Production and characterization of antibodies against the cross-reacting determinant of glycosyl-phosphatidylinositol-anchored acetylcholinesterase.
Dimeric acetylcholinesterase is anchored in the cell membrane by a glycosyl-phosphatidylinositol attached to the C-terminus of the protein. The complex glycan contains an antigenic epitope, the cross-reacting determinant (CRD), which is only revealed after removal of the diradylglycerol by phosphatidylinositol-specific phospholipase C (PI-PLC) but is cryptic in the amphiphilic form. Polyclonal antibodies were raised against the CRD of vertebrate acetylcholinesterase. The purified anti-CRD antibodies recognized only the PI-PLC treated hydrophilic forms of acetylcholinesterase from bovine erythrocytes and Torpedo, and of variant surface glycoprotein from trypanosomes but not the corresponding amphiphilic proteins. Competition experiments showed that inositol-1,2-cyclic phosphate and glucosamine inhibited the binding of the antibodies to the CRD. Furthermore, binding of the anti-CRD antibodies to acetylcholinesterase containing N-methylated glucosamine was markedly reduced. The amphiphilic N-methylated enzyme is less sensitive to digestion with PI-PLC than the non-methylated form. From our results we conclude that inositol-1,2-cyclic phosphate and glucosamine, especially the free amine group of this residue, contribute significantly to the epitope recognized by the anti-CRD antibodies.
Jäger
K
K
Institut für Biochemie und Molekularbiologie, Universität Bern, Switzerland.
Meyer
P
P
Stieger
S
S
Brodbeck
U
U
D000110:Acetylcholinesterase / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D000906:Antibodies; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D003429:Cross Reactions; D000939:Epitopes / Q000032:analysis; D004910:Erythrocyte Membrane / Q000201:enzymology; D005557:Formaldehyde / Q000378:metabolism; D006017:Glycolipids / Q000276:immunology / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols; D007700:Kinetics; D046911:Macromolecular Substances; D008745:Methylation; D010716:Phosphatidylinositols / Q000276:immunology / Q000378:metabolism*; D014101:Torpedo; D010738:Type C Phospholipases / Q000378:metabolism
D000906:Antibodies; D000939:Epitopes; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D046911:Macromolecular Substances; D010716:Phosphatidylinositols; D005557:Formaldehyde; D000110:Acetylcholinesterase; D010738:Type C Phospholipases