Identification and Expression of Acetylcholinesterase in Octopus vulgaris Arm Development and Regeneration: a Conserved Role for ACHE?
- 403 Downloads
Acetylcholinesterase (ACHE) is a glycoprotein with a key role in terminating synaptic transmission in cholinergic neurons of both vertebrates and invertebrates. ACHE is also involved in the regulation of cell growth and morphogenesis during embryogenesis and regeneration acting through its non-cholinergic sites. The mollusk Octopus vulgaris provides a powerful model for investigating the mechanisms underlying tissue morphogenesis due to its high regenerative power. Here, we performed a comparative investigation of arm morphogenesis during adult arm regeneration and embryonic arm development which may provide insights on the conserved ACHE pathways. In this study, we cloned and characterized O. vulgaris ACHE, finding a single highly conserved ACHE hydrophobic variant, characterized by prototypical catalytic sites and a putative consensus region for a glycosylphosphatidylinositol (GPI)-anchor attachment at the COOH-terminus. We then show that its expression level is correlated to the stage of morphogenesis in both adult and embryonic arm. In particular, ACHE is localized in typical neuronal sites when adult-like arm morphology is established and in differentiating cell locations during the early stages of arm morphogenesis. This possibility is also supported by the presence in the ACHE sequence and model structure of both cholinergic and non-cholinergic sites. This study provides insights into ACHE conserved roles during processes of arm morphogenesis. In addition, our modeling study offers a solid basis for predicting the interaction of the ACHE domains with pharmacological blockers for in vivo investigations. We therefore suggest ACHE as a target for the regulation of tissue morphogenesis.
KeywordsAcetylcholinesterase Octopus vulgaris Development Regeneration Molecular modeling
Artificial sea water
Catalytic anionic site
Open reading frame
Peripheral binding site
Root mean square deviation
Real-time quantitative PCR
We thank Jennifer Helm for the help with data collection and Andrea Contestabile for the technical support. We are also grateful to Prof. Jenny Kien for the suggestions and editorial assistance.
Conflict of Interest
The authors declare that they have no conflict of interest.
- 4.Singer M, Davis MH, Arkowitz ES (1960) Acetylcholinesterase activity in the regenerating forelimb of the adult newt, triturus. J Embryol Exp Morpholog 8:98–111Google Scholar
- 13.Berg L, Andersson CD, Artursson E, Hornberg A, Tunemalm AK, Linusson A, Ekstrom F (2011) Targeting acetylcholinesterase: identification of chemical leads by high throughput screening, structure determination and molecular modeling. PLoS One 6(11):e26039. doi: 10.1371/journal.pone.0026039 CrossRefPubMedPubMedCentralGoogle Scholar
- 17.Dvir H, Jiang HL, Wong DM, Harel M, Chetrit M, He XC, Jin GY, Yu GL, Tang XC, Silman I, Bai DL, Sussman JL (2002) X-ray structures of Torpedo californica acetylcholinesterase complexed with (+)-huperzine A and (−)-huperzine B: structural evidence for an active site rearrangement. Biochemistry 41(35):10810–10818CrossRefPubMedGoogle Scholar
- 21.Paz A, Xie Q, Greenblatt HM, Fu W, Tang Y, Silman I, Qiu Z, Sussman JL (2009) The crystal structure of a complex of acetylcholinesterase with a bis-(-)-nor-meptazinol derivative reveals disruption of the catalytic triad. J Med Chem 52(8):2543–2549. doi: 10.1021/jm801657v CrossRefPubMedGoogle Scholar
- 26.Shafferman A, Kronman C, Flashner Y, Leitner M, Grosfeld H, Ordentlich A, Gozes Y, Cohen S, Ariel N, Barak D et al (1992) Mutagenesis of human acetylcholinesterase. Identification of residues involved in catalytic activity and in polypeptide folding. J Biol Chem 267(25):17640–17648PubMedGoogle Scholar
- 27.Fiorito G, Affuso A, Anderson DB, Basil J, Bonnaud L, Botta G, Cole A, D’Angelo L, De Girolamo P, Dennison N, Dickel L, Di Cosmo A, Di Cristo C, Gestal C, Fonseca R, Grasso F, Kristiansen T, Kuba M, Maffucci F, Manciocco A, Mark FC, Melillo D, Osorio D, Palumbo A, Perkins K, Ponte G, Raspa M, Shashar N, Smith J, Smith D, Sykes A, Villanueva R, Tublitz N, Zullo L, Andrews P (2014) Cephalopods in neuroscience: regulations, research and the 3Rs. Invert Neurosci IN. doi: 10.1007/s10158-013-0165-x PubMedGoogle Scholar
- 32.Mackerell ADJ, Feig M, Brooks CL (2004) 3rd Extending the treatment of backbone energetics in protein force fields: limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations. J Comput Chem 25:1400–1415CrossRefPubMedGoogle Scholar
- 34.Sirakov M, Zarrella I, Borra M, Rizzo F, Biffali E, Arnone MI, Fiorito G (2009) Selection and validation of a set of reliable reference genes for quantitative RT-PCR studies in the brain of the Cephalopod Mollusc Octopus vulgaris. BMC Mol Biol 10:70. doi: 10.1186/1471-2199-10-70 CrossRefPubMedPubMedCentralGoogle Scholar
- 35.Tiveron MC, Hirsch MR, Brunet JF (1996) The expression pattern of the transcription factor Phox2 delineates synaptic pathways of the autonomic nervous system. J Neurosci Off J Soc Neurosci 16(23):7649–7660Google Scholar
- 37.Talesa V, Grauso M, Arpagaus M, Giovannini E, Romani R, Rosi G (1999) Molecular cloning and expression of a full-length cDNA encoding acetylcholinesterase in optic lobes of the squid Loligo opalescens: a new member of the cholinesterase family resistant to diisopropyl fluorophosphate. J Neurochem 72(3):1250–1258CrossRefPubMedGoogle Scholar
- 38.Harel M, Kryger G, Rosenberry TL, Mallender WD, Lewis T, Fletcher RJ, Guss JM, Silman I, Sussman JL (2000) Three-dimensional structures of Drosophila melanogaster acetylcholinesterase and of its complexes with two potent inhibitors. Protein Sci Publ Protein Soc 9(6):1063–1072. doi: 10.1110/ps.9.6.1063 CrossRefGoogle Scholar
- 51.Combes D, Fedon Y, Grauso M, Toutant JP, Arpagaus M (2000) Four genes encode acetylcholinesterases in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae. cDNA sequences, genomic structures, mutations and in vivo expression. J Mol Biol 300(4):727–742. doi: 10.1006/jmbi.2000.3917 CrossRefPubMedGoogle Scholar