Aplysia Protein Research Articles

Focus on Brain Proteins and Proteomics

Focus on Brain Proteins and Proteomics

A first partial Aplysia californica proteome

Aplysia proteins have not been studied systematically and it was therefore the aim of the study to carry out protein profiling in ganglia from Aplysia californica (AC). AC ganglia were extirpated, proteins extracted and run on 2DE with subsequent in-gel digestion, followed by identification of proteins by nano-LC-ESI-MS/MS on an ion trap. Proteins were identified based upon a public Aplysia EST database. Out of 408 picked spots, 276 spots were identified corresponding to 172 ESTs and 118 individual proteins. The range of sequence coverage was between 14 and 80% and the average amount of peptides used for the identification of proteins was 9 (from 3 to 24). Mean score for protein identification was 516. Comparison of protein levels between cerebral, pleural, pedal and abdominal ganglia revealed a series of significant differences including: signaling, metabolism, cytoskeleton and structural, redox, chaperone, replication/transcription and electron/proton transport proteins. The generation of a protein map complements transcriptional studies carried out in AC ganglia. The findings provide the basis for investigation into post-translational modifications, splice variants and assist in the generation of antibodies against AC proteins. Moreover, differences in protein expression between ganglia may be valuable for the design of future studies in neurobiology of AC.

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 α-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.

Structural insights into competitive and non‐competitive nicotinic antagonists

Nicotinic acetylcholine receptors (nAChR) undergo conformational changes that allosterically trigger opening of the ion channel. Conus peptide toxins bind with receptor subtype specificity to nAChRs. In particular α-conotoxin ImI binds selectively to the α7 nAChR subtype. Using the soluble acetylcholine binding proteins (AChBP) from Aplysia californica and Lymnaea stagnalis, we recently showed α-conotoxin ImI to be selective for the Aplysia protein. We previously solved the crystal structure of α-conotoxin ImI bound to Aplysia AChBP. Using this model we have identified regions on the apical and membrane side of the competitive binding pocket, which contribute to species specificity. Using site-directed mutagenesis we partially rescued α-conotoxin ImI binding in the Lymnaea protein. The two Conus peptides, α-conotoxins ImI and ImII, differ by only 3 of 11 amino acids, yet they appear to bind to distinct sites on the human α7 nicotinic acetylcholine receptor (nAChR). Additionally, we are exploring techniques for investigating association of α-conotoxin ImII with AChBP, thereby distinguishing its binding parameters and sites from α-conotoxin ImI. (Supported by R37-GM18360, T32_GM07752, and Tobacco-Related Disease Research Program 12DT-0013)

Phosphorylation of eIF4E at a Conserved Serine in Aplysia

We have cloned eIF4E from the marine mollusk, Aplysia californica. The sequence of eIF4E from Aplysia is more similar to vertebrate eIF4Es than to other invertebrate sequences. Aplysia eIF4E is encoded by two tissue-specific RNAs. Antibodies raised to the carboxyl terminus of eIF4E recognize a 29-kDa protein that can bind to 7-methyl-GTP caps. The phosphorylation site identified in mammalian eIF4E is conserved in the Aplysia homologue, and an Aplysia eIF4E fusion protein is phosphorylated well by both Aplysia protein kinase C isoforms. However, protein kinase C phosphorylates both Ser-207 and Thr-208 in vitro, while only Ser-207 is phosphorylated in vivo. We have confirmed that Ser-207 is phosphorylated in vivo by raising a phosphopeptide antibody to this site. This antibody will be useful in determining the signal transduction pathways leading to eIF4E phosphorylation in Aplysia.

An NF-κB-Like Transcription Factor in Axoplasm is Rapidly Inactivated after Nerve Injury inAplysia

We found a protein in Aplysia neurons that has many characteristics of the transcription factor NF-kappaB. Thus, the protein recognized a radiolabeled probe containing the kappaB sequence from the human interferon-beta gene enhancer element (PRDII), and the binding was not affected by PRDIV, an ATF-2 enhancer sequence from the same gene. Binding was efficiently inhibited, however, by nonradioactive oligonucleotides containing H2, the kappaB site from the major histocompatibility complex I gene promotor. In addition, recombinant mammalian IkappaB-alpha, which associates specifically with the P65 subunit of NF-kappaB, inhibited the binding to the PRDII probe in a dose-dependent manner. The nuclear form of the Aplysia protein was constitutively active. Axoplasm, however, contained the constitutively active form as well as a latent form. The latter was activated by treatment with deoxycholate under the same conditions as mammalian NF-kappaB. Based on these findings, we believe the protein to be a homolog of NF-kappaB. To investigate the role of apNF-kappaB in the axon, we crushed the peripheral nerves to the body wall. Surprisingly, there was a rapid loss of apNF-kappaB binding at the crush site and, within 15 min, as far as 2.5 cm along the axon. In contrast, exposing either the intact animal or the nervous system in situ to levels of 5-HT that induce synaptic facilitation did not affect apNF-kappaB activity.

Cloning and characterization of aplycalcin and Aplysia neurocalcin, two new members of the calmodulin superfamily of small calcium-binding proteins.

We report here the sequences of two new proteins from Aplysia, aplycalcin and Aplysia neurocalcin. These proteins belong to a family of calcium-binding proteins, found primarily in vertebrate brain and retina, that have been proposed to play a role in calcium-dependent regulation of enzymes in signal transduction pathways. Like other members of this family, the Aplysia proteins have consensus sequences for myristoylation, bind calcium, and translocate from cytosol to membrane when the calcium level is raised above the resting intracellular concentration. Both proteins are relatively enriched in Aplysia nervous system, but are also found to a significant degree in other tissues. The expression of mRNA for these proteins in Aplysia nervous tissue is regulated during development, roughly paralleling the reported emergence of several forms of synaptic plasticity. The messages are present at low levels in stage 11, show a large increase by late stage 12, and decline to a plateau of approximately 30% of the peak value afterward. On the basis of the properties of these proteins and by analogy with proposed functions of some of the retinal homologues, we suggest that these proteins may play a role in mediating calcium-dependent processes in neuronal function. The presence of both proteins in other tissues may suggest analogous roles for the proteins in other cell types.

Expression and characterization of Aplysia protein kinase C: a negative regulatory role for the E region

The Aplysia nervous system contains two phorbol ester-activated protein kinase C isoforms, the Ca(2+)-activated Apl I and the Ca(2+)-independent Apl II. Short-term applications of the facilitatory transmitter serotonin (5-HT) activates Apl I, but not Apl II. In contrast, Apl II, but not Apl I, can form an autonomous kinase. To investigate the biochemical characteristics of the Aplysia kinases that might underlie their differential activation, we expressed Apl I, Apl II, and two derivatives of Apl II with deletions in the amino-terminal 150 amino acid E region in insect cells using the baculovirus system. Similar to nervous system extracts, expressed Apl II has more autonomous activity than Apl I. Removal of the E region lowered the amount of phosphatidylserine required for activation of Apl II, but did not remove the autonomous kinase activity. In addition, phosphatidylserine vesicles could sediment fusion proteins containing the E region, consistent with a role for the E region in lipid interactions. A partial deletion of the E region modifies activation of Apl II by phorbol esters and oleic acid, suggesting that in the intact enzyme the E region interacts with the phorbol ester-binding domain of the kinase. These results introduce a model whereby the E region acts as a negative regulator of Apl II activation and suggest that this inhibition may explain the inability of short-term applications of 5-HT to activate Apl II.

Structural role of disulfide bridges in the cyclic ADP-ribose related bifunctional ectoenzyme CD38

Human CD38, a type II cell surface glycoprotein, is a bifunctional ectoenzyme catalyzing both ADP-ribosyl cyclase and cyclic ADP-ribose (cADPR) hydrolase reactions. It shares a high degree of sequence homology with the cyclase from Aplysia species and studies of site-directed mutagenesis have recently demonstrated the importance, but not elucidated the role, of several cysteine residues highly conserved between these proteins.N-Ethylmaleimide, iodoacetamide and thiol-oxidizing reagents failed to affect either the cyclase or the weaker hydrolase activity of the Aplysia californica protein. Likewise, these reagents did not impair the two activities of CD38 purified from human erythrocytes. β-mercaptoethanol had no effect on the Aplysia enzyme activities, while it inactivated both the cyclase and the cADPR hydrolase of CD38 by inducing its extensive oligomerization. In intact erythrocytes the β-mercaptoethanol-dependent enzyme inactivation was completely prevented by prior cross-linking of the membrane proteins with glutaraldehyde. These data demonstrate that none of the cysteine residues plays any direct catalytic role in CD38 and Aplysia proteins, and that disulfide bridges are essential for maintaining the monomeric, catalytically active structure of CD38.

Identification of Three Proteins in the Eye of Aplysia, Whose Synthesis Is Altered by Serotonin (5-HT). POSSIBLE INVOLVEMENT OF THESE PROTEINS IN THE OCULAR CIRCADIAN SYSTEM

Previous results using translation inhibitors in the ocular circadian system of Aplysia suggest that protein synthesis may be involved in the light and serotonin (5-HT) entrainment pathways or perhaps in the circadian oscillator. Proteins have been previously identified whose synthesis was altered by treatments of light capable of perturbing the phase of the circadian rhythm in the eye of Aplysia. We extended these studies by investigating the effects of other treatments that perturb the ocular circadian rhythm on protein synthesis. 5-HT altered the synthesis of nine proteins. Interestingly, five of the proteins affected by treatments with 5-HT were previously shown to be affected by treatments with light. Four of the proteins affected by treatments with 5-HT were also affected by treatments with analogs of cAMP, a treatment which mimics the effects of 5-HT on the ocular circadian rhythm. To identify the cellular function of some of these proteins, we obtained their partial amino acid sequences. Based on these sequences and additional characterizations, a 78-kDa, pI 5.6 Aplysia protein appears to be glucose-regulated protein 78/binding protein, and a 36-kDa, pI 5.7 Aplysia protein appears to be porin/voltage-dependent anion channel. Heat shock experiments on Aplysia eyes revealed that yet another one of the Aplysia proteins (70 kDa) affected by 5-HT appears to be a heat-inducible member (heat shock protein 70) of the family of heat shock proteins. These findings suggest that these three identified proteins, together or individually, may be involved in some way in the regulation of the timing of the circadian oscillator in the eye of Aplysia.

Sequencing of proteins from two-dimensional gels by using in situ digestion and transfer of peptides to polyvinylidene difluoride membranes: application to proteins associated with sensitization in Aplysia.

We have developed a method for obtaining partial internal amino acid sequence data from proteins isolated directly from preparative two-dimensional polyacrylamide gels. Proteins from a crude cell homogenate are separated using preparative two-dimensional polyacrylamide gel electrophoresis. Then, the gel is stained with Coomassie blue and the protein spots of interest are cut out. The in situ protein is digested with Staphylococcus aureus V8 protease in a second polyacrylamide gel and the peptides are separated by one-dimensional polyacrylamide gel electrophoresis. The peptides are then electroblotted onto a polyvinylidene difluoride membrane, visualized using Coomassie blue, cut out, and sequenced using an automated gas phase sequencer. Using this method, we have obtained amino acid sequence data for two proteins that are altered after long-term sensitization: actin and Aplysia protein 407. In addition, we have obtained amino acid sequence data for rat protein 425, a protein that appears to be homologous to Aplysia protein 407.

Abundant expression of ras proteins in Aplysia neurons.

We have cloned a DNA fragment from the marine mollusc Aplysia californica, which contains sequences homologous to mammalian ras genes, by screening a genomic library with a viral Ha-ras oncogene probe under conditions of low stringency hybridization. Nucleotide sequencing revealed a putative exon that encodes amino acids sharing 68% homology with residues 5 to 54 of mammalian p21ras polypeptides, and which therefore is likely to encode a ras-like Aplysia protein. The cloned locus, designated Apl-ras, is distinct from the Aplysia rho (ras-homologue) gene and appears to be more closely related to mammalian ras. We used a panel of monoclonal antibodies raised against v-Ha-ras p21 to precipitate an Mr 21,000 protein from extracts of Aplysia nervous tissue, ovotestis, and, to a much lesser degree, buccal muscle. Fluorescence immunocytochemistry revealed that ras-like protein is most abundant in neuronal cell bodies and axon processes, with staining most prominent at plasma membranes. Much less was present in other tissues. The prominence of ras protein in neurons, which are terminally differentiated and non-proliferating, indicates that the control of cell division is not the sole function of this proto-oncogene. The large identified neurons of Aplysia offer the opportunity to examine how ras protein might function in mature nerve cells.

A novel ras-related gene family

We have identified a new family of ras genes, the rho genes, which share several properties with the more classical ras gene family consisting of N-, K-, and H- ras. The rho genes, first isolated from a cDNA library from the abdominal ganglia of Aplysia, encode proteins that share 35% amino acid homology with H- ras. Evolutionarily conserved counterparts of rho have been detected in yeast, in Drosophila, in rat, and in man. Sequence analysis reveals over 85% homology between the human and Aplysia proteins. The ras and rho gene products share several common properties; both are 21,000 daltons, both reveal C-terminal sequences required for membrane attachment, and both show blocks of strong internal homology, suggesting that the two proteins may share common functions but may use these functions in different ways.