1. Proteolytic processing of egg-laying hormone-related precursors in Aplysia. Identification of peptide regions critical for biological activity
G T Nagle, S D Painter, J E Blankenship, A Kurosky J Biol Chem. 1988 Jul 5;263(19):9223-37.
The atrial gland of the marine mollusk Aplysia californica is an exocrine organ that expresses at least three genes belonging to the egg-laying hormone (ELH) family. In order to study the post-translational processing of the ELH-related gene products in the atrial gland and how it compares to the bag cells, peptides were isolated from the atrial gland and chemically characterized. The A- and B-related precursors were each cleaved in vivo to yield several major and minor peptides including peptides A and B and the ELH-related peptide complexes that caused egg laying. About 13% of the peptide complexes were further enzymically processed by the atrial gland to yield smaller fragments, which included A-AP.A-ELH-(15-36), A-AP.[Ala27]A-ELH-(15-36), and A-AP.[Gln23,Ala27]A-ELH-(16-36), where A-AP is an acidic peptide encoded by the A- and B-related genes and A-ELH is an ELH-related peptide encoded by the A gene. These processed peptide fragments were not active in an egg-laying bioassay, indicating that retention of the 14-residue NH2-terminal segment of the A-ELH-related sequence, or some portion thereof, was critical for the induction of egg laying. Other characterized peptides included two novel 13-residue NH2-terminal peptides, A-NTP and B-NTP, representing residues 22-34 of the A and B precursors, respectively. These two peptides occurred adjacent to the signal peptide region in each precursor, and their characterization established the site of signal peptide cleavage to be the Ser21-Gln22 peptide bond of each precursor. Intermediate peptide fragments (A-NTP-peptide A and B-NTP-peptide B) were also identified indicating that there was a specific ordering in the cleavage of peptide bonds during posttranslational processing. Finally, a new 55-residue atrial gland peptide was also isolated that was not a part of any ELH-related precursor characterized to date.
2. An immunohistochemical study of the neuropeptidergic bag cells of Aplysia
A Y Chiu, F Strumwasser J Neurosci. 1981 Aug;1(8):812-26. doi: 10.1523/JNEUROSCI.01-08-00812.1981.
The bag cell neurosecretory system of Aplysia californica synthesizes and releases a neuropeptide, the egg-laying hormone (ELH), which can induce egg-laying behavior. While ELH acts as a neurohormone on distant neuronal and non-neuronal targets, it also can moderate the electrical activity of neurons within the abdominal ganglion. Using antibodies generated against pure ELH, our immunohistochemical study provides direct morphological support for this dual function of ELH by defining the distribution of this neuropeptide within the abdominal ganglion of A. californica. We also find ELH cross-reactivity in the bag cell systems of three other species of Aplysia. In sections of abdominal ganglia stained for ELH by the peroxidase-antiperoxidase method, bag cell clusters were found to be homogeneous populations of ELH-immunoreactive neurons, extending processes in a complex array within the connective tissue sheath overlying the ganglion and the proximal regions of neighboring nerves. This association of neurites with the sheath is the neurohemal organ which facilitates release of ELH into circulating hemolymph. Stained processes also invade the interior of the ganglion in association with septal sheets of connective tissue and as scattered fibers in the neuropil. Local modulation by ELH probably occurs through release from these intraganglionic sites. A few ELH-positive ectopic cells were seen outside of the confines of the bag cell clusters as far displaced as the base of the branchial nerve; they appear to be bag cells which have mis-migrated during development. Specificity of the antiserum was confirmed by the total lack of staining of all other neurons within the abdominal ganglion as well as the absence of staining of bag cells with pre-immune serum. Although th ELHs of Aplysia vaccaria, Aplysia braziliana, and Aplysia dactylomela have not been characterized biochemically, it is known that abdominal ganglion extracts from A. californica can induce egg laying in all three species. We find that antiserum against A. californica ELH stains the bag cell systems of A. vaccaria, A. braziliana, and A. dactylomela and not other parts of their abdominal ganglia. It is likely that this staining represents cross-immunoreactivity of the egg-laying neurohormones from these species which also share cross-bioactivity.
3. Molecular aspects of egg-laying behavior in Aplysia californica
L DesGroseillers Behav Genet. 1990 Mar;20(2):251-64. doi: 10.1007/BF01067793.
The Aplysia neuroendocrine system is a particularly advantageous model for cellular and molecular studies because of the relatively small number and large size of its component neurons. In addition, numerous anatomical and studies have resulted in the assignment of behavioral roles to individual identified neurons. Recombinant DNA techniques have been used to isolate the genes that encode the precursors of peptides involved in egg-laying behavior. The comparison of the egg-laying hormone (ELH) gene family within the genus Aplysia reveals high homologies in the overall structure of the precursors. A well-conserved tetrabasic residue has been shown to be the first endoproteolytic cleavage site of the precursor, giving rise to two intermediates, which are differentially processed and packaged. Some members of the ELH gene family are expressed specifically in the bag cell clusters or the atrial gland, respectively, providing an opportunity to study control of gene expression at the molecular level.
