Structural characterization of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase by fast atom bombardment mass spectrometry.
The glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase (EC 3.1.1.7) is composed of a glycan linked through a glucosamine residue to an inositol phospholipid that is resistant to the action of phosphatidylinositol-specific phospholipase C. Deamination cleavage of the glucosamine with nitrous acid released the inositol phospholipid which was purified by high performance liquid chromatography. Analysis by fast atom bombardment mass spectrometry with negative ion monitoring and by the complementary technique of collision-induced dissociation revealed molecular and daughter ions that indicated a plasmanylinositol with a palmitoyl group on an inositol hydroxyl. The intact membrane anchor was released from reductively methylated human erythrocyte acetylcholinesterase by proteolysis with papain or Pronase, deacylated by base hydrolysis, and purified by high performance liquid chromatography. Positive and negative ion fast atom bombardment mass spectrometry of the major products isolated by high performance liquid chromatography indicated the following structure for the complete glycoinositol phospholipid anchor. (formula; see text) Methylation of free amino groups by reduction with deuterium instead of hydrogen permitted determination of the number of free amino groups in individual fragment ions as further confirmation of structural assignments. The structure of the glycan portion of the human erythrocyte acetylcholinesterase membrane anchor appears to be similar to that described for Trypanosome brucei variant surface glycoprotein MITat 1.4 (variant 117) (Ferguson, M.A.J., Homans, S.W., Dwek, R.A., and Rademacher, T.W. (1988) Science 239, 753-759) except for the absence of a galactose antenna and the presence of a phosphorylethanolamine on the hexose adjacent to glucosamine.
Roberts
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Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
Santikarn
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Reinhold
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Rosenberry
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D000110:Acetylcholinesterase / Q000097:blood*; D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000201:enzymology*; D005944:Glucosamine / Q000378:metabolism; D006017:Glycolipids / Q000302:isolation & purification*; D017261:Glycosylphosphatidylinositols; D006801:Humans; D013058:Mass Spectrometry; D008563:Membrane Lipids / Q000032:analysis*; D009608:Nitrous Acid; D010206:Papain; D010716:Phosphatidylinositols / Q000302:isolation & purification*; D010955:Plasmalogens / Q000302:isolation & purification*; D011402:Pronase
D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D008563:Membrane Lipids; D010716:Phosphatidylinositols; D010955:Plasmalogens; D000110:Acetylcholinesterase; D010206:Papain; D011402:Pronase; D005944:Glucosamine; D009608:Nitrous Acid