A conserved heptapeptide sequence in the waterborne attractin pheromone stimulates mate attraction in Aplysia

Mate attraction in the marine mollusk Aplysia involves long-distance waterborne chemical signaling via the release of the peptide pheromone attractin during egg laying. Aplysia californica attractin attracts conspecifics, reduces the latency to mating, and stimulates hermaphroditic mating. Four additional members of the Aplysia attractin family have recently been characterized from Aplysia brasiliana, Aplysia fasciata, Aplysia depilans, and Aplysia vaccaria. The five sequences differ significantly, but share six cysteine residues and the strictly conserved sequence Ile30-Glu-Glu-Cys-Lys-Thr-Ser36. Attractin is attractive to geographically and evolutionarily distant species, suggesting that the conserved heptapeptide region may be important for mate attraction. Consistent with this prediction, a synthetic constrained cyclic peptide that contains the conserved heptapeptide sequence is significantly attractive in T-maze bioassays. The attractins are the first family of waterborne peptide pheromones characterized in invertebrates and are unique in that family members are not species-specific pheromonal attractants.     

NMR solution structure of attractin,a water-borne protein pheromone from the mollusk Aplysia californica

Water-borne protein pheromones are essential for coordination of reproductive activities in many marine organisms. In this paper, we describe the first structure of a pheromone protein from a marine organism, that of attractin (58 residues) from Aplysia californica. The NMR solution structure was determined from TOCSY, NOESY, and DQF-COSY measurements of recombinant attractin expressed in insect cells. The sequential resonance assignments were done with standard manual procedures. Approximately 90% of the 949 unambiguous NOESY cross-peaks were assigned automatically with simultaneous three-dimensional structure calculation using our NOAH/DIAMOD/FANTOM program suite. The final bundle of energy-refined structures is well-defined, with an average rmsd value to the mean structure of 0.72 +/- 0.12 A for backbone and 1.32 +/- 0.11 A for heavy atoms for amino acids 3-47. Attractin contains two antiparallel helices, made up of residues Ile9-Gln16 and I30-S36. The NMR distance constraints are consistent with the three disulfide bonds determined by mass spectroscopy (C4-C41, C13-C33, and C20-C26), where the first two could be directly determined from NOESY cross-peaks between CH beta protons of the corresponding cysteines. The second helix contains the (L/I)(29)IEECKTS(36) sequence conserved in attractins from five species of Aplysia that could interact with the receptor. The sequence and structure of this region are similar to those of the recognition helix of the Er-11 pheromone of the unicellular ciliate Euplotes raikovi, suggesting a possible common pathway for intercellular communication of these two distinct pheromone families.     

Aplysia seductin is a water-borne protein pheromone that acts in concert with attractin to stimulate mate attraction

Mate attraction in Aplysia involves the long-distance water-borne protein pheromones attractin, enticin, and temptin which are released during egg-laying. Other water-borne pheromones are predicted to act in concert with attractin, enticin, and temptin, but their identities were unknown. We recently identified a highly expressed Aplysia californica albumen gland gene (Alb-23) that encoded a novel protein by differential library screening of an albumen gland cDNA library. To determine whether Alb-23 ('seductin') was a water-borne pheromone, we employed Western blot analysis, purification and expression of albumen gland proteins, immunolocalization studies, pheromone secretion assays, comparative genomics, and behavioral bioassays. Immunoreactive seductin was detected in eluates of egg cordons, indicating that seductin was secreted onto the cordon during egg laying. Aplysia brasiliana seductin was 94% identical to its A.californica homolog. In T-maze attraction assays, the combination of attractin and seductin was significantly attractive to potential mates, whereas either protein alone was not. Data from this and previous studies support the hypothesis that seductin is a water-borne protein pheromone that acts in concert with attractin, enticin, and temptin to attract Aplysia to form and maintain mating aggregations.     

Characterization of Aplysia enticin and temptin, two novel water-borne protein pheromones that act in concert with attractin to stimulate mate attraction

Mate attraction in Aplysia involves a long-distance water-borne signal (attractin) that is released during egg laying. Other pheromones are predicted to be released during egg laying that act in concert with albumen gland attractin to stimulate attraction, but their identities are unknown. To identify other candidate water-borne pheromones, we employed differential library screening of an albumen gland cDNA library, Northern blot analysis, purification, characterization, cloning, and expression of albumen gland proteins, matrix-assisted laser desorption ionization mass spectrometry, pheromone secretion assays, behavioral bioassays, immunolocalization studies, and comparative genomics. Four genes, Alb-23, Alb-24, Alb-69, and Alb-172, were highly expressed in Aplysia californica albumen glands and encoded novel proteins. The products of the Alb-24 ("enticin") and Alb-172 ("temptin") precursors were soluble and highly abundant in albumen gland extracts, whereas Alb-23 and Alb-69 were membrane-associated proteins. A comparative analysis showed that the predicted Aplysia brasiliana enticin and temptin proteins were 90 and 91% identical, respectively, to their A. californica homologs. T-maze attraction bioassay studies have previously demonstrated that egg cordons alone are attractive to Aplysia but that attractin alone is not. In the present study, however, the combination of attractin, enticin, and temptin was found to be significantly attractive to potential mates and doubled the number of animals attracted to this stimulus compared with control animals. The combined data strongly suggest that enticin and temptin are novel candidate water-borne protein pheromones that act in concert with attractin to attract Aplysia to form and maintain egglaying and mating aggregations.     

Newly identified water-borne protein pheromones interact with attractin to stimulate mate attraction in Aplysia

The water-borne protein attractin is a potent sex pheromone involved in forming and maintaining mating and egg-laying aggregations in the marine mollusk Aplysia. Binary blends of attractin and either enticin, temptin, or seductin, three other Aplysia protein pheromones, stimulate mate attraction. The four pheromones are thought to act in concert during egg-laying. The new data presented here show that: (1) the water-borne odor of non-laying Aplysia brasiliana further increases the attractiveness of attractin and of eggs in T-maze bioassays. This suggests that individual Aplysia release additional factors that enhance the effects of attractin, enticin, temptin, and seductin during egg-laying; (2) the N-terminal region of enticin aligns well with the conserved epidermal growth factor (EGF)-like domain of mammalian reproductive proteins known as fertilins, which may mediate intercellular adhesion interactions between eggs and sperm; (3) temptin, according to fold recognition servers, may also have an EGF-like fold. Enticin and temptin also have conserved metal binding sequences that may play a role in their signaling behavior. These results suggest that aspects of mammalian egg-sperm interactions (fertilins) may have evolved from pheromonal signaling mechanisms. We also review the structure, expression, localization, release, and behavioral actions of attractin, enticin, temptin, and seductin.     

Peptide products of the atrial gland are not water-borne reproductive pheromones during egg laying in Aplysia

Mate attraction in Aplysia involves long-distance water-borne signaling via the secretion of the peptide pheromone attractin from the exocrine albumen gland during egg laying. Previous studies have shown that a second exocrine organ, the atrial gland, produces abundant egg-laying hormone (ELH) precursor-related peptides and mollusk-derived growth factor (MDGF), and crude extracts of the atrial gland are attractive in T-maze attraction assays. However, it is not known whether these peptides and proteins are secreted during egg laying. In this report, seawater eluates of freshly laid egg cordons were concentrated and fractionated by C18 RP-HPLC, and the resulting major peaks were examined by amino acid compositional analysis, microsequence analysis, and electrospray mass spectrometry. Concentrated egg cordon eluates were also examined by immunoblot analysis using anti-MDGF antisera as probe. The combined data demonstrated that the atrial gland of Aplysia californica does not secrete detectable levels of either ELH precursor-related peptides or MDGF during egg laying. Although the atrial gland is the last major exocrine organ to make contact with eggs before they are laid, the gland does not appear to secrete water-borne peptide pheromones during egg laying.     

Behavioral characterization of attractin,a water-borne peptide pheromone in the genus aplysia

Pheromones play a significant role in coordinating reproductive activity in many animals, including opisthobranch molluscs of the genus 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 egg cordons. The egg cordons are a source of pheromones that attract other Aplysia to the area, reduce their latency to mating, and induce egg laying. One of these water-borne egg cordon pheromones ("attractin") has been characterized and shown to be attractive in T-maze assays. Attractin is the first water-borne peptide pheromone characterized in invertebrates. In the current studies, behavioral assays were used to better characterize the attraction, and to examine whether attractin can induce mating. Although the two activities could be related (i.e., attraction occurring because animals were looking for a partner), this was not tested. T-maze assays showed that attractin works as part of a bouquet of odors: the peptide is attractive only when Aplysia brasiliana is part of the stimulus. The animal does not need to be a conspecific, perhaps explaining why multiple species may be associated with one aggregation. Native and recombinant attractin are equally attractive, verifying that N-glycosylation at residue 8 is not required for attraction. Mating studies showed that both native and recombinant attractin reduce the latency to mating. The effects are larger when hermaphroditic mating is considered: in addition to reducing latency, attractin doubles the number of pairs mating as hermaphrodites. The effect may result from attractin stimulating both animals to mate as males and would be consistent with behaviors previously seen in the T-maze. Attractin may thus be contributing to the formation of copulatory chains and rings seen in aggregations in the field. These results may be interpreted in two ways: (1). attractin has multiple activities that contribute to the establishment and maintenance of the aggregation; or (2). the induced desire to mate may make attractin attractive when it is presented in conjunction with an animal. In either case, the results open the door for cellular and molecular studies of mechanism of action.     

Aplysia capsulin is localized to egg capsules and egg cordon sheaths and shares sequence homology with Drosophila dec-1 gene products

Differential library screening of an albumen gland cDNA library, Western blot analysis, protein expression, immunolocalization studies, comparative genomics, and secretion assays identified a major Aplysia californica albumen gland protein ('capsulin') that is localized to egg capsules and to the sheaths of the egg cordon. Capsulin shared sequence homology with eggshell proteins encoded by the Drosophila dec-1 gene. The 1790-amino acid A. californica precursor contains 17 repeat sequences that are flanked by basic residue processing sites. The numerous proteolytic processing sites may facilitate the breakdown of capsulin prior to when veliger larvae break out of egg capsules as free-swimming larvae. An Aplysia brasiliana capsulin repeat sequence was 97% identical to its A. californica homolog. Capsulin fragments were not detected in the eluates of egg cordons, suggesting that capsulin is not a candidate water-borne pheromone precursor.     

Peptide and protein pheromones in molluscs

Pheromones have been implicated in the control of a number of behaviors in molluscs, but few peptide pheromones have been characterized in these animals. Peptide pheromones include: (1) a family of water-borne peptide pheromonal attractants (attractins) in the gastropod Aplysia that are released during egg laying and attract other Aplysia to form egg-laying and mating aggregations; (2) a tetrapeptide (ILME) in the cephalopod Sepia that elutes from egg masses and is thought to be involved in the transport of oocytes in the genital tract during egg laying; and (3) a Sepia sperm-attracting peptide (SepSAP; PIDPGVamide) that is released from oocytes during egg laying to facilitate external fertilization.     

Aplysia temptin - the 'glue' in the water-borne attractin pheromone complex

Temptin, a component of the complex of water-borne protein pheromones that stimulate attraction and mating behavior in the marine mollusk Aplysia, has sequence homology to the epidermal growth factor (EGF)-like domains of higher organisms that mediate protein-cell surface contact during fertilization and blood coagulation. In this work, recombinant temptin for structural and functional studies was produced in Escherichia coli using a cold shock promoter and purified by RP-HPLC. CD spectra confirmed a predominantly beta-sheet structure. Two disulfide bonds were determined via limited proteolysis and MS. One internal disulfide (Cys57-Cys77) was predicted from initial alignments with class I EGF-like domains; the second, between Cys18 and Cys103, could protect temptin against proteolysis in seawater and stabilize its interacting surface. A three-dimensional model of temptin was prepared with our MPACK suite, based on the Ca(2+)-binding, EGF-like domain of the extracellular matrix protein fibrillin. Two temptin residues, Trp52 and Trp79, which align with cysteine residues conserved in fibrillins, lie adjacent to and could stabilize the disulfide bonds and a proposed metal-binding loop. The water-borne pheromone attractin in egg cordon eluates is complexed with other proteins. Docking results with our model and the NMR structure of attractin suggest that one face of temptin interacts with the pheromone, perhaps controlling its access to the cellular receptors. Gel shifts confirmed that temptin complexes with wild-type attractin. These results indicate that temptin, analogous to the role of fibrillin in controlling transforming growth factor-beta concentration, modulates pheromone signaling by direct binding to attractin.     

Recombinant production and structural studies of the Aplysia water-borne protein pheromone enticin indicates it has a novel disulfide stabilized fold

Enticin is one of three Aplysia proteins released during egg laying that act in concert with the pheromone attractin to attract other Aplysia and stimulate mating behavior. Whereas the enticin cDNA predicts a 69-residue mature protein, enticin isolated from the albumen gland was found to be posttranslationally processed in vivo by cleavage at Arg(50) residue to generate a smaller 49-residue mature peptide. The Arg(50) cleavage site is conserved in enticin from both Aplysia californica and Aplysia brasiliana. In order to generate sufficient enticin for structural studies, recombinant full-length protein was produced in a soluble form in Escherichia coli using a cold shock promoter-based protein expression system. The enticin cDNA was cloned into the bacterial vector pCold III, and efficiently expressed, as determined by amino acid microsequence and immunoblot analyses. Recombinant enticin, which contained an additional N-terminal 13-residue translation-enhancing element, was purified by reversed-phase HPLC and compared to enticin isolated from the albumen gland. The three disulfide bonds in enticin were characterized by endoproteinase Glu-C proteolysis followed by mass spectrometric characterization of the fragments. The cysteine pairing, for both recombinant and native enticin, was I-II, III-IV, and V-VI, confirming that the protein produced in the bacterial system was correctly folded. The circular dichroism spectrum of the recombinant protein indicated it was predominantly alpha-helical. While this was consistent with fold recognition server results indicating a fold for enticin similar to that of attractin, the disulfide bonding pattern differs. A model for enticin was prepared based on its helical structure and these disulfide constraints.     

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.     

Structural studies on a family of cAMP-binding proteins in the nervous system of Aplysia

Five major cAMP-binding proteins that differ in size and charge have been identified in neurons of Aplysia californica by photoaffinity labeling with [32P]8-N3cAMP. These proteins, which we believe are regulatory subunits of cAMP-dependent protein kinase, all differ from the major cAMP-binding protein of buccal muscle. We have compared the structures of these proteins by peptide mapping after chemical and proteolytic cleavage. These analyses indicate that the five binding proteins from nervous tissue and the major muscle protein are closely related to each other. For example, the three neuronal proteins that are most alike and the cAMP-binding protein from muscle have a similar, if not identical, Mr 20,000 domain that contains the 8-N3cAMP-binding site; beyond this domain they diverge. All six proteins appear to belong to a family in which homologous regions have been conserved to maintain common functions. We suggest that the regions of the molecules that differ mediate special functions such as ticketing to particular compartments of the cell. Evidence for regional assortment of the cAMP-dependent protein kinases according to structural type was afforded by subcellular fractionation of Aplysia nervous tissue; photoaffinity labeling of cytoplasm, cytoskeleton, and membrane fractions demonstrated a differential distribution of the five neuronal cAMP-binding proteins. Selective phosphorylation of specific substrates could be a consequence of the compartmentation of diverse cAMP-dependent kinases.     

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 .     

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.     

Neurocalcin-like immunoreactivity in embryonic stages of the gastropod molluscs Aplysia californica and Lymnaea stagnalis

Abstract. Neurocalcin is a calcium-binding protein that has been localized in neural and non-neural tissues of vertebrates, the arthropod Drosophila melanogaster , and in juveniles and adults of the mollusc Aplysia californica . We examine the distribution of neurocalcin in pre-hatching stages of the molluscs A. californica and Lymnaea stagnalis to elucidate where this calcium-binding protein functions in early development, as well as to localize novel neuronal populations in early stages of ontogeny. Aplysia neurocalcin (ApNc)-like immunoreactivity was localized in shell-secreting cells in embryonic stages of both A. californica and L. stagnalis . In A. californica , central and anterior regions of the embryo were diffusely labeled, as were a few identifiable neurons in veliger stages, On the other hand, in L. stagnalis , ApNc-like immunoreactivity was clearly detected in cells and fibers in the same locations as neuronal elements that have been previously identified very early in development and throughout the embryonic period using techniques to localize specific transmitters and peptides. Furthermore, additional neurons are also identified with anti- ApNc in this species. Establishing the distribution of neurocalcin-like proteins in embryonic stages of these two molluscs provides the first step to understanding the role of such proteins during development.     

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.