Recombinant human acetylcholinesterase is secreted from transiently transfected 293 cells as a soluble globular enzyme
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Coding sequences for the human acetylcholinesterase (HuAChE; EC 126.96.36.199) hydrophilic subunit were subcloned in an expression plasmid vector under the control of cytomegalovirus IE gene enhancer-promoter. The human embryonic kidney cell line 293, transiently transfected with this vector, expressed catalytically active acetylcholinesterase.
The recombinant gene product exhibits biochemical traits similar to native “true” acetylcholinesterase as manifested by characteristic substrate inhibition, aK m of 117µM toward acetylthiocholine, and a high sensitivity to the specific acetylcholinesterase inhibitor BW284C51.
The transiently transfected 293 cells (100 mm dish) produce in 24 hr active enzyme capable of hydrolyzing 1500 nmol acetylthiocholine per min. Eighty percent of the enzymatic activity appears in the cell growth medium as soluble acetylcholinesterase; most of the cell associated activity is confined to the cytosolic fraction requiring neither detergent nor high salt for its solubilization.
The active secreted recombinant enzyme appears in the monomeric, dimeric, and tetrameric globular hydrophilic molecular forms.
In conclusion, the catalytic subunit expressed from the hydrophylic AChE cDNA species has the inherent potential to be secreted in the soluble globular form and to generate polymorphism through self-association.
Key wordsacetylcholinesterase transient transfection 293 cells cytomegalovirus (CMV) promoter
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- Abramson, S. N., Ellisman, M. M., Deerinck, T. J., Maulet, Y., Gentry, M. K., Doctor, B. P., and Taylor, P. (1989). Differences in structure and distribution of the molecular forms of acetylcholinesterase.J. Cell. Biol. 1082301–2311.Google Scholar
- Atack, J. R., Perry, E. K., Bonham, J. R., and Perry, R. H. (1987). Molecular forms of acetylcholinesterase and butyrylcholinesterase in human plasma and cerebrospinal fluid.J. Neurochem. 481845–1850.Google Scholar
- Augustinsson, K. B. (1963). InHandb. Exp. Pharmak. (G. B. Koelle, Ed.), Springer-Verlag, Berlin, Suppl. 15, pp. 89–128.Google Scholar
- Ausubel, M., Brent, R., Kingston, R. E., Moore, D. D., Smith, J. A., Seidman, J. G., and Struhl, K. (eds.) (1987).Current Protocols in Molecular Biology, Wiley Interscience, New York.Google Scholar
- Ben-Aziz, R., Gnatt, A., Prody, C., Lev-Lehman, E., Neville, L., Seidman, S., Ginzberg, D., Soreq, H., Lapidot-Lifson, Y., and Zakut, H. (1990). Differential codon usage and distinct surface probabilities in human acetylcholinesterase. In (F. Bacou, Ed.), Am. Chem. Soc. Books, Washington, D.C. (in press).Google Scholar
- Benoist, C., and Chambon, P. (1981). In vivo sequence requirements of the SV40 early promoter region.Nature 290304–310.Google Scholar
- Brimijoin, S., and Hammond, P. (1988). Butyrylcholinesterase in human brain and acetylcholinesterase in human plasma: Trace enzymes measured by two-site immunoassay.J. Neurochem. 511227–1231.Google Scholar
- Chatonnet, A., and Lockridge, O. (1989). Comparison of butyrylcholinesterase and acetylcholinesterase.Biochem. J. 260 625–634.Google Scholar
- Doctor, B. P., Chapman, T. C., Christner, C. E., Deal, C. C., De La Hoz, M. K., Gentry, R. K., Orget, R. A., Rush, R. S., Smyth, K. K., and Wolfe, A. D. (1990). Complete amino acid sequence of fetal bovine serum acetylcholinesterase and its comparison in various regions with other cholinesterases.FEBS Lett. 266123–127.Google Scholar
- Ellman, G. L., Courtney, K. D., Andres, V., and Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity.Biochem. Pharmacol. 788–95.Google Scholar
- Foecking, M. K., and Hofstetter, H. (1986). Powerful and versatile enhancer-promoter unit for mammalian expression vector.Gene 45101–105.Google Scholar
- Gibney, G. G., MacPhee-Quigley, K., Vedvick, T., Low, M., Taylor, S. S., and Taylor, P. (1988). Divergence in primary structure between the molecular forms of acetylcholinesterase. Biol. Chem.2631140–1145.Google Scholar
- Gnagey, A. L., Forte, M., and Rosenberry, T. L. (1987). Isolation and characterization of acetylcholinesterase from Drosophila.J. Biol. Chem. 26213290–13298.Google Scholar
- Hall, L. M. C., and Spierer, P. (1986). The ace locus of Drosophila melanogaster: Structural gene for acetylcholinesterase with an unusual 5′ leader.ENBO J. 52949–2954.Google Scholar
- Hodgson, A. J., and Chubb, I. W. (1983). Isolation of secretory forms of soluble acetylcholinesterase by using affinity chromatography on Edrophonium Sepharose.J. Neurochem. 41654–662.Google Scholar
- Inestrosa, N., and Perelman, A. (1989). Distribution and anchoring of molecular forms of acetylcholinesterase.TIPS 10325–329.Google Scholar
- Inestrosa, N., Roberts, W. L., Marshall, T. L., and Rosenberry, T. O. (1987). AChE from bovine caudate nucleus is attached to membranes by a novel subunit distinct from those of AChE in other tissues.J. Biol. Chem. 2624441–4444.Google Scholar
- Karnovsky, M. J., and Roots, L. (1964). A direct-coloring thiocholine method for cholinesterases.J. Histochem. Cytochem. 12 219–221.Google Scholar
- Kozak, M. (1984). Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs.Nucleic Acid Res. 12857–872.Google Scholar
- Lazar, M., and Vigny, M. (1980). Modulation of the distribution of acetylcholinesterase molecular forms in a murine neuroblastoma sympathetic ganglion cell hybrid cell line.J. Neurochem. 351067–1079.Google Scholar
- Lockridge, O., Bartels, C. F., Vaughan, T. A., Wong, C. K., Norton, S. E., and Johnson, L. L. (1987). Complete amino acid sequence of human serum cholinesterase.J. Biol. Chem. 262549–557.Google Scholar
- Massoulie, J., and Bon, S. (1982). The molecular forms of cholinesterase and acetylcholinesterase in vertebrates.Annu. Rev. Neurosci. 557–106.Google Scholar
- Maulet, Y., Camp, S., Gibney, Rachinsky, T., Ekstron, T. J., and Taylor, P. (1990). Single gene encodes glycophospholipid-anchored and asymmetric acetylcholinesterase forms: Alternative coding exons contain inverted repeat sequences.Neuron 4289–301.Google Scholar
- Prody, C. A., Zevin-Sonkin, D., Gnatt, A., Golberg, O., and Soreq, H. (1987). Isolation and characterization of full length cDNA clones coding for cholinesterase from fetal human tissues.Proc. Natl. Acad. Sci. USA 843555–3559.Google Scholar
- Ralston, J. C., Rush, R. S., Doctor, B. P., and Wolfe, A. D. (1985). Acethylcholinesterase from fetal bovine serum, purification and characterization of soluble G4 enzymeJ. Biol. Chem. 2604312–4318.Google Scholar
- Raconczay, Z., and Brimijoin, S. (1988). Monoclonal antibodies to human brain acetylcholinesterase: Production and application.Cell. Mol. Neurobiol. 885–93.Google Scholar
- Reiner, R. (1990). Mechanism of substrate inhibition of acetylcholinesterase. In (F. Bacou, Ed.), Am. Chem. Soc. Books, Washington D.C. (in press).Google Scholar
- Rosenberry, T. L., and Scoggin, D. M. (1984). Structure of human erythrocyte acetylcholinesterase. Characterization of intersubunit disulfide bonding and detergent interactions.J. Biol. Chem. 2595643–5652.Google Scholar
- Schumacher, M., Camp, S., Maulet, Y., Newton, M., MacPhee-Quigley, K., Taylor, S. S., Fridman, T., and Taylor, P. (1986). Primary structure of Torpedo californica acetylcholinesterase deduced from its cDNA sequence.Nature 319407–409.Google Scholar
- Seidman, S., and Soreq, H. (1990). Coinjection ofXenopus oocytes with cDNA-produced and native mRNAs: A molecular biological approach to the tissue specific processing of human cholinesterase.Int. Rev. Neurobiol. (in press).Google Scholar
- Sikorav, J. L., Krejci, E., and Massoulie, J. (1987). cDNA sequence of Torpedo marmaorata acetylcholinesterase: Primary structure of the precursor of a catalytic subunit; Existence of multiple 5′-untranslated regions.EMBO J. 61865–1873.Google Scholar
- Sikorav, J. L., Duval, N., Anselmet, A., Bon, S., Krejci, E., Legay, E., Osterlund, M., Reimund, B., and Massoulie, J. (1988). Complex alternative splicing of acetylcholinesterase transcripts in Torpedo electric organ: Primary structure of the precursor of the glycolipid-anchored dimeric form.EMBO J. 72983–2993.Google Scholar
- Silman, I., and Futerman, A. H., (1987). Models of attachment of acetylcholinesterase to the surface of membranes.Eur. J. Bochem. 17011–22.Google Scholar
- Soreq, H., and Prody, C. A. (1989). Sequence similarities between human acetylcholinesterase and related proteins: Putative implications for therapy of anticholinesterase intoxication. InComputer-Assisted Modelling of Receptor Ligand Interactions (A. Golombeck and R. Rein, Eds.), Alan R. Liss, New York, pp. 347–359.Google Scholar
- Soreq, H., Seidman, S., Dreyfus, P. A., Zevin-Sonkin, D., and Zakut, H. (1989). Expression of tissue specific assembly of human butyrylcholinesterase in microinjectedXenopus laevis oocytes.J. Biol. Chem. 26410608–10613.Google Scholar
- Soreq, H., Ben Aziz, R., Prody, C., Gnatt, A., Neville, A., Lieman-Hurwitz, J., Lev-Lehman, E., Ginzberg, D., Seidman, S., Lapidot-Lifson, Y., and Zakut, H. (1990). Molecular cloning and construction of the coding region for human acetylcholinesterase reveals a G,C, attenuating structure.Proc. Natl. Acad. Sci. (in press).Google Scholar
- Southern, P. L., and Berg, P. (1982). Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter.Mol. Appl. Genet. 1327–341.Google Scholar
- Younkin, S. G., Rosenstein, C., Collins, P. L., and Rosenberry, T. L. (1982). Cellular localization of the molecular forms of acetylcholinesterase in rat diaphragm.J. Biol. Chem. 25713630–13637.Google Scholar
- Wigler, M., Silverstein, S., Lee, L. S., Pellicer, A., Cheng, Y. C., and Axel, R. (1977). Transfer of purified herpes virus thymidine kinase gene to cultured cells.Cell 11223–232.Google Scholar