Alport alloantibodies but not Goodpasture autoantibodies induce murine glomerulonephritis: protection by quinary crosslinks locking cryptic ?3(IV) collagen autoepitopes in vivo.
ABSTRACT: The noncollagenous (NC1) domains of alpha3alpha4alpha5(IV) collagen in the glomerular basement membrane (GBM) are targets of Goodpasture autoantibodies or Alport posttransplant nephritis alloantibodies mediating rapidly progressive glomerulonephritis. Because the autoepitopes but not the alloepitopes become cryptic upon assembly of alpha3alpha4alpha5NC1 hexamers, we investigated how the accessibility of B cell epitopes in vivo influences the development of glomerulonephritis in mice passively immunized with human anti-GBM Abs. Alport alloantibodies, which bound to native murine alpha3alpha4alpha5NC1 hexamers in vitro, deposited linearly along the mouse GBM in vivo, eliciting crescentic glomerulonephritis in Fcgr2b(-/-) mice susceptible to Ab-mediated inflammation. Goodpasture autoantibodies, which bound to murine alpha3NC1 monomer and dimer subunits but not to native alpha3alpha4alpha5NC1 hexamers in vitro, neither bound to the mouse GBM in vivo nor induced experimental glomerulonephritis. This was due to quinary NC1 crosslinks, recently identified as sulfilimine bonds, which comprehensively locked the cryptic Goodpasture autoepitopes in the mouse GBM. In contrast, non-crosslinked alpha3NC1 subunits were identified as a native target of Goodpasture autoantibodies in the GBM of squirrel monkeys, a species susceptible to Goodpasture autoantibody-mediated nephritis. Thus, crypticity of B cell autoepitopes in tissues uncouples potentially pathogenic autoantibodies from autoimmune disease. Crosslinking of alpha3alpha4alpha5NC1 hexamers represents a novel mechanism averting autoantibody binding and subsequent tissue injury by posttranslational modifications of an autoantigen.
Project description:Alport post-transplant nephritis (APTN) is an aggressive form of anti-glomerular basement membrane disease that targets the allograft in transplanted patients with X-linked Alport syndrome. Alloantibodies develop against the NC1 domain of ?5(IV) collagen, which occurs in normal kidneys, including renal allografts, forming distinct ?345(IV) and ?1256(IV) networks. Here, we studied the roles of these networks as antigens inciting alloimmunity and as targets of nephritogenic alloantibodies in APTN. We found that patients with APTN, but not those without nephritis, produce two kinds of alloantibodies against allogeneic collagen IV. Some alloantibodies targeted alloepitopes within ?5NC1 monomers, shared by ?345NC1 and ?1256NC1 hexamers. Other alloantibodies specifically targeted alloepitopes that depended on the quaternary structure of ?345NC1 hexamers. In Col4a5-null mice, immunization with native forms of allogeneic collagen IV exclusively elicited antibodies to quaternary ?345NC1 alloepitopes, whereas alloimmunogens lacking native quaternary structure elicited antibodies to shared ?5NC1 alloepitopes. These results imply that quaternary epitopes within ?345NC1 hexamers may initiate alloimmune responses after transplant in X-linked Alport patients. Thus, ?345NC1 hexamers are the culprit alloantigen and primary target of all alloantibodies mediating APTN, whereas ?1256NC1 hexamers become secondary targets of anti-?5NC1 alloantibodies. Reliable detection of alloantibodies by immunoassays using ?345NC1 hexamers may improve outcomes by facilitating early, accurate diagnosis.
Project description: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.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.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.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.)
Project description:Defective assembly of alpha 3 alpha 4 alpha 5(IV) collagen in the glomerular basement membrane causes Alport syndrome, a hereditary glomerulonephritis progressing to end-stage kidney failure. Assembly of collagen IV chains into heterotrimeric molecules and networks is driven by their noncollagenous (NC1) domains, but the sites encoding the specificity of these interactions are not known. To identify the sites directing quaternary assembly of alpha 3 alpha 4 alpha 5(IV) collagen, correctly folded NC1 chimeras were produced, and their interactions with other NC1 monomers were evaluated. All alpha1/alpha 5 chimeras containing alpha 5 NC1 residues 188-227 replicated the ability of alpha 5 NC1 to bind to alpha3NC1 and co-assemble into NC1 hexamers. Conversely, substitution of alpha 5 NC1 residues 188-227 by alpha1NC1 abolished these quaternary interactions. The amino-terminal 58 residues of alpha3NC1 encoded binding to alpha 5 NC1, but this interaction was not sufficient for hexamer co-assembly. Because alpha 5 NC1 residues 188-227 are necessary and sufficient for assembly into alpha 3 alpha 4 alpha 5 NC1 hexamers, whereas the immunodominant alloantigenic sites of alpha 5 NC1 do not encode specific quaternary interactions, the findings provide a basis for the rational design of less immunogenic alpha 5(IV) collagen constructs for the gene therapy of X-linked Alport patients.
Project description:Goodpasture syndrome is an autoimmune vascular disease associated with kidney and lung failure, with pathogenic circulating autoantibodies targeted to a set of discontinuous epitope sequences within the noncollagenous domain-1 (NC1) of the alpha3 chain of type IV collagen (alpha3(IV)NC1), the Goodpasture autoantigen. We demonstrate that basement membrane extracted NC1 domain preparations from Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio do not bind Goodpasture autoantibodies, while Xenopus laevis, chicken, mouse and human alpha3(IV)NC1 domains bind autoantibodies. The alpha3(IV) chain is not present in C elegans and Drosophila melanogaster, but is first detected in the Danio rerio. Interestingly, native Danio rerio alpha3(IV)NC1 does not bind Goodpasture autoantibodies. Next, we cloned, sequenced, and generated recombinant Danio rerio alpha3(IV)NC1 domain. In contrast to recombinant human alpha3(IV)NC1 domain, there was complete absence of autoantibody binding to recombinant Danio rerio alpha3(IV)NC1. Three-dimensional molecular modeling from existing x-ray coordinates of human NC1 domain suggest that evolutionary alteration of electrostatic charge and polarity due to the emergence of critical serine, aspartic acid, and lysine residues, accompanied by the loss of asparagine and glutamine, contributes to the emergence of the 2 major Goodpasture epitopes on the human alpha3(IV)NC1 domain, as it evolved from the Danio rerio over 450 million years.
Project description:Autoantibodies mediate organ destruction in multiple autoimmune diseases, yet their origins in patients remain poorly understood. To probe the genetic origins and structure of disease-associated autoantibodies, we engrafted immunodeficient mice with human CD34+ hematopoietic stem cells and immunized with the non-collagenous-1 (NC1) domain of the alpha3 chain of type IV collagen. This antigen is expressed in lungs and kidneys and is targeted by autoantibodies in anti-glomerular basement membrane (GBM) nephritis and Goodpasture syndrome (GPS), prototypic human organ-specific autoimmune diseases. Using Epstein Barr virus transformation and cell fusion, six human anti-alpha3(IV)NC1 collagen monoclonal autoantibodies (mAb) were recovered, including subsets reactive with human kidney and with epitopes recognized by patients' IgG. Sequence analysis reveals a long to exceptionally long heavy chain complementarity determining region3 (HCDR3), the major site of antigen binding, in all six mAb. Mean HCDR3 length is 25.5 amino acids (range 20-36), generated from inherently long DH and JH genes and extended regions of non-templated N-nucleotides. Long HCDR3 are suited to forming noncontiguous antigen contacts and to binding recessed, immunologically silent epitopes hidden from conventional antibodies, as seen with self-antigen crossreactive broadly neutralizing anti-HIV Ig (bnAb). The anti-alpha3(IV)NC1 collagen mAb also show preferential use of unmutated variable region genes that are enriched among human chronic lymphocytic leukemia antibodies that share features with natural polyreactive Ig. Our findings suggest unexpected relationships between pathogenic anti-collagen Ig, bnAb, and autoreactive Ig associated with malignancy, all of which arise from B cells expressing unconventional structural elements that may require transient escape from tolerance for successful expansion.
Project description:Goodpasture antigen, the non-collagenous domain of ?3 chain of type IV collagen [?3(IV)NC1], is the target antigen of anti-glomerular basement membrane (GBM) antibodies. The pathogenicity of T cell epitopes is not elucidated clearly. In this study, we aim to define the nephritogenic T cell epitopes and its critical amino acid residues. Twenty-four overlapping linear peptides were synthesized covering the whole sequence of human ?3(IV)NC1. Wistar-Kyoto rats were immunized with linear peptides, and experimental autoimmune glomerulonephritis was evaluated. Critical amino acid was identified by the loss of nephritogenic function after each amino acid substitution by alanine. Of the 24 peptides, P14 (?3<sub>127-148</sub> ) could induce 90.5% (19/21) of WKY rats developing anti-GBM glomerulonephritis with proteinuria, elevated serum urea and creatinine, IgG linear deposit on GBM and substantial (in average 82.4 ± 5.6%) crescent formation in glomeruli. Lymphocytes of immunized rats proliferated in response to ?3<sub>127-148</sub> and ?3(IV)NC1 in vitro. Sera of these rats recognized ?3<sub>127-148</sub> and later on together with intact human ?3(IV)NC1. Antibodies towards ?3<sub>127-148</sub> and intact ?3(IV)NC1 could also be detected from the kidney elutes. These antibodies showed no cross-reaction with each other, which implies intramolecular epitope spreading during disease progress. After sequential amino acid substitution, the ?3<sub>127-148</sub> with substitution of tryptophan<sub>136</sub> , isoleucine<sub>137</sub> , leucine<sub>139</sub> or tryptophan<sub>140</sub> lost its nephritogenicity. Human ?3<sub>127-148</sub> is a nephritogenic T cell epitope in WKY rats, with the critical amino acids as W<sub>136</sub> I<sub>137</sub> xL<sub>139</sub> W<sub>140</sub> . These findings might facilitate future investigation on microbial aetiology and potential specific immunotherapy of anti-GBM disease.
Project description:Background: Goodpasture's disease (GP) is mediated by autoantibodies that bind the glomerular and alveolar basement membrane, causing rapidly progressive glomerulonephritis with or without pulmonary hemorrhage. The autoantibodies bind neoepitopes formed upon disruption of the quaternary structure of ?345NC1 hexamer, a critical structural domain of ?345 collagen IV scaffolds. Hexamer disruption leads to a conformational changes that transitions ?3 and ?5NC1 subunits into immunogens, however, the trigger remains unknown. This contrasts with another anti-GBM disease, Alports' post-transplant nephritis (APTN), where the pathogenic alloantibody binds directly to native NC1 hexamer. The current report includes the first study of antigenic specificity and allo-incompatability in anti-GBM disease occurring after allogeneic haematopoietic stem cell transplant (HSCT). Results: The anti-GBM antibodies were found to be directed predominantly against the EA epitope of the ?3 NC1 monomer of collagen IV and developed rapidly in patient serum reaching peak level within 5 weeks. Autoantibody binding to native ?345NC1 hexamer was minimal; however, binding was greatly increased upon dissociation of the native hexamer. There were no polymorphic genetic differences between donor and recipient collagen IV genes which would be predicted to cause a significant NC1 conformational change or to provide a target for antibody binding. Both patient and donor possessed the Goodpasture's susceptibility HLA-allele DRB1 * 1501. Conclusions: The current report includes the first in-depth study of allo-incompatability and antigenic specificity in anti-GBM disease occurring after allogeneic haematopoietic stem cell transplant (HSCT). No polymorphic genetic differences were identified between donor and recipient collagen IV genes which would be predicted to provide a target for antibody binding. Furthermore, autoantibody binding to native ?345NC1 hexamer was minimal, increasing greatly upon dissociation of the native hexamer, resembling wild-type GP diseases and marking this as the first example of a post-HSCT conformeropathy.
Project description:Anti-glomerular basement membrane nephritis and Goodpasture syndrome result from autoantibody (Ab)-mediated destruction of kidney and lung. Ab target the noncollagenous 1 (NC1) domain of alpha3(IV) collagen, but little is known about Ab origins or structure. This ignorance is due in part to the inability to recover monoclonal Ab by transformation of patients' blood cells. The aim of this study was to assess the suitability of two humanized models for this purpose.NOD-scid-gamma immunodeficient mice were engrafted either with human CD34+ hematopoietic stem cells (HSC) (Hu-HSC mice) and immunized with alpha3(IV)NC1 collagen containing the Goodpasture epitopes or with nephritis patients' peripheral blood leukocytes (PBL) (Hu-PBL mice). After in vivo immune cell development and/or expansion, recovered human B cells were Epstein Barr virus (EBV)-transformed, screened for antigen (Ag) binding, electrofused with a mouse-human heterohybridoma, subcloned, and human Ab RNA sequenced by PCR after reverse transcription to cDNA. Flow cytometry was used to assess human B cell markers and differentiation in Hu-PBL mice.Sequence analysis of a human Ab derived from an immunized Hu-HSC mouse and reactive with alpha3(IV)NC1 collagen reveals that it is encoded by unmutated heavy and light chain genes. The heavy chain complementarity determining region 3, a major determinant of Ag binding, contains uncommon motifs, including an N-region somatically-introduced highly hydrophobic tetrapeptide and dual cysteines encoded by a uniquely human IGHD2-2 Ab gene segment that lacks a murine counterpart. Comparison of human and mouse autoantibodies suggests that structurally similar murine Ab may arise by convergent selection. In contrast to the Hu-HSC model, transformed human B cells are rarely recovered from Hu-PBL mice, in which human B cells terminally differentiate and lose expression of EBV receptor CD21, thus precluding their transformation and recovery.Hu-HSC mice reveal that potentially pathogenic B cells bearing unmutated Ig receptors reactive with the NC1 domain on alpha3(IV) collagen can be generated in, and not purged from, the human preimmune repertoire. Uniquely human gene elements are recruited to generate the antigen binding site in at least a subset of these autoantibodies, indicating that humanized models may provide insights inaccessible using conventional mouse models.
Project description:The autoantigen in Goodpasture disease is the noncollagenous domain of alpha3 type IV collagen [alpha3(IV)NC1]. We previously demonstrated that IL-12p40(-/-) mice are protected from experimental autoimmune anti-glomerular basement membrane (anti-GBM) glomerulonephritis, seemingly defining a role for IL-12 in this disease; however, the recent identification of IL-23, a heterodimer composed of IL-12p40 and IL-23p19 subunits, raises the possibility that IL-23, rather than IL-12, may modulate this disease instead. We immunized wild-type, IL-12p35(-/-) (IL-12 deficient, IL-23 intact), IL-12p40(-/-) (deficient in both IL-12 and IL-23), and IL-23p19(-/-) (IL-12 intact, IL-23 deficient) mice with recombinant mouse alpha3(IV)NC1. Wild-type mice developed autoreactivity to alpha3(IV)NC1: Humoral responses, cellular responses, renal histologic abnormalities, leukocyte accumulation, autoantibody deposition, and IL-17A mRNA expression (a cytokine produced by the IL-23-maintained Th17 subset). IL-23 but not IL-12 was detected in the immune system. Regardless of the presence of IL-12, mice deficient in IL-23 were protected, but mice with IL-23 were not. Both IL-23-deficient strains exhibited lower autoantibody titers, reduced cellular reactivity, diminished cytokine production (IFN-gamma [Th1], IL-17A [Th17], TNF, and monocyte chemoattractant protein 1), and less renal disease and glomerular IgG deposition. The deficient responses in the absence of IL-23 were not due to increased regulatory T cells; IL-12p40(-/-) and IL-23p19(-/-) mice did not show increased proportions of CD4(+)CD25(+)FoxP3(+) cells or IL-10 levels early in the immune response. In conclusion, autoreactivity to the Goodpasture antigen is directed primarily by IL-23, absence of which results in hyporeactivity including but extending beyond a deficient Th17 response.
Project description: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.