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Gregg T. Nagle - One of the best experts on this subject based on the ideXlab platform.
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Conservation of the egg-laying hormone neuropeptide and Attractin pheromone in the spotted sea hare, Aplysia dactylomela.
Peptides, 2009Co-Authors: Scott F Cummins, Gregg T. Nagle, Parinyaporn Nuurai, Bernard M. DegnanAbstract:In the marine opisthobranch mollusc, Aplysia, secreted peptides and proteins play an essential role in egg laying and mate attraction. Aplysia californica egg laying is initiated by secretion of the egg-laying hormone (ELH) peptide while mate attraction is made possible by protein pheromones, such as Attractin, released into the surrounding seawater with the egg cordon. In this study, we investigated the existence of similar egg-laying hormone and Attractin products in the spotted sea hare, Aplysia dactylomela, a species that is widely distributed in almost all tropical and temperate oceans, including Australia's Great Barrier Reef. Immunological analysis revealed that an ELH-like transmitter is present within bag cell somata and processes of the abdominal ganglion. A molecular genetic approach found that the ELH precursor mRNA is synthesized in the abdominal ganglia and encodes a 36-residue peptide (dELH) that is cleaved from the prohormone prior to secretion. It is most closely related to A. californica and A. brasiliana ELH (91.7% identical). We also found that A. dactylomela synthesize an Attractin pheromone in the albumen gland that is released during egg laying. The gene encodes a 58-residue mature protein that is 74.9% similar to A. californica Attractin. We demonstrate that an increase in seawater temperature can disrupt Attractins higher order interactions, such as those with the pheromone temptin, and accelerates Attractin degradation. Together, these findings further expands our understanding of pheromone intermolecular interactions and presents an opportunity for further study of how increases in sea water temperature may affect this important marine communication system.
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Characterization of Aplysia Alb-1, a candidate water-borne protein pheromone released during egg laying.
Peptides, 2007Co-Authors: Scott F Cummins, Bernard M. Degnan, Gregg T. NagleAbstract:Pheromones are known to be important to the innate behavior of marine animals. Attraction in Aplysia involves the long-distance water-borne protein pheromones Attractin, enticin, temptin and seductin, which are released from the albumen gland during egg laying. Other pheromones are predicted to act in concert with these pheromones, but their identities are unknown. To identify additional pheromone candidates, we employed differential library screening of an albumen gland cDNA library, RT-PCR, recombinant protein expression, rhinophore contraction bioassays and immunocytochemistry. Alb-1 is expressed in the Aplysia californica albumen gland and encodes a novel protein that does not share significant sequence identity with any proteins in the database. RT-PCR analysis detected Alb-1 transcripts in the albumen gland, exocrine atrial gland and ovotestis. The Alb-1 precursor has a signal peptide sequence followed by a predicted 101-residue protein sequence containing eight cysteine residues. Recombinant protein expression, RP-HPLC, microsequence analysis and MALDI mass spectrometry analyses demonstrated that mature recombinant Alb-1 was processed at a paired basic residue site to generate an N-terminal and C-terminal protein fragment; this was consistent with immunoblot observations on purified albumen gland extracts. In rhinophore contraction (twitch) bioassays, the recombinant N-terminal protein induced rhinophore contractions whereas the C-terminal protein did not. An antibody generated to the N-terminal protein was used for immunocytochemical and immunoblot analyses and demonstrated that this protein is present in albumen gland secretory cells, egg cordons and egg eluates. Overall, the data suggest that Alb-1 may be processed in the albumen gland and that the Alb-11–56 protein released during egg laying may serve a pheromonal function in concert with Attractin, enticin, temptin and seductin.
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Aplysia temptin - the 'glue' in the water-borne Attractin pheromone complex.
The FEBS journal, 2007Co-Authors: Scott F Cummins, Gregg T. Nagle, Jonathan V Sweedler, Bernard M. Degnan, Fang Xie, Melissa R. De Vries, Suresh P. Annangudi, Milind Misra, Catherine H ScheinAbstract: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 Ca2+-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.
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Newly identified water-borne protein pheromones interact with Attractin to stimulate mate attraction in Aplysia.
Peptides, 2005Co-Authors: Scott F Cummins, Amy E. Nichols, Catherine H Schein, Gregg T. NagleAbstract: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.
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Molluscan Attractins, a family of water-borne protein pheromones with interspecific attractiveness.
Peptides, 2005Co-Authors: Scott F Cummins, Catherine H Schein, Werner Braun, Gregg T. NagleAbstract:The marine mollusk Aplysia releases the water-borne pheromone Attractin during egg laying. This small protein stimulates the formation and maintenance of mating and egg-laying aggregations. Attractin has been characterized from five Aplysia species: A. californica, A. brasiliana, A. fasciata, A. vaccaria, and A. depilans. We describe here the isolation of Attractin from Bursatella leachii, and show that it belongs to the same protein family. The pattern of residue conservation, especially the six invariant cysteines, suggests that all of these Attractins have a common fold. The nuclear magnetic resonance solution structure of A. californica Attractin contains two antiparallel α-helices, the second of which contains the heptapeptide sequence IEECKTS that has been implicated in Attractin function. Synthetic peptides containing this IEECKTS region are attractive, and mutating surface exposed charged residues within this region of Attractin abolishes Attractin activity. This suggests that the second helix is an essential part of the receptor-binding interface. In contrast to the peptide pheromonal attractants in amphibians, which are species specific, the Attractins are, to our knowledge, the first water-borne peptide or protein pheromone family in invertebrates and vertebrates that are not species specific.
Scott F Cummins - One of the best experts on this subject based on the ideXlab platform.
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Identification of an Attractin-like pheromone in the mucus-secreting hypobranchial gland of the abalone Haliotis asinina linnaeus.
Journal of Shellfish Research, 2010Co-Authors: Chitraporn Kuanpradit, Scott F Cummins, Bernard M. Degnan, Prapee Sretarugsa, Peter J. Hanna, Prasert Sobhon, Jittipan ChavadejAbstract:Pheromones are chemicals used to communicate between animals of the same species, and are thought to be used by most marine animals. With limited vision, abalone primarily sense their world chemically, and pheromones may play an important role in settlement, attraction, recognition, alarm, and reproduction. Despite this, there has been no detailed investigation into pheromone substances, both in their precise biochemical nature or pheromonal function. In this study, we investigated the presence of pheromonelike substances from the hypobranchial gland of the abalone Haliotis asinina using bioassays, immunohistochemistry, Western blotting, and reverse-phase high-performance liquid chromatography (RP-HPLC). The hypobranchial gland of many prosobranchial marine molluscs has been classified as a sex auxiliary gland releasing unknown substances during spawning. In our study, cephalic tentacle assays demonstrated that the cell extracts of the hypobranchial gland contain chemical cues that are sensed by conspecifics. An antibody against the sea slug “Attractin” pheromone was used as a probe to localize a similar protein in the mucin-secreting cells of the epithelial lining the hypobranchial gland of both male and female abalone. The approximate molecular weight of this abalone Attractin-like protein is 30 kDa in both males and females. Fractionation of hypobranchial gland extracts by C5 RP-HPLC could not selectively purify this protein, and no sex-specific differences were observed. We predict that the Attractin-like protein could be one of a number of important proteins involved in maturation, aggregation, and/or spawning behavior of abalone. In future research, additional hypobranchial gland components will be tested further for these types of behavior.
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Conservation of the egg-laying hormone neuropeptide and Attractin pheromone in the spotted sea hare, Aplysia dactylomela.
Peptides, 2009Co-Authors: Scott F Cummins, Gregg T. Nagle, Parinyaporn Nuurai, Bernard M. DegnanAbstract:In the marine opisthobranch mollusc, Aplysia, secreted peptides and proteins play an essential role in egg laying and mate attraction. Aplysia californica egg laying is initiated by secretion of the egg-laying hormone (ELH) peptide while mate attraction is made possible by protein pheromones, such as Attractin, released into the surrounding seawater with the egg cordon. In this study, we investigated the existence of similar egg-laying hormone and Attractin products in the spotted sea hare, Aplysia dactylomela, a species that is widely distributed in almost all tropical and temperate oceans, including Australia's Great Barrier Reef. Immunological analysis revealed that an ELH-like transmitter is present within bag cell somata and processes of the abdominal ganglion. A molecular genetic approach found that the ELH precursor mRNA is synthesized in the abdominal ganglia and encodes a 36-residue peptide (dELH) that is cleaved from the prohormone prior to secretion. It is most closely related to A. californica and A. brasiliana ELH (91.7% identical). We also found that A. dactylomela synthesize an Attractin pheromone in the albumen gland that is released during egg laying. The gene encodes a 58-residue mature protein that is 74.9% similar to A. californica Attractin. We demonstrate that an increase in seawater temperature can disrupt Attractins higher order interactions, such as those with the pheromone temptin, and accelerates Attractin degradation. Together, these findings further expands our understanding of pheromone intermolecular interactions and presents an opportunity for further study of how increases in sea water temperature may affect this important marine communication system.
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Characterization of Aplysia Alb-1, a candidate water-borne protein pheromone released during egg laying.
Peptides, 2007Co-Authors: Scott F Cummins, Bernard M. Degnan, Gregg T. NagleAbstract:Pheromones are known to be important to the innate behavior of marine animals. Attraction in Aplysia involves the long-distance water-borne protein pheromones Attractin, enticin, temptin and seductin, which are released from the albumen gland during egg laying. Other pheromones are predicted to act in concert with these pheromones, but their identities are unknown. To identify additional pheromone candidates, we employed differential library screening of an albumen gland cDNA library, RT-PCR, recombinant protein expression, rhinophore contraction bioassays and immunocytochemistry. Alb-1 is expressed in the Aplysia californica albumen gland and encodes a novel protein that does not share significant sequence identity with any proteins in the database. RT-PCR analysis detected Alb-1 transcripts in the albumen gland, exocrine atrial gland and ovotestis. The Alb-1 precursor has a signal peptide sequence followed by a predicted 101-residue protein sequence containing eight cysteine residues. Recombinant protein expression, RP-HPLC, microsequence analysis and MALDI mass spectrometry analyses demonstrated that mature recombinant Alb-1 was processed at a paired basic residue site to generate an N-terminal and C-terminal protein fragment; this was consistent with immunoblot observations on purified albumen gland extracts. In rhinophore contraction (twitch) bioassays, the recombinant N-terminal protein induced rhinophore contractions whereas the C-terminal protein did not. An antibody generated to the N-terminal protein was used for immunocytochemical and immunoblot analyses and demonstrated that this protein is present in albumen gland secretory cells, egg cordons and egg eluates. Overall, the data suggest that Alb-1 may be processed in the albumen gland and that the Alb-11–56 protein released during egg laying may serve a pheromonal function in concert with Attractin, enticin, temptin and seductin.
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Aplysia temptin - the 'glue' in the water-borne Attractin pheromone complex.
The FEBS journal, 2007Co-Authors: Scott F Cummins, Gregg T. Nagle, Jonathan V Sweedler, Bernard M. Degnan, Fang Xie, Melissa R. De Vries, Suresh P. Annangudi, Milind Misra, Catherine H ScheinAbstract: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 Ca2+-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.
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Newly identified water-borne protein pheromones interact with Attractin to stimulate mate attraction in Aplysia.
Peptides, 2005Co-Authors: Scott F Cummins, Amy E. Nichols, Catherine H Schein, Gregg T. NagleAbstract: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.
Catherine H Schein - One of the best experts on this subject based on the ideXlab platform.
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Aplysia temptin - the 'glue' in the water-borne Attractin pheromone complex.
The FEBS journal, 2007Co-Authors: Scott F Cummins, Gregg T. Nagle, Jonathan V Sweedler, Bernard M. Degnan, Fang Xie, Melissa R. De Vries, Suresh P. Annangudi, Milind Misra, Catherine H ScheinAbstract: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 Ca2+-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.
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Newly identified water-borne protein pheromones interact with Attractin to stimulate mate attraction in Aplysia.
Peptides, 2005Co-Authors: Scott F Cummins, Amy E. Nichols, Catherine H Schein, Gregg T. NagleAbstract: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.
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Molluscan Attractins, a family of water-borne protein pheromones with interspecific attractiveness.
Peptides, 2005Co-Authors: Scott F Cummins, Catherine H Schein, Werner Braun, Gregg T. NagleAbstract:The marine mollusk Aplysia releases the water-borne pheromone Attractin during egg laying. This small protein stimulates the formation and maintenance of mating and egg-laying aggregations. Attractin has been characterized from five Aplysia species: A. californica, A. brasiliana, A. fasciata, A. vaccaria, and A. depilans. We describe here the isolation of Attractin from Bursatella leachii, and show that it belongs to the same protein family. The pattern of residue conservation, especially the six invariant cysteines, suggests that all of these Attractins have a common fold. The nuclear magnetic resonance solution structure of A. californica Attractin contains two antiparallel α-helices, the second of which contains the heptapeptide sequence IEECKTS that has been implicated in Attractin function. Synthetic peptides containing this IEECKTS region are attractive, and mutating surface exposed charged residues within this region of Attractin abolishes Attractin activity. This suggests that the second helix is an essential part of the receptor-binding interface. In contrast to the peptide pheromonal attractants in amphibians, which are species specific, the Attractins are, to our knowledge, the first water-borne peptide or protein pheromone family in invertebrates and vertebrates that are not species specific.
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structural and functional analysis of aplysia Attractins a family of water borne protein pheromones with interspecific attractiveness
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Sherry D Painter, Amy E. Nichols, Scott F Cummins, David B G Akalal, Catherine H Schein, Werner Braun, John S Smith, Abraham J Susswein, Miriam Levy, Pamela A C M De BoerAbstract:Mate attraction in Aplysia involves a long-distance water-borne signal (the protein pheromone Attractin), which is released during egg laying. Aplysia californica Attractin attracts species that produce closely related Attractins, such as Aplysia brasiliana, whose geographic distribution does not overlap that of A. californica. This finding suggests that other mollusks release Attractin-related pheromones to form and maintain breeding aggregations. We describe four additional members of the Attractin family: A. brasiliana, Aplysia fasciata, Aplysia depilans (which aggregates with A. fasciata aggregations), and Aplysia vaccaria (which aggregates with A. californica aggregations). On the basis of their sequence similarity with A. californica Attractin, the Attractin proteins fall into two groups: A. californica, A. brasiliana, and A. fasciata (91-95% identity), and A. depilans and A. vaccaria (41-43% identity). The sequence similarity within the Attractin family, the conserved six cysteines, and the compact fold of the NMR solution structure of A. californica Attractin suggest a common fold for this pheromone family containing two antiparallel helices. The second helix contains the IEECKTS sequence conserved in Aplysia Attractins. Mutating surface-exposed charged residues within this heptapeptide sequence abolishes Attractin activity, suggesting that the second helix is an essential part of the receptor-binding interface.
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NMR solution structure of Attractin, a water-borne protein pheromone from the mollusk Aplysia californica.
Biochemistry, 2003Co-Authors: Ravindranath Garimella, Gregg T. Nagle, Krishna Rajarathnam, Sherry D Painter, Catherine H Schein, Werner BraunAbstract: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.
Amy E. Nichols - One of the best experts on this subject based on the ideXlab platform.
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Newly identified water-borne protein pheromones interact with Attractin to stimulate mate attraction in Aplysia.
Peptides, 2005Co-Authors: Scott F Cummins, Amy E. Nichols, Catherine H Schein, Gregg T. NagleAbstract: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.
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Aplysia seductin is a water-borne protein pheromone that acts in concert with Attractin to stimulate mate attraction.
Peptides, 2005Co-Authors: Scott F Cummins, Amy E. Nichols, Carrie J. Warso, Gregg T. NagleAbstract:Abstract 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.
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characterization of aplysia enticin and temptin two novel water borne protein pheromones that act in concert with Attractin to stimulate mate attraction
Journal of Biological Chemistry, 2004Co-Authors: Scott F Cummins, Amy E. Nichols, Andinet Amare, Amanda B Hummon, Jonathan V Sweedler, Gregg T. NagleAbstract:Abstract 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.
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structural and functional analysis of aplysia Attractins a family of water borne protein pheromones with interspecific attractiveness
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Sherry D Painter, Amy E. Nichols, Scott F Cummins, David B G Akalal, Catherine H Schein, Werner Braun, John S Smith, Abraham J Susswein, Miriam Levy, Pamela A C M De BoerAbstract:Mate attraction in Aplysia involves a long-distance water-borne signal (the protein pheromone Attractin), which is released during egg laying. Aplysia californica Attractin attracts species that produce closely related Attractins, such as Aplysia brasiliana, whose geographic distribution does not overlap that of A. californica. This finding suggests that other mollusks release Attractin-related pheromones to form and maintain breeding aggregations. We describe four additional members of the Attractin family: A. brasiliana, Aplysia fasciata, Aplysia depilans (which aggregates with A. fasciata aggregations), and Aplysia vaccaria (which aggregates with A. californica aggregations). On the basis of their sequence similarity with A. californica Attractin, the Attractin proteins fall into two groups: A. californica, A. brasiliana, and A. fasciata (91-95% identity), and A. depilans and A. vaccaria (41-43% identity). The sequence similarity within the Attractin family, the conserved six cysteines, and the compact fold of the NMR solution structure of A. californica Attractin suggest a common fold for this pheromone family containing two antiparallel helices. The second helix contains the IEECKTS sequence conserved in Aplysia Attractins. Mutating surface-exposed charged residues within this heptapeptide sequence abolishes Attractin activity, suggesting that the second helix is an essential part of the receptor-binding interface.
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A conserved heptapeptide sequence in the waterborne Attractin pheromone stimulates mate attraction in Aplysia
Peptides, 2004Co-Authors: Scott F Cummins, Amy E. Nichols, Krishna Rajarathnam, Gregg T. NagleAbstract: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 Ile 30 -Glu-Glu-Cys-Lys-Thr-Ser 36 . 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. © 2004 Elsevier Inc. All rights reserved.
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structural and functional analysis of aplysia Attractins a family of water borne protein pheromones with interspecific attractiveness
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Sherry D Painter, Amy E. Nichols, Scott F Cummins, David B G Akalal, Catherine H Schein, Werner Braun, John S Smith, Abraham J Susswein, Miriam Levy, Pamela A C M De BoerAbstract:Mate attraction in Aplysia involves a long-distance water-borne signal (the protein pheromone Attractin), which is released during egg laying. Aplysia californica Attractin attracts species that produce closely related Attractins, such as Aplysia brasiliana, whose geographic distribution does not overlap that of A. californica. This finding suggests that other mollusks release Attractin-related pheromones to form and maintain breeding aggregations. We describe four additional members of the Attractin family: A. brasiliana, Aplysia fasciata, Aplysia depilans (which aggregates with A. fasciata aggregations), and Aplysia vaccaria (which aggregates with A. californica aggregations). On the basis of their sequence similarity with A. californica Attractin, the Attractin proteins fall into two groups: A. californica, A. brasiliana, and A. fasciata (91-95% identity), and A. depilans and A. vaccaria (41-43% identity). The sequence similarity within the Attractin family, the conserved six cysteines, and the compact fold of the NMR solution structure of A. californica Attractin suggest a common fold for this pheromone family containing two antiparallel helices. The second helix contains the IEECKTS sequence conserved in Aplysia Attractins. Mutating surface-exposed charged residues within this heptapeptide sequence abolishes Attractin activity, suggesting that the second helix is an essential part of the receptor-binding interface.
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NMR solution structure of Attractin, a water-borne protein pheromone from the mollusk Aplysia californica.
Biochemistry, 2003Co-Authors: Ravindranath Garimella, Gregg T. Nagle, Krishna Rajarathnam, Sherry D Painter, Catherine H Schein, Werner BraunAbstract: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.
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Behavioral Characterization of Attractin, a Water-Borne Peptide Pheromone in the Genus Aplysia
The Biological bulletin, 2003Co-Authors: Sherry D Painter, Bret Clough, Sara Black, Gregg T. NagleAbstract: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 ...
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Peptide products of the atrial gland are not water-borne reproductive pheromones during egg laying in Aplysia.
Peptides, 2003Co-Authors: David B G Akalal, Sherry D Painter, Scott F Cummins, Gregg T. NagleAbstract: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.
