The distribution of pancreatic polypeptide in the nervous system and gut of the blowfly,Calliphora vomitoria (Diptera)

Summary The distribution of a neuropeptide, previously shown to have the same or a very similar amino acid composition as vertebrate pancreatic polypeptide (PP), has been studied in the nervous system and gut of the blowfly, Calliphora vomitoria. Neurones immunoreactive to a bovine PP antiserum occur in the thoracic and abdominal ganglionic components of the central nervous system, in addition to the brain and suboesophageal ganglion. Pancreatic polypeptide appears to be relayed from its cells of origin to a neurohaemal organ in the dorsal sheath of the thoracic ganglion. PP immunoreactivity is also found in cells of the hypocerebral ganglion of the stomatogastric nervous system and in associated nerve fibres. The mid-gut contains PP-positive material in flask-shaped cells of its epithelial lining.     

Neuroendocrine peptides (insulin, pancreatic polypeptide, neuropeptide Y, galanin, somatostatin, substance P, and neuropeptide gamma) from the desert tortoise, Gopherus agassizii.

The traditional view that Testudines (tortoises and turtles) should be regarded as the surviving clade of the anapsid reptiles rather than classified with the diapsid reptiles (snakes, lizards, and crocodiles) has recently been challenged. Neuropeptide Y, neuropeptide gamma, and somatostatin-14 were isolated from an extract of the brain, substance P and galanin from an extract of the intestine, and insulin and pancreatic polypeptide from an extract of the pancreas of the desert tortoise, Gopherus agassizii. Despite that crocodilians did not appear until the late Triassic, the amino acid sequences of the tortoise peptides resemble those of the American alligator quite closely. The primary structures of neuropeptide Y, somatostatin-14, and neuropeptide gamma are the same in tortoise and alligator. The primary structures of substance P, insulin, galanin, and pancreatic polypeptide in the two species differ by 1, 3, 5, and 8 amino acid residues, respectively. Although fewer neurohormonal peptides from squamates (lizards and snakes) have been characterized, the primary structures of neuropeptide gamma, insulin, and pancreatic polypeptide from the Burmese python and the desert tortoise differ by 3, 8, and 18 residues, respectively. The data suggest, therefore, a closer phylogenetic relationship between Testudines and Crocodilians than that derived from 'classical' analyses based on morphological criteria and the fossil record.     

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).     

Characterization of an amidated form of pancreatic polypeptide from the daddy sculpin (Cottus scorpius)

The primary structure of pancreatic polypeptide from the teleostean fish, Cottus scorpius (daddy sculpin) was established as: YPPQPESPGGNASPEDWAKYHAAVRHYVNLITRQRYNH2 The presence of a COOH-terminally alpha-amidated amino acid was established using an HPLC method of general applicability. Although the peptide shows strong homology towards anglerfish pancreatic polypeptide (86%), homology towards porcine peptide YY (PYY) (61%) and porcine neuropeptide Y (NPY) (61%) was greater than towards porcine pancreatic polypeptide (PP) (47%). This result supports suggestions that the gene duplication events which led to PP, NPY and PYY formation took place after the time of divergence of fish and mammals.     

Isolation and partial sequencing of a pancreatic polypeptide-like neuropeptide from the liver fluke, Fasciola hepatica.

1. A pancreatic polypeptide (PP)-immunoreactive neuropeptide has been isolated and partially sequenced from the liver fluke, Fasciola hepatica. 2. Gel permeation chromatography of an acid ethanol extract of cattle flukes showed that the peptide is similar in size to mammalian (bovine) PP. 3. The Fasciola peptide was purified to homogeneity by means of reverse-phase HPLC, employing different column chemistries. 4. The purified peptide was sequenced using automated gas-phase Edman degradation and the first 24 amino acid residues determined.     

Isolation and partial characterization of pancreatic polypeptide-like material in the brain of the blowfly Calliphora vomitoria.

Using 10(6) flies (5 kg of heads) a pancreatic polypeptide-like material has been partially purified from the blowfly Calliphora vomitoria. The isolated material was eluted on Sephadex G-50 similarly to bovine pancreatic polypeptide and had an RF on polyacrylamide-gel electrophoresis that was identical with that of the bovine hormone. The material diluted linearly and showed parallelism with bovine standards in a bovine pancreatic polypeptide immunoassay. In specificity controls the immunoreactivity was not abolished by trasylol and no cross-reactivity was discerned in assay for glucagon, proangiotensin and cyclic AMP. These data suggest that the pancreatic polypeptide material in the brain of the blowfly has close structural similarity to the mammalian hormone.     

Complete cDNA and gene sequence of the developmentally regulated arylphorin of Calliphora vicina and its homology to insect hemolymph proteins and arthropod hemocyanins.

Two cDNA libraries were prepared from poly(A)+ RNA isolated from fat bodies of last instar larvae of the blowfly Calliphora vicina. The libraries were probed with a genomic clone containing the coding sequence for an arylphorin subunit. Two cDNA clones as well as the genomic clone were mapped and their nucleotide sequences were determined. This revealed the presence of an open reading frame corresponding to a polypeptide with 759 amino acid residues. The deduced primary structure of Calliphora arylphorin and hemolymph proteins of other insect species and arthropod hemocyanine show nearly 30% identity. Highly conserved regions could be also identified.     

Isolation and partial sequencing of a pancreatic polypeptide-like neuropeptide from the liver fluke,Fasciola hepatica

1. A pancreatic polypeptide (PP)-immunoreactive neuropeptide has been isolated and partially sequenced from the liver fluke, Fasciola hepatica.2. Gel permeation chromatography of an acid ethanol extract of cattle flukes showed that the peptide is similar in size to mammalian (bovine) PP.3. The Fasciola peptide was purified to homogeneity by means of reverse-phase HPLC, employing different column chemistries.4. The purified peptide was sequenced using automated gas-phase Edman degradation and the first 24 amino acid residues determined.     

Isolation of peptide hormones from the pancreas of the bullfrog (Rana catesbeiana). Amino acid sequences of pancreatic polypeptide, oxyntomodulin, and two glucagon-like peptides

Insulin, pancreatic polypeptide, glucagon, oxyntomodulin, and two distinct glucagon-like peptides were isolated from acidic ethanol extracts of bullfrog pancreas by gel filtration followed by high pressure liquid chromatography. The amino acid sequences of pancreatic polypeptide, oxyntomodulin, and both glucagon-like peptides were determined. Frog pancreatic polypeptide contains 36 amino acid residues and has a COOH-terminal phenylalaninamide. It is more homologous with human pancreatic polypeptide (61%) than other characterized members of this family of peptides. Frog glucagon has an amino acid composition identical to the NH2-terminal 29 residues of the larger, more abundant oxyntomodulin and was not sequenced. The finding of a single form of glucagon and oxyntomodulin, but two glucagon-like peptides in frog pancreas extract is similar to that found or deduced for mammals.     

Structure of a precursor to human pancreatic polypeptide

We have isolated mRNA from a human pancreatic islet cell tumor and have identified among the cell-free translation products a precursor of pancreatic polypeptide with an approximate Mr = 11,000. Recombinant DNA molecules encoding this precursor were selected from a cDNA library prepared from the islet tumor mRNA. From the nucleotide sequences of cDNAs encoding the precursor, we have deduced the complete amino acid sequence of pre-propancreatic polypeptide. These sequences encode a protein consisting of 95 amino acid residues with a Mr = 10,432. The sequence of human pancreatic polypeptide occurs in the middle of the precursor and is flanked at its carboxyl terminus by a 27-amino acid sequence which is similar to a peptide previously isolated from canine pancreatic islets. At the amino terminus of the precursor is a probable leader sequence which is rich in hydrophobic residues. A smaller pancreatic polypeptide-related protein was generated in cell-free translations of mRNA supplemented with microsomal membranes. Sequential Edman degradations of this smaller peptide indicate that the sequence of pancreatic polypeptide is located at the amino terminus of the prohormone.     

Accelerated evolution of the pituitary adenylate cyclase-activating polypeptide precursor gene during human origin

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide abundantly expressed in the central nervous system and involved in regulating neurogenesis and neuronal signal transduction. The amino acid sequence of PACAP is extremely conserved across vertebrate species, indicating a strong functional constraint during the course of evolution. However, through comparative sequence analysis, we demonstrated that the PACAP precursor gene underwent an accelerated evolution in the human lineage since the divergence from chimpanzees, and the amino acid substitution rate in humans is at least seven times faster than that in other mammal species resulting from strong Darwinian positive selection. Eleven human-specific amino acid changes were identified in the PACAP precursors, which are conserved from murine to African apes. Protein structural analysis suggested that a putative novel neuropeptide might have originated during human evolution and functioned in the human brain. Our data suggested that the PACAP precursor gene underwent adaptive changes during human origin and may have contributed to the formation of human cognition.     

Isolation and identification of islet amyloid polypeptide in normal human pancreas

To identify islet amyloid polypeptide (IAPP) present in normal human pancreas, we isolated the peptide from a soluble peptide fraction of amyloid deposit-free pancreata of two non-diabetic patients by using reverse-phase high performance liquid chromatography coupled with a radioimmunoassay specific for human IAPP. IAPP(1-37) and IAPP(17-37) were isolated and their complete amino acid sequences were determined up to the C-terminus. Identification of IAPP in normal human pancreas suggests the possible biological function of IAPP as a novel pancreatic hormone in humans.     

Neuropeptide Y-related peptides from the pancreas of a teleostean (eel), holostean (bowfin) and elasmobranch (skate) fish

Homologous peptides belonging to the pancreatic polypeptide (PP) family were isolated from the pancreas of a teleostean fish, the American eel (Anguilla rostrata), an holostean fish, the bowfin (Amia calva) and an elasmobranch fish, the skate (Raja rhina), and their primary structures were determined. The peptides show stronger homology to neuropeptide Y, particularly in their COOH-terminal regions, than to peptide YY or pancreatic polypeptide and contain an alpha-amidated COOH-terminal tyrosine residue. The skate peptide Tyr-Pro-Pro-Lys-Pro-Glu-Asn-Pro-Gly-Asp10-Asp-Ala-Ala-Pro-Glu-Glu- Leu-Ala-Lys- Tyr20-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu30-Ile-Thr-Arg- Gln-Arg-Tyr-NH2 represents the first member of the PP family to be isolated from a cartilaginous fish. The primary structure of the pancreatic PP family peptide has been more strongly conserved among the phylogenetically more ancient holostean and elasmobranch fishes than among the teleosts. A comparison of the primary structures of all PP family peptides supports the hypothesis and evolution has acted to conserve features of tertiiary structure in the molecules (e.g., the polyproline- and alpha-helices) rather than individual amino acid residues.     

The amino acid sequence of pancreatic spasmolytic polypeptide

The sequence of porcine pancreatic spasmolytic polypeptide has been established by a variety of techniques including manual as well as automatic sequencing of fragments resulting from the cleavage of reduced and S-carboxymethylated pancreatic spasmolytic polypeptide with trypsin, chymotrypsin, clostripain, cyanogen bromide and formic acid. The N- and C-terminal sequences were established using pyroglutamate amino-peptidase and carboxypeptidase A, respectively. Pancreatic spasmolytic polypeptide contains 106 amino acid residues in a single chain with seven S-S bridges and a pyroglutamyl blocked N-terminal. The alignment of the sequences representing amino acids 14-49 and 63-98 shows pair-wise identical amino acid residues in 18 out of 36 positions, indicating that these two "domains" have been derived from a common gene.     

Peptide YY. Structure of the precursor and expression in exocrine pancreas

Peptide YY is a 36-residue gastrointestinal hormone which inhibits both pancreatic and gastric secretion. We have isolated a cDNA encoding the peptide YY precursor by screening a rat intestinal lambda gt11 cDNA library with an antiserum directed against the porcine hormone. The nucleotide sequence of the cDNA encodes a 98-residue protein (molecular weight, 11, 121) which has an amino acid sequence identical to that of porcine peptide YY. Rat peptide YY is preceded immediately by a signal sequence and followed by a cleavage-amidation sequence Gly-Lys-Arg plus 31 additional amino acids. Thus the peptide YY precursor is similar in structure to that of two related peptides, pancreatic polypeptide and neuropeptide Y. RNA blot hybridizations reveal that the peptide YY gene is much more actively expressed in pancreas than previously realized. In situ hybridizations localized peptide YY cells exclusively to the exocrine pancreas. The abundance of peptide YY in one of its target organs, the pancreas, suggests a paracrine mechanism for peptide YY in regulating pancreatic enzyme secretion.     

Purification and primary structure of ostrich pancreatic polypeptide

Pancreatic polypeptide has been isolated from ostrich pancreas by gel filtration, ion exchange chromatography and high pressure liquid chromatography. The ostrich peptide contains 36 amino acids and has an amino acid composition similar to pancreatic polypeptide of other avian species. The primary structure of ostrich pancreatic polypeptide differs from that of the chicken peptide only at residues 3 and 18 where the ostrich peptide contains an alanine and a valine residue compared to the serine and isoleucine residues found in those positions in the chicken peptide.     

A critical examination of the occurrence of FMRFamide immunoreactivity in the brain of guinea pig and rat

Summary Several reports (cf. Weber et al. (1981) Science 214: 1248–1251) have described the extensive occurrence, in rat brain, of material immunologically related to the molluscan neuropeptide FMRFamide. We have reexamined these data in guinea pig and rat, using six different antisera to FMRFamide. Immunoreactive perikarya and fibres were found to be distributed throughout the rodent brain (Table 1). This distribution was roughly similar to that found by Weber and coworkers. However, solid-phase absorption of the antisera with bovine pancreatic polypeptide, which shares an arginine and an amidated aromatic amino acid (RYamide) with FMRFamide, showed that staining in most regions could be due to crossreactivity with bovine pancreatic polypeptide-like (or neuropeptide Y-like) material. Double-labelling experiments with antisera to FMRFamide and bovine pancreatic polypeptide led to the same conclusion. The only structures where no apparent crossreactivity occurred were perikarya and fibres in the nucleus dorsomedialis, ventromedialis, periventricularis and paraventricularis hypothalami and fibres in the area lateralis hypothalami, nucleus parabrachialis, substantia grisea centralis mesencephali, various parts of the formatio reticularis, and spinal cord. Hence only these structures might contain material which is more related to the molluscan retrapeptide.Used one-letter abbreviations of amino acids D aspartic acid - F phenylalanine - G glycine - H histidine - L leucine - M methionine - P proline - R arginine - T threonine - V valine - W tryptophan - Y tyrosine     

Immunohistochemical detection of secretoneurin, a novel neuropeptide endoproteolytically processed from secretogranin II, in normal human endocrine and neuronal tissues

Summary An antiserum raised against a synthetic peptide derived from the primary amino sequence of rat secretogranin II (chromogranin C) was used for immunological (quantitative radioimmunoassay analysis) and immunohistochemical studies of normal human endocrine and nervous tissues. This antibody recognized a novel and biologically active neuropeptide which was coined as secretoneurin. In endocrine tissues, secretoneurin was mainly co-localized with chromogranin A and B with some exceptions (e.g., parathyroid gland). Secretoneurin was demonstrated immunohistochemically in the adrenal medulla, thyroid C cells, TSH- and FSH/LH-produting cells of the anterior pituitary, A and B cells of pancreatic islets, in endocrine cells of the gastrointestinal tract and the bronchial mucosa, and the prostate. Immunoreactivity determined by radioimmunoassay analysis revealed high secretoneurin levels in the anterior and posterior pituitary and lower levels in pancreatic and thyroid tissue. A strong secretoneurin immunoreactivity was also found in ganglion cells of the submucdsal and myenteric plexus of the gastrointestinal tract, and in ganglionic cells of dorsal root ganglia, peripheral nerves, and ganglion cells of the adrenal medulla. Thus, secretoneurin may serve as a useful marker of gangliocytic/neuronal differentiation.     

Mosaic evolution of prepropancreatic polypeptide

Pancreatic polypeptide, a 36-amino acid peptide hormone, is synthesized in pancreatic islets of Langerhans and acts as a regulator of pancreatic and gastrointestinal functions. We isolated cDNA clones encoding rat pancreatic polypeptide precursor from an islet cDNA library and determined their nucleic acid sequences. Rat pancreatic polypeptide was found to be flanked on the amino terminus by a putative signal peptide and on the carboxyl terminus by Gly-Lys-Arg followed by a 30-amino acid peptide. Nucleotide and amino acid sequences of the signal peptide and the pancreatic polypeptide of the rat were highly homologous to those of the human (Boel, E., Schwartz, T. W., Norris, K. E., and Fill, N. P. (1984) EMBO J. 3, 909-912). On the other hand, the rat carboxyl-terminal peptide differed markedly from the corresponding domain of the human precursor and did not contain any sequence similar to the icosapeptide, which has so far been known to be a second stable product from mammalian pancreatic polypeptide precursors (Schwartz, T. W., Hansen, H. F., Hakanson, R., Sundler, F., and Tager, H. S. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 708-712). The mosaicism of sequence conservation and divergence in prepropancreatic polypeptides may be a unique example in the evolution of prohormones.