Identification and molecular cloning of a neuropeptide Y homolog that produces prolonged inhibition in Aplysia neurons.

The neuroendocrine bag cell neurons of the marine mollusk Aplysia produce prolonged inhibition that lasts for more than 2 hr. We purified a peptide from the abdominal ganglion that mimics this inhibition. Mass spectrometry and microsequence analysis indicate that the peptide is 40 aa long and is amidated at its carboxyl terminus. It is highly homologous to vertebrate neuropeptide Y (NPY) and other members of the pancreatic polypeptide family. As determined from cloned cDNA, the gene coding for the precursor protein shares a common structural organization with genes encoding precursors of the vertebrate family. The peptides may therefore have arisen from a common ancestral gene. Bag cell neurons are immunoreactive for Aplysia NPY, and Northern blot analysis indicates that as with its vertebrate counterparts, the peptide is abundantly expressed in the CNS. This suggests that peptides related to NPY may have important functions in the nervous system of Aplysia as well as in other invertebrates.     

Release of pedal peptide from Aplysia neurons in primary culture

Pedal peptide (Pep) is a modulatory neuropeptide that is predominantly synthesized in a group of neurons on the dorsal surfaces of the pedal ganglia of Aplysia. Following the determination that Pep is the major peptide selectively present in these neurons in situ, primary cell culture of single Pep-neurons was used to study the release of this neuropeptide. Individual Pep-neurons were grown in culture where they extended many branched neurites with large varicosities. Immunocytology revealed that these newly grown varicosities were intensely Pep immunoreactive. Cultured Pep-neurons, grown in a medium containing radiolabeled methionine, synthesized labeled Pep and transported it into their regenerated neurites. Finally, these neurons released radiolabeled Pep in a calcium- and stimulation-dependent fashion. These results, taken together with previous findings, strongly support the proposition that Pep is a transmitter in Aplysia.     

Planarian peptidylglycine-hydroxylating monooxygenase, a neuropeptide processing enzyme, colocalizes with cytochrome b561 along the central nervous system

Planarians are one of the simplest animal groups with a central nervous system. Their primitive central nervous system produces large quantities of a variety of neuropeptides, of which many are amidated at their C terminus. In vertebrates, peptide amidation is catalyzed by two enzymes [peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxylglycine alpha-amidating lyase] acting sequentially. In mammals, both enzymatic activities are contained within a single protein that is encoded by a single gene. By utilizing PCR with degenerate oligonucleotides derived from conserved regions of PHM, we succeeded in cloning a full-length cDNA encoding planarian PHM. The deduced amino acid sequence showed full conservation of five His residues and one Met residue, which bind two Cu atoms that are essential for the activity of PHM. Northern blot analysis confirmed the expression of a PHM mRNA of the expected size. Distribution of the mRNA was analyzed by in situ hybridization, showing specific expression in neurons with two morphologically distinct structures, a pair of the ventral nerve cords and the brain. The distribution of PHM was very similar to that of cytochrome b561. This indicates that the ascorbate-related electron transfer system operates in the planarian central nervous system to support the PHM activity and that it predates the emergence of Plathelminthes in the evolutionary history.     

Modulation of growth of Aplysia neurons by an endogenous lectin.

We have purified and characterized a galactose-binding lectin from the gonads of the mollusk Aplysia californica that modulates neurite outgrowth from cultured Aplysia neurons. Agglutination of sheep red blood cells (RBC) by this lectin, termed Aplysia gonad lectin (AGL), is inhibited strongly by galactose and to a lesser extent by fucose. On SDS-PAGE, AGL appears as a single species with a molecular weight of 34 kD under reducing conditions, and 65 kD under nonreducing conditions. This suggests that AGL is a disulfide-linked dimer in its native state. Amino terminal sequence analysis of purified AGL indicates a similarity to another galactose-binding lectin, phytohemagglutinin-E (E-PHA), found in red kidney beans. By using polyclonal antibodies prepared against AGL, we have found that the lectin is present in the gonads and eggs but not in other tissues of adult Aplysia californica. We have examined biological actions of AGL on Aplysia neurons growing in primary cell culture. AGL affects several properties of these neurons. The addition of 100 nM AGL to cultured neurons enhances neurite outgrowth from the cell soma, resulting in a greater number of primary processes. In addition, AGL acts as a neurotrophic agent, increasing neurite viability in vitro. This trophic effect is not seen with concanavalin A (con A), another lectin known to affect several properties of cultured Aplysia neurons. The results are consistent with the suggestion that AGL may play a role in neuronal differentiation and/or maintenance of viability.     

Levitide, a neurohormone-like peptide from the skin of Xenopus laevis. Peptide and peptide precursor cDNA sequences

A novel peptide, levitide, less than Glu-Gly-Met-Ile-Gly-Thr-Leu-Thr-Ser-Lys-Arg-Ile-Lys-Gln-NH2 has been isolated from skin secretions of the South African frog Xenopus laevis and sequenced by fast atom bombardment mass spectrometry. Synthetic oligonucleotides were used as probes to screen a X. laevis skin cDNA library for species coding for preprolevitide. Two such clones were detected and their sequences are reported here. Preprolevitide is 88 residues long, exhibits a putative signal sequence at the amino terminus, and contains the levitide peptide at the carboxyl terminus. The levitide precursor shows a striking nucleotide and amino acid (86%) sequence homology with the precursor of xenopsin, a biologically active octapeptide from Xenopus skin, and also encodes a 25-residue amphipathic peptide that is released by processing at a single arginine residue.     

The structure of anchorin CII, a collagen binding protein isolated from chondrocyte membrane

cDNA clones for anchorin CII (Mr = 34,000), a collagen-binding protein, were isolated from a lambda gt 11 cDNA library prepared from chick cartilage mRNA. Several overlapping clones were characterized which gave rise to an open reading frame coding for 329 residues and a 3'-untranslated segment of 500 base pairs. The clones were identified as coding for anchorin by hybrid select translation analysis and by comparing the deduced amino acid sequence with the sequence of 10 tryptic peptides of the protein. A hydrophobic domain of 25 residues interrupted with 3 polar residues was identified with the carboxyl-terminal portion. There was no evidence for an aminoterminal signal peptide. Northern analysis revealed that the 5' probe hybridizes to a single 1.7-kilobases (kb) mRNA species, whereas the 3' probe hybridizes to two mRNA species of 1.7 kb and 5 kb, which are present in many cells including chondrocytes, crop cells, and fibroblasts. The level of anchorin mRNA in chick embryo fibroblasts was increased by infection with Rous sarcoma virus.     

Abundant expression of ras proteins in Aplysia neurons

We have cloned a DNA fragment from the marine mollusc Aplysia californica, which contains sequences homologous to mammalian ras genes, by screening a genomic library with a viral Ha-ras oncogene probe under conditions of low stringency hybridization. Nucleotide sequencing revealed a putative exon that encodes amino acids sharing 68% homology with residues 5 to 54 of mammalian p21ras polypeptides, and which therefore is likely to encode a ras-like Aplysia protein. The cloned locus, designated Apl-ras, is distinct from the Aplysia rho (ras-homologue) gene and appears to be more closely related to mammalian ras. We used a panel of monoclonal antibodies raised against v-Ha-ras p21 to precipitate an Mr 21,000 protein from extracts of Aplysia nervous tissue, ovotestis, and, to a much lesser degree, buccal muscle. Fluorescence immunocytochemistry revealed that ras-like protein is most abundant in neuronal cell bodies and axon processes, with staining most prominent at plasma membranes. Much less was present in other tissues. The prominence of ras protein in neurons, which are terminally differentiated and non-proliferating, indicates that the control of cell division is not the sole function of this proto-oncogene. The large identified neurons of Aplysia offer the opportunity to examine how ras protein might function in mature nerve cells.     

Sequence of small cardioactive peptide A: a second member of a class of neuropeptides in Aplysia

Previous studies have shown that the nervous system and other tissues of molluscs contain a number of peptides that potently excite molluscan hearts. Two such peptides, termed small cardioactive peptides A and B (SCPA and SCPB) are present in large quantities in the nervous system of Aplysia. These peptides are widely distributed within the CNS and peripheral tissues and have been found to be potent modulators of synaptic transmission in Aplysia. SCPB has previously been purified from nervous tissue and sequenced. In this paper, we report the purification of SCPA and propose its sequence. This sequence was confirmed by comparing the chromatographic properties of native SCPA (labelled in organ culture) with a synthetic peptide that has the proposed sequence. A significant proportion of the sequence of the two SCPs is conserved, indicating that they are members of the same peptide class, a finding that is consistent with the recent observation that the two peptide sequences are present in a single precursor.     

Distribution of the Aplysia cardioexcitatory peptide,NdWFamide, in the central and peripheral nervous systems of Aplysia

NdWFamide is an Aplysia cardioexcitatory tri-peptide containing D-tryptophan. To investigate the roles of this peptide, we examined the immunohistochemical distribution of NdWFamide-positive neurons in Aplysia tissues. All the ganglia of the central nervous system (CNS) contained NdWFamide-positive neurons. In particular, two left upper quadrant cells in the abdominal ganglion, and the anterior cells in the pleural ganglion showed extensive positive signals. NdWFamide-positive processes were observed in peripheral tissues, such as those of the cardio-vascular system, digestive tract, and sex-accessory organs, and in the connectives or neuropils in the CNS. NdWFamide-positive neurons were abundant in peripheral plexuses, such as the stomatogastric ring. To examine the NdWFamide contents of tissues, we fractionated peptidic extracts from the respective tissues by reversed-phase high-pressure liquid chromatography and then assayed the fractions by competitive enzyme-linked immunosorbent assay. A fraction corresponding to the retention time of synthetic NdWFamide contained the most immunoreactivity, indicating that the tissues contained NdWFamide. The prevalence of the NdWFamide content was roughly in the order: abdominal ganglion >heart >gill >blood vessels >digestive tract. In most of the tissues containing NdWFamide-positive nerves, NdWFamide modulated the motile activities of the tissues. Thus, NdWFamide seems to be a versatile neurotransmitter/modulator of Aplysia and probably regulates the physiological activities of this animal.     

Aplysia brasiliana neurons R3-R14: primary structure of the myoactive histidine-rich basic peptide and its prohormone

Neurons R3-R14 of the marine mollusc Aplysia are model neuroendocrine cells thought to regulate cardiovascular activity in vivo. The cells express a gene encoding three peptides--peptides I, II and the histidine-rich basic peptide (HRBP)--each of which has been chemically characterized in Aplysia californica. In the studies presented here, HRBP and its prohormone (proHRBP) were purified from A. brasiliana abdominal ganglion extracts by reversed-phase high-performance liquid chromatography and characterized by amino acid compositional and sequence analyses. ProHRBP was an 85-residue peptide whose sequence was: NH2-Glu-Glu-Val-Phe-Asp-Asp-Thr-Asp-Val-Gly-Asp-Glu-Leu-Thr-Asn-Ala-Leu- Glu-Ser - Val-Leu-Thr-Asp-Leu-Lys-Asp-Lys-Arg-Asp-Ala-Glu-Glu-Pro-Ser-Ala-Phe-Met- Thr-Arg - Leu-Arg-Arg-Gln-Val-Ala-Gln-Met-His-Ile-Trp-Arg-Ala-Asn-His-Asp-Arg-His- His-Ser - Thr-Gly-Ser-Gly-Arg-His-Ser-Arg-Phe-Leu-Thr-Arg-Asn-Arg-Tyr-Gly-Gly-Gly- His-Leu - Ser-Asp-Ala-COOG. It differed from A. californica pro-HRBP at seven of the 85 positions. Compositional and sequence analyses demonstrated that A. brasiliana HRBP was a 43-residue peptide corresponding to residues 43 through 85 of proHRBP, and that a significant proportion of the isolated peptide possessed a blocked NH2 terminus. Although this sequence differed from that of A. californica HRBP at five of 43 residues, the two peptides were approximately equipotent in inducing contractions of A. californica crop muscle in vitro, suggesting that the substituted residues may not be critical for biological activity.     

Aplysia californica neurons R3-R14: primary structure of the myoactive histidine-rich basic peptide and peptide I

The R3-R14 neurons of the marine mollusc Aplysia are neuroendocrine cells that express a gene encoding peptides I, II and histidine-rich basic peptide (HRBP), a myoactive peptide that excites Aplysia heart and enhances gut motility in vitro. Peptide II has been chemically characterized (35), but the complete primary structures of peptide I and HRBP have not been established by amino acid sequence analysis. HRBP, peptide I, and the prohormone (proHRBP) were therefore purified from acid extracts of Aplysia californica neural tissue using sequential gel filtration and reverse-phase high-performance liquid chromatography and chemically characterized. Amino acid sequence analysis demonstrated that HRBP was a 43-residue peptide whose sequence was: less than Glu-Val-Ala-Gln-Met-His-Val-Trp-Arg-Ala-Val-Asn-His-Asp-Arg-Asn-His-Gly- Thr-Gly - Ser-Gly-Arg-His-Gly-Arg-Phe-Leu-Ile-Arg-Asn-Arg-Tyr-Arg-Tyr-Gly-Gly-Gly- His-Leu - Ser-Asp-Ala-COOH. Compositional and sequence analyses of peptide I and proHRBP demonstrated that peptide I was a 26-residue peptide with the following sequence: NH2-Glu-Glu-Val-Phe-Asp-Asp-Thr-Asp-Val-Gly-Asp-Glu-Leu-Thr-Asn-Ala- Leu-Glu-Ser-Val-Leu-Thr-Asp-Phe-Lys-Asp-COOH. These results demonstrated that the pro-HRBP sequence predicted by nucleotide sequence analysis of a cDNA clone (24) was in fact synthesized in R3-R14 neurons. Hydrophilicity and hydrophobicity profiles of preproHRBP, combined with charge distribution profiles and predictive secondary structural analysis, showed that cleavage at dibasic sequences was strongly associated with peaks of hydrophilicity in alpha-helical regions of the preprohormone.     

Repetitive firing triggers clustering of Kv2.1 potassium channels in Aplysia neurons

The Kv2.1 gene encodes a highly conserved delayed rectifier potassium channel that is widely expressed in neurons of the central nervous system. In the bag cell neurons of Aplysia, Kv2.1 channels contribute to the repolarization of action potentials during a prolonged afterdischarge that triggers a series of reproductive behaviors. Partial inactivation of Aplysia Kv2.1 during repetitive firing produces frequency-dependent broadening of action potentials during the afterdischarge. We have now found that, as in mammalian neurons, Kv2.1 channels in bag cell neurons are localized to ring-like clusters in the plasma membrane of the soma and proximal dendrites. Either elevation of cyclic AMP levels or direct electrical stimulation of afterdischarge rapidly enhanced formation of these clusters on the somata of these neurons. In contrast, injection of a 13-amino acid peptide corresponding to a region in the C terminus that is required for clustering of Kv2.1 channels produced disassociation of the clusters, resulting in a more uniform distribution over the somata. Voltage clamp recordings demonstrated that peptide-induced dissociation of the Kv2.1 clusters is associated with an increase in the amplitude of delayed rectifier current and a shift of activation toward more negative potentials. In current clamp recording, injection of the unclustering peptide reduced the width of action potentials and reduced frequency-dependent broadening of action potentials. Our results suggest that rapid redistribution of Kv2.1 channels occurs during physiological changes in neuronal excitability.     

The primary structure of rat ribosomal protein S5. A ribosomal protein present in the rat genome in a single copy.

The amino acid sequence of the rat 40 S ribosomal subunit protein S5 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed by the determination, directly from the protein, of 17 residues near the NH2 terminus. S5 has 204 amino acids; the molecular weight is 22,863. The protein designated S5a has the same amino acid sequence as S5 except that it lacks the NH2-terminal 5 residues. It is not known whether the conversion of a portion of S5 to S5a is physiological or fortuitous. The mRNA for S5 has about 820 nucleotides. Hybridization of the S5 cDNA to digests of nuclear DNA indicates that the rat genome has only a single copy of the gene; this is in distinction to the mouse and human genomes which have three to six copies of the S5 gene. Rat ribosomal protein S5 is related to the eubacteria, the arachaebacteria, and the chloroplast family of S7 ribosomal proteins. There is a peptide of 16 residues at the carboxyl terminus of S5 that is highly conserved in 18 species spanning the three kingdoms and chloroplasts.     

Rat preprocarboxypeptidase H. Cloning, characterization, and sequence of the cDNA and regulation of the mRNA by corticotropin-releasing factor

Carboxypeptidase H is a putative post-translational processing enzyme which removes basic amino acid residues from intermediates during protein hormone biosynthesis. A 2.2-kilobase pair cDNA was shown to contain the complete amino acid sequence of rat carboxypeptidase H. The deduced amino acid sequence revealed that the enzyme was synthesized as preprocarboxypeptidase H, a precursor form of 476 amino acid residues. Preprocarboxypeptidase H contained a putative hydrophobic signal peptide and a short propeptide which contained 5 adjacent Arg residues at its C terminus. Northern blot analysis identified a single carboxypeptidase H mRNA of approximately 2.3 kilobases in brain, pituitary, and heart, as well as in mouse AtT20 cells. No carboxypeptidase H mRNA was detected in rat liver, spleen, kidney, lung, and mammary gland. Sequence analysis of cDNAs obtained from different rat tissues suggested that a single mRNA encodes an identical carboxypeptidase in several tissues. Treatment of AtT20 cells with dexamethasone decreased the levels of both carboxypeptidase H and preproopiomelanocortin (POMC) mRNAs by approximately 30%. Exposure of the dexamethasone-treated cells to corticotropin-releasing factor effected a 2- to 3-fold increase in the carboxypeptidase H and POMC mRNA levels relative to those of dexamethasone-treated cells exposed to control medium. This suggests that the mRNA levels of POMC and one of its putative post-translational processing enzymes, carboxypeptidase H, are co-regulated by corticotropin-releasing factor and steroid hormones.     

The dopamine D2 receptor: two molecular forms generated by alternative splicing.

Cloned human dopamine D2 receptor cDNA was isolated from a pituitary cDNA library and found to encode an additional 29 amino acid residues in the predicted intracellular domain between transmembrane regions 5 and 6 relative to a previously described rat brain D2 receptor. Results from polymerase chain reactions as well as in situ hybridization revealed that mRNA encoding both receptor forms is present in pituitary and brain of both rat and man. The larger form was predominant in these tissues and, as shown in the rat, expressed by dopaminergic and dopaminoceptive neurons. Analysis of the human gene showed that the additional peptide sequence is encoded by a separate exon. Hence, the two receptor forms are generated by differential splicing possibly to permit coupling to different G proteins. Both receptors expressed in cultured mammalian cells bind [3H]spiperone with high affinity and inhibit adenylyl cyclase, as expected of the D2 receptor subtype.     

Profiling 26,000 Aplysia californica neurons by single cell mass spectrometry reveals neuronal populations with distinct neuropeptide profiles

Neuropeptides are a chemically diverse class of cell-to-cell signaling molecules that are widely expressed throughout the central nervous system, often in a cell-specific manner. While cell-to-cell differences in neuropeptides is expected, it is often unclear how exactly neuropeptide expression varies among neurons. Here we created a microscopy-guided, high-throughput single cell matrix-assisted laser desorption/ionization mass spectrometry approach to investigate the neuropeptide heterogeneity of individual neurons in the central nervous system of the neurobiological model Aplysia californica, the California sea hare. In all, we analyzed more than 26,000 neurons from 18 animals and assigned 866 peptides from 66 prohormones by mass matching against an in silico peptide library generated from known Aplysia prohormones retrieved from the UniProt database. Louvain–Jaccard (LJ) clustering of mass spectra from individual neurons revealed 40 unique neuronal populations, or LJ clusters, each with a distinct neuropeptide profile. Prohormones and their related peptides were generally found in single cells from ganglia consistent with the prohormones’ previously known ganglion localizations. Several LJ clusters also revealed the cellular colocalization of behaviorally related prohormones, such as an LJ cluster exhibiting achatin and neuropeptide Y, which are involved in feeding, and another cluster characterized by urotensin II, small cardiac peptide, sensorin A, and FRFa, which have shown activity in the feeding network or are present in the feeding musculature. This mass spectrometry–based approach enables the robust categorization of large cell populations based on single cell neuropeptide content and is readily adaptable to the study of a range of animals and tissue types.     

Molecular cloning of S-protein, a link between complement, coagulation and cell-substrate adhesion.

cDNA clones coding for human S-protein have been isolated using monoclonal antibodies to screen a cDNA library in pEX. These clones are shown to be authentic S-protein clones on the basis of sequence, composition and immunological criteria. The complete open reading frame sequence for S-protein has been determined and shows it to be a single polypeptide chain of 459 amino acids preceded by a cleaved leader peptide of 19 residues. No evidence was found for polymorphism of S-protein suggesting that different molecular weight forms arise by proteolytic degradation. Of the first 44 amino-terminal residues 42 are identical with the so-called somatomedin B peptide suggesting that S-protein is the somatomedin B precursor. Striking homology is found in the rest of the sequence with the serum spreading factor, vitronectin, which has also been shown to contain somatomedin B sequences at its amino terminus. We conclude that S-protein and vitronectin are identical and discuss the relevance of this finding to the coagulation and complement pathways.     

Identification of an Acidic Dipeptide, β-Aspartylglycine, in the CNS of Aplysia

Abstract: A novel dipeptide, (β-aspartylglycine (β-DG), has been isolated from tissues of the marine gastropod mollusc Aplysia californica. This compound was detected only in Aplysia and not in other molluscs, such as Helix or Mercenaria , or in lobster or frog. Among the Aplysia tissues, the highest levels of β-DG were in nervous tissue and in the reproductive tract. β-DG was assayed by HPLC as the o -phthaldialdehyde derivative and found to be present in all individual, identified neurons at a concentration of approximately 40 pmol/μg protein. The peptide was identified as β-DG by gas chromatography-mass spectrometry (GCMS) using trimethylsilyl derivatives prepared before and after acid hydrolysis. It was further characterized as the β-isomer by TLC, including R_f, atypical blue-gray color with ninhydrin, and a violet color with Cu²⁺-ninhydrin. A fractionation scheme is described whereby acid-soluble tissue constituents can be divided into acidic, neutral, and basic components using mini ion-exchange columns. This partial purification prior to TLC analysis was necessary to remove compounds that interfered with the isolation of β-DG.     

Evidence for conservation of ferritin sequences among plants and animals and for a transit peptide in soybean

Ferritin is a large multisubunit protein that stores iron in plants, animals, and bacteria. In animals, the protein is mainly cytoplasmic and is highly conserved, while in plants ferritin is found in chloroplasts and other plastids. Ferritin is synthesized in plants as a larger precursor of the mature subunit. There is no sequence information for ferritin from plants, except an NH2-terminal peptide of 35 residues which shows little similarity to any known ferritin sequences or transit peptides (Laulhere, J. P., Laboure, A. M., and Briat, J. F. (1989) J. Biol. Chem. 264, 3629-3635). To understand the genetic origin and the location of ferritin synthesis in plant cells, as well as the structure of ferritin from plants, we have sequenced both CNBr peptides from pea seed ferritin and nucleotides of a soybean hypocotyl ferritin cDNA, identified using a frog ferritin cDNA as a probe. Comparison of pea and soybean sequences showed an identity of 89%. Alignment of the plant ferritin sequences with animal ferritins showed 55-65% sequence identity in the common regions. However, a peptide of 28 amino acids extended the NH2 terminus of the plant ferritins. Furthermore, the cDNA encoded additional amino acids which appear to be a transit peptide. None of the sequences in soybean ferritin were found in the tobacco chloroplast genome, suggesting, as does the transit peptide, a nuclear location of ferritin gene(s) in plants. Plant ferritin mRNA is 400-500 nucleotides longer than animal ferritin mRNAs, a difference accounted for in part by the extra peptides encoded. The size of soybean ferritin mRNA was constant in different tissues but expression varied in different tissues (leaf greater than hypocotyl). Thus, higher plants and animal ferritins display sequence homology and differential tissue expression. An ancient, common progenitor apparently gave rise to contemporary eukaryotic ferritins after specific modifications, e.g. transport to plasmids.