A Role for Collagen IV Cross-links in Conferring Immune Privilege to the Goodpasture Autoantigen

Abstract

Thedetailed structural basis for the cryptic nature (crypticity) of a B cell epitope harbored by an autoantigen is unknown. Because the immune system may be ignorant of the existence of such “cryptic” epitopes, their exposure could be an important feature in autoimmunity. Here we investigated the structural basis for the crypticity of the epitopes of the Goodpasture autoantigen, the ? 3 4 5 noncollagenous-1 (NC1) hexamer, a ? ? globular domain that connects two triple-helical molecules of the ? 3 4 5 collagen IV network. The NC1 hexamer occurs in ? ? two isoforms as follows: the M-isoform composed of monomer subunits in which the epitopes are accessible to autoantibodies, and the D-isoform composed of both monomer and dimer sub- units in which the epitopes are cryptic. The D-isoform was char- acterized with respect to quaternary structure, as revealed by mass spectrometry of dimer subunits, homology modeling, and molecular dynamics simulation. The results revealed that the D-isoform contains two kinds of cross-links as follows: S-hy- droxylysyl-methionine and S-lysyl-methionine cross-links, which stabilize the ? 3 5-heterodimers and ? ? 4 4-homodimers, ? respectively. Construction and analysis of a three-dimensional model of the D-isoform of the ? 3 4 5 NC1 hexamer revealed ? ? that crypticity is a consequence of the following: (a) sequestra- tion of key residues between neighboring subunits that are sta- bilized by domain-swapping interactions, and (b) by cross-link- ing of subunits at the trimer-trimer interface, which stabilizes the structural integrity of theNC1hexamer and protects against binding of autoantibodies.Thesequestrated epitopes and cross- linked subunits represent a novel structuralmechanismfor con- ferring immune privilege at the level of quaternary structure. Perturbation of the quaternary structure may be a key factor in the etiology of Goodpasture disease.