Monoclonal antibodies against brain acetylcholinesterases which recognize the subunits bearing the hydrophobic anchor.
Monoclonal antibodies were raised against amphiphilic detergent-soluble (DS) acetylcholinesterase (AChE) from human brain caudate nucleus. Three mAb, 132-4 (IgG1), 132-5 (IgG1) and 132-6 (IgG3), specific for brain DS-AChE were selected and subcloned. These mAb reacted with native as well as heat-denatured and SDS-denatured DS-AChE, indicating that the epitopes to which mAb bound are continuous determinants. The mAb cross-reacted with DS-AChE from bovine and mouse brain and with brain DS-AChE from river trout (Salmo trutta forma fario) and lake trout (Salmo trutta forma lacustris). No cross-reaction was detected with the following antigens: salt-soluble (SS) AChE from bovine brain, glycophospholipid-anchored AChE from human and bovine erythrocytes, DS-butyrylcholinesterase and SS-butyrylcholinesterase (BtChE) from the brains of human and bovine, DS-BtChE from chicken and BtChE from human serum. Deglycosylation of brain DS-AChE with N-glycosidase F did not abolish the binding of mAb to DS-AChE. After reduction of brain DS-AChE by dithiothreitol, the mAb no longer reacted with the antigen, indicating that a disulfide bridge is important for the epitope. Monomerization of brain DS-AChE by trypsin and limited proteinase K treatment also abolished the binding of mAb to DS-AChE. Sucrose-density-gradient centrifugation showed that mAb reacted only with native tetrameric forms, but not with dimeric and monomeric forms. Western blot, after SDS/PAGE under non-reducing conditions, showed that mAb reacted with those subunits carrying the hydrophobic anchor (i.e. tetramers, trimers and heavy dimers) but not with those devoid of it (light dimers or monomers). Since mAb 132-4, 132-5 and 132-6 recognized DS-AChE from fish up to mammalian brain in the evolutionary tree, it is concluded that the epitope to which these mAb bind, is conserved in nature.
Liao
J
J
Institute of Biochemistry and Molecular Biology, University of Bern, Switzerland.
Mortensen
V
V
Nørgaard-Pedersen
B
B
Koch
C
C
Brodbeck
U
U
D000110:Acetylcholinesterase / Q000737:chemistry / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002421:Caudate Nucleus / Q000201:enzymology*; D003429:Cross Reactions; D003902:Detergents; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007124:Immunoenzyme Techniques; D051379:Mice; D010084:Oxidation-Reduction; D013045:Species Specificity; D014337:Trout
D000911:Antibodies, Monoclonal; D003902:Detergents; D000939:Epitopes; D000110:Acetylcholinesterase; D004229:Dithiothreitol