Hydrophobic amino acid residues are critical for the immunodominant epitope of the Goodpasture autoantigen. A molecular basis for the cryptic nature of the epitope.
Goodpasture (GP) autoimmune disease is caused by autoantibodies to type IV collagen that bind to the glomerular basement membrane, causing rapidly progressing glomerulonephritis. The immunodominant GP(A) autoepitope is encompassed by residues 17-31 (the E(A) region) within the noncollagenous (NC1) domain of the alpha 3(IV) chain. The GP epitope is cryptic in the NC1 hexamer complex that occurs in the type IV collagen network found in tissues and inaccessible to autoantibodies unless the hexamer dissociates. In contrast, the epitope for the Mab3 monoclonal antibody is also located within the E(A) region, but is fully accessible in the hexamer complex. In this study, the identity of residues that compose the GP(A) autoepitope was determined, and the molecular basis of its cryptic nature was explored. This was achieved using site-directed mutagenesis to exchange the alpha3(IV) residues in the E(A) region with the corresponding residues of the homologous but non-immunoreactive alpha1(IV) NC1 domain and then comparing the reactivity of the mutated chimeras with GP(A) and Mab3 antibodies. It was shown that three hydrophobic residues (Ala(18), Ile(19), and Val(27)) and Pro(28) are critical for the GP(A) autoepitope, whereas two hydrophilic residues (Ser(21) and Ser(31)) along with Pro(28) are critical for the Mab3 epitope. These results suggest that the cryptic nature of the GP(A) autoepitope is the result of quaternary interactions of the alpha 3, alpha 4, and alpha 5 NC1 domains of the hexamer complex that bury the one or more hydrophobic residues. These findings provide critical information for understanding the etiology and pathogenesis of the disease as well as for designing drugs that would mimic the epitope and thus block the binding of GP autoantibodies to autoantigen.
David
M
M
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
Borza
D B
DB
Leinonen
A
A
Belmont
J M
JM
Hudson
B G
BG
D000595:Amino Acid Sequence; D019867:Anti-Glomerular Basement Membrane Disease / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology; D001324:Autoantigens / Q000737:chemistry* / Q000276:immunology; D003094:Collagen / Q000737:chemistry* / Q000276:immunology; D024141:Collagen Type IV; D006801:Humans; D016056:Immunodominant Epitopes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D013329:Structure-Activity Relationship
D001323:Autoantibodies; D001324:Autoantigens; D024141:Collagen Type IV; D016056:Immunodominant Epitopes; C057112:type IV collagen alpha3 chain; D003094:Collagen