Glycine-extended anglerfish peptide YG (aPY) a neuropeptide Y (NPY) homologue may be a precursor of a biologically active peptide

The 37 residue peptide YG (aPY), isolated from anglerfish endocrine pancreas, bears distinct sequence homology to the pancreatic polypeptide family of hormones. However, instead of a carboxyl-terminal tyrosine-amide, aPY has a free carboxyl-terminus ending with glycine. Towards studying the structure-activity relationship of this hormone, we have synthesized aPY by solid phase methodology using Boc-amino acid derivatives and phenylacetamidomethyl resin. The crude peptide was purified to homogeneity in 20% yield by reversed phase chromatography. The purified peptide had the expected amino acid composition and sequence, and was found to be identical with the natural aPY by analytical HPLC and peptide mapping of proteolytic digests. Neither the snythetic nor the natural aPY exhibited the characteristic vasoconstrictor activity of the related pancreatic polypeptide family of hormones. However, [Des37-Gly]-aPY, isolated from the anglerfish pancreas, caused vasoconstriction in rats. Based on these results and by analogy to the glycine-extended gastrin peptides, it may be suggested that aPY is a precursor of a biologically active peptide, namely [Des37-Gly]-aPY-amide.     

Isolation and sequence of rat peptide YY and neuropeptide Y

Rat peptide YY and rat neuropeptide Y have been isolated in parallel from colon and brain extracts respectively, using salt fractionation, gel filtration chromatography, cation-exchange HPLC, and reverse phase phenyl-silica HPLC. Immunoreactivity was identified using a combination of 3 NPY immunoassays which exhibit differing cross-reactivities for PYY (90%, less than 0.01% and 30% respectively). The yield at the final purification step was 1.2 nmol rPYY and 0.5 nmol rNPY. Half of each purified peptide was subjected to complete microsequence analysis. This showed that while rat NPY was structurally identical to human NPY, the sequence of PYY from rat colon was the same as porcine PYY isolated from extracts of duodenum.     

Isolation and characterization of neuropeptide Y from porcine intestine

The isolation and primary structure of intestinal neuropeptide Y (NPY) is described. The peptide was purified from porcine intestinal extracts using a chemical assay and radioimmunoassay for NPY. The amino acid sequence of this peptide is: Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala- Arg-Tyr-Tyr- Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH2. This the structure of intestinal NPY is identical to the NPY of brain origin.     

Isolation and structure of the principal products of preproglucagon processing, including an amidated glucagon-like peptide

The principal products derived from in vivo processing of anglerfish preproglucagon II were isolated and their structures determined. The structures were confirmed by a combination of automated Edman degradation, amino acid analysis, and fast atom bombardment mass spectrometry. The peptide corresponding to anglerfish preproglucagon II-(22-49) (numbering from the amino terminus of preproglucagon) was isolated intact and defines the site of signal cleavage to be between Gln-21 and Met-22. Glucagon from the anglerfish preproglucagon gene II was found to correspond to preproglucagon II-(52-80) (numbering from the amino terminus). Three forms of a glucagon-like peptide derived from preproglucagon II were also isolated. The structure of the longest form was consistent with the sequence of preproglucagon II-(89-122) deduced from the cDNA, His-Ala-Asp-Gly-Thr-Tyr-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Gln-Asp-Gln-Ala- Ala-Lys-Asp-Phe-Val-Ser-Trp-Leu-Lys-Ala-Gly-Arg-Gly-Arg-Arg-Glu. The carboxyl-terminal portion deduced from the cDNA remains intact in this form. A second form, preproglucagon II-(89-119) appears to result from proteolytic processing of the major form at the two adjacent arginine residues occurring at the carboxyl terminus. This second form has a glycine residue at its carboxyl terminus and is processed to the third form (preproglucagon II-(89-118)) which contains a carboxyl-terminal arginineamide. Radiolabeling studies in primary tissue culture support the observation that glucagon (preproglucagon II-(52-80], preproglucagon II-(89-122), and preproglucagon II-(89-119) are products of proglucagon processing in vivo.     

Isolation and structure elucidation of the major photodegradation products of loteprednol etabonate

Photodegradation of loteprednol etabonate (5), a steroid anti-inflammatory drug, in the solid state, in aqueous suspension, and in aqueous acetonitrile solution has been investigated. Analysis by HPLC showed that the profile of photodegradation products in the solid state was qualitatively similar to that in the aqueous suspension, although the profile in the aqueous acetonitrile solution was considerably different. The major photodegradation products were isolated from the aqueous suspension and the aqueous acetonitrile solution by using preparative reversed-phase HPLC and their structures were elucidated on the basis of spectroscopic data. Photolysis in the solid state and in aqueous suspension yielded three rearrangement products, chloromethyl 17alpha-ethoxycarbonyloxy-11beta-hydroxy-5alpha-methyl-2-oxo-19-norandrosta-1(10),3-diene-17beta-carboxylate (8), chloromethyl 17alpha-ethoxycarbonyloxy-11beta-hydroxy-1-methyl-3-oxo-6(5-->10alpha)-abeo-19-norandrosta-1,4-diene-17beta-carboxylate (9), and chloromethyl 1beta,11beta-epoxy-17alpha-ethoxycarbonyloxy-2-oxo-10alpha-androsta-4-ene-17beta-carboxylate (10). In aqueous acetonitrile solution, 10 was the major product, however, 8 and 9 were not obtained. Pathways for the formation of these compounds from loteprednol etabonate (5) are proposed.     

Structure of a peptide from coho salmon endocrine pancreas with homology to neuropeptide Y

The amino acid sequence of a peptide isolated from the Pacific salmon (Oncorhynchus kisutch) endocrine pancreas has been determined. This simple 36 residue peptide is a member of the pancreatic polypeptide family. It contains a C-terminal tyrosinamide and is more homologous with porcine neuropeptide Y (NPY) (83%) and peptide YY (75%) than any of the previously characterized pancreatic polypeptides (PP). This peptide appears to be the major but not the only representative of this family of peptides present in the endocrine pancreas of this fish. This peptide is referred to as salmon pancreatic polypeptide (salmon PP).     

Partial processing of the neuropeptide Y precursor in transfected CHO cells

The activation of regulatory peptides by post-translational modification of their biosynthetic precursors is generally thought to occur only in neuroendocrine cells. We have selected clones of Chinese hamster ovary cells, a non-neuroendocrine cell line, which were transfected with a eukaryotic expression vector coding for the precursor for neuropeptide Y. Although the majority of the immunoreactive NPY was found in the form of pro-NPY, some degree of intracellular proteolytic processing of the precursor occurred in all clones. Part of the intracellular NPY immunoreactivity was even correctly amidated. Extracellular degradation of pro-NPY in the tissue culture medium generated immunoreactivity which corresponded in size to NPY. It is concluded that precursor processing can occur in non-neuroendocrine cells both as a biological process within the cells and as apparent processing, degradation in the tissue culture medium.     

The primary structure and tissue distribution of an amphibian neuropeptide Y.

Neuropeptide Y (NPY) has been isolated and sequenced from brain extracts of the European common frog, Rana temporaria. Plasma desorption mass spectroscopy of the purified peptide indicated a molecular mass of 4243.3 Da which was in agreement with that deduced from the sequence (4243.7 Da), incorporating a C-terminal amide. The primary structure of frog NPY was established as: YPSKPDNPGEDAPAEDMAKYYSALRHYINLITRQRY-NH2. Frog NPY contains a single, highly-conservative amino acid substitution (Lys for Arg at residue 19) with respect to human NPY. NPY immunoreactivity was localised exclusively in nerves within the brain, pancreas and gastrointestinal tract and reverse-phase HPLC of extracts of these tissues resolved a single immunoreactive peptide of identical retention time in each case. The primary structure of NPY has therefore been highly-conserved over a considerable evolutionary time-span.     

Solubilization and affinity purification of the Y2 receptor for neuropeptide Y and peptide YY from rabbit kidney.

Neuropeptide Y (NPY) is an important neuropeptide in both central and peripheral neurones whereas peptide YY (PYY) is a gut hormone present in endocrine cells in the lower bowel. Both peptides interact with multiple binding sites that have been further classified into Y1 and Y2 receptors. We have solubilized native Y2 receptors both from basolateral membranes of proximal convoluted tubules from rabbit kidney and from rat hippocampal membranes. Solubilization of functional Y2 receptors was obtained with both 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin and resulted in each case in a single class of high affinity binding sites. The soluble receptor retained the binding specificity for different peptides and long C-terminal fragments of NPY exhibited by membrane preparations. Gel filtration of solubilized receptors resulted in a single peak of specific PYY binding activity corresponding to Mr = 350,000 whereas affinity labeling revealed a major band of Mr = 60,000. Since this binding activity was inhibited by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) the Y2 receptor is probably solubilized as a receptor complex containing a G-protein along with the ligand binding protein. Y2 receptor binding sites from kidney tubular membranes were purified to homogeneity by a three-step procedure employing Mono S cation-exchange adsorption, affinity chromatography on wheat germ lectin-agarose beads, and affinity chromatography on NPY-Affi-Gel. Electrophoresis and silver staining of the final receptor preparation revealed a single protein with Mr = 60,000 whereas gel filtration showed a single peak at approximately Mr = 60,000. The purified protein can be affinity labeled with [125I-Tyr36]PYY, indicating that the Mr = 60,000 protein contains the ligand binding site of the Y2 receptor, and this binding is not affected by GTP gamma S. Scatchard transformation of binding data for the purified Y2 receptors was compatible with a single class of binding sites with Kd = 76 pM. The purified Y2 receptors retain their binding properties with regard to affinity and specificity for different members of the pancreatic polypeptide-fold peptide family. The specific activity of purified Y2 receptors was calculated to approximately 14.7 nmol of ligand binding/mg of receptor protein, which is consistent with the theoretical value (16.6 nmol/mg) for a pure Mr = 60,000 protein binding one PYY molecule. Purification to homogeneity thus reveals the Y2 receptor as an Mr = 60,000 glycoprotein.     

Identification of a Drosophila brain-gut peptide related to the neuropeptide Y family.

A neuropeptide F (NPF) was isolated from the fruit fly, Drosophila mellanogaster, based on a radioimmunoassay for a gut peptide from the corn earworm, Helicoverpa zea. A partial sequence was obtained from the fly peptide, and a genomic sequence coding for NPF was cloned after inverse polymerase chain reaction and shown to exist as a single genomic copy. The encoded, putative prepropeptide can be processed into an amidated NPF with 36 residues that is related to invertebrate NPF's and the neuropeptide Y family of vertebrates. In situ hybridization and immunocytochemistry showed that Drosophila NPF was expressed in the brain and midgut of fly larvae and adults.     

Zebrafish genes for neuropeptide Y and peptide YY reveal origin by chromosome duplication from an ancestral gene linked to the homeobox cluster

Neuropeptide Y (NPY) and peptide YY (PYY) are related 36-amino acid peptides. NPY is widely distributed in the nervous system and has several physiological roles. PYY serves as an intestinal hormone as well as a neuropeptide. We report here cloning of the npy and pyy genes in zebrafish (Danio rerio). NPY differs at only one to four amino acid positions from NPY in other jawed vertebrates. Zebrafish PYY differs at three positions from PYY from other fishes and at 10 positions from mammals. In situ hybridization showed that neurons containing NPY mRNA have a widespread distribution in the brain, particularly in the telencephalon, optic tectum, and rhombencephalon. PYY mRNA was found mainly in brainstem neurons, as reported previously for vertebrates as divergent as the rat and the lamprey, suggesting an essential role for PYY in these neurons. PYY mRNA was observed also in the telencephalon. These results were confirmed by immunocytochemistry. As in the human, the npy gene is located adjacent to homeobox (hox) gene cluster A (copy a in zebrafish), whereas the pyy gene is located close to hoxBa. This suggests that npy and pyy arose from a common ancestral gene in a chromosomal duplication event that also involved the hox gene clusters. As zebrafish has seven hox clusters, it is possible that additional NPY family genes exist or have existed. Also, the NPY receptor system seems to be more complex in zebrafish than in mammals, with at least two receptor genes without known mammalian orthologues.     

Cryptic peptide derived from the rat neuropeptide FF precursor affects G-proteins linked to opioid receptors in the rat brain

Recently, we reported the discovery of a novel amino acid sequence derived from the NPFF precursor NAWGPWSKEQLSPQA, which blocked the expression of conditioned place preference induced by morphine and reversed the antinociceptive activity of morphine (5mg/kg, s.c.) in the tail-immersion test in rats. Here, we name it as NPNA (Neuropeptide NA from its flanking amino acid residues). The synthetic peptide influenced the expression of mRNA coding for Galpha(i1), (i2), and (i3) subunits. The results provide further evidence that yet another bioactive sequence might be present within the NPFF precursor.     

Aspects of the molecular structure and dynamics of neuropeptide Y.

Human neuropeptide Y (hNPY) and the Q34-->P34 mutant (P34-hNPY) have been characterized by CD spectroscopy. hNPY self-associates in aqueous solution with a dimerization constant in the micromolar range. The self-association correlates with an increase in secondary-structure content which was studied as a function of concentration, temperature and pH. The effects of temperature were measured in water (5-84 degrees C) and in ethanediol/water (2 : 1) (-90 degrees to +90 degrees C). A single-residue mutation, Q34-->P34, affects the pH, thermal and self-association properties of NPY. The CD results are correlated with photochemically induced dynamic nuclear polarization NMR experiments which show that the tyrosines at the interface between two monomer units present limited accessibility to a photoreactive dye. An equilibrium state is described, involving a PP-fold monomer form and a handshake dimer form, that accommodates the physicochemical properties of NPY.     

Identification of neuropeptide Y cleavage products in human blood to improve metabolic stability

Regulatory, receptor-binding peptides are considered as the agents of choice for diagnostic imaging and therapy of cancers, because their receptors are overexpressed in various human cancer cells. It has been recently indicated that there is a putative role of NPY in breast tumors. The expression of the two best-investigated NPY receptor subtypes, Y1 and Y2, in breast tissue shows predominant occurrence of the Y1 receptor subtype in tumors, whereas Y2 receptors are found in nonproliferative tissue. To investigate the usefulness of NPY analogs for tumor diagnosis and therapy, we investigated the metabolic stability of receptor-selective NPY analogs in human blood plasma. NPY analogs were synthesized by Fmoc/t-Bu solid-phase strategy. Prior to the cleavage of peptides from the resin, they were labeled with 5(6)-carboxyfluorescein (CF) either at the N-terminus or at the side chain of Lys4. For the metabolic stability study, the digestion of peptides was monitored by HPLC and the cleavage products were identified by MALDI-ToF mass spectrometry. The data showed that full-length [Phe7, Pro34]NPY analogs, which show high binding affinity to Y1 receptors are enzymatically more stable than centrally truncated analogs, which show high binding affinity to Y2 receptors. Furthermore, the N-terminally CF-labeled Y1 and Y2 receptor-selective peptides were found to be enzymatically more resistant than their counterparts containing the CF label at Lys4 side chain.     

Design of the Y1-receptor-selective cyclic peptide based on the C-terminal sequence of neuropeptide Y.

We designed four cyclic peptides which are mimics of the C-terminal region of human neuropeptide Y (NPY) on the basis of the structural model of NPY. One of these cyclic peptides, c[D-Cys29-L-Cys34]NPY Ac-29-36 (YM-42454), exhibited significantly higher affinity for the Y1-receptor than the corresponding C-terminal linear fragment, NPY Ac-28-36. Interestingly, YM-42454 showed binding affinity for the Y1-receptor in spite of the lack of the N-terminal sequence of NPY, whereas it did not show any binding affinity for the Y2-receptor. This conformationally restricted Y1-selective peptide would provide some insights into the bioactive conformation of the C-terminal region of NPY.     

Autoradiographic localization of peptide YY and neuropeptide Y binding sites in the medulla oblongata

Peptide YY is a highly potent emetic when given intravenously in dogs. We hypothesized that the area postrema, a small brain stem nucleus that acts as a chemoreceptive trigger zone for vomiting and lies outside the blood-brain barrier, might have receptors that PYY would bind to, in order to mediate the emetic response. We prepared [125I]PYY and used autoradiography to show that high affinity binding sites for this ligand were highly localized in the area postrema and related nuclei of the dog medulla oblongata. Furthermore, the distribution of [125I]PYY binding sites in the rat medulla oblongata was very similar to that in the dog; the distribution of [125I]PYY binding sites throughout the rat brain was seen to be similar to the distribution of [125I]NPY binding sites.     

Synthesis and biological properties of 4-norleucine-neuropeptide Y; secondary structure of neuropeptide Y

Neuropeptide Y (NPY) is a 36 amino acid peptide amide isolated from porcine brain. The NPY analog, 4-norleucine-NPY was synthesized by a solid-phase method and purified to homogeneity in 20% yield by reverse-phase chromatography. Investigation of the biological properties indicated that the analog is an agonist of NPY. Secondary structural analyses revealed that NPY and the analog exhibited predominantly alpha-helical and beta-sheet structures, respectively; however, experiments in trifluoroethanol indicated that the analog has the potential of assuming an alpha-helical structure. Based on circular dichroism (CD), Raman spectroscopy and Chou-Fasman analyses, a model has been proposed for the secondary structure of NPY.     

NMR solution structure of the precursor for carnobacteriocin B2, an antimicrobial peptide from Carnobacterium piscicola.

Type IIa bacteriocins, which are isolated from lactic acid bacteria that are useful for food preservation, are potent antimicrobial peptides with considerable potential as therapeutic agents for gastrointestinal infections in mammals. They are ribosomally synthesized as precursors with an N-terminal leader, typically 18-24 amino acid residues in length, which is cleaved during export from the producing cell. We have chemically synthesized the full precursor of carnobacteriocin B2, precarnobacteriocin (preCbnB2), which has a C-terminal amide rather than a carboxyl, and also produced preCbnB2(1-64), which is missing two amino acid residues at the C-terminus (Arg65 and Pro66), via expression in Escherichia coli as a maltose-binding protein fusion that is then cut with Factor Xa. PreCbnB2(1-64) is readily labeled with (15)N and (13)C for NMR studies using the latter approach. Multidimensional NMR analysis of preCbnB2(1-64) shows that, like the parent bacteriocin, it exists as a random coil in water but assumes a defined conformation in water/trifluoroethanol mixtures. In 70 : 30 trifluoroethanol/water, the 3D structure of the preCbnB2 section corresponding to the mature bacteriocin is essentially the same as reported previously by us for carnobacteriocin B2 (CbnB2). This structure maintains the highly conserved alpha-helix corresponding to residues 20-38 of CbnB2 that is believed to be responsible for interaction with a target receptor in sensitive cells, including Listeria monocytogenes. PreCbnB2 also has a second alpha-helix from residues 3-13 (i.e. -15 to -5 relative to CbnB2) in the leader section of the peptide. This helix appears to be conserved in related type IIa bacteriocin precursors based on sequence analysis. It is likely to be a key recognition element for export and processing, and is probably responsible for the considerably reduced antimicrobial activity of preCbnB2. The latter effect may assist the producing cell in avoiding the toxic effects of the bacteriocin. This is the first 3D structure determined for a prebacteriocin from lactic acid bacteria.