Summary
-
1.
We describe two simple procedures for the rapid identification of certain structural features of glycolipid anchors in acetylcholinesterases (AChEs).
-
2.
Treatment with alkaline hydroxylamine (that cleaves ester-linked acyl chains but not ether-linked alkyl chains) converts molecules possessing adiacylglycerol, but not those with analkylacylglycerol, into hydrophilic derivatives. AChEs in human and bovine erythrocytes possess an alkylacylglycerol (Robertset al., J. Biol. Chem. 263:18766–18775, 1988;Biochem. Biophys. Res. Commun. 150:271–277, 1988) and are not converted to hydrophilic dimers by alkaline hydroxylamine. Amphiphilic dimers of AChE fromDrosophila, from mouse erythrocytes, and from the human erythroleukaemia cell line K562 also resist the treatment with hydroxylamine and likely possess a terminal alkylacylglycerol. This indicates that the cellular pool of free glycolipids used as precursors of protein anchors is distinct from the pool of membrane phosphatidylinositols (which contain diacylglycerols).
-
3.
Pretreatment with alkaline hydroxylamine is required to render the amphiphilic AChE from human erythrocytes susceptible to digestion byBacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC) (Toutantet al., Eur. J. Biochem. 180:503–508, 1989). We show here that this is also the case for the AChE from mouse erythrocytes, which therefore likely possesses an additional acyl chain in the anchor that prevents the action of PI-PLC.
-
4.
In two sublines of K562 cells (48 and 243), we observed that AChE either was directly susceptible to PI-PLC (243) or required a prior deacylation by alkaline hydroxylamine (48). This suggests that glycolipid anchors in AChE of K562-48 cells, but not those in AChE of K562-243 cells, contain the additional acylation demonstrated in AChE from human erythrocytes. These observations illustrate the cell specificity (and the lack of species-specificity) of the structure of glycolipid anchors.
Similar content being viewed by others
References
Arpagaus, M., and Toutant, J. P. (1985). Polymorphism of acetylcholinesterase in adultPieris brassicae heads. Evidence for detergent-insensitive and Triton X100-interacting forms.Neurochem. Int. 7793–804.
Barbet, J., and Pierres, M. (1987). Thy-1 solubilization from mouse T cells by phosphatidylinositolspecific phospholipase C: Biochemical and antigenic characterization.Ann. Inst. Pasteur/Immunol. 138531–547.
Bon, S., Toutant, J. P., Méflah, K., and Massoulié, J. (1988). Amphiphilic and non-amphiphilic forms ofTorpedo cholinesterases. II. Electrophoretic variants, phosphatidylinositolphospholipase C sensitive and insensitive forms.J. Neurochem. 51 786–794.
Davitz, M. A., Low, M. G., and Nussenzweig, V. (1986). Release of decay-accelerating factor (DAF) from the cell membrane by phosphatidylinositol-specific phospholipase C (PIPLC). Selective modification of a complement regulatory protein.J. Exp. Med. 1631150–1161.
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.
Espinoza, B., Tarrab-Hazdai, R., Silman, I., and Arnon, R. (1988). Acetylcholinesterase inSchistosoma mansoni is anchored to the membrane via covalently attached phosphatidylinositol.Mol. Biochem. Parasitol. 29171–179.
Ferguson, M. A. J., Homans, S. W., Dwek, R. A., and Rademacher, T. W. (1988). Glycosylphosphatidylinositol moiety that anchorsTrypanosoma brucei variant surface glycoprotein to the membrane.Science 239753–759.
Fournier, D., Bergé, J. B., Cardoso de Almeida, M. L., and Bordier, C. (1988). Acetylcholinesterase fromMusca domestica andDrosophila melanogaster are linked to membranes by a glycophospholipid anchor sensitive to an endogenous phospholipase.J. Neurochem. 501158–1163.
Futerman, A. H., Low, M. G., and Silman, I. (1983). A hydrophobic dimer of acetylcholinesterase fromTorpedo californica electric organ is solubilized by phosphatidylinositol-specific phospholipase C.Neurosci. Lett. 4085–89.
Futerman, A. H., Low, A. G., Michelson, D. M., and Silman, I. (1985). Solubilization of membrane-bound acetylcholinesterase by a phosphatidylinositol-specific phospholipase C.J. Neurochem. 451487–1494.
Futerman, A. H., Raviv, D., Michaelson, D. M., and Silman, I. (1987). Differential susceptibility to phosphatidylinositol-specific phospholipase C of acetylcholinesterase in excitable tissues of embryonic and adultTorpedo ocellata.Mol. Brain Res. 2105–112.
Garcia, L., Verdière-Sahuqué, M., Dreyfus, P., Nicolet, M., and Rieger, F. (1988). A dimeric form of acetylcholinesterase anchored through a glycolipid in mouse skeletal muscle.Neurochem. Int. 13327–332.
Haas, R., Brandt, P. T., Knight, J., and Rosenberry, T. L. (1986). Identification of amine components in a glycolipid membrane-binding domain at the C-terminus of human erythrocyte acetylcholinesterase.Biochemistry 253098–3105.
Haas, R., Marshall, T. L., and Rosenberry, T. L. (1988).Drosophila acetylcholinesterase: Demonstration of a glycoinositol phospholipid anchor and an endogenous proteolytic cleavage.Biochemistry 276453–6457.
He, H. T., Barbet, J., Chaix, J. C., and Goridis, C. (1986). Phosphatidylinositol is involved in the membrane attachment of NCAM-120, the smallest component of the neural cell adhesion molecule.EMBO J. 52489–2494.
Helenius, A., and Simons, K. (1977). Charge shift electrophoresis: Simple method for distinguishing between amphiphilic and hydrophilic proteins in detergent solution.Proc. Natl. Acad. Sci. USA 74529–532.
Low, M. G. (1989). Glycosyl-phosphatidylinositol: A versatile anchor for cell surface proteins.FASEB J. 31600–1608.
Low, M. G., and Finean, J. B. (1977). Non-lytic release of acetylcholinesterase from erythrocytes by a phosphatidylinositol-specific phospholipase C.FEBS Lett. 82143–146.
Low, M. G., Stiernberg, J., Waneck, G. L., Flaveil, R. A., and Kincade, P. W. (1988). Cell-specific heterogeneity in sensitivity of phosphatidylinositol-anchored membrane antigens to release by phospholipase C.J. Immunol. Meth. 113101–111.
Massoulié, J., and Toutant, J.-P. (1988). Vertebrate cholinesterases: Structure and type of interaction.Handbook Exp. Pharmacol. 86167–224.
Medof, M. E., Walter, E. I., Roberts, W. L., Haas, R., and Rosenberry, T. L. (1986). Decay accelerating factor of complement is anchored to cells by a C-terminal glycolipid.Biochemistry 256740–6747.
Roberts, W. L., Kim, B. H., and Rosenberry, T. L. (1987). Differences in the glycolipid membrane anchors of bovine and human erythrocyte acetylcholinesterases.Proc. Natl. Acad. Sci. USA 847817–7821.
Roberts, W. L., Myher, J. J., Kuksis, A., Low, M. G., and Rosenberry, T. L. (1988a). Lipid analysis of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase. Palmitoylation of inositol results in resistance to phosphatidylinositol-specific phospholipase C.J. Biol. Chem. 26318766–18775.
Roberts, W. L., Santikrn, S., Reinhold, V. R., and Rosenberry, T. L. (1988b). Structural characterization of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase by fast atom bombardment mass spectrometryJ. Biol. Chem. 26318776–18784.
Roberts, W. L., Myher, J. J., Kuksis, A., and Rosenberry, T. L. (1988c). Alkylacylglycerol molecular species in the glycoinositol phospholipid membrane anchor of bovine erythrocyte acetylcholinesterase.Biochem. Biophys. Res. Commun. 150271–277.
Rosenberry, T. L., and Scoggin, D. M. (1984). Structure of human erythrocyte acetylcholinesterase. Characterization of intersubunit disulfide bonding and detergent interaction.J. Biol. Chem. 2595643–5652.
Rosenberry, T. L., Toutant, J. P., Haas, R., and Roberts, W. L. (1989). Identification of glycoinositol phospholipid anchors of membrane proteins.Meth. Cell Biol. 32231–255.
Sadoul, K., Meyer, A., Low, M. G., and Schachner, M. (1986). Release of the 120 kDa component of the mouse neural cell adhesion molecule N-CAM from cell surfaces by phosphatidylinositolspecific phospholipase C.Neurosci. Lett. 72341–346.
Selvaraj, P., Rosse, W. F., Silber, R., and Springer, T. A. (1988). The major Fc receptor in blood has a phosphatidylinositol anchor and is deficient in paroxysmal nocturnal haemoglobinuria.Nature 333565–567.
Silman, I., and Futerman, A. H. (1987). Modes of attachment of acetylcholinesterase to the surface membrane.Eur. J. Biochem. 17011–22.
Simmons, D., and Seed, B. (1988). The Fc receptor of natural killer cells is a phospholipid-linked membrane protein.Nature 333568–570.
Stieger, A., Cardoso de Almeida, M. L., Blatter, M. C., Brodbeck, U., and Bordier, C. (1986). The membrane-anchoring systems of vertebrate acetylcholinesterase and variant surface glycoproteins of African trypanosomes share a common antigenic determinantFEBS Lett. 199182–186.
Taguchi, R., Asahi, Y., and Ikezawa, H. (1980). Purification and properties of phosphatidylinositolspecific phospholipase C ofBacillus thuringiensis.Biochim. Biophys. Acta 61948–57.
Taguchi, R., Suzuki, K., Nakabayashi, T., and Ikezawa, H. (1984). Acetylcholinesterase release from mammalian erythrocytes by phosphatidylinositol-specific phospholipase C ofBacillus thuringiensis and characterization of the released enzyme.J. Biochem. 96437–446.
Toutant, J. P. (1989). Insect acetylcholinesterase: Catalytic properties, tissue distribution and molecular forms.Prog. Neurobiol. 32423–446.
Toutant, J. P., Arpagaus, M., and Fournier, D. (1988). Native molecular forms of head acetylcholinesterase from adultDrosophila melanogaster. Quaternary structure and hydrophobic character.J. Neurochem. 50209–218.
Toutant, J. P., Roberts, W. L., Murray, N. R., and Rosenberry, T. L. (1989). Conversion of human erythrocyte acetylcholinesterase from an amphiphilic to a hydrophilic form by phosphatidylinositol-specific phospholipase C and serum phospholipase D.Eur. J. Biochem. 180503–508.
Toutant, J. P., Richards, M. K., Krall, J. A., and Rosenberry, T. L. (1990). Molecular forms of acetylcholinesterase in two sublines of human K562 cells. Sensitivity of resistance to phosphatidylinositol-specific phospholipase C and biosynthesis.Eur. J. Biochem. 18731–38.
Villeval, J. L., Pelicci, P. G., Tabilio, A., Titeux, M., Henri, A., Houesche, F., Thomopoulos, P., Vainchenker, W., Garbaz, M., Rochant, H., Breton-Gorius, J., Edwards, P. A. W., and Testa, U. (1983). Erythroid differentiation of K562 cells. Effect of hemin, butyrate and TPA induction.Exp. Cell Res. 146428–435.
Walter, E. I., Toutant, J. P., Rosenberry, T. L., Tykocinski, M. L., and Medof, M. E. (1988). Regulation of the susceptibility of decay accelerating factor's anchor to phosphatidylinositolspecific phospholipase C enzymatic cleavage. Abstracts Parasitology Meeting, NIH, Bethesda, Md.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Toutant, JP., Krall, J.A., Richards, M.K. et al. Rapid analysis of glycolipid anchors in amphiphilic dimers of acetylcholinesterases. Cell Mol Neurobiol 11, 219–230 (1991). https://doi.org/10.1007/BF00712811
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00712811