Molecular architecture of the Goodpasture autoantigen in anti-GBM nephritis

Abstract

Background: In Goodpasture's disease, circulating autoantibodies bind to the noncollagenous-1 (NC1) domain of type IV collagen in the glomerular basement membrane (GBM). The specificity and molecular architecture of epitopes of tissue-bound autoantibodies are unknown. Alport's post-transplantation nephritis, which is mediated by alloantibodies against the GBM, occurs after kidney transplantation in some patients with Alport's syndrome. We compared the conformations of the antibody epitopes in Goodpasture's disease and Alport's post-transplantation nephritis with the intention of finding clues to the pathogenesis of anti-GBM glomerulonephritis.

Methods: We used an enzyme-linked immunosorbent assay to determine the specificity of circulating autoantibodies and kidney-bound antibodies to NC1 domains. Circulating antibodies were analyzed in 57 patients with Goodpasture's disease, and kidney-bound antibodies were analyzed in 14 patients with Goodpasture's disease and 2 patients with Alport's post-transplantation nephritis. The molecular architecture of key epitope regions was deduced with the use of chimeric molecules and a three-dimensional model of the alpha345NC1 hexamer.

Results: In patients with Goodpasture's disease, both autoantibodies to the alpha3NC1 monomer and antibodies to the alpha5NC1 monomer (and fewer to the alpha4NC1 monomer) were bound in the kidneys and lungs, indicating roles for the alpha3NC1 and alpha5NC1 monomers as autoantigens. High antibody titers at diagnosis of anti-GBM disease were associated with ultimate loss of renal function. The antibodies bound to distinct epitopes encompassing region E(A) in the alpha5NC1 monomer and regions E(A) and E(B) in the alpha3NC1 monomer, but they did not bind to the native cross-linked alpha345NC1 hexamer. In contrast, in patients with Alport's post-transplantation nephritis, alloantibodies bound to the E(A) region of the alpha5NC1 subunit in the intact hexamer, and binding decreased on dissociation.

Conclusions: The development of Goodpasture's disease may be considered an autoimmune "conformeropathy" that involves perturbation of the quaternary structure of the alpha345NC1 hexamer, inducing a pathogenic conformational change in the alpha3NC1 and alpha5NC1 subunits, which in turn elicits an autoimmune response. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases.)

Figure 1. Classic Kidney Lesions in Goodpasture’s Disease, and the Immunoreactivity of Circulating and Kidney-Bound Goodpasture Autoantibodies to Six Noncollagenous-1 Domain Monomers of Human Collagen IV

The specimen at left in Panel A (Jones’s silver stain) shows cellular crescents (arrows) and necrosis of glomerular tufts (arrowheads), features of glomerulonephritis mediated by anti–glomerular-basement-membrane (GBM) antibodies; the specimen at right shows a glomerulus with crescent and linear staining of the GBM with fluorescein-labeled antihuman IgG antibody. Panels B, C, and D show the reactivity of serum from a total of 57 patients with Goodpasture’s disease, grouped according to noncollagenous-1 (NC1) specificity. In Panel B, serum samples from 12 patients react only with α3NC1. In Panel C, samples from 12 different patients react with α3NC1 and α5NC1. In Panel D, samples from 33 different patients react with α1NC1, α3NC1, α4NC1, and α5NC1. These findings differed significantly from the findings in serum samples from 18 healthy volunteers, which showed non-reactivity (P<0.05). Panel E shows the binding of autoantibodies eluted from the kidneys of 14 patients with Goodpasture’s disease. Significant binding was detected only to the α3NC1, α5NC1, and α4NC1 domains, with less binding to the last than to the first two (P<0.05). Normal kidney eluates from 3 patients without Goodpasture’s disease were nonreactive with all NC1 domains. In Panels B through E, the circles indicate values in individual patients, the solid horizontal lines indicate medians, and the dotted horizontal lines indicate means plus 3 SD for normal samples.

Figure 2. Characterization and Epitope Mapping of Circulating Goodpasture Autoantibodies Specific to the α3 and α5 Noncollagenous-1 Domains

Autoantibodies were preincubated with various concentrations of the monomer α3 or α5 noncollagenous-1 (NC1) domain, and binding to immobilized antigens α3NC1 and α5NC1 was measured with the use of an enzyme-linked immunosorbent assay (ELISA). Panels A and B show means (±SE) for relative binding, expressed as a percentage of binding in the absence of NC1 monomers in solution, for α3NC1 and α5NC1 IgG antibodies, respectively, from seven patients with Goodpasture’s disease. Binding of the α3NC1 IgG antibodies to immobilized α3NC1 was strongly inhibited in the presence of soluble α3NC1 (solid circles) but not α5NC1 (open circles) (Panel A). The α5NC1 IgG antibodies had a lower affinity for α5NC1 (Panel B). Panel C shows the extent of binding of α3NC1 and α5NC1 IgG antibodies to NC1 hexamers from native glomerular basement membrane (N-GBM) and dissociated GBM (D-GBM). IgG antibodies from individual serum samples from patients with Goodpasture’s disease are represented by circles and medians by horizontal lines. Panel D shows the alignment of the α1NC1 and α5NC1 amino acid sequences corresponding to the E_A and E_B regions of the α3NC1 domain. Residues that differ from those in α1NC1 (bold) and residues that were mutated in α5 chimeras (bold red) are shown. Panel E shows means (±SE) for the inhibition of the binding of circulating α5NC1-IgG antibodies from the seven patients with Goodpasture’s disease to the α5NC1 domain. E_A-α5 chimeras are represented by solid triangles, and E_B-α5 chimeras by open triangles. The monomers α5NC1 (open circles) and α1NC1 (solid circles) were included as positive and negative controls, respectively. In Panels A, B, and E, I bars denote standard errors for seven α5NC1 antibodies.

Figure 3. Comparison of Kidney- and Lung-Bound Autoantibodies from Patients with Goodpasture’s Disease and Alloantibodies from Patients with Alport’s Post-Transplantation Nephritis

Panels A and B show the extent to which kidney-bound Goodpasture autoantibodies bind to the E_A and E_B chimeras of the α3 noncollagenous-1 (NC1) and α5NC1 domains. Individual patients with Goodpasture’s disease are represented by circles, background binding to α1NC1 by dotted lines, and median values for groups that are different from the background by horizontal lines (P<0.05). Panel C shows the specificity of kidney-bound alloantibodies for the α3/α5NC1 monomer and epitope in samples from two patients with Alport’s post-transplantation nephritis (APTN). Panel D shows the binding of circulating, kidney-bound, and lung-bound autoantibodies to native glomerular basement membrane (N-GBM) and dissociated GBM (D-GBM) NC1 hexamers in samples from one patient with Goodpasture’s disease and two patients with APTN. Normal human IgG (hIgG) does not bind to NC1 hexamers. Panel E shows the binding of circulating antibodies from 27 patients with Goodpasture’s disease and kidney-bound autoantibodies from 14 patients with Goodpasture’s disease to N-GBM and D-GBM NC1 hexamers. Individual patients are represented by circles, and medians for each group by horizontal lines. Panel F shows the positive correlation between the immunoreactivity of the α3NC1 and α5NC1 monomers as revealed by simultaneous enzyme-linked immunosorbent assay (ELISA) for all 57 patients with Goodpasture’s disease (Spearman’s correlation coefficient, 0.852; P<0.001). Data points representing individual patients are fitted to the exponential curve. Panel G shows the levels of α3NC1 (triangles) and α5NC1 (circles) autoantibodies in serum from 17 patients with Goodpasture’s disease who had functioning native kidneys and 21 patients who were dependent on dialysis at 6-month follow-up. P values are based on the Mann–Whitney U test.

Figure 4. Topology of the E_A and E_B Regions in the α345 Noncollagenous-1 Hexamer, Structural Determinants for the Binding of Alport Alloantibodies and Goodpasture Autoantibodies In Vitro, and Accessible Surface Area of the E_A-α3 and E_A-α5 Regions of the Noncollagenous-1 Hexamer

The α345 noncollagenous-1 (NC1) hexamer is composed of two trimeric caps, each consisting of α3NC1 (red), α4NC1 (blue), and α5NC1 (green) subunits (Panel A). Two of the six sulfilimine bonds (S = N) that stabilize the trimer–trimer interface are shown (light yellow). The location and structure of the four homologous regions are also shown: E_A (yellow) and E_B (orange) in the α3NC1 subunit, and E_A (pink) and E_B (purple) in the α5NC1 subunit. Three regions, E_A and E_B in α3NC1 and E_A in α5NC1, become critical parts of the neoepitopes for Goodpasture autoantibodies. The topology of the E_A regions in α3NC1 and α5NC1 is similar, as indicated in the ribbon diagrams (Panel A, bottom), with the characteristic folding pattern of a β-sheet stabilized with a disulfide bond. Ala¹⁹, Gln²⁴, and Gln²⁸ (pink) within the E_A region of α5NC1, exposed in the α345NC1 hexamer, are candidates for the binding of Alport alloantibodies (Panel B, bottom right). In contrast, Leu²⁷ and Val²⁹ (gray) are sequestered by their lateral interaction with the α4NC1 domain, and when exposed as a result of hexamer dissociation, they become critical to the binding of Goodpasture autoantibodies. Dissociation of the sulfilimine-cross-linked hexamer into α35 dimer subunits is concomitant with a conformational change that results in the formation of the neoepitopes encompassing the E_A regions of the α5NC1 and α3NC1 monomers and the binding of their respective autoantibodies (Panel B, bottom left). The accessible surface area of the E_A-α3 region (Panel C, top) and the E_A-α5 region (Panel C, bottom) was calculated for a probe, which mimics the antibody molecule (radius, 9 Å); the area of individual residues in the α345NC1 hexamer (black bars) and the α3NC1/α5NC1 model monomers (gray bars) is shown. An increase in the surface area of the monomers indicates that residues are buried in the hexamer (Val²⁷ and Leu²⁹ in E_A-α3 and Leu²⁷ and Val²⁹ in E_A-α5). In contrast, residues with similar areas within the hexamer and monomers are exposed in the hexamer (Ala¹⁹, Gln²⁴, and Gln²⁸ in E_A-α5).

Figure 5. Conformational Diversity and Differential Reactivity of α345 Noncollagenous-1 Hexamers of the Glomerular Basement Membrane

The diagram shows a portion of the collagen IV network with the α345 noncollagenous-1 (NC1) hexamer tethered to the triple-helical domain. The different possible NC1 conformers shown are the cross-linked form stabilized by sulfilimine bonds (conformer 1 [C-1]), the non-cross-linked form (C-2), and the form in which the NC1 hexamers are dissociated into trimers (C-3). In Goodpasture’s disease the latter may undergo a conformational change resulting in the formation of neoepitopes shown as white squares on the α3NC1 (red) and α5NC1 (green) subunits of C-4, eliciting antibody formation and subsequent binding to conformers C-3 and C-4. Conformers C-1 and C-2 have the potential to be transformed into the pathogenic conformer C-4.