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.     

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.     

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.     

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.     

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.     

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.     

A water-borne sex pheromone and trail following in the apple snail, Pomacea canaliculata

We investigated whether individuals of the apple snail Pomaceacanaliculata were attracted by conspecifics or follow mucustrails of other individuals. The snails' behaviour was studiedby a series of choice experiments in a T-maze and in Petri dishes.Both males and females chose the side with a snail of the oppositesex significantly more frequently than the control side withouta snail. Males were attracted by water conditioned with femalesmore frequently than unconditioned water, whereas females didnot show a preference for male-conditioned water. Moreover,juveniles were not attracted by water conditioned with a male,a female and a juvenile. These data indicate that males wereattracted by female odour, which contains one or more water-bornesex pheromones. In addition, both males and females follow mucustrails of snails of the opposite sex. Since females also followedtrails of females, the function of trail following is not necessarilyrelated to reproduction. In these experiments P. canaliculatadid not distinguish the direction of the trail. (Received 10 March 2007; accepted 12 July 2007)     

SEPALLATA3: the 'glue' for MADS box transcription factor complex formation

Background  

Plant MADS box proteins play important roles in a plethora of developmental processes. In order to regulate specific sets of target genes, MADS box proteins dimerize and are thought to assemble into multimeric complexes. In this study a large-scale yeast three-hybrid screen is utilized to provide insight into the higher-order complex formation capacity of the Arabidopsis MADS box family. SEPALLATA3 (SEP3) has been shown to mediate complex formation and, therefore, special attention is paid to this factor in this study.     

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.     

Oviposition aggregation pheromone in the Simulium damnosum complex

Abstract. Communal oviposition by the Simulium damnosum complex of Afrotropical blackflies (Diptera: Simuliidae) was investigated under controlled laboratory conditions, using wild-caught flies in Sierra Leone. Volatile compounds emitted by Simulium eggs were trapped using a closed collection system, and their attractiveness to gravid flies was tested in a two-choice behavioural bioassay. Significantly more female blackflies oviposited on substrates baited with freshly laid eggs (100% chose the baited substrate), or with the volatiles collected from freshly laid eggs (85% chose the baited substrate), in preference to the relevant control substrates. Substrates baited with volatiles from 12-h-old eggs were not significantly more attractive than controls (only 31% chose the baited substrates; P = 0.33). Gas chromatographic analysis of the egg volatiles consistently showed two peaks emanating from fresh eggs, but significantly lower amounts from 12-h-old eggs ( P <0.05). A novel system for collecting the volatiles from this and other blackfly species, as they laid eggs on a substrate in flowing water, is described. Volatiles collected using this method showed identical gas chromatographic profiles to those of fresh eggs alone, indicating that the flies themselves produced no other volatile chemical signals during oviposition. Evidently communal oviposition by S. damnosum s.l . was mediated by a pheromone emanating from fresh eggs. The role of pheromone-mediated egg aggregation in blackfly ecology is discussed, and its possible manipulation is considered.     

Absence-like and tonic seizures in aspartoacylase/attractin double-mutant mice.

The Spontaneously Epileptic Rat (SER), a double-mutant for tremor and zitter mutations, shows spontaneous occurrences of absence-like and tonic seizures. Several lines of evidence suggest that the combined effect of Aspa and Atrn mutations is the most likely cause of the epileptic phenotype of the SER. To address this issue, we produced a new double-mutant mouse line carrying both homozygous Aspa-knockout and Atrn(mg-3J) mutant alleles. The Aspa/Atrn double-mutant mice exhibited absence-like and tonic seizures that were characterized by the appearance of 5-7 Hz spike-wave-like complexes and low voltage fast waves on EEGs. These results demonstrate directly that the simultaneous loss of the Aspa and Atrn gene functions causes epileptic seizures in the mouse and suggest that both Aspa and Atrn deficiencies might be responsible for epileptic seizures in the SER.     

Cholinergic receptors in the Aplysia gill

Acetylcholine has been suggested as a neurotransmitter released in the Aplysia gill by peripheral afferents of central neurons and by peripheral neurons within the gill. The perfused gill, isolated from the abdominal ganglion, was examined. At concentrations greater than 1 microM, acetylcholine elicited a slowly developing tonic contraction of the afferent vein that reversed upon washout. This effect was observed on both quiescent and active preparations. At concentrations less than 1 microM, acetylcholine perfusion resulted in a reduction of gill tone. The excitatory effect of acetylcholine was reduced 80 and 60% by the cholinergic antagonists atropine and hexamethonium, respectively. The acetylcholine-evoked contraction was potentiated 2.5-fold when curare was coinfused. Carbachol did not mimic the excitatory effects of acetylcholine. At all concentrations examined (1-100 microM), carbachol infusion reduced baseline tension, the amplitude of spontaneous contractions and contractions evoked by FMRFamide and dopamine. Contractions evoked by perfusion of p-chlorophenylthiocyclic AMP were greatly reduced when carbachol was added to the perfusate. Further addition of curare reversibly blocked carbachol inhibition of the cyclic AMP-evoked contractions. These findings suggest that excitatory and inhibitory cholinergic receptors are involved in the regulation of gill contractile behavior by acetylcholine.     

PI4KII activity-dependent Golgi complex targeting of Aplysia phosphodiesterase 4 long-form mutant

The compartmentalization of cAMP by specifically targeted phosphodiesterases (PDEs) contributes to signal regulation in defined regions of cells. We previously demonstrated that the 20 N-terminal amino acids of Aplysia PDE4 (ApPDE4) long-form (L(N20)) and the two mutants of L(N20) were localized to the Golgi complex. However, the molecular mechanisms underlying the Golgi complex targeting of ApPDE4 long-form and its mutated forms are not clear. In the present study, we show that the Golgi complex targeting of L(N20/C14,15S)-enhanced green fluorescent protein (EGFP) was antimycin A-, phenylarsine oxide (PAO)-, and adenosine-sensitive, but insensitive to high concentrations of wortmannin. On the other hand, the Golgi complex targeting of L(N20)-EGFP and L(N20/C3,14S)-EGFP was antimycin A- and PAO-insensitive. These results suggest that the Golgi-localized lipid kinase protein, phosphatidylinositol 4-kinase type II alpha (PI4KIIα), the activity of which is inhibited by PAO and adenosine, but not by high concentrations of wortmannin, is likely involved in the Golgi complex targeting of L(N20/C14,15S)-EGFP. In addition, subcellular localization of L(N20/C14,15S)-EGFP, but not L(N20)-EGFP or L(N20/C3,14S)-EGFP, was changed from the Golgi complex only to both the endoplasmic reticulum (ER) and the Golgi complex following treatment with a palmitoylation inhibitor, 2-bromo palmitate. Taken together, our results suggest that L(N20/C14,15S)-EGFP, but not L(N20)-EGFP or L(N20/C3,14S)-EGFP, is localized to the Golgi complex in a PI4KII activity- and palmitoylation-dependent manner. Therefore, phosphatidylinositol 4-phosphate (PI4P) generated by PI4KIIα at the Golgi complex might play a key role in the Golgi complex targeting of L(N20/C14,15S)-EGFP.     

Oviposition pheromone in the Simulium damnosum complex: biological activity of chemical fractions from gravid ovaries

Abstract. Communal oviposition in the Afrotropical blackfly species complex Simulium damnosum Theobald (Diptera: Simuliidae) is mediated by a pheromone emitted by freshly laid eggs. Previously, two compounds (designated peaks A and B) emanating from fresh eggs were shown to be associated with attractiveness to gravid blackflies in bioassay. The present study investigated the role of these compounds by testing the responses of wild-caught Simulium yahense in Ghana to fractionated hexane extracts of gravid ovaries prepared by gas chromatography (GC). Although the fractions were prepared from Sierra Leonean Simulium leonense , GC analysis of the emissions from fresh S.yahense eggs showed that the volatile blends of both species were similar. When tested in a two-choice bioassay, 66% of ovipositing blackflies chose the substrate baited with a mixture of the four fractions recombined. In a series of bioassays testing responses to the four individual fractions presented with a control in a multiple-choice arrangement, only fraction 3 (containing peaks A and B) attracted significantly more ovipositions than the other fractions and control. However, fraction 3 failed to elicit a significant response when presented as the sole attractant with a control in a two-choice bioassay. It was concluded that fraction 3 , though mainly responsible for mediating aggregated oviposition by S.yahense , was acting in tandem with additional cues, probably further chemicals, which remain to be isolated and characterized.     

Serial synapses in Aplysia

Serial synapses occur between small profiles in the neuropil of Aplysia abdominal ganglion. Material was fixed in phosphate buffered OsO₄, embedded in epon, and sections were stained with uranyl acetate and lead citrate. A class of synapses had the following characteristics: (1) synaptic vesicles clustered against the presynaptic membrane, (2) a widened extracellular space of about 20 nm containing electron-dense material, (3) straightening of the pre- and postsynaptic membranes, and (4) no postsynaptic membrane specialization. Some density between the presynaptic membrane and the adjacent synaptic vesicles was occasionally observed. Synapses occurred between small profiles in the neuropil (typical profile diameters were 1–3 m?m). In this sample of approximately 100 synapses, four serial synapses were identified. The serial synaptic profiles were all small. In addition to the finding of serial synapses, 40% of the postsynaptic profiles contained vesicles similar to the synaptic vesicles seen in presynaptic profile. Serial synapses may be the anatomical substrate of presynaptic inhibition and facilitation and of dishabituation.