A Role for Collagen IV Cross-links in Conferring Immune Privilege to the Goodpasture Autoantigen: STRUCTURAL BASIS FOR THE CRYPTICITY OF B CELL EPITOPES

Roberto M Vanacore,Amy-Joan L Ham,Jean-Philippe Cartailler,Munirathinam Sundaramoorthy,Parvin Todd,Vadim Pedchenko,Yoshikazu Sado,Dorin-Bogdan Borza,Billy G Hudson

doi:10.1074/jbc.m803451200

Abstract

The detailed 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 alpha3alpha4alpha5 noncollagenous-1 (NC1) hexamer, a globular domain that connects two triple-helical molecules of the alpha3alpha4alpha5 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 subunits in which the epitopes are cryptic. The D-isoform was characterized 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-hydroxylysyl-methionine and S-lysyl-methionine cross-links, which stabilize the alpha3alpha5-heterodimers and alpha4alpha4-homodimers, respectively. Construction and analysis of a three-dimensional model of the D-isoform of the alpha3alpha4alpha5 NC1 hexamer revealed that crypticity is a consequence of the following: (a) sequestration of key residues between neighboring subunits that are stabilized by domain-swapping interactions, and (b) by cross-linking of subunits at the trimer-trimer interface, which stabilizes the structural integrity of the NC1 hexamer and protects against binding of autoantibodies. The sequestrated epitopes and cross-linked subunits represent a novel structural mechanism for conferring immune privilege at the level of quaternary structure. Perturbation of the quaternary structure may be a key factor in the etiology of Goodpasture disease.

Highlights

Autoimmune disease results from a failure of the adaptive immune system to differentiate between self- and nonself-antigens
The crypticity of Goodpasture disease (GP) epitopes is a feature of the quaternary structure of the ␣3␣4␣5 NC1 hexamer, which occurs in M- and D-isoforms
We proposed a quaternary structure of the ␣3␣4␣5 NC1 hexamer based on a composition of (␣3)2(␣4)2(␣5)2, which allows for a total of 18 possible combinations of different NC1 subunit arrangements [32]

Summary

Introduction

Autoimmune disease results from a failure of the adaptive immune system to differentiate between self- and nonself-antigens. Immunization with ␣3 NC1 monomer and dimer subunits, having exposed B cell epitopes, induces antibody-mediated glomerulonephritis in rats, rabbits, and mice (10 –13). Their cryptic equivalents, ␣3-containing NC1 hexamers, are nonpathogenic (10 –12). Immunization with intact thyrotropin receptor, in which the epitope is cryptic, does not induce an immunogenic response Another example is anti-phospholipid disease where the autoantigen is plasma ␤2-glycoprotein I in which the cryptic epitope is sequestered by a carbohydrate moiety [16, 17]. Interactions with cell membrane phospholipids induce a conformational change in ␤2-glycoprotein I structure that exposes the cryptic epitope by relocating the carbohydrate moiety away from the autoantigen, allowing binding of pathogenic autoantibodies

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Conclusion