Journal conference 10.09.01 Molecular Architecture of the Goodpasture Autoantigen in Anti-GBM Nephritis Vadim Pedchenko, Ph.D., Olga Bondar, Ph.D., Agnes B. Fogo, M.D., Roberto Vanacore, Ph.D., Paul Voziyan, Ph.D., A. Richard Kitching, M.D., Ph.D., Jorgen Wieslander, M.D., Ph.D., Clifford Kashtan, M.D., Dorin-Bogdan Borza, Ph.D., Eric G. Neilson, M.D., Curtis B. Wilson, M.D., and Billy G. Hudson, Ph.D. N Engl J Med July 22, 2010;363:343-54. by the Tuberculosis Network European Trials Group (TBNET) R2. Shin A-ri / Prof. Ihm Chun gyoo

Korea National Diabetes Program Background Goodpasture’s disease : organ-specific autoimmune disease characterized by rapidly progressive glomerulonephritis, pulmonary hemorrhage, glomerular pathological findings that include linear deposits of antibodies along the GBM. Alport’s post-transplantation nephritis (APTN) : mediated by alloantibodies against the GBM, occurs after KTP in some patients (3-5%) with Alport’s syndrome, result in allograft loss. Target GBM antigen for circulating antibodies identified as noncollagenous-1 (NC1) domain of α3 chain of collagen IV in GBM, assembled into collagen IV a/w α3, α4, and α5 chains, forming α345NC1 hexamer Korea National Diabetes Program Mediated by the deposition of alloantibodies to the α3NC1 & α5NC1 domains in response to the “foreign” α345 collagen network that is absent in the kidneys of patients with Alport’s syndrome but, present in the renal allograft

Korea National Diabetes Program ddddk Background Goodpasture’s disease α345NC1 hexamer is targeted by Ab that arise in Goodpasture’s dis & APTN But, the specificity & molecular architecture of epitopes of tissue-bound autoantibodies are unknown Korea National Diabetes Program Jones’s silver stain fluorescein-labeled antihuman IgG antibody

Korea National Diabetes Program ddddk Purpose Goodpasture’s disease ; autoAb require hexamer dissociation to unmask hidden epitopes Alport’s post-transplantation nephritis ; alloantibodies bind epitopes exposed on native hexamer Compare the conformation of the antibody epitopes Korea National Diabetes Program Intention of finding clues to the pathogenesis of anti-GBM glomerulonephritis

Korea National Diabetes Program Methods Serum and tissue samples - Serum samples ; circulating antibodies 57 patients with anti-GBM glomerulonephritis samples were collected before plasma exchange or immunosuppressive drug treatment was initiated - Tissue samples ; kidney-bound antibodies 14 patients with Goodpasture’s disease, 2 patients with Alport’s post -transplantation nephritis Used an enzyme-linked immunosorbent assay to determine the specificity of circulating autoantibodies and kidney-bound antibodies to NC1 domains Molecular architecture of key epitope regions deduced with the use of chimeric molecules and a three-dimensional model of the α345NC1 hexamer. Korea National Diabetes Program

Korea National Diabetes Program Results 1. Clinical data 2. Specificity of Circulating and Kidney-Bound Abs 3. Circulating α3NC1 & α5NC1 AutoAb / Epitope mapping Goodpasture’s Ab 4. Epitope mapping for kidney-bound autoAb and alloAb 5. Association of α3NC1 and α5NC1 autoantibodies with dis. activity 6. Three-dimensional structure of the α345NC1 hexamer 7. Conformational diversity and differential reactivity Korea National Diabetes Program

1. Clinical data Clinical data - retrospective study - serum samples from 57 patients with Goodpasture’s disease median age of patients at the time of Dx : 59 yrs 22 pts (41%) had positive results for myeloperoxidase ANCA lung involvement : 46 pts / overt lung hemorrhage : 12 pts - Follow-up information was available for 50 of the 57 patients after 6 months 17 pts (34%) remains alive with stable kidney function, 21 pts (42%) were treated with dialysis, and 12 pts (24%) had died. Linezolid 복용기간이 길어질수록 복용 용량이 많을 수록 부작용의 빈도가 높아짐.

2. Specificity of Circulating (B,C,D) and Kidney-Bound (E) Abs Linezolid 복용기간이 길어질수록 복용 용량이 많을 수록 부작용의 빈도가 높아짐. Horizontal line ; median Dotted line ; mean+3SD for NL sample

3. Circulating α3NC1, α5NC1 AutoAb (A) / Epitope mapping Goodpasture’s Ab Linezolid 복용기간이 길어질수록 복용 용량이 많을 수록 부작용의 빈도가 높아짐. Extent of binding of α3NC1 & α5NC1 IgG Ab to NC1 hexamers from N-GBM & D-GBM. α1NC1 & α5NC1 amino acid sequences corresponding to EA & EB regions of α3NC1

4. Epitope mapping for kidney-bound autoAbs and alloAbs Linezolid 복용기간이 길어질수록 복용 용량이 많을 수록 부작용의 빈도가 높아짐. Binding of circulating, kidney & lung-bound autoantibodies to N-GBM & D-GBM NC1 hexamers in samples from 1 Goodpasture pts & 2 APTN pts. c.f) hIgG : normal human IgG Circulating antibodies from 27 Goodpasture pts & kidney eluates from 14 other Goodpasture pts

5. Association of α3NC1 and α5NC1 autoantibodies with disease activity Linezolid 복용기간이 길어질수록 복용 용량이 많을 수록 부작용의 빈도가 높아짐. α3NC1 α5NC1

6. Three-dimensional structure of the α345NC1 hexamer Linezolid 복용기간이 길어질수록 복용 용량이 많을 수록 부작용의 빈도가 높아짐.

7. Conformational diversity and differential reactivity of α345 NC1 hexamers of GBM Linezolid 복용기간이 길어질수록 복용 용량이 많을 수록 부작용의 빈도가 높아짐.

Korea National Diabetes Program Conclusion The development of Goodpasture’s disease may be considered an autoimmune “conformeropathy” that involves perturbation of α345NC1 hexamer, inducing a pathogenic conformational change in the α3NC1 and α5NC1 subunits, which in turn elicits an autoimmune response. Korea National Diabetes Program