Molecular identification of candidate chemoreceptor genes and signal transduction components in the sensory epithelium of Aplysia.

An ability to sense and respond to environmental cues is essential to the survival of most marine animals. How water-borne chemical cues are detected at the molecular level and processed by molluscs is currently unknown. In this study, we cloned two genes from the marine mollusk Aplysia dactylomela which encode multi-transmembrane proteins. We have performed in situ hybridization that reveals expression and spatial distribution within the long-distance chemosensory organs, the rhinophores. This finding suggests that they could be receptors involved in binding water-borne chemicals and coupling to an intracellular signal pathway. In support of this, we found expression of a phospholipase C and an inositol trisphosphate receptor in the rhinophore sensory epithelia and possibly distributed within outer dendrites of olfactory sensory neurons. In Aplysia, mate attraction and subsequent reproduction is initiated by responding to a cocktail of water-borne protein pheromones released by animal conspecifics. We show that the rhinophore contraction in response to pheromone stimulants is significantly altered following phospholipase C inhibition. Overall, these data provide insight into the molecular components of chemosensory detection in a mollusk. An important next step will be the elucidation of how these coordinate the detection of chemical cues present in the marine environment and activation of sensory neurons.

Lymnaea EGF and gigantoxin I, novel invertebrate members of the epidermal growth factor family.

In this review, we compare the sequence and structural relationships of two epidermal growth factor (EGF) family related proteins that have recently been discovered in invertebrate species. The first is L-EGF, a secreted growth factor from the gastropod mollusk Lymnaea stagnalis. The second is a peptide toxin (Gigantoxin I), isolated from the sea anenome Stichodactyla giganteus, which can paralyze crabs. L-EGF and Gigantoxin I share striking sequence similarity with mammalian erbB1 receptor ligands, including most of the essential receptor binding sites. Intriguingly, L-EGF's tertiary structure resembles more the structure of the EGF-like domain of coagulation factors. That is, the secondary and tertiary structure of L-EGF indicates the presence of a double-stranded beta-sheet but also suggests that this protein, in contrast to all other erbB1 ligands, contains a calcium-binding domain. One of the most remarkable features of L-EGF and Gigantoxin I however, is the indication that these protein are synthesized as non-membrane bound secreted peptides. This feature sets L-EGF and Gigantoxin I apart from all other members of the EGF family or EGF-like proteins identified thus far. We discuss sequence similarities and dissimilarities in the light of indications that, despite the more than 600 million years of phylogenetic distance separating both these invertebrates from mammals, Gigantoxin I and L-EGF retain some affinity for the mammalian erbB-family of receptors. Considering that mammalian EGF and its family members are frequently implicated in neoplastic diseases, the increasing number of identified and characterized invertebrate EGF family members may provide valuable leads in the design of erbB receptor antagonists.

Mollusk-derived growth factor and the new subfamily of adenosine deaminase-related growth factors.

Peptide and protein growth factors play critical roles in the control of proliferation, differentiation and survival of most, if not all, cell types. In this review, we describe a newly isolated growth factor from Aplysia californica, mollusk derived growth factor (MDGF), that is a member of the adenosine deaminase-related growth factor (ADGF) subfamily. Other known subfamily members from a range of invertebrate and vertebrate species include: insect-derived growth factor, Drosophila ADGFs, tsetse salivary growth factors, insect adenosine deaminases (ADAs; Lutzomyia, Culex, Aedes, Anopheles), and cat eye syndrome critical region gene 1 (CECR1) in humans, pigs, and zebrafish. ADGFs from vertebrates and invertebrates contain both an ADA domain and a novel N-terminal region of about 100 amino acids. Catalytic residues involved in ADA activity are conserved in ADGFs, and inhibitors of ADA can block ADGF activity. ADA enzymatic activity has been shown, by inhibitor and site-directed mutagenesis studies, to be related to the ability of ADGFs from many species to stimulate cell proliferation. The available evidence suggests that the conversion of adenosine to inosine (or their analogs) is important for the mitogenic actions of ADGFs. Future investigations of this novel subfamily should lead to the identification of their receptors.

Mollusk-derived growth factor: cloning and developmental expression in the central nervous system and reproductive tract of Aplysia.

We have isolated and characterized an atrial gland cDNA that corrects the previously reported sequence for Aplysia atrial gland granule-specific antigen (AGSA), a glycoprotein of unknown function. We designated the protein mollusk-derived growth factor (MDGF) to distinguish the revised sequence from AGSA and to emphasize its similarity to an insect-derived growth factor (IDGF). We describe MDGF mRNA expression that suggests a possible role during embryonic development and CNS injury repair.

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.

Neuropeptide amidation: cloning of a bifunctional alpha-amidating enzyme from Aplysia.

One of the most common mechanisms of posttranslational modifications to generate biologically active (neuro)peptides is the process of peptide alpha-amidation. The only enzyme known to catalyze this important modification is peptidylglycine alpha-amidating monooxygenase (PAM): a (bifunctional) zymogen, giving rise to a monooxygenase (PHM) and a lyase (PAL). The highly peptidergic central nervous system and endocrine system of the marine mollusk Aplysia has homologs of various mammalian peptide processing enzymes, including furin, Afurin2, prohormone convertase 1 (PC1), PC2, carboxypeptidase E (CPE) and CPD. Previously, it has been shown that the abdominal ganglion of Aplysia, which contains approximately 800 peptidergic bag cell neurons, contains the highest specific alpha-amidating activity. We have identified and cloned multiple overlapping central nervous system and bag cell cDNAs that encode a predicted 748-residue protein that is a member of the PAM family. The protein sequence contains the contiguous sequence of the catalytic domains of PHM and PAL, clearly demonstrating the existence of bifunctional Aplysia PAM, the first invertebrate PAM zymogen with an organization similar to that in vertebrates. None of the characterized clones encoded the so-called exon A domain between the PHM and PAL domains. Furthermore, in a specific search by reverse transcription-polymerase chain reaction of RNA from multiple tissues we could only detect exon A-less transcripts. PAM expression was detected in the central nervous system, and in several endocrine and exocrine organs. Aplysia PAM is a candidate prohormone processing enzyme that plays an important role in the processing of Aplysia prohormones in the secretory pathway.

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.

Expression of the L-type Ca(2+) channel in AtT-20 cells is regulated by cyclic AMP.

Activation of adenylyl cyclase by corticotropin-releasing hormone (CRH) stimulates secretion of adrenocorticotropin (ACTH) in rat anterior pituitary corticotropes and in the murine AtT-20 cell line. The stimulation of secretion is mediated by cAMP and is largely dependent on Ca(2+) influx through voltage-gated L-type Ca(2+) channels. To investigate whether CRH and cAMP also increase expression of the L-type Ca(2+) channel in AtT-20 cells, an RNase protection assay was used to measure the alpha(1C) mRNA that encodes the pore-forming subunit of the L-type Ca(2+) channel. The alpha(1C) mRNA level was measured by autoradiographic densitometry and normalized to the beta-actin mRNA level in the same sample. The alpha(1C) mRNA was not changed by 24-hour treatment with CRH (10-500 nM). A 24-hour treatment with 1 mM 8Br-cAMP significantly increased the alpha(1C) mRNA by 40% over its control. The stimulatory effect was blocked by 2 microM actinomycin D and was, therefore, dependent on gene transcription. The measured half-life of the alpha(1C) mRNA, after inhibition of transcription, was 4.7 +/- 0.3 h in control and 5.2 +/- 0.6 h in the presence of 8Br-cAMP. Thus the 8Br-cAMP- induced increase in alpha(1C) mRNA could be due to an increase in alpha(1C) gene transcription or to a transcriptionally regulated increase in a protein that helps to stabilize alpha(1C) mRNA. Finally, to determine if the increased mRNA was followed by an increase in production of L-type Ca(2+) channels, the binding of [(3)H]PN200-110 to Ca(2+) channel proteins was assayed in AtT-20 membrane fragments. 8Br-cAMP increased [(3)H]PN200-110 binding sites by 32% (B(max) 36.0 +/- 1.2 fmol/mg protein in control vs. 47.4 +/- 3.2 fmol/mg protein in 8Br-cAMP-treated cells) but did not change the K(d). These studies show that both alpha(1C) mRNA and L-type Ca(2+) channel protein are increased in AtT-20 cells by cAMP.

Cold stress and corticotropin-releasing hormone induced changes in messenger ribonucleic acid for the alpha(1)-subunit of the L-type Ca(2+) channel in the rat anterior pituitary and enriched populations of corticotropes.

In response to stress, adrenocorticotropin (ACTH) is secreted from anterior pituitary corticotropes. Corticotropin-releasing hormone (CRH) is a potent stimulator of ACTH secretion. The CRH stimulation of secretion is mediated by cAMP and is largely dependent on Ca(2+) influx through voltage-gated L-type Ca(2+) channels. This study was designed to investigate whether the expression of L-type Ca(2+) channels in the rat anterior pituitary and in corticotropes is regulated by acute stress and CRH. RNase protection assays were used to quantify alpha(1C) mRNA of the L-type Ca(2+) channel. The alpha(1C) mRNA levels from stressed rats increased by 31% in anterior pituitaries of rats after 30 min of exposure to cold stress. Neither 60 min cold stress nor 30 min restraint stress had an effect on alpha(1C) mRNA levels. When alpha(1C) mRNA was detected by in situ hybridization in a population of corticotropes enriched to 90%, 0.5 nM CRH (3 h) stimulated a 36% increase in the average area of label/cell and a 10% increase in the average density of label. Our results suggest that (1) the expression of alpha(1C) subunit mRNA of L-type Ca(2+) channels is increased in the rat anterior pituitary with a stress-specific response that might reflect an increase both in thyrotropes and corticotropes (both are known to be stimulated by cold stress), and (2) the CRH-mediated increase in alpha(1C) mRNA expression in individual rat corticotropes, in vitro, supports the hypothesis that some of the increase in vivo is due to changes in corticotropes.

Cloning and expression of Aplysia carboxypeptidase D, a candidate prohormone-processing enzyme.

Many peptide hormones in a variety of species are produced from larger precursors by limited proteolysis at basic amino acid-containing sites. The marine mollusc Aplysia has homologs of mammalian peptide-processing enzymes, including furin, prohormone convertase 1 (PC1), PC2, and carboxypeptidase E (CPE). A novel neuronal Aplysia enzyme was recently identified that was most closely related to carboxypeptidase D (CPD; Fan and Nagle, DNA Cell Biol. 15, 937-945, 1996), a second carboxypeptidase thought to be present in the secretory pathway and to contribute to peptide hormone processing. We have identified and cloned multiple overlapping bag-cell neuron cDNAs that encode two proteins that are members of the CPD family. Sequence analyses demonstrate that the longer CPD protein (1446 residues) contains an N-terminal signal peptide and four carboxypeptidase-like domains; the third and fourth domains are not predicted to form active enzymes, as several critical residues are absent. The shorter CPD protein is predicted to contain two active carboxypeptidase-like domains. Northern blot analysis identified a major Aplysia CPD mRNA (5.3 kb) and several smaller minor transcripts in central nervous system tissue. The CPD was purified from Aplysia ovotestis using a method previously developed for mammalian CPD. The purified Aplysia CPD binds antisera raised against regions of the protein encoded by the Aplysia cDNA clone, as well as an antiserum raised against duck CPD. The enzymatic properties of purified Aplysia CPD are generally similar to those of mammalian CPD. Aplysia CPD is a candidate prohormone-processing enzyme that may play a role in the processing of Aplysia prohormones in the secretory pathway.

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.

Characterization of Aplysia carboxypeptidase E.

Carboxypeptidase E (CPE) is involved in the biosynthesis of peptide hormones and neurotransmitters. To determine whether a recently reported Aplysia californica cDNA encodes a CPE-like enzyme, this cDNA was expressed in the baculovirus system. The Aplysia CPE is optimal at pH 5.5-6.5 and is inhibited by chelating agents and by the sulfhydryl reagent p-chloromercuriphenyl sulfonate. The effect of divalent cations and active site-directed inhibitors on enzyme activity are generally similar for Aplysia and rat CPE. Western blot analysis using antisera to the N- and C-terminal regions of the Aplysia CPE show that the Aplysia CPE is present in atrial glands and ovotestis. This Aplysia CPE is purified on a p-aminobenzoyl-Arg Sepharose affinity column under conditions that selectively purify rat CPE. Taken together, these results suggest that the previously cloned cDNA represents a CPE-like enzyme that is expressed in Aplysia tissue.

Molecular cloning of Aplysia neuronal cDNAs that encode carboxypeptidases related to mammalian prohormone processing enzymes.

The bag cell neurons of Aplysia synthesize an egg-laying hormone (ELH) precursor that initially is cleaved into two fragments in the Golgi apparatus, and the fragments are differentially packaged in separate granule populations and further processed. Aplysia Afurin, Afurin2, prohormone convertase 1 (PC1), and PC2 are thought to be involved in the posttranslational processing of the ELH prohormone. In the present study, we have cloned Aplysia neuronal cDNAs that encode an enzyme most closely related to mammalian carboxypeptidase E (CPE), a peptide hormone processing enzyme that removes basic residues during prohormone processing. Northern blot analysis identified a single Aplysia CPE mRNA (approximately 5.2 kb) in central nervous system tissue. The C-terminal region of Aplysia CPE contains amphiphilic alpha-helices that may serve as a hydrophobic membrane anchor. A novel neuronal Aplysia enzyme was also identified by the polymerase chain reaction that was most closely related to the carboxypeptidase D (CPD)-related duck protein gp180 and the Drosophila silver gene carboxypeptidases. Aplysia CPE and the CPD-related enzyme are candidate processing enzymes that may play a role in the processing of the ELH prohormone and other Aplysia prohormones.

Presence of gonadotropin-releasing hormone (GnRH) receptor mRNA in rat myenteric plexus cells.

Idiopathic neuromuscular disease of the gastrointestinal tract (functional bowel disease) is thought to result from the malfunction of neurons within the enteric nervous system. Gonadotropin-releasing hormone (GnRH) analogs have recently been shown to organize the disordered motility patterns typical in these patients and to produce significant, long-term symptomatic improvement. To determine whether GnRH analogs might bind to an endogenous enteric nervous system GnRH receptor, reverse transcription-polymerase chain reaction (RT-PCR) was performed using cultured neonatal rat enteric neuron RNA and rat GnRH receptor primers. A PCR product of the predicted size was cloned and nucleotide sequence analysis demonstrated that the myenteric plexus PCR product encoded a portion of the GnRH receptor sequence previously identified in rat pituitary. These results suggest that cells in the myenteric plexus express GnRH receptors that may bind exogenously administered GnRH analogs. The expression of GnRH receptors in enteric neurons would provide an explanation for the effectiveness of GnRH analogs in treatment of idiopathic neuromuscular disease of the gastrointestinal tract.

Molecular cloning of prohormone convertase 1 from the atrial gland of Aplysia.

We have screened an Aplysia atrial gland cDNA library using a prohormone convertase (PC)1 probe prepared by polymerase chain reaction (PCR) and have isolated an Aplysia PC1-related full-length 3.6-kb cDNA clone. The cDNA sequence (3,565 bp) encoded a putative preproendoprotease (APC1) of 703 amino acid residues that showed considerable sequence identity with other eukaryotic PC1s, and indicated a high degree of sequence identity with an Aplysia nervous system PC sequence (aPC1B). Northern blot analysis of atrial gland RNA identified two APC1 transcripts of 3.9 kb and 5.0 kb. APC1 is a candidate PC that may play an important role in the processing of egg-laying hormone (ELH)-related precursors in atrial gland secretory cells and represents one of the first examples of PC1 expression in an exocrine tissue.

Molecular cloning and cellular localization of a furin-like prohormone convertase from the atrial gland of Aplysia.

Prohormone convertases (PCs) are Ca(2+)-dependent subtilisin-related endoproteases that have been implicated in the post-translational processing of prohormones and other proproteins. Furin is an ubiquitously expressed PC that has been shown to hydrolyze a wide variety of precursor proteins in secretory pathways. We have screened an Aplysia atrial gland cDNA library using a furin probe prepared by polymerase chain reaction (PCR) and have isolated an Aplysia furin-related 6.7-kb cDNA partial clone that was truncated on the 5' end. The remaining 5' atrial gland furin nucleotide sequence was obtained by two stages of reverse transcription PCR. The final composite nucleotide sequence of the atrial gland furin cDNA was 7,837 bp in length. This sequence encoded a putative preproendoprotease (Afurin2) of 824 amino acid residues that was related to other eukaryotic furins, and showed a high sequence identity with a recently reported Aplysia nervous system furin-like sequence. In situ hybridization demonstrated extensive expression of Afurin2 in atrial gland secretory cells. The cDNA clone contained a relatively long 3' untranslated region of 5,230 nucleotides that included a microsatellite repeat region (TG)n. The characterized Aplysia Afurin2 is a candidate PC that may play an important role in the processing of egg-laying hormone (ELH)-related precursors in the secretory cells of the atrial gland. In addition, comparative structural studies of Afurin2, together with previously reported localization studies, argue for the occurrence of a furin-like convertase within secretory granules.