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The Goodpasture Autoantigen

MAPPING THE MAJOR CONFORMATIONAL EPITOPE(S) OF α3(IV) COLLAGEN TO RESIDUES 17–31 AND 127–141 OF THE NC1 DOMAIN*
  • Kai-Olaf Netzer
    Footnotes
    Affiliations
    From the Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160 and the
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  • Anu Leinonen
    Affiliations
    From the Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160 and the
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  • Ariel Boutaud
    Affiliations
    From the Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160 and the
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  • Dorin-Bogdan Borza
    Affiliations
    From the Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160 and the
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  • Parvin Todd
    Affiliations
    From the Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160 and the
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  • Sripad Gunwar
    Affiliations
    From the Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160 and the
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  • Jan P.M. Langeveld
    Affiliations
    Institute for Animal Science and Health (ID-DLO), P. O. Box 65, 8200 AB Lelystad, The Netherlands
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  • Billy G. Hudson
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160
    Affiliations
    From the Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160 and the
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant DK 18381 (to B. G. H.) and grants from the Fritz Thyssen Stiftung and the American Heart Association, Kansas Affiliate, Grant KS-94-F-5 (to K.-O. N.) A preliminary report of this work has been presented at the 30th Annual Meeting of the American Society of Nephrology (Netzer K.-O., Gunwar, S., Boutaud, A., Leinonen, A. and Hudson, B.G. (1997) J. Am. Soc. Nephrol. 8,462 (abstr.)).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.
    ‡ Present address: Medizinische Klinik I, Kliniken Köln-Merheim, Lehrkrankenhaus der Universität zu Köln, Köln, Germany.
Open AccessPublished:April 16, 1999DOI:https://doi.org/10.1074/jbc.274.16.11267
      The Goodpasture (GP) autoantigen has been identified as the α3(IV) collagen chain, one of six homologous chains designated α1–α6 that comprise type IV collagen (Hudson, B. G., Reeders, S. T., and Tryggvason, K. (1993) J. Biol. Chem. 268, 26033–26036). In this study, chimeric proteins were used to map the location of the major conformational, disulfide bond-dependent GP autoepitope(s) that has been previously localized to the noncollagenous (NC1) domain of α3(IV) chain. Fourteen α1/α3 NC1 chimeras were constructed by substituting one or more short sequences of α3(IV)NC1 at the corresponding positions in the non-immunoreactive α1(IV)NC1 domain and expressed in mammalian cells for proper folding. The interaction between the chimeras and eight GP sera was assessed by both direct and inhibition enzyme-linked immunosorbent assay. Two chimeras, C2 containing residues 17–31 of α3(IV)NC1 and C6 containing residues 127–141 of α3(IV)NC1, bound autoantibodies, as did combination chimeras containing these regions. The epitope(s) that encompasses these sequences is immunodominant, showing strong reactivity with all GP sera and accounting for 50–90% of the autoantibody reactivity toward α3(IV)NC1. The conformational nature of the epitope(s) in the C2 and C6 chimeras was established by reduction of the disulfide bonds and by PEPSCAN analysis of overlapping 12-mer peptides derived from α1- and α3(IV)NC1 sequences. The amino acid sequences 17–31 and 127–141 in α3(IV)NC1 have thus been shown to contain the critical residues of one or two disulfide bond-dependent conformational autoepitopes that bind GP autoantibodies.
      Goodpasture (GP)
      The abbreviations used are: GP, Goodpasture; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; NC1, the noncollagenous domain of type IV collagen; PCR, polymerase-chain reaction; CMV, cytomegalovirus; EA and EB , α3(IV)NC1 residues 17–31 and 127–141, respectively.
      1The abbreviations used are: GP, Goodpasture; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; NC1, the noncollagenous domain of type IV collagen; PCR, polymerase-chain reaction; CMV, cytomegalovirus; EA and EB , α3(IV)NC1 residues 17–31 and 127–141, respectively.
      autoimmune disease is characterized by pulmonary hemorrhage and/or rapidly progressing glomerulonephritis (
      • Wilson C.
      • Dixon F.
      ). Tissue injury is mediated by anti-basement membrane antibodies that bind alveolar and glomerular basement membranes. The target autoantigen of basement membranes has been identified as the α3(IV) collagen chain, one of six homologous chains designated α1–α6 that comprise type IV collagen (
      • Hudson B.G.
      • Reeders S.T.
      • Tryggvason K.
      ). In the glomerular basement membrane, the α3(IV) chain exists in a supramolecular network along with the α4(IV) and α5(IV) chains (
      • Gunwar S.
      • Ballester F.
      • Noelken M.E.
      • Sado Y.
      • Ninomiya Y.
      • Hudson B.G.
      ). The α3(IV) chain is composed of a long collagenous domain of 1410 amino acids and a non-collagenous (NC1) domain of 232 residues at the carboxyl terminus (
      • Mariyama M.
      • Leinonen A.
      • Mochizuki T.
      • Tryggvason K.
      • Reeders S.T.
      ).
      The GP autoepitope(s) has been localized to the NC1 domain of the α3(IV) chain (
      • Butkowski R.J.
      • Langeveld J.P.
      • Wieslander J.
      • Hamilton J.
      • Hudson B.G.
      ,
      • Saus J.
      • Wieslander J.
      • Langeveld J.P.
      • Quinones S.
      • Hudson B.G.
      ). Antibodies that bind to the NC1 domain of other α(IV) chains may be found in some Goodpasture patients (
      • Kalluri R.
      • Wilson C.B.
      • Weber M.
      • Gunwar S.
      • Chonko A.M.
      • Neilson E.G.
      • Hudson B.G.
      ,
      • Dehan P.
      • Weber M.
      • Zhang X.
      • Reeders S.T.
      • Foidart J.M.
      • Tryggvason K.
      ), but they only account for about 10% of autoreactivity (
      • Hellmark T.
      • Brunmark C.
      • Trojnar J.
      • Wieslander J.
      ). The autoepitope(s) in the α3(IV)NC1 domain appears to be conformational, because reduction of disulfide bonds abolishes most of the binding (
      • Hellmark T.
      • Brunmark C.
      • Trojnar J.
      • Wieslander J.
      ,
      • Wieslander J.
      • Bygren P.
      • Heinegard D.
      ,
      • Kalluri R.
      • Gunwar S.
      • Reeders S.T.
      • Morrison K.C.
      • Mariyama M.
      • Ebner K.E.
      • Noelken M.E.
      • Hudson B.G.
      ). The identification of the precise amino acid residues that constitute this epitope(s) is important for understanding the etiology and pathogenesis of the GP disease and for the development of diagnostic and therapeutic agents. Several groups have attempted to map the location of the autoepitope(s) by using short linear peptides (
      • Hellmark T.
      • Brunmark C.
      • Trojnar J.
      • Wieslander J.
      ,
      • Kalluri R.
      • Gunwar S.
      • Reeders S.T.
      • Morrison K.C.
      • Mariyama M.
      • Ebner K.E.
      • Noelken M.E.
      • Hudson B.G.
      ,
      • Kefalides N.A.
      • Ohno N.
      • Wilson C.B.
      • Fillit H.
      • Zabriski J.
      • Rosenbloom J.
      ,
      • Levy J.B.
      • Turner A.N.
      • George A.J.
      • Pusey C.D.
      ,
      • Levy J.B.
      • Coulthart A.
      • Pusey C.D.
      ) or by site-directed mutagenesis of the α3(IV)NC1 domain expressed in Escherichia coli (
      • Kalluri R.
      • Sun M.J.
      • Hudson B.G.
      • Neilson E.G.
      ). Although linear sequences have been identified that bind GP antibodies, these findings are at variance with each other. Moreover, prior studies have not addressed whether these linear sequences constitute the major conformational, disulfide bond-dependent epitope(s).
      The aim of this study was to identify the α3(IV)NC1 amino acid sequences that form the thus far elusive conformational GP epitope(s). To circumvent the limitations of previous approaches, we pursued an epitope mapping strategy based on chimeric proteins. This approach has been specifically developed and successfully used to map conformational epitopes (
      • Hsia R.
      • Beals T.
      • Boothroyd J.C.
      ) or autoepitopes (
      • Henriksson E.W.
      • Pettersson I.
      ). We hypothesized that the α3(IV)NC1 regions most likely to form the autoepitope(s) are those most divergent from the other homologous α(IV) chains. A series of chimeric α1/α3(IV)NC1 domains were constructed in which these candidate α3(IV)NC1 sequences replaced the corresponding sequences in the non-immunoreactive α1(IV)NC1. The chimeras were expressed in mammalian cells for correct protein folding and disulfide bond formation. We report that two specific sequences, α3(IV)NC1 residues 17–31 and 127–141, contain the critical residues of one or two disulfide bond-dependent conformational GP autoepitopes within the α3(IV)NC1 domain.

      DISCUSSION

      In the present study, a new strategy based on chimeric proteins was employed to map regions within α3(IV)NC1 that constitute theconformational epitope(s) for GP autoantibodies. This novel approach has two methodological improvements over previous work. Unlike in peptide-based epitope mapping, short α3(IV)NC1 candidate regions (<15 residues) were grafted onto an inert α1(IV)NC1 framework and expressed in mammalian cells to ensure native folding. The resulting chimeras were assayed for “gain-of-function,” i.e.capacity to bind autoantibodies, in contrast with previous site-directed mutagenesis studies (
      • Kalluri R.
      • Sun M.J.
      • Hudson B.G.
      • Neilson E.G.
      ) that relied on a “loss-of-function” of the protein expressed in E. coli. The results from 14 different chimeras revealed two previously unidentified regions, designated EA andEB (residues 17–31 and 127–141 of α3(IV)NC1, respectively), that strongly bound autoantibodies from eight GP patients. Together, EA andEB accounted for 50–90% (on average 68%) of autoreactivity to α3(IV)NC1.
      Among the six candidate regions evaluated in this study, regionsE A and E B clearly exhibited a distinct capacity to bind GP antibodies by Western blots, direct ELISA, and inhibition ELISA. The six regions were selected based on the following: (a) autoantibodies preferentially bind the α3(IV)NC1 domain but not the other five homologous NC1 domains of type IV collagen; (b) therefore, regions of substantial sequence divergence between α3(IV)NC1 and the other NC1 domains confer antibody binding to the former. The four regions that were found non-reactive (i.e. those substituted in C1,C3, C4, and C5 chimeras) further distinguish EA and EB as the primary regions for the GP epitope. It is significant that theEA and EB regions are homologous (47% sequence identity) and are located at corresponding positions in the two homologous NC1 subdomains (
      • Netzer K.
      • Suzuki K.
      • Itoh Y.
      • Hudson B.G.
      • Khalifah R.G.
      ), but they are noncontiguous. EA and EBcould represent two separate and distinct epitopes or a single epitopeEAB, in which EA andEB are held in close proximity to each other by the disulfide bonds. In either case, the complete epitope(s) probably includes additional residues from other regions, less critical for binding. So far, the x-ray crystallographic structures of other protein-antibody complexes have revealed noncontiguous epitopes of 15–22 amino acids that belong to several surface loops (
      • Laver W.G.
      • Air G.M.
      • Webster R.G.
      • Smith-Gill S.J.
      ,
      • Jones S.
      • Thornton J.M.
      ).
      Our results demonstrate that regions EA andEB reproduce very well the authentic GP epitopes in the α3(IV)NC1 domain. Most remarkably, EAand EB form conformational epitopes that require intact disulfide bonds to bind GP antibodies, as demonstrated by loss of GP immunoreactivity of the C2, C6, andC2·6 chimeras upon reduction (Fig. 7). The majority of GP autoantibodies appears to recognize conformational epitopes in α3(IV)NC1 (
      • Hellmark T.
      • Brunmark C.
      • Trojnar J.
      • Wieslander J.
      ,
      • Wieslander J.
      • Bygren P.
      • Heinegard D.
      ,
      • Kalluri R.
      • Gunwar S.
      • Reeders S.T.
      • Morrison K.C.
      • Mariyama M.
      • Ebner K.E.
      • Noelken M.E.
      • Hudson B.G.
      ), but epitope mapping studies have not addressed until now the nature of the epitopes found (see below). Further demonstrating the good mimicry of the original epitope(s), the chimeras produced significant inhibition of GP sera at concentrations in the range of 10−8m, comparable with α3(IV)NC1 domain. In contrast, linear α3(IV)NC1 peptides produced a comparable effect in inhibition ELISA only at concentrations 100–1000-fold higher (
      • Kalluri R.
      • Gunwar S.
      • Reeders S.T.
      • Morrison K.C.
      • Mariyama M.
      • Ebner K.E.
      • Noelken M.E.
      • Hudson B.G.
      ,
      • Levy J.B.
      • Coulthart A.
      • Pusey C.D.
      ).
      The EA and EB regions have not been previously identified by peptide-based epitope mapping (
      • Hellmark T.
      • Brunmark C.
      • Trojnar J.
      • Wieslander J.
      ,
      • Kalluri R.
      • Gunwar S.
      • Reeders S.T.
      • Morrison K.C.
      • Mariyama M.
      • Ebner K.E.
      • Noelken M.E.
      • Hudson B.G.
      ,
      • Kefalides N.A.
      • Ohno N.
      • Wilson C.B.
      • Fillit H.
      • Zabriski J.
      • Rosenbloom J.
      ,
      • Levy J.B.
      • Turner A.N.
      • George A.J.
      • Pusey C.D.
      ,
      • Levy J.B.
      • Coulthart A.
      • Pusey C.D.
      ). As shown here, this was due to the inability of peptide scanning procedures to reliably identify the conformational GP epitope(s). An intrinsic tendency of peptide-based methods to identify sequential epitopes has already been noted (
      • Schwab C.
      • Twardek A.
      • Lo T.P.
      • Brayer G.D.
      • Bosshard H.R.
      ). Thus, the α1(IV)NC1 framework of the chimeras is instrumental for adoption of the native conformation by EA andEB, and in addition, it may contribute auxiliary residues for binding. It is likely that the previous reports have largely identified linear GP epitopes, which constitute a minority (about 5% of the reactivity against α3(IV)NC1). Furthermore, various linear sequences were found reactive in different studies, suggesting heterogeneity of the linear epitopes. In contrast, the chimera-based approach has successfully identified the critical regions of one or two immunodominant, conformational GP epitope(s) that were consistently recognized by all autoimmune sera used in this work.
      A report published at the time of submission of this study (
      • Ryan J.J.
      • Mason P.J.
      • Pusey C.D.
      • Turner N.
      ) also used α1/α3 chimeras, but larger regions of α3(IV)NC1 were swapped (the amino-terminal 54 amino acids, the carboxyl-terminal 63 amino acids, and the intervening 115 amino acids, respectively). The conformational nature of the epitopes identified was not explored. Our more fine analysis is in full agreement with the broader conclusions of that study, namely that the amino-terminal third of α3(IV)NC1 accounts for most immunoreactivity with GP sera.
      Region EA clearly represents an immunodominant epitope. It was recognized strongly and consistently by all sera analyzed, whereas EB reacted significantly (>10%) with only half of the sera. This may be due to the higher divergence of EA (eight distinct amino acids) compared with EB (five distinct amino acids). The existence of an immunodominant epitope explains the considerable cross-inhibition between GP sera from different patients or between GP sera and certain monoclonal antibodies (
      • Levy J.B.
      • Turner A.N.
      • George A.J.
      • Pusey C.D.
      ,
      • Hellmark T.
      • Johansson C.
      • Wieslander J.
      ,
      • Pusey C.D.
      • Dash A.
      • Kershaw M.J.
      • Morgan A.
      • Reilly A.
      • Rees A.J.
      • Lockwood C.M.
      ).EA and EB may well be the counterpart of the shared structural determinants on the GP antibodies, found by using an anti-idiotype antibody against anti-α3(IV) IgG (
      • Meyers K.E.
      • Kinniry P.A.
      • Kalluri R.
      • Neilson E.G.
      • Madaio M.P.
      ).
      In summary, two specific homologous sequences in α3(IV)NC1 have been identified for the first time to be part of one or two disulfide bond-dependent, conformational and immunodominant GP autoepitopes. This finding provides new knowledge to investigate further the pathogenesis of GP disease. It has recently been shown that α3(IV)NC1 but not α1(IV)NC1 can induce experimental GP disease in mice (
      • Sado Y.
      • Boutaud A.
      • Kagawa M.
      • Naito I.
      • Ninomiya Y.
      • Hudson B.G.
      ). A very important question, relevant for the identification of the nephritogenic epitope(s) in α3(IV)NC1, is whether any of the α1/α3 chimeras can induce experimental GP syndrome. In myasthenia gravis, another autoimmune disease, the immunodominant epitope on the acetylcholine receptor (known as “MIR” or main immunogenic region) was also pathogenic (
      • Tzartos S.J.
      • Cung M.T.
      • Demange P.
      • Loutrari H.
      • Mamalaki A.
      • Marraud M.
      • Papadouli I.
      • Sakarellos C.
      • Tsikaris V.
      ). By providing a highly specific target, the new identification of an immunodominant GP epitope should be useful for the development of more specific therapeutic approaches, such as use of vaccines to induce tolerance or the manipulation of the idiotype network.

      Acknowledgments

      This work was initiated after helpful discussions with Dr. K. Hilgers, Erlangen, Germany. We thank Dr. R. Khalifah for helpful discussions and critical reading of this manuscript. We thank Midwest Organ Bank for providing the human kidneys.

      REFERENCES

        • Wilson C.
        • Dixon F.
        Berner B. Rector F. The Kidney. 3rd Ed. W. B. Saunders Co., Philadelphia1986: 800-889
        • Hudson B.G.
        • Reeders S.T.
        • Tryggvason K.
        J. Biol. Chem. 1993; 268: 26033-26036
        • Gunwar S.
        • Ballester F.
        • Noelken M.E.
        • Sado Y.
        • Ninomiya Y.
        • Hudson B.G.
        J. Biol. Chem. 1998; 273: 8767-8775
        • Mariyama M.
        • Leinonen A.
        • Mochizuki T.
        • Tryggvason K.
        • Reeders S.T.
        J. Biol. Chem. 1994; 269: 23013-23017
        • Butkowski R.J.
        • Langeveld J.P.
        • Wieslander J.
        • Hamilton J.
        • Hudson B.G.
        J. Biol. Chem. 1987; 262: 7874-7877
        • Saus J.
        • Wieslander J.
        • Langeveld J.P.
        • Quinones S.
        • Hudson B.G.
        J. Biol. Chem. 1988; 263: 13374-13380
        • Kalluri R.
        • Wilson C.B.
        • Weber M.
        • Gunwar S.
        • Chonko A.M.
        • Neilson E.G.
        • Hudson B.G.
        J. Am. Soc. Nephrol. 1995; 6: 1178-1185
        • Dehan P.
        • Weber M.
        • Zhang X.
        • Reeders S.T.
        • Foidart J.M.
        • Tryggvason K.
        Nephrol. Dial. Transplant. 1996; 11: 2215-2222
        • Hellmark T.
        • Brunmark C.
        • Trojnar J.
        • Wieslander J.
        Clin. Exp. Immunol. 1996; 105: 504-510
        • Wieslander J.
        • Bygren P.
        • Heinegard D.
        Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1544-1548
        • Kalluri R.
        • Gunwar S.
        • Reeders S.T.
        • Morrison K.C.
        • Mariyama M.
        • Ebner K.E.
        • Noelken M.E.
        • Hudson B.G.
        J. Biol. Chem. 1991; 266: 24018-24024
        • Kefalides N.A.
        • Ohno N.
        • Wilson C.B.
        • Fillit H.
        • Zabriski J.
        • Rosenbloom J.
        Kidney Int. 1993; 43: 94-100
        • Levy J.B.
        • Turner A.N.
        • George A.J.
        • Pusey C.D.
        Clin. Exp. Immunol. 1996; 106: 79-85
        • Levy J.B.
        • Coulthart A.
        • Pusey C.D.
        J. Am. Soc. Nephrol. 1997; 8: 1698-1705
        • Kalluri R.
        • Sun M.J.
        • Hudson B.G.
        • Neilson E.G.
        J. Biol. Chem. 1996; 271: 9062-9068
        • Hsia R.
        • Beals T.
        • Boothroyd J.C.
        Mol. Microbiol. 1996; 19: 53-63
        • Henriksson E.W.
        • Pettersson I.
        J. Autoimmun. 1997; 10: 559-568
        • Mayer U.
        • Poschl E.
        • Nischt R.
        • Specks U.
        • Pan T.C.
        • Chu M.L.
        • Timpl R.
        Eur. J. Biochem. 1994; 225: 573-580
        • Sambrook J.
        Fritsch E.F. Maniatis T. 2nd Ed. Molecular Cloning: A Laboratory Manual. 3. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 16.32-16.40
        • Gill S.C.
        • von Hippel P.H.
        Anal. Biochem. 1989; 182: 319-326
        • Sado Y.
        • Boutaud A.
        • Kagawa M.
        • Naito I.
        • Ninomiya Y.
        • Hudson B.G.
        Kidney Int. 1998; 53: 664-671
        • Neilson E.G.
        • Kalluri R.
        • Sun M.J.
        • Gunwar S.
        • Danoff T.
        • Mariyama M.
        • Myers J.C.
        • Reeders S.T.
        • Hudson B.G.
        J. Biol. Chem. 1993; 268: 8402-8405
        • Wieslander J.
        • Kataja M.
        • Hudson B.G.
        Clin. Exp. Immunol. 1987; 69: 332-340
        • Gunwar S.
        • Ballester F.
        • Kalluri R.
        • Timoneda J.
        • Chonko A.M.
        • Edwards S.J.
        • Noelken M.E.
        • Hudson B.G.
        J. Biol. Chem. 1991; 266: 15318-15324
        • Laemmli U.K.
        Nature. 1970; 227: 680-685
        • Geysen H.M.
        • Meloen R.H.
        • Barteling S.J.
        Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3998-4002
        • Netzer K.
        • Suzuki K.
        • Itoh Y.
        • Hudson B.G.
        • Khalifah R.G.
        Protein Sci. 1998; 7: 1340-1351
        • Penades J.R.
        • Bernal D.
        • Revert F.
        • Johansson C.
        • Fresquet V.J.
        • Cervera J.
        • Wieslander J.
        • Quinones S.
        • Saus J.
        Eur. J. Biochem. 1995; 229: 754-760
        • Fox J.W.
        • Mayer U.
        • Nischt R.
        • Aumailley M.
        • Reinhardt D.
        • Wiedemann H.
        • Mann K.
        • Timpl R.
        • Krieg T.
        • Engel J.
        • Chu M.
        EMBO J. 1991; 10: 3137-3146
        • Yurchenco P.D.
        • Quan Y.
        • Colognato H.
        • Mathus T.
        • Harrison D.
        • Yamada Y.
        • O'Rear J.J.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10189-10194
        • Hellmark T.
        • Johansson C.
        • Wieslander J.
        Kidney Int. 1994; 46: 823-829
        • Laver W.G.
        • Air G.M.
        • Webster R.G.
        • Smith-Gill S.J.
        Cell. 1990; 61: 553-556
        • Jones S.
        • Thornton J.M.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13-20
        • Schwab C.
        • Twardek A.
        • Lo T.P.
        • Brayer G.D.
        • Bosshard H.R.
        Protein Sci. 1993; 2: 175-182
        • Pusey C.D.
        • Dash A.
        • Kershaw M.J.
        • Morgan A.
        • Reilly A.
        • Rees A.J.
        • Lockwood C.M.
        Lab. Invest. 1987; 56: 23-31
        • Meyers K.E.
        • Kinniry P.A.
        • Kalluri R.
        • Neilson E.G.
        • Madaio M.P.
        Kidney Int. 1998; 53: 402-407
        • Tzartos S.J.
        • Cung M.T.
        • Demange P.
        • Loutrari H.
        • Mamalaki A.
        • Marraud M.
        • Papadouli I.
        • Sakarellos C.
        • Tsikaris V.
        Mol. Neurobiol. 1991; 5: 1-29
        • Ryan J.J.
        • Mason P.J.
        • Pusey C.D.
        • Turner N.
        Clin. Exp. Immunol. 1998; 113: 17-27