Aplysia attractin: biophysical characterization and modeling of a water-borne pheromone.

Attractin, a 58-residue protein secreted by the mollusk Aplysia californica, stimulates sexually mature animals to approach egg cordons. Attractin from five different Aplysia species are approximately 40% identical in sequence. Recombinant attractin, expressed in insect cells and purified by reverse-phase high-performance liquid chromatography (RP-HPLC), is active in a bioassay using A. brasiliana; its circular dichroism (CD) spectrum indicates a predominantly alpha-helical structure. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) characterization of proteolytic fragments identified disulfide bonds between the six conserved cysteines (I-VI, II-V, III-IV, where the Roman numeral indicates the order of occurrence in the primary sequence). Attractin has no significant similarity to any other sequence in the database. The protozoan Euplotes pheromones were selected by fold recognition as possible templates. These diverse proteins have three alpha-helices, with six cysteine residues disulfide-bonded in a different pattern from attractin. Model structures with good stereochemical parameters were prepared using the EXDIS/DIAMOD/FANTOM program suite and constraints based on sequence alignments with the Euplotes templates and the attractin disulfide bonds. A potential receptor-binding site is suggested based on these data. Future structural characterization of attractin will be needed to confirm these models.

Structure, localization and potential role of a novel molluscan trypsin inhibitor in Lymnaea.

Eggs and egg masses of the freshwater gastropod mollusc Lymnaea provide a microenvironment for developing embryos. Secretions of the exocrine albumen gland of Lymnaea are packaged in the eggs of an egg mass before the eggs are laid externally. The perivitelline fluid that directly surrounds individual oocytes is the main source of nutrition for developing embryos. During early stages of development, the perivitelline fluid is initially internalized by pinocytosis and degraded by lysosomes; in later stages, the embryo ingests the fluid. We previously found that the albumen gland produces large amounts of Lymnaea epidermal growth factor. The albumen gland also appears to produce significant amounts of a novel Lymnaea trypsin inhibitor (LTI), a second peptide that was purified and characterized from Lymnaea albumen gland extracts. The primary structure was determined by microsequence analysis, mass spectrometry, and C-terminal sequence analysis, and showed that LTI is a 57-residue glycosylated peptide. Comparison of the LTI sequence with other known serine protease inhibitors indicates that LTI is a member of the bovine pancreatic trypsin inhibitor family. Reverse phase-high performance liquid chromatography, microsequence analysis, mass spectrometry, and immunocytochemistry demonstrated that abundant amounts of intact LTI are packaged in egg masses. The presence of a trypsin inhibitor in the perivitelline fluid compartment of the egg mass may minimize digestion of peptides and proteins in the perivitelline fluid that are important for the development of the embryo, for example, Lymnaea epidermal growth factor.

Neurotrophic actions of a novel molluscan epidermal growth factor.

The mammalian epidermal growth factor (EGF) is expressed in the developing and adult CNS, and it has been implicated in the control of cell proliferation, differentiation, and neurotrophic events. Despite extensive evolutionary conservation of the EGF motif in a range of different types of proteins, secreted EGF homologs with neurotrophic actions have not been reported in invertebrates. In this study, we present a novel member of the family of EGF-like growth factors, an EGF homolog from the mollusc Lymnaea stagnalis (L-EGF), and we demonstrate that this protein has neurotrophic activity. Purified L-EGF is a 43-residue peptide and retains the typical structural characteristics of the EGF motif. The L-EGF cDNA reveals a unique precursor organization. In contrast to the multidomain mammalian EGFs, it consists of only two domains, a signal peptide and a single EGF motif. Conspicuously, the L-EGF precursor lacks a transmembrane domain, setting it apart from all other members of the EGF-family. L-EGF mRNA is expressed throughout embryonic development, in the juvenile CNS, but not in the normal adult CNS. However, expression in the adult CNS is upregulated after injury, suggesting a role of L-EGF in repair functions. This notion is supported by the observation that L-EGF evokes neurite outgrowth in specific adult Lymnaea neurons in vitro, which could be inhibited by an EGF receptor tyrosine kinase inhibitor. In conclusion, our findings further substantiate the notion that the EGF family has an early phylogenetic origin, and our data support a neurotrophic role for L-EGF during development and injury repair.

Excitatory synaptogenesis between identified Lymnaea neurons requires extrinsic trophic factors and is mediated by receptor tyrosine kinases.

Neurotrophic factors have well established roles in neuronal development and adult synaptic plasticity, but their precise role in synapse formation has yet to be determined. This paper provides the first direct evidence that neurotrophic factors in brain conditioned medium (CM) differentially regulate excitatory and inhibitory synapse formation. Somata of identified presynaptic and postsynaptic neurons were isolated from the CNS of Lymnaea and were cultured in a soma-soma configuration in the presence (CM) or absence [defined medium (DM)] of trophic factors. In DM, excitatory synapses did not form. When they were paired in CM or in DM containing Lymnaea epidermal growth factor (EGF); however, all presynaptic neurons reestablished their specific excitatory synapses, which had electrical properties similar to those seen in vivo. CM-induced formation of excitatory synapses required transcription and de novo protein synthesis, as indicated by the observations that synapse formation was blocked by the protein synthesis inhibitor anisomycin and the protein transcription blocker actinomycin D; the CM factor was inactivated by boiling. They were also blocked by receptor tyrosine kinase inhibitors (lavendustin A, genistein, K252a, and KT5926) but not by inactive analogs (genistin and lavendustin B), suggesting that the effect was mediated by receptor tyrosine kinases. These results, together with our previously published data, demonstrate that trophic factors are required for excitatory, but not inhibitory, synapse formation and extends the role of EGF from cell proliferation, neurite outgrowth, and survival to excitatory synapse formation.

Characterization of Aplysia attractin, the first water-borne peptide pheromone in invertebrates.

Although animals in the genus Aplysia are solitary during most of the year, they form breeding aggregations during the reproductive season. The aggregations contain both mating and egg-laying animals and are associated with masses of egg cordons. The egg cordons are a source of pheromones that establish and maintain the aggregation, but none of the pheromonal factors have been chemically characterized. In these studies, specimens of Aplysia were induced to lay eggs, the egg cordons collected and eluted, and the eluates fractionated by C18 reversed-phase HPLC. Four peak fractions were bioassayed in a T-maze. All four increased the number of animals attracted to a nonlaying conspecific and were thus subjected to compositional and microsequence analysis. Each contained the same NH2-terminal peptide sequence. The full-length peptide ("attractin") was isolated from the albumen gland, a large exocrine organ that packages the eggs into a cordon. The complete 58-residue sequence was obtained, and it matched that predicted by an albumen gland cDNA. Mass spectrometry showed that attractin is 21 wt.% carbohydrate as the result of N-linked glycosylation. T-maze bioassays confirmed that the full-length peptide is attractive. Attractin is the first water-borne peptide pheromone characterized in molluscs, and the first in invertebrates.

Molecular cloning of a cDNA encoding the neuropeptides APGWamide and cerebral peptide 1: localization of APGWamide-like immunoreactivity in the central nervous system and male reproductive organs of Aplysia.

While much is known about the neural and endocrine mechanisms that control egg laying in the gastropod mollusk Aplysia, relatively little is known about the regulation of male reproductive activity in this simultaneous hermaphrodite. In the present study, we have cloned and sequenced a cDNA that encodes a precursor protein, the predicted posttranslational processing of which presumably generates nine copies of the neuropeptide Ala-Pro-Gly-Trp-NH2 (APGWamide), five connecting peptide sequences, and a C-terminal peptide. The sequence of one connecting peptide is identical to the previously characterized cerebral peptide 1. Northern blot analysis identified two major APGWamide mRNA transcripts (approximately 1.3 kb, approximately 2.4 kb), which were present in central nervous system ganglia, but were most abundant in the right cerebral and right pedal ganglia. Immunohistochemical studies using sexually mature Aplysia demonstrated that the vast majority of APGWamide-like immunoreactivity was localized in 30-40 neurons along the anterior and medial margins of the right cerebral ganglion and in a cluster of 15-20 neurons in the right pedal ganglion. A total of only about ten immunoreactive neurons were located in other ganglia. Immunohistochemistry also demonstrated that APGWamide was present in the reproductive organs that participate in the storage or transport of sperm, including the small hermaphroditic duct (site of sperm storage before mating), the white hemiduct (also known as the copulatory duct), and penial complex. As a group, these data suggest that APGWamide may play a role in regulating male reproductive function in Aplysia, as it does in other gastropods.

Molecular cloning of a cDNA encoding a potential water-borne pheromonal attractant released during Aplysia egg laying.

Recently deposited egg cordons are a source of water-borne pheromones that attract the marine mollusk Aplysia into breeding aggregations and coordinate male and female reproductive behavior within the aggregation. A potential pheromonal attractant has been isolated from egg cordon eluates and the peptide partially characterized [S.D. Painter, B. Clough, X. Fan, G.T. Nagle, Soc. Neurosci. Abstr., Vol. 22 (1996) 837]. Using this information, we have cloned an Aplysia albumen gland cDNA that encodes a precursor protein containing a single copy of the full-length peptide, and demonstrated that there are abundant levels of pheromone mRNA transcripts (0.8 and 2.5 kb) in the albumen gland. This is consistent with the reported function of the gland (i.e. packaging the eggs into a cordon for deposition), with behavioral studies showing that the albumen gland is a potential source of attractants, and more recent biochemical studies in which the full-length peptide has been isolated from the albumen gland. This is the first candidate peptide pheromone in mollusks and the first in invertebrates. The pheromonal regulatory system in Aplysia may provide a model system for examining the structural characteristics of peptide pheromones.

Microanatomy courses in U.S. and Canadian medical schools, 1991-92.

BACKGROUND: Many medical schools are reevaluating their approaches to teaching the basic and clinical sciences, yet there is little systematic information available about how specific courses are being taught. METHOD: A detailed questionnaire was sent to 139 U.S. and Canadian medical schools to obtain information about microanatomy courses in 1991-92. RESULTS: Overall, 114 (82%) of the schools responded. The responses showed that microanatomy courses were including more cell biology and related material in their curricula, and that many were coordinating subject presentation with other basic science courses. The courses relied primarily on lecture-based teaching for non-laboratory material and structured laboratory exercises for laboratory material. New approaches, such as small-group, problem-solving sessions, were being introduced slowly; most of the courses used faculty-provided learning objectives. Computer-aided instruction had been introduced at a small number of schools, usually in the form of self-instructional packages or reviews. Multiple-choice written examinations and short-answer practical examinations were usually used to assess student knowledge; computer-based testing was rare. CONCLUSION: The microanatomy curricula tended to be relatively conventional. The primary barrier to the introduction of computer-aided instruction may be the wide-spread use of other self-instructional tools that are effective and less expensive to develop and maintain. Because of the trend toward fewer student contact hours, the adoption of problem-based learning at the course level could have a significant negative impact on the amount of microanatomy material that could be covered.

Coordination of Reproductive Activity in Aplysia: Peptide Neurohormones, Neurotransmitters, and Pheromones Encoded by the Egg-Laying Hormone Family of Genes.

Pheromones play a significant role in coordinating reproductive activity in the marine opisthobranch mollusk Aplysia. Although solitary during most of the year, these simultaneous hermaphrodites gather into breeding aggregations during the reproductive season. The aggregations contain both mating and egg-laying animals, and are associated with masses of recently deposited egg cordons. Behavioral studies suggest that cordon-derived pheromonal factors are primarily responsible for establishing and maintaining the aggregations. Egg-laying animals are more attractive than sexually mature, but nonlaying, conspecifics and have a shorter mean latency to mating; egg cordons and egg-cordon eluates, when placed in the surrounding seawater, enhance the attractiveness of nonlaying animals and reduce their mean latency to mating. Similar effects are observed when extracts of the atrial gland are placed in the seawater, suggesting that secretory products of this oviductal exocrine organ may function as sexual pheromones. Biochemical analyses indicate that there may be multiple attractants in atrial gland extracts, and that at least one of these (A-NTP) is a peptide encoded by the A gene. The A gene belongs to a small family of structurally related genes that are expressed in a tissue-specific manner. Another member of the family, the egg-laying hormone (ELH) gene, is expressed in the neuroendocrine bag cells. Peptide products of the ELH gene act as neurohormones and nonsynaptic neurotransmitters, initiating egg laying and coordinating its associated behaviors. Peptide products of a family of genes may thus act internally and externally to coordinate both male and female reproductive activities.

Relative Contributions of the Egg Layer and Egg Cordon to Pheromonal Attraction and the Induction of Mating and Egg-laying Behavior in Aplysia.

Many species of the opisthobranch mollusk Aplysia form breeding aggregations during the reproductive season. The aggregations contain both mating and egg-laying animals, and are associated with egg cordons. Although pheromones play a significant role in developing and maintaining the aggregations, little is known about the active factors. Behavioral studies have shown that egg-laying animals are more attractive than nonlaying animals, have shorter latencies to mating, and induce conspecifics to lay eggs. As a first step toward isolating and chemically characterizing the active factors, we examined the relative importance of the egg layer and egg cordon as sources of pheromonal activity in Aplysia brasiliana. T-maze experiments showed that both animal-derived and cordon-derived factors are attractive, and that the animal-derived factors are specifically associated with egg layers. Extracts of the atrial gland--an exocrine organ secreting into the oviduct--increased the attractiveness of nonlaying animals when placed in the surrounding seawater, suggesting that the "cordon-derived" aggregation pheromones may be products of the atrial gland. Mating studies showed that both animal-derived and cordon-derived factors induce mating, and that the animal-derived factors are associated with both egg layers and nonlayers. In contrast, neither animal-derived nor cordon-derived factors induced egg laying. Comparable results were obtained with either one or two animals in the chamber, suggesting that the accessibility of a potential mate did not influence the results. The lack of effect may result from the low-probability nature of egg-laying activity.

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).

A calfluxin-related peptide is present in the bag cells and atrial gland of Aplysia.

The caudodorsal cells of Lymnaea and the bag cells of Aplysia are neuroendocrine cells whose peptide products have homologous functions, i.e., regulation of egg deposition. One of the Lymnaea products, calfluxin, increases cytosolic and hence mitochondrial calcium concentrations in secretory cells of the albumen gland, an exocrine organ that secretes perivitellin fluid around the egg cells during packaging; the changes can be visualized at the ultrastructural level for quantification with the pyroantimonate precipitation technique and are correlated with changes in the secretory and biosynthetic activity in the gland. Comparable studies have now been carried out with Aplysia and indicate that the bag cells of A. californica and A. brasiliana also contain a factor with calfluxin-related activity, and that the factor is not the egg-laying hormone (ELH). The bag cell factor does not affect mitochondrial calcium levels in the Lymnaea albumen gland, and synthetic calfluxin does not affect the Aplysia gland. Thus, although the bag cell and caudodorsal cell peptides have the same activity in their respective genera, the sequences have diverged sufficiently during the course of evolution to preclude cross-reactivity. Calfluxin-related activity was also detected in the atrial gland of A. californica and the atrial gland-like epithelium of A. brasiliana, two exocrine organs in the oviduct that express genes structurally related to the bag cell ELH gene. It is postulated that the active atrial gland factors may be peptides A and B.

Localization of immunoreactive alpha-bag-cell peptide in the central nervous system of Aplysia.

The bag cells of the marine mollusc Aplysia are well-characterized neuroendocrine cells that initiate egg laying, but the natural stimulus triggering bag-cell activity has not been determined. As a first step toward identifying central neurons that might provide synaptic or neurohormonal input onto the bag-cell network, antibodies specific for alpha-bag-cell peptide (alpha-BCP) were generated. This peptide belongs to a small family of structurally related peptides that can elicit bag-cell activity in vitro. Antibody specificity was established by immunodot assay and preabsorption studies: immunocytochemical labeling was abolished in each ganglion when the antibodies were preincubated with either alpha-BCP-thyroglobulin conjugate or alpha-BCP-(1-8) but was not affected by preincubation with thyroglobulin or thyroglobulin-thyroglobulin conjugate. The antibodies specifically labeled the bag cells in the abdominal ganglion and ectopic bag cells in both the abdominal and right pleural ganglia. The ectopic bag cells were similar to conventional bag cells in size and morphology, but varied in number and location among preparations. In the cerebral ganglion, the antibodies labeled a bilaterally symmetrical pair of cell clusters, containing approximately ten cells each, on the dorsal surface of the ganglion. The cerebral cells were smaller than bag cells, were constant in location, and sent their processes into the neuropil rather than the connective tissue sheath. Immunoreactive processes were observed in the neuropils of the cerebral, pleural, and pedal ganglia and among the axons of the cerebropedal, cerebropleural, and pleurovisceral connectives. No immunoreactive cell bodies were observed in the buccal or pedal ganglia. Identical patterns of labeling were observed in Aplysia californica, A. brasiliana, and A. dactylomela. The distribution of immunoreactive cell bodies within the circumesophageal ganglia of all three species thus parallels the distribution of receptive sites for the in vitro induction of bag-cell activity by atrial gland peptide B, a peptide structurally related to alpha-BCP. These observations suggest that the immunoreactive cells identified in these studies, or a subset of them, may be involved in the physiological induction of bag-cell activity. Since low doses of alpha-BCP have additional inhibitory actions on the bag cells, however, it is possible that the identified cells could play a more complex role in the regulation of bag-cell activity.

Post-translational processing in model neuroendocrine systems: precursors and products that coordinate reproductive activity in Aplysia and Lymnaea.

The regulation of egg-laying behavior in the hermaphroditic marine mollusc Aplysia has been an important model system that has provided insight into the cellular and molecular bases of neuroendocrine function. The advantages of this system derive primarily from the unique characteristics of the bag cells, two clusters of neurosecretory cells whose peptide products initiate and coordinate egg deposition. In particular, the large number of bag cell neurons in a single animal and their relative isolation from other elements of the nervous system facilitate biochemical analyses of the peptides synthesized and released by the cells. Moreover, the episodic nature of bag cell electrical activity, combined with the relative simplicity of the Aplysia nervous system and the stereotypic nature of the behaviors exhibited during egg deposition, provide unusually favorable opportunities for investigating neuropeptide action and function. This review will focus on neuropeptide synthesis and processing in the bag cells and in other organs expressing structurally related genes.

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.

Induction of copulatory behavior in Aplysia: atrial gland factors mimic the excitatory effects of freshly deposited egg cordons.

Egg laying in the marine mollusc Aplysia is induced and coordinated by peptide products of the egg-laying hormone (ELH) gene expressed in the neuroendocrine bag cells of the central nervous system. At least three structurally related genes, belonging to the ELH family but distinct from the ELH gene, are expressed in the atrial gland, an exocrine organ of unknown function that secretes into the oviduct of Aplysia. The experiments described in this report were designed to test the hypothesis that the atrial gland gene products serve a pheromonal function for the animal, coordinating reproductive behavior among individuals. Our studies showed that there was a significantly shorter latency to copulation when an Aplysia was paired with an animal that was actively laying eggs than when it was paired with a sexually mature but nonlaying animal. Moreover, the addition of extracts or homogenates of the atrial gland to the seawater surrounding two nonlaying animals reduced the latency to mating compared to animals exposed only to seawater or to homogenates of other regions of the reproductive tract, including oviduct. These results suggest that atrial gland products, secreted onto the egg cordon as it passes through the oviduct, may play a pheromonal role and induce mating behavior between individuals. Experiments are in progress to determine whether the active atrial gland factor(s) are products of the ELH-family genes expressed in the gland.

Peptide B induction of bag-cell activity in Aplysia: localization of sites of action to the cerebral and pleural ganglia.

The bag cells of the marine mollusc Aplysia are model neuroendocrine cells involved in the initiation of egg laying and its associated behaviors, but the natural stimulus triggering bag-cell activity is not known. The atrial gland of A. californica, an exocrine organ in the reproductive tract, contains two structurally related peptides (A and B) which can induce an afterdischarge in vitro, and these peptides can be used to probe the central nervous system for sites where extrinsic excitatory input onto the bag-cell system might occur. These sites were identified in a series of lesion and ablation experiments. The entire central nervous system was removed from an animal and placed in a chamber with two compartments which could be independently perfused, allowing peptides (atrial gland extract or purified peptide B) to be selectively applied to specific regions of the nervous system while bag-cell activity was monitored using extracellular suction electrodes. Afterdischarges were consistently and reliably induced when peptides were applied to the cerebral ganglion, the pleural ganglia, the cerebropleural connectives, or the rostral 10-15% of the pleurovisceral connectives, provided that an intact neuronal pathway connected the site of peptide application with the bag cells. In contrast, afterdischarges were never observed when peptides were selectively applied to the buccal or pedal ganglia and only rarely observed when applied to the abdominal ganglion and caudal pleurovisceral connectives. These results support the hypothesis that bag-cell processes and/or neuron(s) presynaptically excitatory to the bag cells are located in the pleural and cerebral ganglia and narrow the region of the central nervous system where the critical initiator element(s) can be identified.

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 .

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.

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.