Antiidiotypic antibody recognizes an amiloride binding domain within the alpha subunit of the epithelial Na+ channel.
We previously raised an antibody (RA6.3) by an antiidiotypic approach which was designed to be directed against an amiloride binding domain on the epithelial Na+ channel (ENaC). This antibody mimicked amiloride in that it inhibited transepithelial Na+ transport across A6 cell monolayers. RA6.3 recognized a 72-kDa polypeptide in A6 epithelia treated with tunicamycin, consistent with the size of nonglycosylated Xenopus laevis alphaENaC. RA6.3 specifically recognized an amiloride binding domain within the alpha-subunit of mouse and bovine ENaC. The deduced amino acid sequence of RA6.3 was used to generate a three-dimensional model structure of the antibody. The combining site of RA6.3 was epitope mapped using a novel computer-based strategy. Organic residues that potentially interact with the RA6.3 combining site were identified by data base screening using the program LUDI. Selected residues docked to the antibody in a manner corresponding to the ordered linear array of amino acid residues within an amiloride binding domain on the alpha-subunit of ENaC. A synthetic peptide spanning this domain inhibited the binding of RA6.3 to alphaENaC. This analysis provided a novel approach to develop models of antibody-antigen interaction as well as a molecular perspective of RA6.3 binding to an amiloride binding domain within alphaENaC.
Kieber-Emmons
T
T
Department of Pathology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Lin
C
C
Foster
M H
MH
Kleyman
T R
TR
D000584:Amiloride / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D003001:Cloning, Molecular; D003198:Computer Simulation; D018076:DNA, Complementary / Q000737:chemistry; D053503:Epithelial Sodium Channels; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D015222:Sodium Channels / Q000276:immunology* / Q000378:metabolism; D014982:Xenopus laevis
D000888:Antibodies, Anti-Idiotypic; D018076:DNA, Complementary; D053503:Epithelial Sodium Channels; D015222:Sodium Channels; D000584:Amiloride