Isolation and structure of a novel C-terminally amidated opioid peptide, amidorphin, from bovine adrenal medulla

Biologically active peptide hormones and neurotransmitters have been shown to be enzymatically liberated from larger, inactive precursor molecules by tissue-specific post-translational processing, particularly at the typical cleavage signals of paired basic residues. Subsequent N-terminal or C-terminal modifications may be of importance in regulating the biological activities of these peptides. C-terminal alpha-amidation is considered to be essential for the biological function of several non-opioid peptides. Here we present the isolation and structure of a novel C-terminally amidated opioid peptide, amidorphin, from bovine adrenal medulla. Amidorphin and the recently isolated octapeptide metorphamide (adrenorphin) are the only endogenous opioid peptides in mammals known to possess a C-terminal amide group. The amino acid sequence of amidorphin corresponds to the sequence 104-129 of bovine proenkephalin A. Very high concentrations of amidorphin were detected in bovine adrenal medulla and in a further endocrinological system, the hypothalamic-neurohypophyseal axis. Amidorphin may therefore be considered to be a major gene product of the opioid peptide precursor proenkephalin A in these endocrine tissues.     

Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid

A variety of hypophysiotropic peptides or proteins have been reported to be present in mammalian gonads. Inhibin, a hormone that under most circumstances selectively suppresses the secretion of follicle-stimulating hormone (FSH) but not luteinizing hormone (LH), has been isolated from the gonadal fluids of several species and characterized as a heterodimeric protein consisting of alpha- and beta-polypeptides associated by disulphide bonds. The complete amino-acid sequences of the precursors of porcine and human inhibin alpha-subunits and two distinct porcine inhibin beta-subunits (beta A and beta B) have been deduced from complementary DNA sequences. Gonadotropin releasing peptides have also been found in the gonad and have generally been shown to be active in radioreceptor assays for gonadotropin releasing hormone (GnRH) but to exhibit different chromatographic and immunological characteristics from those of GnRH. During our purification of inhibin from porcine follicular fluid, we noted fractions that could stimulate the secretion of FSH by cultured anterior pituitary cells. We report here the purification of an FSH releasing protein (FRP) and its characterization by SDS-polyacrylamide gel electrophoresis under non-reducing and reducing conditions and by partial sequence analysis. Our results indicate that porcine gonadal FRP is a homodimer consisting of two inhibin beta A-chains linked by disulphide bonds. FRP is highly potent (50% effective concentration (EC50) approximately 25 pM) in stimulating the secretion and biosynthesis of FSH but not of LH or any other pituitary hormone. In contrast to the effects of GnRH and other reported gonadal gonadotropin releasing fractions, the action of FRP is not mediated by GnRH receptors.     

Predominant naturally processed peptides bound to HLA-DR1 are derived from MHC-related molecules and are heterogeneous in size.

Peptides bound to class I molecules are 8-10 amino acids long, and possess a binding motif representative of peptides that bind to a given class I allele. In the only published study of naturally processed peptides bound to class II molecules (mouse I-Ab and I-Eb), these peptides were longer (13-17 amino acids) and had heterogenous carboxy terminals but precise amino-terminal truncations. Here we report the characterization of acid-eluted peptides bound to HLA-DR1 by high-performance liquid chromatography, mass spectrometry and microsequencing analyses. The relative molecular masses of the peptides varied between 1,602 and 2,996 (13-25 residues), the most abundant individual M(r) values being between 1,700 and 1,800, corresponding to an average peptide length of 15 residues. Complete sequence data were obtained for twenty peptides derived from five epitopes, of which all but one were from self proteins. These peptides represented sets nested at both the N- and C-terminal ends. Binding experiments confirmed that all of the isolated peptides had high affinity for the groove of DR1. Alignment of the peptides bound to HLA-DR1 and the sequences of 35 known HLA-DR1-binding peptides revealed a putative motif. Although peptides bound to class II molecules may have some related features (due to the nonpolymorphic HLA-DR alpha-chain), accounting for degenerate binding to different alleles, particular amino acids in the HLA-DR beta-chains presumably define allelic specificity of peptide binding.     

Pancreastatin, a novel pancreatic peptide that inhibits insulin secretion

In mammalian tissues the C-terminal amide structure has been found to occur only in neuroactive or hormonally-active peptides. About half known neuropeptide and peptide hormones have this unique chemical feature. Using a chemical detection method, a search for previously unknown peptides that possess the C-terminal amide structure in extracts of brain and intestine was carried out and a number of novel neuropeptides and hormonal peptides, designated neuropeptide Y, PHI, peptide YY, galanin and neuropeptide K were isolated. We recently performed a similar search in porcine pancreas and found a high concentration of a peptide having a glycine amide at its C-terminus. Here we report the isolation, primary structure and biological activity of this novel peptide. The 49-residue peptide strongly inhibits glucose-induced insulin release from the isolated perfused pancreas and was therefore named pancreastatin. It may be important in the regulation of insulin secretion and in the pathogenesis and treatment of diabetes mellitus.     

Purification and cloning of amyloid precursor protein beta-secretase from human brain

Proteolytic processing of the amyloid precursor protein (APP) generates amyloid beta (Abeta) peptide, which is thought to be causal for the pathology and subsequent cognitive decline in Alzheimer's disease. Cleavage by beta-secretase at the amino terminus of the Abeta peptide sequence, between residues 671 and 672 of APP, leads to the generation and extracellular release of beta-cleaved soluble APP, and a corresponding cell-associated carboxy-terminal fragment. Cleavage of the C-terminal fragment by gamma-secretase(s) leads to the formation of Abeta. The pathogenic mutation K670M671-->N670L671 at the beta-secretase cleavage site in APP, which was discovered in a Swedish family with familial Alzheimer's disease, leads to increased beta-secretase cleavage of the mutant substrate. Here we describe a membrane-bound enzyme activity that cleaves full-length APP at the beta-secretase cleavage site, and find it to be the predominant beta-cleavage activity in human brain. We have purified this enzyme activity to homogeneity from human brain using a new substrate analogue inhibitor of the enzyme activity, and show that the purified enzyme has all the properties predicted for beta-secretase. Cloning and expression of the enzyme reveals that human brain beta-secretase is a new membrane-bound aspartic proteinase.     

Structural basis of latency in plasminogen activator inhibitor-1.

Human plasminogen activator inhibitor-1 (PAI-1) is the fast-acting inhibitor of tissue plasminogen activator and urokinase and is a member of the serpin family of protease inhibitors. Serpins normally form complexes with their target proteases that dissociate very slowly as cleaved species and then fold into a highly stable inactive state in which the residues that flank the scissile bond (P1 and P1';) are separated by about 70 A. PAI-1 also spontaneously folds into a stable inactive state without cleavage; this state is termed 'latent' because inhibitory activity can be restored through denaturation and renaturation. Here we report the structure of intact latent PAI-1 determined by single-crystal X-ray diffraction to 2.6 A resolution. The three-dimensional structure reveals that residues on the N-terminal side of the primary recognition site are inserted as a central strand of the largest beta sheet, in positions similar to the corresponding residues in the cleaved form of the serpin alpha 1-proteinase inhibitor (alpha 1-PI). Residues C-terminal to the recognition site occupy positions on the surface of the molecule distinct from those of the corresponding residues in cleaved serpins or in the intact inactive serpin homologue, ovalbumin, and its cleavage product, plakalbumin. The structure of latent PAI-1 is similar to one formed after cleavage in other serpins, and the stability of both latent PAI-1 and cleaved serpins may be derived from the same structural features.     

A novel analgesic dipeptide from bovine brain is a possible Met-enkephalin releaser.

It is generally accepted that morphine exerts its analgesic effect by binding to specific opiate receptors in the brain and spinal cord. Since Hughes et al. isolated and identified two endogenous pentapeptides, Met- and Leu-enkephalin, from the brain and found that they acted as agonists at opiate receptors, alpha-, beta- and gamma-endorphins, larger peptides than enkephalins and having morphine-like activity, have been identified in either the brain or pituitary of various species. Several studies have demonstrated that enkephalins possess analgesic properties and that they are distributed in the pain-mediated pathways in the central nervous system. These findings suggest that enkephalins are important neurotransmitters or neuromodulators regulating pain transmission. We now report the isolation of a novel substance which has a Met-enkephalin releasing action. Our findings suggest the possibility of a regulating mechanism for the release of endogenous opioid peptides, especially Met-enkephalin.     

Cloning of a novel gene encoding human thioredoxin-like protein

mRNA differential display (DDRT-PCR) has been used to analyze different human fetal brain tissues of different developmental stages (13- and 33-week). According to the sequence of one EST obtained in this assay, a pair of primers have been designed to screen the arrayed human fetal brain cDNA library. A1 .2-kb cDNA clone has been found. This cDNA consists of an 867 bp open reading frame, a 132 bp 5' untranslated sequence and a 209 bp 3' untranslated sequence with a typical polyadenylation signal. The coding region predicts a protein of 289 amino acids. Its N-terminal of 105 residues is highly homologous to human thioredoxin, while no homology has been found in the databases with its C-terminal of 184 residues. Its N-terminal region also contains the conserved active site sequence CGPC (Cys-Gly-Pro-Cys) of thioredoxin. It was named human Thioredoxin-like gene (hTRXL).     

A synthetic peptide that is a bombesin receptor antagonist

The tetradecapeptide bombesin was originally isolated from frog skin. Bombesin-like peptides have since been detected in mammalian gastrointestinal tract, brain and lung. These peptides have potent pharmacological effects on the central nervous system; they cause contraction of intestinal, uterine and urinary tract smooth muscle; and stimulate the release of other peptides including gastrin, cholecystokinin, motilin, pancreatic polypeptide, neurotensin, insulin, enteroglucagon, prolactin and growth hormone. Specific plasma membrane receptors for bombesin have been demonstrated on pancreatic acinar cells, brain membranes and pituitary cells. Studies defining the physiological importance of bombesin have been impeded by the lack of a bombesin receptor antagonist. Here we describe experiments which demonstrate that a peptide originally described as a substance P receptor antagonist, [D-Arg, D-Pro, D-Trp, Leu ]substance P, is also a bombesin receptor antagonist. This peptide competitively inhibits the ability of bombesin to stimulate enzyme secretion from dispersed pancreatic acini, and also inhibits the action of other peptides that interact with the bombesin receptor.     

Most of the coding region of rat ACTH beta--LPH precursor gene lacks intervening sequences

The peptide hormones ACTH, beta-endorphin, alpha- and beta-melanotropin(MSH) and possibly gamma-MSH are synthesized in the pituitary gland by the processing of a 32,000-molecular weight (MW) polypeptide called proopiomelanocortin (POMC). The existence of a further precursor (pre form) to POMC containing an additional N-terminal 'leader' peptide has been suggested by analysis of the in vitro translation products of poly(A)-containing RNA from AtT-20 cells, a mouse ACTH-producing cell line of pituitary origin. Nakanishi et al. cloned and sequenced a cDNA copy of the bovine prePOMC mRNA. This sequence confirmed the known structure of the carboxyl half of POMC and revealed the presence of a new MSH-like moiety, gamma-MSH, within the 16,000-MW amino half of the precursor (16K fragment). Recent experiments have suggested that this peptide may act in synergy with ACTH to increase corticosterone and aldosterone production in vivo and in vitro. We have now isolated from a rat genomic DNA library a segment of a DNA encoding most of POMC, using as probe a mouse 144-base pair cloned cDNA fragment encoding beta-MSH and beta-endorphin. The cloned rat gene is one of two (or more) closely related POMC genes. The DNA sequence obtained shows that the cloned POMC gene is not interrupted by any intervening sequence (IVS) between the codon for amino acid 19 and the presumptive poly(A) addition site. This region of POMC encodes all the biologically active peptides mentioned above. The DNA sequence encoding the putative gamma-MSH and the coding sequence that precedes it are highly conserved between rat and cow. This may indicate an as yet unrecognized biological function(s) for the NH2-terminal portion of the 16K fragment.     

Formation of amyloid-like fibrils in COS cells overexpressing part of the Alzheimer amyloid protein precursor

A pathological hallmark of Alzheimer's disease is the deposition of amyloid fibrils in the brain. The principal component of the amyloid fibril is beta/A4 protein, which is derived from a large membrane-bound glycoprotein, Alzheimer amyloid protein precursor (APP). Although the deposition of amyloid is thought to result from the aberrant processing of APP, the detailed molecular mechanisms of amyloidogenesis remain unclear. A C-terminal fragment of APP which spans the beta/A4 and cytoplasmic domains has a tendency to self-aggregate. In an attempt to establish a cultured-cell model for amyloid fibril formation, we have transfected COS-1 cells with complementary DNA encoding the C-terminal 100 residues of APP. In the perinuclear regions of a small population of DNA-transfected cells, we observed inclusion-like deposits which showed a strong immunohistochemical reaction towards an anti-C-terminal APP antibody or an anti-beta/A4 amyloid core-specific antibody. Electron microscope observations of the inclusion-carrying cells revealed an accumulation of amyloid-like fibrils of 8-22 nm diameter near and on the nuclear membrane. The fibrils showed a beaded or helical structure, and reacted positively with the anti-C-terminus antibody by immunoelectron microscopy. These results suggest that the formation of amyloid fibrils is an inherent characteristic of the C-terminal peptide of APP. The present system provides a suitable model for the molecular dissection of the process of brain amyloidogenesis.     

A new natriuretic peptide in porcine brain

Atrial natriuretic peptide (ANP), a hormone secreted from mammalian atria, regulates the homoeostatic balance of body fluid and blood pressure. ANP-like immunoreactivity is also present in the brain, suggesting that the peptide functions as a neuropeptide. We report here identification in porcine brain of a novel peptide of 26 amino-acid residues, eliciting a pharmacological spectrum very similar to that of ANP, such as natriuretic-diuretic, hypotensive and chick rectum relaxant activities. The complete amino-acid sequence determined for the peptide is remarkably similar to but definitely distinct from the known sequence of ANP, indicating that the genes for the two are distinct. Thus, we have designated the peptide 'brain natriuretic peptide' (BNP). The occurrence of BNP with ANP in mammalian brain suggests the possibility that the physiological functions so far thought to be mediated by ANP may be regulated through a dual mechanism involving both ANP and BNP.     

Alzheimer's disease. Molecular consequences of presenilin-1 mutation

Alzheimer's disease is characterized by accumulation in the brain of a family of insoluble amyloid peptides (Abeta peptides), which are produced as a result of the normal processing of beta-amyloid precursor protein (beta-APP). Russo et al. claim that a truncated Abeta peptide that lacks the first ten amino acids accumulates in the brains of patients carrying a mutant form of pre-senilin 1 (PS1), a protein that is involved in cleavage of beta-APP. However, we have found that this same species is also overrepresented in Alzheimer's patients with mutations in beta-APP itself. Our findings do not support the conclusion of Russo et al. that pathogenic PS1 mutations may control cleavage of beta-APP by beta-secretase.     

Somatostatin stimulates Ca(2+)-activated K+ channels through protein dephosphorylation.

The neuropeptide somatostatin inhibits secretion from electrically excitable cells in the pituitary, pancreas, gut and brain. In mammalian pituitary tumour cells somatostatin inhibits secretion through two distinct pertussis toxin-sensitive mechanisms. One involves inhibition of adenylyl cyclase, the other an unidentified cyclic AMP-independent mechanism that reduces Ca2+ influx by increasing membrane conductance to potassium. Here we demonstrate that the predominant electrophysiological effect of somatostatin on metabolically intact pituitary tumour cells is a large, sustained increase in the activity of the large-conductance Ca(2+)- and voltage-activated K+ channels (BK). This action of somatostatin does not involve direct effects of Ca2+, cAMP or G proteins on the channels. Our results indicate instead that somatostatin stimulates BK channel activity through protein dephosphorylation.     

Differential activation by atrial and brain natriuretic peptides of two different receptor guanylate cyclases

Alpha atrial natriuretic peptide (alpha-ANP) and brain natriuretic peptide are homologous polypeptide hormones involved in the regulation of fluid and electrolyte homeostasis. These two natriuretic peptides apparently share common receptors and stimulate the intracellular production of cyclic GMP as a second messenger. Molecular cloning has defined two types of natriuretic peptide receptors: the ANP-C receptor of relative molecular mass (Mr) 60-70,000 (60-70 K), which is not coupled to cGMP production and may function in the clearance of ANP and the ANP-A receptor of Mr 120-140 K, which is a membrane form of guanylate cyclase in which ligand binding to the extracellular domain activates the cytoplasmic domain of the enzyme. Here we report the cloning and expression of a second human natriuretic peptide-receptor guanylate cyclase, the ANP-B receptor. The ANP-B receptor is preferentially activated by porcine brain natriuretic peptide rather than human alpha-ANP, whereas the ANP-A receptor responds similarly to both natriuretic peptides. These observations may have important implications for our understanding of the central and peripheral control of cardiovascular homeostasis.     

Human preprovasoactive intestinal polypeptide contains a novel PHI-27-like peptide, PHM-27

Vasoactive intestinal polypeptide (VIP), a 28-amino acid peptide originally isolated from porcine duodenum, is present not only in gastrointestinal tissues but also in neural tissues, possibly as a neurotransmitter, and exhibits a wide range of biological actions (for example, relaxation of smooth muscle, stimulation of intestinal water and electrolyte secretion and release of insulin, glucagon and several anterior pituitary hormones). As the structure of porcine and bovine VIP shows several similarities to those of mammalian glucagon, secretin and gastric inhibitory peptide (GIP), VIP is considered to be a member of the glucagon-secretin family. Recently, we have found that VIP is synthesized from a precursor, pro-VIP (molecular weight (Mr) 17,500), in human neuroblastoma cells and that the primary translation product of the mRNA encoding VIP is prepro-VIP (Mr 20,000). In an attempt to elucidate the primary structure of the precursor, we have now cloned the DNA sequence complementary to the mRNA coding for human VIP and analysed the nucleotide sequence. The entire amino acid sequence of the precursor, deduced from the nucleotide sequence, indicates that the precursor protein contains not only VIP but also a novel peptide of 27 amino acids. The peptide, designated PHM-27, differs by only 2 amino acids from PHI-27, a peptide recently isolated from porcine intestine, and is also closely related in sequence to VIP.     

大熊猫肌红蛋白的纯化及一级结构的研究

本文从大熊猫骨骼肌分离纯化了肌红蛋白,鉴定了纯度,测定了分子量及氨基酸组成。确定了该蛋白的N端及C端氨基酸。采用CNBr裂解的方法,用HPLC分离得到4个肽段,分别测定了大熊猫肌红蛋白1—52位,56—77位及132—153位氨基酸的排列顺序。大熊猫肌红蛋白共含153个氨基酸,本文已完成了96个氨基酸的序列测定。用此结果与食肉目有关动物已知肌红蛋白的氨基酸顺序进行比较,获得了有意义的结论。     

The protein-coding sequence of the bovine ACTH-beta-LPH precursor gene is split near the signal peptide region

The pituitary hormones corticotropin (ACTH) and beta-lipotropin (beta-LPH) are formed from a large common precursor. Recently, we have elucidated the whole primary structure of the bovine ACTH-beta-LPH precursor (designated alternatively as preproopiocortin) by determining the nucleotide sequence of cloned DNA complementary to the mRNA coding for the precursor protein. The amino acid sequence assigned has disclosed a characteristic repetitive structure of the ACTH-beta-LPH precursor. The repetitive units of the precursor protein each contain a melanotropin (MSH) sequence (alpha-, beta- or gamma-MSH) as well as other peptide components such as beta-endorphin and corticotropin-like intermediate lobe peptide (CLIP). The repetitive units as well as their peptide components are each bounded by paired basic amino acid residues, which apparently represent the sites of proteolytic processing. Several studies have confirmed the translational initiation site and protein structure assigned (see also ref. 11 and refs therein). In view of the recent knowledge about the organization of eukaryotic genes (see refs 12, 13 for reviews), it would be of particular interest to investigate the relationship between the repetitive structure of the ACTH-beta-LPH precursor containing different functional components and the arrangement of the protein-coding sequence in its gene. We have now isolated and characterized bovine genomic DNA fragments encoding this precursor protein and have demonstrated that the protein sequence is encoded by two non-consecutive DNA segments. An intron (intervening sequence) of approximately 2.2 kilobase pairs separates the smaller exon (mRNA-coding sequence), which contains the gene sequence encoding the signal peptide, from the larger exon, which contains the gene sequence for most of the protein structure, including the known biologically active component peptides.     

Regulation of pro-opiomelanocortin biosynthesis and processing by transplantation immunity

It is more than thirty years since Billingham and Medawar showed that adrenocorticotrophic hormone (ACTH) and cortisol can prolong the survival of skin allografts. It has since become clear that glucocorticoid hormones are critically involved in the regulation of immunity. The level of glucocorticoids secreted in response to antigenic challenge corresponds to the magnitude of the immune response and in general reaches immunosuppressive levels. Interestingly, not all immune responses enhance ACTH and glucocorticoid hormone production. In transplantation immunity, the reverse seems to be true: circulating glucocorticoid levels at the time of skin graft rejection are lower than control levels. Because beta-endorphin and ACTH originate from the same prohormone, pro-opiomelanocortin (POMC), and are closely related in their tissue-specific processing and coordinate release, we have investigated the role of pituitary beta-endorphin in transplantation immunity. We report here that POMC biosynthesis and processing in the pars intermedia, but not in the anterior pituitary, can be regulated by T cell-specific factors secreted in animals undergoing transplantation immunity.