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Quaternary Organization of the Goodpasture Autoantigen, the α3(IV) Collagen Chain

SEQUESTRATION OF TWO CRYPTIC AUTOEPITOPES BY INTRAPROTOMER INTERACTIONS WITH THE α4 AND α5 NC1 DOMAINS*
  • Dorin-Bogdan Borza
    Correspondence
    To whom correspondence should be addressed: Dept. of Medicine, Vanderbilt University Medical Center, Division of Nephrology, S-3223 Medical Center North, Nashville, TN 37232. Tel.: 615-322-2089; Fax: 615-343-7156; E-mail:
    Affiliations
    From the Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232, the
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  • Olga Bondar
    Affiliations
    From the Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232, the
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  • Parvin Todd
    Affiliations
    From the Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232, the
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  • Munirathinam Sundaramoorthy
    Affiliations
    From the Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232, the
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  • Yoshikazu Sado
    Affiliations
    Division of Immunology, Shigei Medical Research Institute, Okayama 701, Japan, and the
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  • Yoshifumi Ninomiya
    Affiliations
    Department of Molecular Biology and Biochemistry, Okayama University Medical School, Okayama 700, Japan
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  • Billy G. Hudson
    Affiliations
    From the Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232, the
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  • Author Footnotes
    * This work was supported by Postdoctoral Fellowship 9920539Z from the American Heart Association-Kansas Affiliate (to D. B. B.); Grants R37 DK18381 (to B. G. H.), P01 DK53763 (to B. G. H.), and R01 DK63925 (to M. S.) from the National Institutes of Health; and by Grant-in-aid for Scientific Research (B) 14370434 from the Japan Society for the Promotion of Science (to Y. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:August 21, 2002DOI:https://doi.org/10.1074/jbc.M207769200
      Goodpasture's (GP) disease is caused by autoantibodies that target the α3(IV) collagen chain in the glomerular basement membrane (GBM). Goodpasture autoantibodies bind two conformational epitopes (EA and EB) located within the non-collagenous (NC1) domain of this chain, which are sequestered within the NC1 hexamer of the type IV collagen network containing the α3(IV), α4(IV), and α5(IV) chains. In this study, the quaternary organization of these chains and the molecular basis for the sequestration of the epitopes were investigated. This was accomplished by physicochemical and immunochemical characterization of the NC1 hexamers using chain-specific antibodies. The hexamers were found to have a molecular composition of (α3)2(α4)2(α5)2 and to contain cross-linked α3-α5 heterodimers and α4-α4 homodimers. Together with association studies of individual NC1 domains, these findings indicate that the α3, α4, and α5 chains occur together in the same triple-helical protomer. In the GBM, this protomer dimerizes through NC1-NC1 domain interactions such that the α3, α4, and α5 chains of one protomer connect with the α5, α4, and α3 chains of the opposite protomer, respectively. The immunodominant Goodpasture autoepitope, located within the EA region, is sequestered within the α3α4α5 protomer near the triple-helical junction, at the interface between the α3NC1 and α5NC1 domains, whereas the EB epitope is sequestered at the interface between the α3NC1 and α4NC1 domains. The results also reveal the network distribution of the six chains of collagen IV in the renal glomerulus and provide a molecular explanation for the absence of the α3, α4, α5, and α6 chains in Alport syndrome.
      GBM
      glomerular basement membrane
      ELISA
      enzyme-linked immunosorbent assay
      GP
      Goodpasture
      IgG
      immunoglobulin G
      NC1
      the noncollagenous domain of type IV collagen
      r-
      recombinant
      EA and EB
      the α3(IV)NC1 residues 17–31 and 127–141, respectively, that encompass the epitopes of human GP autoantibodies
      mAb
      monoclonal antibody
      The glomerular basement membrane (GBM)1 is a key component of the kidney ultrafiltration barrier. Type IV collagen, the main structural component of the GBM, has been implicated in several glomerular diseases. Goodpasture's (GP) disease is caused by anti-GBM autoantibodies that bind to the type IV collagen networks of the GBM, and, in some patients, of the alveolar basement membranes (
      • Wilson C.B.
      • Borza D.B.
      • Hudson B.G.
      ). The bound antibodies induce an inflammatory response causing rapidly progressive glomerulonephritis and pulmonary hemorrhage. The GP autoantibodies are specifically targeted to the non-collagenous domain (NC1) of the α3(IV) chain, the “Goodpasture autoantigen,” one of the six chains that comprise the collagen IV family (
      • Saus J.
      • Wieslander J.
      • Langeveld J.P.
      • Quinones S.
      • Hudson B.G.
      ). Two conformational GP epitopes, designated EA and EB, have been localized within the α3(IV) NC1 domain at residues 17–31 and 127–141, respectively (
      • Netzer K.O.
      • Leinonen A.
      • Boutaud A.
      • Borza D.B.
      • Todd P.
      • Gunwar S.
      • Langeveld J.P.
      • Hudson B.G.
      ). The GP epitopes are sequestered in the type IV collagen network of native basement membranes by quaternary interactions among NC1 domains (
      • Borza D.B.
      • Netzer K.O.
      • Leinonen A.
      • Todd P.
      • Cervera J.
      • Saus J.
      • Hudson B.G.
      ).
      How the α3(IV) chain is organized in the collagen IV network of GBM is unknown. Collagen IV networks are composed of triple-helical protomers that are connected at the carboxyl termini by NC1-to-NC1 interactions, forming dimers of protomers, and at the amino termini by interactions involving the 7 S domain, forming tetramers (
      • Timpl R.
      • Wiedemann H.
      • van Delden V.
      • Furthmayr H.
      • Kuhn K.
      ,
      • Yurchenco P.D.
      • Furthmayr H.
      ). In the GBM, an α1·α2(IV) network and a distinct α3·α4·α5(IV) network have been identified based on differential solubilization with pseudolysin (
      • Gunwar S.
      • Ballester F.
      • Noelken M.E.
      • Sado Y.
      • Ninomiya Y.
      • Hudson B.G.
      ) and analysis of collagenase-solubilized NC1 hexamers (
      • Boutaud A.
      • Borza D.B.
      • Bondar O.
      • Gunwar S.
      • Netzer K.O.
      • Singh N.
      • Ninomiya Y.
      • Sado Y.
      • Noelken M.E.
      • Hudson B.G.
      ). The protomer and network organization of α1(IV) and α2(IV) chains has been well established and confirmed by the recent determination of the crystal structure of the [(α1)2α2]2(IV) NC1 hexamer (
      • Sundaramoorthy M.
      • Meiyappan M.
      • Todd P.
      • Hudson B.G.
      ,
      • Than M.E.
      • Henrich S.
      • Huber R.
      • Ries A.
      • Mann K.
      • Kuhn K.
      • Timpl R.
      • Bourenkov G.P.
      • Bartunik H.D.
      • Bode W.
      ). In contrast, little is known about the organization of α3(IV), α4(IV), and α5(IV) chains, i.e. which combinations of three chains form protomers, what is the relative position of chains within protomers, and which kinds of protomers associate through NC1-to-NC1 interactions. This information is essential for understanding the molecular basis of GP disease.
      The GP epitopes are sequestered within the NC1 hexamer structure of the α3·α4·α5(IV) network and inaccessible for autoantibody binding unless the hexamer dissociates (
      • Borza D.B.
      • Netzer K.O.
      • Leinonen A.
      • Todd P.
      • Cervera J.
      • Saus J.
      • Hudson B.G.
      ,
      • Wieslander J.
      • Langeveld J.
      • Butkowski R.
      • Jodlowski M.
      • Noelken M.
      • Hudson B.G.
      ). Unmasking the GP epitopes is thought to be critical for etiology and pathogenesis of GP disease, but the molecular basis for the epitope sequestration is not known (
      • Kalluri R.
      • Sun M.J.
      • Hudson B.G.
      • Neilson E.G.
      ). Based on the identification of hydrophobic residues in the epitope of the immunodominant GPA autoantibodies, it has been proposed that the epitope is buried at the interface between interacting NC1 domains (
      • David M.
      • Borza D.B.
      • Leinonen A.
      • Belmont J.M.
      • Hudson B.G.
      ). An analysis of the homologous α1·α2(IV) NC1 hexamer structure revealed that the GP epitopes are located adjacent to the triple-helical domain and to the interfaces between NC1 monomers within a protomer (
      • Sundaramoorthy M.
      • Meiyappan M.
      • Todd P.
      • Hudson B.G.
      ,
      • Than M.E.
      • Henrich S.
      • Huber R.
      • Ries A.
      • Mann K.
      • Kuhn K.
      • Timpl R.
      • Bourenkov G.P.
      • Bartunik H.D.
      • Bode W.
      ).
      In the present study, the quaternary organization of the α3(IV) chain in relation to the α4(IV) and α5(IV) chains in the network was investigated to determine: (a) which chains coexist and their relative position in the triple-helical protomers, (b) how the protomers are connected through the NC1 domains, and (c) how the GP epitopes are sequestered. The results unambiguously identify a single quaternary organization of the α3·α4·α5 NC1 hexamer revealing that the α3, α4, and α5 chains coexist in a single protomer that self-associates through NC1-NC1 domain interactions to form a network. Moreover, the immunodominant GP epitope, located within the EA region, is sequestered at the α3-α5 NC1 interface within the protomer, which partially buries residues required for autoantibody binding, whereas the EB epitope is sequestered at the α3-α4 NC1 interface.
      Based on these findings, we propose that the designation of Goodpasture autoantigen should henceforth refer to the α3·α4·α5(IV) triple-helical protomer, rather than to the α3(IV) collagen chain, as this molecules reflects both the organization and the cryptic property of the autoantigen in the GBM.

      Acknowledgments

      Human kidneys not suitable for transplantation were kindly provided by Midwest Organ Bank, Kansas City, for preparation of human GBM. Larry Howell contributed the artwork in Fig. 7 B.

      References

        • Wilson C.B.
        • Borza D.B.
        • Hudson B.G.
        Theofilopoulos A.N. Bona A.C. The Molecular Pathology of Autoimmune Diseases. 2nd Ed. Gordon & Breach Science Publishers/Harwood Academic Publishers, Newark, NJ2002: 981-1010
        • Saus J.
        • Wieslander J.
        • Langeveld J.P.
        • Quinones S.
        • Hudson B.G.
        J. Biol. Chem. 1988; 263: 13374-13380
        • Netzer K.O.
        • Leinonen A.
        • Boutaud A.
        • Borza D.B.
        • Todd P.
        • Gunwar S.
        • Langeveld J.P.
        • Hudson B.G.
        J. Biol. Chem. 1999; 274: 11267-11274
        • Borza D.B.
        • Netzer K.O.
        • Leinonen A.
        • Todd P.
        • Cervera J.
        • Saus J.
        • Hudson B.G.
        J. Biol. Chem. 2000; 275: 6030-6037
        • Timpl R.
        • Wiedemann H.
        • van Delden V.
        • Furthmayr H.
        • Kuhn K.
        Eur. J. Biochem. 1981; 120: 203-211
        • Yurchenco P.D.
        • Furthmayr H.
        Biochemistry. 1984; 23: 1839-1850
        • Gunwar S.
        • Ballester F.
        • Noelken M.E.
        • Sado Y.
        • Ninomiya Y.
        • Hudson B.G.
        J. Biol. Chem. 1998; 273: 8767-8775
        • Boutaud A.
        • Borza D.B.
        • Bondar O.
        • Gunwar S.
        • Netzer K.O.
        • Singh N.
        • Ninomiya Y.
        • Sado Y.
        • Noelken M.E.
        • Hudson B.G.
        J. Biol. Chem. 2000; 275: 30716-30724
        • Sundaramoorthy M.
        • Meiyappan M.
        • Todd P.
        • Hudson B.G.
        J. Biol. Chem. 2002; 277: 31142-31153
        • Than M.E.
        • Henrich S.
        • Huber R.
        • Ries A.
        • Mann K.
        • Kuhn K.
        • Timpl R.
        • Bourenkov G.P.
        • Bartunik H.D.
        • Bode W.
        Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6607-6612
        • Wieslander J.
        • Langeveld J.
        • Butkowski R.
        • Jodlowski M.
        • Noelken M.
        • Hudson B.G.
        J. Biol. Chem. 1985; 260: 8564-8570
        • Kalluri R.
        • Sun M.J.
        • Hudson B.G.
        • Neilson E.G.
        J. Biol. Chem. 1996; 271: 9062-9068
        • David M.
        • Borza D.B.
        • Leinonen A.
        • Belmont J.M.
        • Hudson B.G.
        J. Biol. Chem. 2001; 276: 6370-6377
        • Wieslander J.
        • Kataja M.
        • Hudson B.G.
        Clin. Exp. Immunol. 1987; 69: 332-340
        • Sado Y.
        • Boutaud A.
        • Kagawa M.
        • Naito I.
        • Ninomiya Y.
        • Hudson B.G.
        Kidney Int. 1998; 53: 664-671
        • Ninomiya Y.
        • Kagawa M.
        • Iyama K.
        • Naito I.
        • Kishiro Y.
        • Seyer J.M.
        • Sugimoto M.
        • Oohashi T.
        • Sado Y.
        J. Cell Biol. 1995; 130: 1219-1229
        • Borza D.B.
        • Bondar O.
        • Ninomiya Y.
        • Sado Y.
        • Naito I.
        • Todd P.
        • Hudson B.G.
        J. Biol. Chem. 2001; 276: 28532-28540
        • Sado Y.
        • Kagawa M.
        • Kishiro Y.
        • Sugihara K.
        • Naito I.
        • Seyer J.M.
        • Sugimoto M.
        • Oohashi T.
        • Ninomiya Y.
        Histochem. Cell Biol. 1995; 104: 267-275
        • Gunwar S.
        • Saus J.
        • Noelken M.E.
        • Hudson B.G.
        J. Biol. Chem. 1990; 265: 5466-5469
        • Seki T.
        • Naito I.
        • Oohashi T.
        • Sado Y.
        • Ninomiya Y.
        Histochem. Cell Biol. 1998; 110: 359-366
        • Weber S.
        • Dolz R.
        • Timpl R.
        • Fessler J.H.
        • Engel J.
        Eur. J. Biochem. 1988; 175: 229-236
        • Langeveld J.P.
        • Wieslander J.
        • Timoneda J.
        • McKinney P.
        • Butkowski R.J.
        • Wisdom Jr., B.J.
        • Hudson B.G.
        J. Biol. Chem. 1988; 263: 10481-10488
        • Timoneda J.
        • Gunwar S.
        • Monfort G.
        • Saus J.
        • Noelken M.E.
        • Hudson B.G.
        Connect. Tissue Res. 1990; 24: 169-186
        • Kleppel M.M.
        • Fan W.
        • Cheong H.I.
        • Michael A.F.
        J. Biol. Chem. 1992; 267: 4137-4142
        • Johansson C.
        • Butkowski R.
        • Wieslander J.
        J. Biol. Chem. 1992; 267: 24533-24537
        • Dolz R.
        • Engel J.
        • Kuhn K.
        Eur. J. Biochem. 1988; 178: 357-366
        • Netzer K.O.
        • Suzuki K.
        • Itoh Y.
        • Hudson B.G.
        • Khalifah R.G.
        Protein Sci. 1998; 7: 1340-1351
        • Heidet L.
        • Cai Y.
        • Guicharnaud L.
        • Antignac C.
        • Gubler M.C.
        Am. J. Pathol. 2000; 156: 1901-1910
        • Nakanishi K.
        • Yoskikawa N.
        • Iijima K.
        • Nakamura H.
        J. Am. Soc. Nephrol. 1996; 7: 938-945
        • Hino S.
        • Takemura T.
        • Sado Y.
        • Kagawa M.
        • Oohashi T.
        • Ninomiya Y.
        • Yoshioka K.
        Pediatr. Nephrol. 1996; 10: 742-744
        • Kashtan C.E.
        • Kleppel M.M.
        • Gubler M.C.
        Contrib. Nephrol. 1996; 117: 142-153
        • Peissel B.
        • Geng L.
        • Kalluri R.
        • Kashtan C.
        • Rennke H.G.
        • Gallo G.R.
        • Yoshioka K.
        • Sun M.J.
        • Hudson B.G.
        • Neilson E.G.
        • Zhou J.
        J. Clin. Invest. 1995; 96: 1948-1957
        • Gubler M.C.
        • Knebelmann B.
        • Beziau A.
        • Broyer M.
        • Pirson Y.
        • Haddoum F.
        • Kleppel M.M.
        • Antignac C.
        Kidney Int. 1995; 47: 1142-1147
        • Nomura S.
        • Naito I.
        • Fukushima T.
        • Tokura T.
        • Kataoka N.
        • Tanaka I.
        • Tanaka H.
        • Osawa G.
        Am. J. Kidney Dis. 1998; 31: E4
        • Hellmark T.
        • Burkhardt H.
        • Wieslander J.
        J. Biol. Chem. 1999; 274: 25862-25868