Isolation and primary structure of the califins, three biologically active egg-laying hormone-like peptides from the atrial gland of Aplysia californica

The atrial gland of the marine mollusk Aplysia californica contains several biologically active peptides that are thought to be important in reproductive function. In the present study, three novel peptides, which we named califin A, B, and C, were purified from extracts of atrial glands by high performance liquid chromatography, and their primary structures were determined. Each consists of a 36-residue subunit bound by a single disulfide bond to an 18-residue subunit. The large subunits differ from each other by one or two residues, whereas the small subunits are identical. The large subunits are 78-83% homologous to egg-laying hormone (ELH), a 36-residue peptide synthesized by the neuroendocrine bag cells of Aplysia. Like ELH, the califins excite LB and LC cells of the abdominal ganglion and cause egg laying when injected into sexually mature animals. Based on previously described DNA sequence data, each califin is likely to be derived from one of several precursor proteins that are encoded by members of the ELH gene family. Califin A is encoded on the peptide A precursor, and califin B may be encoded on the peptide B precursor. No gene encoding califin C has been sequenced. Because peptides A and B are also biologically active, the precursors encoding them and califins A and B are polyproteins. The possible role of atrial gland peptides as pheromones is discussed.     

The bag cell egg-laying hormones of Aplysia brasiliana and Aplysia californica are identical

Egg laying in the marine molluscan genus Aplysia is elicited by an egg-laying hormone (ELH) which induces ovulation and acts on central neurons to effect egg-laying behavior. ELH, isolated from the A. californica bag cells, and three ELH-related peptides, isolated from the A. californica atrial gland, have been chemically characterized, yet relatively little is known about homologous peptides in other Aplysia species. In these studies, the primary structure of A. brasiliana ELH was determined. Bag cell clusters were extracted in an acidic solution, and the peptides purified by sequential gel filtration and reversed-phase HPLC; ELH was identified by bioassay. Amino acid compositional and sequence analyses demonstrated that the neurohormone was a 36-residue peptide whose sequence was identical to that of A. californica ELH: NH2-Ile-Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-Thr-Glu- Gln-Ile- Arg-Glu-Arg-Gln-Arg-Tyr-Leu-Ala-Asp-Leu-Arg-Gln-Arg-Leu-Leu-Glu-Lys-COOH .     

Delta-bag cell peptide from the egg-laying hormone precursor of Aplysia. Processing, primary structure, and biological activity

The bag cells of the marine mollusk Aplysia express a gene encoding a 271-residue egg-laying hormone (ELH) precursor that is processed into at least nine peptide products. Four of the peptides have been identified in bag cell releasates and are known to act as nonsynaptic neurotransmitters in the abdominal ganglion. The isolation, primary structure, and proposed biological activity of a fifth peptide product (delta-bag cell peptide (delta-BCP)) from the ELH precursor are described. delta-BCP was established to be a 39-residue peptide: NH2-Asp-Gln-Asp-Glu-Gly-Asn-Phe-Arg-Arg-Phe-Pro-Thr-Asn-Ala-Val-Ser-Met- Ser-Ala-Asp- Glu-Asn-Ser-Pro-Phe-Asp-Leu-Ser-Asn-Glu-Asp-Gly-Ala-Val-Tyr-Gln-Arg- Asp-Leu-COOH. This sequence corresponds to residues 81-119 of the ELH prohormone and shares sequence identity with atrial gland peptides A and B. Significantly, synthetic delta-BCP stimulated Ca2+ uptake into mitochondria of secretory cells in the albumin gland in vitro, suggesting that the peptide regulates the cellular release of perivitelline fluid by the gland. Similar results were obtained with purified peptide A and a shorter version of delta-BCP (delta-BCP-(14-33)). These results indicate that delta-BCP belongs to a family of structurally related peptides with similar pharmacological activities that center at a conserved region of sequence corresponding to delta-BCP-(14-33).     

Evidence for the expression of three genes encoding homologous atrial gland peptides that cause egg laying in Aplysia

Three peptide complexes which can induce egg laying in Aplysia were isolated from the atrial gland of the marine mollusc Aplysia californica and chemically characterized. Amino acid sequence analyses established the covalent structures, including disulfide assignments, of all three dimeric complexes. Each complex consisted of an identical 18-residue peptide (A-AP) which was disulfide-bonded to a 36-residue peptide that was homologous to bag cell egg-laying hormone (ELH). The primary structure of A-AP was determined to be: NH2-Asp-Ser-Asp-Val-Ser-Leu-Phe-Asn-Gly-Asp-Leu-Leu-Pro-Asn-Gly-Arg-Cys- Ser-COOH. The primary structure of one of the three ELH-related peptides (A-ELH) was determined to be NH2-Ile-Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-Thr-Glu- Gln-Ile-Gln-Ala-Arg-Arg-Arg-Cys-Leu-Asp-Ala-Leu-Arg-Gln-Arg-Leu-Leu-Asp- -Leu-COOH. The two other ELH-related peptides, [Ala27]A-ELH and [Gln23, Ala27]A-ELH, differed from A-ELH at 1 and 2 residues, respectively. Both [Ala27] A-ELH and [Gln23, Ala27]A-ELH were novel peptide sequences representing products of as yet uncharacterized genes within the ELH family. These structural studies provide the first direct chemical evidence that an 18-residue peptide (A-AP) derived from a polypeptide precursor encoded by the A gene, as predicted from nucleotide sequence analysis, occurs in the atrial gland; the Cys17 residue of A-AP is disulfide-bonded to Cys25 of A-ELH; and A-AP also occurs disulfide-bonded to two additional, previously undescribed ELH-related peptides, [Ala27]A-ELH and [Gln23, Ala27]A-ELH.     

Distribution of alpha CDCP-immunoreactive neurons in the central nervous system of the snail Helix aspersa.

alpha CDCP is a neuropeptide produced by the caudodorsal cells of Lymnaea stagnalis and encoded by the genes of the egg-laying hormone (ELH). The use of a polyclonal antiserum raised against alpha CDCP resulted in the detection of about 800 immunoreactive neurons in the parietal ganglia and a small population (60 cells) in the cerebral ganglia of Helix aspersa. As the genes of ELH are well conserved among the gastropod species, these data designate the parietal ganglia as a putative source for the egg-laying hormone in Helix aspersa.     

Proteolytic processing of egg-laying hormone-related precursors in Aplysia. Identification of peptide regions critical for biological activity

The atrial gland of the marine mollusk Aplysia californica is an exocrine organ that expresses at least three genes belonging to the egg-laying hormone (ELH) family. In order to study the post-translational processing of the ELH-related gene products in the atrial gland and how it compares to the bag cells, peptides were isolated from the atrial gland and chemically characterized. The A- and B-related precursors were each cleaved in vivo to yield several major and minor peptides including peptides A and B and the ELH-related peptide complexes that caused egg laying. About 13% of the peptide complexes were further enzymically processed by the atrial gland to yield smaller fragments, which included A-AP.A-ELH-(15-36), A-AP.[Ala27]A-ELH-(15-36), and A-AP.[Gln23,Ala27]A-ELH-(16-36), where A-AP is an acidic peptide encoded by the A- and B-related genes and A-ELH is an ELH-related peptide encoded by the A gene. These processed peptide fragments were not active in an egg-laying bioassay, indicating that retention of the 14-residue NH2-terminal segment of the A-ELH-related sequence, or some portion thereof, was critical for the induction of egg laying. Other characterized peptides included two novel 13-residue NH2-terminal peptides, A-NTP and B-NTP, representing residues 22-34 of the A and B precursors, respectively. These two peptides occurred adjacent to the signal peptide region in each precursor, and their characterization established the site of signal peptide cleavage to be the Ser21-Gln22 peptide bond of each precursor. Intermediate peptide fragments (A-NTP-peptide A and B-NTP-peptide B) were also identified indicating that there was a specific ordering in the cleavage of peptide bonds during posttranslational processing. Finally, a new 55-residue atrial gland peptide was also isolated that was not a part of any ELH-related precursor characterized to date.     

Multiple neuropeptides derived from a common precursor are differentially packaged and transported

The ELH prohormone is proteolytically processed into at least nine peptides which govern egg-laying behavior in Aplysia. Quantitative immunocytochemistry demonstrates that peptides derived from the prohormone are packaged into distinct vesicle classes. Further experiments suggest the segregation occurs via a rapid initial proteolytic cleavage of the prohormone followed by sorting at the trans Golgi. Egg-laying hormone (ELH) immunoreactivity is localized to the cell body and processes, while bag cell peptide (BCP) immunoreactivity is greater in the cell body. Steady state levels of the amino-terminal set of peptides including the BCPs are 3- to 8-fold lower than the carboxy-terminal cleavage products, such as ELH. Thus, intracellular packaging and routing of the peptides cleaved from a single prohormone regulate their localization and levels in these neurons.     

Atrial gland cells synthesize a family of peptides that can induce egg laying inAplysia

Endogenous peptides induce egg laying in the marine mollusc Aplysia in two ways: egg-laying hormone (ELH) from the neuroendocrine bag cells acts directly, causing the release of eggs from the ovotestis; peptides A and B from the atrial gland act indirectly, activating the bag cells to release ELH. Another atrial gland peptide (egg-releasing hormone; ERH) is a structural and functional hybrid of ELH and peptides A and B; it can act both directly and indirectly to induce egg laying. Atrial glands were incubated in a mixture of 3H-amino acids for 18 h, and the biosynthetically labelled peptides isolated using sequential Sephadex G-50 column chromatography and isoelectric focusing. Radiolabelled peaks were localized and bioassayed in intact animals. Bioactive peaks were then characterized functionally using two additional assays: egg laying in bag cell-less animals (ELH-like peptides) and in vitro induction of bag cell discharge (A- and B-like peptides). ERH-like molecules are active in both assays. Homogeneity of bioactive IEF peaks was assessed by SDS-PAGE. Sephadex G-50 gel filtration of biosynthetically labelled atrial gland extracts reveals two major peptide peaks. Peak D (apparent Mr 6,000) is strongly radiolabelled and contains most of the egg-laying activity, but has a low absorbance at 274 nm. Peak E (apparent Mr 3,500) is weakly labelled and contains a small proportion of the total egg-laying activity, but has a large absorbance at 274 nm. Isoelectric focusing of radiolabelled peptides in peak D reveals seven distinct ELH-like species (pI 5.5, 7.5, 8.5, 8.7, 8.9, 9.1, 9.4), and two peaks (pI 5.9, 8.1) that have both ELH-like and A-/B-like activity. The pI 8.1 peak may result from the comigration of peptide A with ERH or with an unidentified ELH-like peptide. It is not yet clear whether the pI 5.9 activity results from comigration of distinct peptides or from the presence of a previously uncharacterized ERH-like molecule. Isoelectric focusing of radiolabelled peptides in peak E reveals five distinct ELH-like species (pI 7.3, 8.5, 8.7, 9.1, 9.4), and one peak (pI 8.9) with both ELH-like and A-/B-like activity. The pI 8.9 peak may result from the comigration of an ELH-like peptide with peptide B. Three of the ELH-like peptides (pI 8.5, 8.9, 9.1) found in peak E are probably identical to the ELH-like peptides found at the same pI's in peak D.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for gene conversion between the phosphoglycerate kinase genes of Trypanosoma brucei

Trypanosoma brucei contains a tandem array of three genes for phosphoglycerate kinase (PGKase), genes A, B and C, each coding for a different protein. We have compared allelic variants of this gene array and find evidence for gene conversion between the three genes. Near the 3' end, the different alleles and gene B contain a variable sequence that is similar to the corresponding sequence in either gene A or gene C. This sequence is flanked by glycine triplets that are conserved in all PGKases from bacteria to mammals. The triplets are encoded by (GGT)n, resulting in sequences that resemble the recombination-promoting chi-sites of Escherichia coli. Upstream of the variable sequence, there is an area of 800 base-pairs in which genes A, B and C are highly homologous; in all three genes this region ends with a sharp boundary at which gene B again shows segmental homology with both genes A and C. These results suggest that repeated gene conversion events partially erase the differences between genes A, B and C that arise in evolution and suggest that chi-like sequences may act as recombinational hotspots in protozoa such as T. brucei.     

Gene for parathyroid hormone-like peptide is on mouse chromosome 6

The single-copy parathyroid hormone-like peptide gene (Pthlh) was assigned to mouse chromosome 6 using a rat PTHLH cDNA as hybridization probe in the Southern blot analysis of DNAs isolated from a panel of mouse x Chinese hamster cell hybrids. The mouse parathyroid hormone gene (Pth) has previously been assigned to mouse chromosome 7 and the PTHLH and PTH genes have also been shown to be on different chromosomes in human and rat. Therefore, despite significant amino-terminal sequence homology between the PTHLH and PTH peptides, as well as similarities in the structural organization of the human PTHLH and PTH genes, the genes encoding these peptides have discrete chromosomal locations in the mouse, rat, and man.     

Specific identification of an authentic clone for mammalian tyrosinase

Tyrosinase, the critical enzyme to melanin pigmentation in mammals, occurs as a series of isozymic forms, which have been previously regarded as different stages in processing of a single precursor form. Recently, three different cDNA clones have been identified which may encode tyrosinase, they share extensive sequence homology but are distinct; two of them have been mapped to genetic loci which regulate different aspects of melanogenesis. Since direct confirmation of the authentic tyrosinase sequence has proven impossible by conventional protein sequencing strategies, we have approached the identification of the tyrosinase gene by synthesizing peptides encoded by the putative genes and preparing antibodies to those peptides. By use of pulse-chase labeling and immunoprecipitation analyses, and by enzymatic determinations, pMT4 (which maps to the brown b locus in mice) is shown to encode a molecule with tyrosinase catalytic activity which is biochemically identical with authentic tyrosinase. However, our results raise the possibility that other gene products may contribute to melanogenesis by one or more melanogenic activities.     

A large pheromone and receptor gene complex determines multiple B mating type specificities in Coprinus cinereus.

Pheromone signaling plays an essential role in the mating and sexual development of mushroom fungi. Multiallelic genes encoding the peptide pheromones and their cognate 7-transmembrane helix (7-TM) receptors are sequestered in the B mating type locus. Here we describe the isolation of the B6 mating type locus of Coprinus cinereus. DNA sequencing and transformation analysis identified nine genes encoding three 7-TM receptors and six peptide pheromone precursors embedded within 17 kb of mating type-specific sequence. The arrangement of the nine genes suggests that there may be three functionally independent subfamilies of genes each comprising two pheromone genes and one receptor gene. None of the nine B6 genes showed detectable homology to corresponding B gene sequences in the genomic DNA from a B3 strain, and each of the B6 genes independently alter B mating specificity when introduced into a B3 host strain. However, only genes in two of the B6 groups were able to activate B-regulated development in a B42 host. Southern blot analysis showed that these genes failed to cross-hybridize to corresponding genes in the B42 host, whereas the three genes of the third subfamily, which could not activate development in the B42 host, did cross-hybridize. We conclude that cross-hybridization identifies the same alleles of a particular subfamily of genes in different B loci and that B6 and B42 share alleles of one subfamily. There are an estimated 79 B mating specificities: we suggest that it is the different allele combinations of gene subfamilies that generate these large numbers.     

Sequence of heat-labile enterotoxin of Escherichia coli pathogenic for humans.

We determined the complete nucleotide sequence of the toxB gene (375 base pairs in length), which encodes the B subunit of heat-labile enterotoxin produced from Escherichia coli pathogenic for humans (hLT). The amino acid sequence of the B subunit of hLT was deduced from the nucleotide sequence. Consequently, it has become possible to study the homology between the B subunits of three similar toxins: hLT, LT produced from E. coli pathogenic for piglets (pLT), and cholera toxin (the latter two sequences have been reported by others). The three B subunits are all 103 amino acids in length. A comparison of the toxB gene and the eltB gene, which encodes the B subunit of pLT, showed a 98% homology at the nucleotide level and a 95% homology at the amino acid (of a precursor) level, indicating the possibility that the two genes share a common ancestor. With respect to the B-subunit sequences, the homologies between hLT and pLT, between hLT and cholera toxin, and between pLT and cholera toxin were 96, 81, and 79%, respectively. Several large common sequences are conserved by the three peptides. In contrast, no sequences are present in both pLT and cholera toxin but missing in hLT.