Epitope mapping in cell surface proteins by site-directed masking: defining the structural elements of NTPDase3 inhibition by a monoclonal antibody.
We adapted the method of epitope mapping by site-directed masking, which was described for purified soluble antigens [Paus,D. and Winter,G. (2006) Proc. Natl Acad. Sci. USA, 103, 9172-9177.], to map the binding site of an inhibitory monoclonal antibody on the cell surface protein ecto-nucleotidase NTPDase3. Using homology modeling, we built a 3D structure of NTPDase3 and designed 21 single cysteine mutations distributed over the surface of the enzyme. The mutant proteins were expressed in cells, biotinylated with a cysteine-specific reagent, and then extracted with detergent and immobilized on streptavidin-coated plates. Tethering NTPDase3 via cysteine residues located in a surface patch near the active site cleft masked the epitope and blocked antibody binding, as evaluated by enzyme inhibition assay and by ELISA. We then constructed 18 single alanine substitution mutations within the defined patch and found that W403A, D414A, E415A and R419A decreased the inhibitory effect of the antibody, whereas the double mutation W403A/R419A abolished both antibody binding and enzyme inhibition, suggesting the critical role of these residues for interaction with the antibody. Lack of competition between the antibody and a non-hydrolyzable substrate analog AMPPCP, as well as location of the epitope adjacent to the active site, suggest a noncompetitive mechanism of inhibition by steric hindrance. The described technique should be useful for systematic epitope mapping in cell membrane proteins for which either a 3D structure is available, or a sufficiently accurate 3D model can be obtained by homology modeling.
Ivanenkov
Vasily V
VV
Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, PO Box 670575, Cincinnati, OH 45267-0575, USA.
Crawford
Patrick A
PA
Toyama
Aimi
A
Sévigny
Jean
J
Kirley
Terence L
TL
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D001666:Binding Sites, Antibody; D001710:Biotin / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D051381:Rats
D000911:Antibodies, Monoclonal; D004800:Enzymes, Immobilized; D000939:Epitopes; D001710:Biotin; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D003545:Cysteine