A combined ELISA-immunoelectron microscopic approach for topological mapping of membrane protein epitopes: application to the nicotinic acetylcholine receptor.
Identification of epitope localization on either side of the lipid membrane by immunoelectron microscopy constitutes an intrinsic powerful method of structure determination for membrane proteins. We have developed a method allowing measurement and observation, under almost identical experimental conditions, of the binding of monoclonal antibodies (MAb) to membrane-bound acetylcholine receptor from Torpedo marmorata electric tissue. This method, based on ELISA and electron microscopy of negatively stained specimens, was developed with MAb of known epitope specificity. With native membrane fragments, we found that MAb bound to extracellular epitopes in a stoichiometric manner, whereas almost no binding was detected for intracellular epitopes. The treatment based on tissue homogenization in the presence of Zn2+ ions and sucrose resulted in the formation of large, stable openings, rendering accessible about 25% of intracellular epitopes. Electron microscopic observations showed a clear distinction between antibody binding to either intracellular or extracellular epitopes, both with native and Zn(2+)-treated membranes. In addition, the binding of one antibody directed against an extracellular epitope was strikingly dependent on the packing density of acetylcholine receptor molecules, thus enabling us to further distinguish between two levels of accessibility for extracellular epitopes. The method presented here is of general application for studies of epitope mapping in membrane proteins.
Schmutz
M
M
Laboratoire de Génétique Moléculaire des Eucaryotes, CNRS, Unité 184 de Biologie Moléculaire et de Génie Génétique, INSERM, Faculté de Médecine, Strasbourg, France.
Kling
D
D
Tzartos
S
S
Brisson
A
A
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002463:Cell Membrane Permeability; D003902:Detergents; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism / Q000648:ultrastructure; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D008565:Membrane Proteins / Q000378:metabolism*; D016253:Microscopy, Immunoelectron / Q000379:methods*; D011978:Receptors, Nicotinic / Q000378:metabolism*; D014101:Torpedo / Q000378:metabolism*; D015032:Zinc
D000911:Antibodies, Monoclonal; D003902:Detergents; D000939:Epitopes; D008565:Membrane Proteins; D011978:Receptors, Nicotinic; D015032:Zinc; D000109:Acetylcholine