino acid sequence homologies among rat, human, and mouse PC1, PC2, and furin are consistent with each being a highly conserved but distinct member of a larger family of mammalian subtilisin-like proteases. PC1 and PC2 mRNAs show a restricted distribution among rat tissues and cultured cell lines, consistent with a role in tissue-specific peptide processing; the occurrence of furin mRNA among these tissues and cell lines is much more widespread, being high in many nonneuroendocrine tissues. In the neurointermediate pituitary, PC1 and PC2 mRNAs are strikingly regulated in response to dopaminergic agents, in parallel with mRNAs for POMC, peptidylglycine alpha-amidating monooxygenase, and carboxypeptidase-H. In AtT-20 cells, PC1 mRNA is coregulated with POMC and peptidylglycine alpha-amidating monooxygenase mRNAs in response to CRH and glucocorticoids. When the endogenous PC1 mRNA level in AtT-20 cells is significantly and specifically decreased by stable expression of antisense RNA to PC1, biosynthetic labeling of newly synthesized POMC-derived peptides shows a substantial blockade of normal POMC processing. These data are consistent with a role for PC1 protein in endoproteolysis, either as a processing endoprotease or as the activator of the actual processing endoprotease(s).     

5' sequence of porcine and rat pro-opiomelanocortin mRNA. One porcine and two rat forms

We have determined the nucleotide sequences of the 5' noncoding regions and the regions encoding the first 56 amino acids of rat and porcine pro-opiomelanocortin (POMC) mRNA. We accomplished this by sequencing cDNA produced by elongating specific DNA primers hybridized to neurointermediate pituitary mRNA. The nucleotide sequence of the 5' region of rat POMC mRNA fills a gap in our knowledge of the structure of this mRNA and the protein it codes for. While we have observed only a single porcine POMC mRNA, two different rat POMC mRNAs are detected. The rat POMC mRNAs differ by a 30-base insertion/deletion in their 5' noncoding regions. Its position and sequence suggest that it is the result of alternate modes of intron removal during RNA splicing.     

Human joining peptide: a proopiomelanocortin product secreted as a homodimer

The human (h) POMC gene sequence predicts a 30 amino acid joining peptide (JP) separating the N-terminal fragment [POMC(1-76) or hNT] and ACTH within their common precursor. We used an anti-serum directed against the amidated COOH-terminal end of mouse JP to develop a RIA for the predicted hJP molecule. Immunoreactive JP was detected in tissue extracts from human normal pituitary, ACTH-secreting pituitary- and nonpituitary tumors, and in plasma from patients with ACTH hypersecretory syndromes. Its molar concentration was of the same order of magnitude as, and correlated with, that of the other POMC peptides. Gel exclusion chromatography in 1% formic acid and 6 M guanidine-HCl revealed a predominant immunoreactive material with an apparent mol wt of ca. 6000. After reduction with dithiothreitol this material was recovered in an elution volume identical to that of purified hJP and corresponding to a mol wt of ca. 3000. These data show that POMC processing generates a COOH terminally amidated hJP predominantly secreted as a homodimer, probably through disulfide bonding between the single Cys9 residue of two molecules.     

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.     

Prothrombin fragment 1 X 2 X 3, a major product of prothrombin activation in human plasma

The conversion of the blood coagulation zymogen prothrombin to thrombin is associated with the production of several cleavage intermediates and products. In contrast to earlier studies of prothrombin cleavage in chemically defined systems, the current investigation examines the fragmentation of human prothrombin in normal plasma. Radiolabeled prothrombin was added to platelet-poor relipidated normal human plasma, and clotting was initiated with the addition of Ca(II) and kaolin. Analysis of the radiolabeled prothrombin cleavage products by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and beta-mercaptoethanol identified a heretofore unobserved product of prothrombin activation with an apparent molecular weight of 45,000. This product was identified as fragment 1 X 2 X 3, the NH2-terminal 286 amino acids of prothrombin. The product was isolated from a prothrombin digest by immunoaffinity chromatography using anti-prothrombin:Ca(II) antibodies and by preparative gel electrophoresis. Its amino-terminal sequence is identical to that of prothrombin. Digestion of this product with either Factor Xa or thrombin yields, at a minimum, fragment 1 X 2 and fragment 1. Amino-terminal sequence analysis of the products obtained by digestion with Factor Xa of the unknown activation product indicated 3 amino acid residues at each cycle consistent with the presence of fragment 1, fragment 2, and fragment 3. To unambiguously identify the COOH-terminal amino acid sequence of the product, its factor Xa digestion products were separated by reverse-phase high performance liquid chromatography. Edman degradation of one peptide revealed the complete sequence of fragment 3. On this basis, we identify the Mr 45,000 polypeptide as fragment 1 X 2 X 3 and indicate that it is a prominent product of prothrombin conversion to thrombin when activation occurs in plasma.     

Mammalian neural and endocrine pro-protein and pro-hormone convertases belonging to the subtilisin family of serine proteinases.

Conversion of pro-hormones and precursor proteins into biologically active peptides and proteins involves the concerted action of a number of convertases and post-translation modification enzymes. The identification of the yeast convertase kexin as a prototype processing enzyme led to the discovery of the mammalian convertase designated furin, PC1 and PC2. Whereas furin is ubiquitously expressed, PC1 and PC2 are found only in endocrine and neural tissues and cell lines. In man and mouse, the genes coding for furin, PC1 and PC2 reside on three different chromosomes. The analysis of the intracellular processing of PC1 and PC2 and the removal of their pro-segment is presented, together with a summary of the cleavage specificity of these enzymes for precursors such as pro-opiomelanocortin (POMC) and human pro-renin. The distinct tissue distribution of PC1 and PC2 and their coregulation with POMC in the pituitary neurointermediate lobe adds credence to their physiological role as convertases involved in the tissue-specific processing of precursor proteins.     

An in vivo characterization of the cleavage site specificity of the insulin cell prohormone processing enzymes

Most peptide hormones and neurotransmitters are synthesized as larger precursor proteins, which are post-translationally processed to mature bioactive products. An early event in prohormone maturation is endoproteolytic cleavage, occurring usually at pairs of basic amino acids (e.g. Lys-Arg). Since many of the characteristics of a prohormone endoprotease are unknown, distinguishing these enzymes from other cellular proteases in vitro has been difficult. In this report, the substrate specificity of a model prohormone processing system, the insulinoma cell line Rin m5F, was characterized in vivo to establish a set of criteria by which putative proinsulin endoproteases may be assessed. To determine the role of composition of the paired basic amino acid site in directing cleavage, a series of mutant prohormones containing altered cleavage sites was constructed and expressed in Rin m5F cells. Proopiomelanocortin (POMC) was used as a substrate since this prohormone was previously shown to be processed by these cells. To control for positional effects, all four permutations of lysine and arginine (Lys-Arg, Arg-Arg, Arg-Lys, and Lys-Lys) were introduced at both the efficiently processed cleavage site separating the ACTH and beta-lipotropin (beta-LPH) domains of POMC and at the inefficiently processed site in the beta-endorphin sequence near the COOH-terminus of the precursor. His-Arg and Met-Arg sites were also introduced at the ACTH/beta-LPH junction to assess the requirement for paired lysines and arginines. Identification of POMC-derived peptides demonstrated efficient processing of Lys-Arg and inefficient processing of Lys-Lys and Arg-Lys sites at both positions in the prohormone. The Arg-Arg sequence, however, was processed in a position-dependent manner, being efficiently cleaved between ACTH and beta-LPH but only about 50% processed within beta-endorphin. His-Arg was not cleaved in Rin m5F cells, although surprisingly Met-Arg was partially processed. These results indicate a strict preference of the insulinoma prohormone endoprotease(s) for paired basic amino acids ending in arginine, but that processing efficiency of some sequences may be modulated by location within the precursor molecule.     

Differential glycosylation of N-POMC1-77 regulates the production of gamma 3-MSH by purified pro-opiomelanocortin converting enzyme. A possible mechanism for tissue-specific processing.

The amino terminus of bovine pro-opiomelanocortin (N-POMC^1–77) is partially processed in the intermediate lobe of the pituitary to N-POMC^1–49 and lys-γ₃ -melanotropin. Two pools of N-POMC^1–77 were isolated which were differentially glycosylated at threonine⁴⁵, while N-POMC^1–49 isolated from bovine intermediate lobe extracts existed in a non-glycosylated form. This suggested that differential O-linked glycosylation of N-POMC^1–77 may regulate cleavage at the Arg⁴⁹-Lys⁵⁰ processing site. We tested this hypothesis by incubating N-POMC^1–77 glycoforms with purified pro-opiomelanocortin converting enzyme. Only non-O-glycosylated N-POMC^1–77 and O-glycosylated N-POMC^1–77 with truncated oligosaccharide sidechains were sensitive to cleavage and generated predominantly lys-γ₃ -melanotropin, identified by high-performance liquid chromatography. These data provide the first functional evidence to support a role for differential O-linked glycosylation in the regulation of the processing of the N-terminus of bovine POMC.     

Post-translational processing of preprotachykinins. Isolation of protachykinin-(1-37)-peptide from human adrenal-medullary phaeochromocytoma tissue.

The biosynthetic precursors of the mammalian tachykinins, alpha-, beta-and gamma-preprotachykinins, contain a common N-terminal region of 74 amino acids. A polyclonal antiserum was raised against a synthetic peptide representing N-tyrosylated beta-preprotachykinin-(48-56)-peptide as predicted from the nucleotide sequence of cloned DNA complementary to human beta-preprotachykinin mRNA. By using this antiserum in radioimmunoassay, a single immunoreactive peptide was identified in an extract of a human pheochromocytoma that produced substance P and neurokinin A. Partial microsequencing and determination of the amino acid composition of the peptide indicated identity with preprotachykinin-(20-56)-peptide. Thus the data demonstrate that the Ala19-Glu20 bond in preprotachykinin is the site of cleavage of the signal peptide.