Molecular Neurobiology

, Volume 52, Issue 1, pp 45–56 | Cite as

Identification and Expression of Acetylcholinesterase in Octopus vulgaris Arm Development and Regeneration: a Conserved Role for ACHE?

  • Sara Maria Fossati
  • Simona Candiani
  • Marie-Therese Nödl
  • Luca Maragliano
  • Maria Pennuto
  • Pedro Domingues
  • Fabio Benfenati
  • Mario Pestarino
  • Letizia Zullo


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.


Acetylcholinesterase Octopus vulgaris Development Regeneration Molecular modeling 





Artificial sea water




Catalytic anionic site


Catalytic triad




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.

Supplementary material

12035_2014_8842_MOESM1_ESM.xlsx (22 kb)
Fig. S1 (XLSX 21 kb)
12035_2014_8842_MOESM2_ESM.xls (28 kb)
Fig. S2 (XLS 28 kb)
12035_2014_8842_Fig8_ESM.jpg (1.2 mb)
Fig. S3

(JPEG 1201 kb)

12035_2014_8842_Fig9_ESM.jpg (471 kb)
Fig. S4

(JPEG 471 kb)

12035_2014_8842_Fig10_ESM.gif (78 kb)
Fig. S5

(GIF 78 kb)

12035_2014_8842_MOESM3_ESM.tif (18.6 mb)
High Resolution Image (TIFF 19032 kb)
12035_2014_8842_MOESM4_ESM.docx (42 kb)
ESM 1 (DOCX 42 kb)
12035_2014_8842_MOESM5_ESM.avi (6.2 mb)
Movie S1 (AVI 6338 kb)


  1. 1.
    Jiang H, Zhang XJ (2008) Acetylcholinesterase and apoptosis. A novel perspective for an old enzyme. FEBS J 275(4):612–617. doi: 10.1111/j.1742-4658.2007.06236.x CrossRefPubMedGoogle Scholar
  2. 2.
    Soreq H, Seidman S (2001) Acetylcholinesterase—new roles for an old actor. Nat Rev Neurosci 2:294–302CrossRefPubMedGoogle Scholar
  3. 3.
    Layer PG, Willbold E (1995) Novel functions of cholinesterases in development, physiology and disease. Prog Histochem Cytochem 29(3):1–94PubMedGoogle Scholar
  4. 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
  5. 5.
    Lenique PM, Feral JP (1976) A mechanism of action of neurotransmitters on the regeneration of the planarian worm Dugesia tigrina. Role of acetylcholine as a negative feed-back. Acta Zool 57:1–5CrossRefGoogle Scholar
  6. 6.
    Srivatsan M, Peretz B (1997) Acetylcholinesterase promotes regeneration of neurites in cultured adult neurons of Aplysia. Neuroscience 77(3):921–931CrossRefPubMedGoogle Scholar
  7. 7.
    Lauder JM, Schambra UB (1999) Morphogenetic roles of acetylcholine. Environ Health Perspect 107:65–69CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Vogel-Hopker A, Sperling LE, Layer PG (2012) Co-opting functions of cholinesterases in neural, limb and stem cell development. Protein Pept Lett 19(2):155–164CrossRefPubMedGoogle Scholar
  9. 9.
    Dori A, Soreq H (2006) ARP, the cleavable C-terminal peptide of “readthrough” acetylcholinesterase, promotes neuronal development and plasticity. J Mol Neurosci MN 28(3):247–255. doi: 10.1385/JMN:28:3:247 CrossRefPubMedGoogle Scholar
  10. 10.
    Paraoanu LE, Steinert G, Klaczinski J, Becker-Rock M, Bytyqi A, Layer PG (2006) On functions of cholinesterases during embryonic development. J Mol Neurosci MN 30(1–2):201–204. doi: 10.1385/JMN:30:1:201 CrossRefPubMedGoogle Scholar
  11. 11.
    Falugi C, Aluigi MG (2012) Early appearance and possible functions of non-neuromuscular cholinesterase activities. Front Mol Neurosci 5:54. doi: 10.3389/fnmol.2012.00054 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Behra M, Cousin X, Bertrand C, Vonesch JL, Biellmann D, Chatonnet A, Strähle U (2002) Acetylcholinesterase is required for neuronal and muscular development in the zebrafish embryo. Nat Neurosci 5:111–118CrossRefPubMedGoogle Scholar
  13. 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
  14. 14.
    Massoulié J, Anselmet A, Bon S, Krejci E, Legay C, Morel N, Simon S (1999) The polymorphism of acetylcholinesterase: post-translational processing, quaternary associations and localization. Chem Biol Interact 119–120:29–42CrossRefPubMedGoogle Scholar
  15. 15.
    Massoulié J (2002) The origin of the molecular diversity and functional anchoring of cholinesterases. Neuro Signals 11(3):130–143CrossRefPubMedGoogle Scholar
  16. 16.
    Hicks D, John D, Makova NZ, Henderson Z, Nalivaeva NN, Turner AJ (2011) Membrane targeting, shedding and protein interactions of brain acetylcholinesterase. J Neurochem 116(5):742–746. doi: 10.1111/j.1471-4159.2010.07032.x CrossRefPubMedGoogle Scholar
  17. 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
  18. 18.
    Pezzementi L, Johnson K, Tsigelny I, Cotney J, Manning E, Barker A, Merritt S (2003) Amino acids defining the acyl pocket of an invertebrate cholinesterase. Comp Biochem Physiol B Biochem Mol Biol 136(4):813–832CrossRefPubMedGoogle Scholar
  19. 19.
    Silman I, Sussman JL (2008) Acetylcholinesterase: how is structure related to function? Chem Biol Interact 175(1–3):3–10. doi: 10.1016/j.cbi.2008.05.035 CrossRefPubMedGoogle Scholar
  20. 20.
    Pierleoni A, Martelli PL, Casadio R (2008) PredGPI: a GPI-anchor predictor. BMC Bioinforma 9:392. doi: 10.1186/1471-2105-9-392 CrossRefGoogle Scholar
  21. 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
  22. 22.
    Dvir H, Silman I, Harel M, Rosenberry TL, Sussman JL (2010) Acetylcholinesterase: from 3D structure to function. Chem Biol Interact 187(1–3):10–22. doi: 10.1016/j.cbi.2010.01.042 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Pegan K, Matkovic U, Mars T, Mis K, Pirkmajer S, Brecelj J, Grubic Z (2010) Acetylcholinesterase is involved in apoptosis in the precursors of human muscle regeneration. Chem Biol Interact 187:96–100CrossRefPubMedGoogle Scholar
  24. 24.
    Xie J, Jiang H, Wan YH, Du AY, Guo KJ, Liu T, Ye WY, Niu X, Wu J, Dong XQ, Zhang XJ (2011) Induction of a 55 kDa acetylcholinesterase protein during apoptosis and its negative regulation by the Akt pathway. J Mol Cell Biol 3(4):250–259. doi: 10.1093/jmcb/mjq047 CrossRefPubMedGoogle Scholar
  25. 25.
    Sussman JL, Harel M, Frolow F, Oefner C, Goldman A, Toker L, Silman I (1991) Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein. Science 253(5022):872–879CrossRefPubMedGoogle Scholar
  26. 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. 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
  28. 28.
    Arnold JM (1965) Normal embryonic stages of the squid, Loligo pealii (Lesueur). Biol Bull 128(1):24–32CrossRefGoogle Scholar
  29. 29.
    Xu Y, Colletier JP, Weik M, Jiang H, Moult J, Silman I, Sussman JL (2008) Flexibility of aromatic residues in the active-site gorge of acetylcholinesterase: X-ray versus molecular dynamics. Biophys J 95(5):2500–2511. doi: 10.1529/biophysj.108.129601 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Eswar N, John B, Mirkovic N, Fiser A, Ilyin VA, Pieper U, Stuart AC, Marti-Renom MA, Madhusudhan MS, Yerkovich B, Sali A (2003) Tools for comparative protein structure modeling and analysis. Nucleic Acids Res 31(13):3375–3380CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Shen MY, Sali A (2006) Statistical potential for assessment and prediction of protein structures. Protein Sci Publ Protein Soc 15(11):2507–2524. doi: 10.1110/ps.062416606 CrossRefGoogle Scholar
  32. 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
  33. 33.
    Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, Villa E, Chipot C, Skeel RD, Kale L, Schulten K (2005) Scalable molecular dynamics with NAMD. J Comput Chem 26(16):1781–1802. doi: 10.1002/jcc.20289 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 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. 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
  36. 36.
    Lee PN, Callaerts P, De Couet HG, Martindale MQ (2003) Cephalopod Hox genes and the origin of morphological novelties. Nature 424(6952):1061–1065. doi: 10.1038/nature01872 CrossRefPubMedGoogle Scholar
  37. 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. 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
  39. 39.
    Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425PubMedGoogle Scholar
  40. 40.
    Guindon S, Lethiec F, Duroux P, Gascuel O (2005) PHYML online—a web server for fast maximum likelihood-based phylogenetic inference. Nucleic Acids Res 33(Web Server issue):W557–W559. doi: 10.1093/nar/gki352 CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Pang YP (2006) Novel acetylcholinesterase target site for malaria mosquito control. PLoS One 1:e58. doi: 10.1371/journal.pone.0000058 CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Fossati SM, Carella F, De Vico G, Benfenati F, Zullo L (2013) Octopus arm regeneration: role of acetylcholinesterase during morphological modification. JEMBE 447:93–99CrossRefGoogle Scholar
  43. 43.
    Futerman AH, Low MG, Ackermann KE, Sherman WR, Silman I (1985) Identification of covalently bound inositol in the hydrophobic membrane-anchoring domain of Torpedo acetylcholinesterase. Biochem Biophys Res Commun 129(1):312–317CrossRefPubMedGoogle Scholar
  44. 44.
    Massoulié J, Perrier N, Noureddine H, Liang D, Bon S (2008) Old and new questions about cholinesterases. Chem Biol Interact 175:30–44CrossRefPubMedGoogle Scholar
  45. 45.
    Taylor P (1991) The cholinesterases. J Biol Chem 266(7):4025–4028PubMedGoogle Scholar
  46. 46.
    Massoulié J, Pezzementi L, Bon S, Krejci E, Vallette FM (1993) Molecular and cellular biology of cholinesterases. Prog Neurobiol 41(1):31–91CrossRefPubMedGoogle Scholar
  47. 47.
    Harel M, Kleywegt GJ, Ravelli RB, Silman I, Sussman JL (1995) Crystal structure of an acetylcholinesterase-fasciculin complex: interaction of a three-fingered toxin from snake venom with its target. Structure 3(12):1355–1366CrossRefPubMedGoogle Scholar
  48. 48.
    Grisaru D, Sternfeld M, Eldor A, Glick D, Soreq H (1999) Structural roles of acetylcholinesterase variants in biology and pathology. Eur J Biochem/FEBS 264(3):672–686CrossRefGoogle Scholar
  49. 49.
    Massoulie J, Anselmet A, Bon S, Krejci E, Legay C, Morel N, Simon S (1998) Acetylcholinesterase: C-terminal domains, molecular forms and functional localization. J Physiol Paris 92(3–4):183–190CrossRefPubMedGoogle Scholar
  50. 50.
    Grauso M, Culetto E, Combes D, Fedon Y, Toutant JP, Arpagaus M (1998) Existence of four acetylcholinesterase genes in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae. FEBS Lett 424(3):279–284CrossRefPubMedGoogle Scholar
  51. 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
  52. 52.
    Cossu G, Eusebi F, Grassi F, Wanke E (1987) Acetylcholine receptor channels are present in undifferentiated satellite cells but not in embryonic myoblasts in culture. Dev Biol 123(1):43–50CrossRefPubMedGoogle Scholar
  53. 53.
    Yamamoto M, Shimazaki Y, Shigeno S (2003) Atlas of the embryonic brain in the pygmy squid, Idiosepius paradoxus. Zool Sci 20(2):163–179CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Sara Maria Fossati
    • 1
  • Simona Candiani
    • 2
  • Marie-Therese Nödl
    • 1
  • Luca Maragliano
    • 1
  • Maria Pennuto
    • 1
    • 3
  • Pedro Domingues
    • 4
  • Fabio Benfenati
    • 1
    • 5
  • Mario Pestarino
    • 2
  • Letizia Zullo
    • 1
  1. 1.Department of Neuroscience and Brain TechnologiesIstituto Italiano di TecnologiaGenoaItaly
  2. 2.DISTAVUniversity of GenovaGenoaItaly
  3. 3.Dulbecco Telethon Institute, Lab of Neurodegenerative Diseases, CIBIOUniversity of TrentoTrentoItaly
  4. 4.Centro Oceanográfico de VigoInstituto Español de OceanografíaVigoSpain
  5. 5.Department of Experimental MedicineUniversity of GenovaGenoaItaly

Personalised recommendations