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• W2034215619 abstract "Goodpasture (GP) disease is an autoimmune disorder in which autoantibodies against the α3(IV) chain of type IV collagen bind to the glomerular and alveolar basement membranes, causing progressive glomerulonephritis and pulmonary hemorrhage. Two major conformational epitope regions have been identified on the noncollagenous domain of type IV collagen (NC1 domain) of the α3(IV) chain as residues 17–31 (EA) and 127–141 (EB) (Netzer, K.-O. et al. (1999)J. Biol. Chem. 274, 11267–11274). To determine whether these regions are two distinct epitopes or form a single epitope, three GP sera were fractionated by affinity chromatography on immobilized NC1 chimeras containing the EA and/or the EB region. Four subpopulations of GP antibodies with distinct epitope specificity for the α3(IV)NC1 domain were thus separated and characterized. They were designated GPA, GPB, GPAB, and GPX, to reflect their reactivity with EA only, EB only, both regions, and neither, respectively. Hence, regions EA and EB encompass critical amino acids that constitute three distinct epitopes for GPA, GPB, and GPAB antibodies, respectively, whereas the epitope for GPX antibodies is located in a different unknown region. The GPA antibodies were consistently immunodominant, accounting for 60–65% of the total immunoreactivity to α3(IV)NC1; thus, they probably play a major role in pathogenesis. Regions EA and EB are held in close proximity because they jointly form the epitope for Mab3, a monoclonal antibody that competes for binding with GP autoantibodies. All GP epitopes are sequestered in the hexamer configuration of the NC1 domain found in tissues and are inaccessible for antibody binding unless dissociation of the hexamer occurs, suggesting a possible mechanism for etiology of GP disease. GP antibodies have the capacity to extract α3(IV)NC1 monomers, but not dimers, from native human glomerular basement membrane hexamers, a property that may be of fundamental importance for the pathogenesis of the disease. Goodpasture (GP) disease is an autoimmune disorder in which autoantibodies against the α3(IV) chain of type IV collagen bind to the glomerular and alveolar basement membranes, causing progressive glomerulonephritis and pulmonary hemorrhage. Two major conformational epitope regions have been identified on the noncollagenous domain of type IV collagen (NC1 domain) of the α3(IV) chain as residues 17–31 (EA) and 127–141 (EB) (Netzer, K.-O. et al. (1999)J. Biol. Chem. 274, 11267–11274). To determine whether these regions are two distinct epitopes or form a single epitope, three GP sera were fractionated by affinity chromatography on immobilized NC1 chimeras containing the EA and/or the EB region. Four subpopulations of GP antibodies with distinct epitope specificity for the α3(IV)NC1 domain were thus separated and characterized. They were designated GPA, GPB, GPAB, and GPX, to reflect their reactivity with EA only, EB only, both regions, and neither, respectively. Hence, regions EA and EB encompass critical amino acids that constitute three distinct epitopes for GPA, GPB, and GPAB antibodies, respectively, whereas the epitope for GPX antibodies is located in a different unknown region. The GPA antibodies were consistently immunodominant, accounting for 60–65% of the total immunoreactivity to α3(IV)NC1; thus, they probably play a major role in pathogenesis. Regions EA and EB are held in close proximity because they jointly form the epitope for Mab3, a monoclonal antibody that competes for binding with GP autoantibodies. All GP epitopes are sequestered in the hexamer configuration of the NC1 domain found in tissues and are inaccessible for antibody binding unless dissociation of the hexamer occurs, suggesting a possible mechanism for etiology of GP disease. GP antibodies have the capacity to extract α3(IV)NC1 monomers, but not dimers, from native human glomerular basement membrane hexamers, a property that may be of fundamental importance for the pathogenesis of the disease. Goodpasture enzyme-linked immunosorbent assay glomerular basement membrane guanidinium hydrochloride noncollagenous domain of type IV collagen Tris-buffered saline α3(IV)NC1 residues 17–31 and 127–141, respectively Goodpasture (GP)1disease is a paradigm for autoantibody-mediated, organ-specific autoimmune diseases. The disease is produced by autoantibodies to type IV collagen, the major component of basement membranes. Binding of autoantibodies to glomerular and/or alveolar basement membranes causes rapidly progressing glomerulonephritis and pulmonary hemorrhage, respectively (1.Wilson C. Dixon F. Berner B. Rector F. The Kidney. 3rd Ed. W. B. Saunders Co., Philadelphia1986: 800-889Google Scholar). The basement membrane autoantigen has been identified as the α3(IV) chain (2.Butkowski R.J. Langeveld J.P. Wieslander J. Hamilton J. Hudson B.G. J. Biol. Chem. 1987; 262: 7874-7877Abstract Full Text PDF PubMed Google Scholar, 3.Saus J. Wieslander J. Langeveld J.P. Quinones S. Hudson B.G. J. Biol. Chem. 1988; 263: 13374-13380Abstract Full Text PDF PubMed Google Scholar, 4.Kalluri R. Wilson C.B. Weber M. Gunwar S. Chonko A.M. Neilson E.G. Hudson B.G. J. Am. Soc. Nephrol. 1995; 6: 1178-1185Crossref PubMed Google Scholar), one of the six homologous α chains that make up type IV collagen (5.Hudson B.G. Reeders S.T. Tryggvason K. J. Biol. Chem. 1993; 268: 26033-26036Abstract Full Text PDF PubMed Google Scholar). The GP autoepitope(s) has been mapped to the noncollagenous domain (NC1), composed of 232 amino acids at the C terminus of the α3(IV) chain. A strategy based on chimeric proteins has been recently used by several groups to localize the immunodominant GP epitope(s) (6.Ryan J.J. Mason P.J. Pusey C.D. Turner N. Clin. Exp. Immunol. 1998; 113: 17-27Crossref PubMed Scopus (37) Google Scholar, 7.Hellmark T. Segelmark M. Unger C. Burkhardt H. Saus J. Wieslander J. Kidney Int. 1999; 55: 936-944Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 8.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: 11267-11274Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). In this approach, α3(IV) sequences have been substituted at the corresponding positions into the homologous but nonimmunoreactive α1(IV)NC1 domain. The use of α1(IV) as inert scaffolding is required to preserve the three-dimensional structure of conformational GP epitopes. By swapping relatively large portions between α1(IV)NC1 and α3(IV)NC1 domains (thirds of the molecule), one study has found that GP immunoreactivity maps exclusively to the N-terminal 54 amino acids of α3(IV)NC1 (6.Ryan J.J. Mason P.J. Pusey C.D. Turner N. Clin. Exp. Immunol. 1998; 113: 17-27Crossref PubMed Scopus (37) Google Scholar), whereas another (7.Hellmark T. Segelmark M. Unger C. Burkhardt H. Saus J. Wieslander J. Kidney Int. 1999; 55: 936-944Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) has reported that both the N-terminal 85 amino acids and the central portion of α3(IV)NC1 domain (amino acids 86–175) react with GP autoantibodies. We have further narrowed down the location of the conformational GP epitopes of α3(IV)NC1 to two short amino acid sequences: residues 17–31, designatedEA , and 127–141, designatedEB (8.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: 11267-11274Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Whether EA and EB sequences form two distinct conformational epitopes or belong to a single epitope is unknown. Moreover, an intriguing homology that exists between the EA and EB regions (7 identical residues out of 15 and similar location in the two homologous half-domains of NC1) raises an additional possibility, that autoantibodies to one epitope could cross-react with the other epitope. A detailed characterization of the GP epitopes is important for understanding of the etiology and pathogenesis of the disease and may facilitate the design of specific therapeutic approaches aimed at removing or neutralizing the pathogenic antibodies. The purpose of the present study was to determine whether theEA and EB regions are two distinct epitopes or form a single epitope. Three GP sera were fractionated by affinity chromatography on immobilized NC1 chimeras containing the EA and/or theEB region. Four subpopulations of GP antibodies with specificity for distinct epitopes within the α3(IV)NC1 domain were thus separated and characterized, proving thatEA and EB regions constitute separate GP epitopes. These two regions, held in close proximity in the folded α3(IV)NC1 domain, contain certain amino acid residues critical for GP autoantibody binding that are sequestered in the hexamer configuration of the NC1 domain found in tissues. This cryptic property is likely important for the etiology of the disease. Recombinant human α1–3(IV)NC1 domains and α1/α3 chimeras containing a N-terminal FLAG sequence were expressed in human embryonic kidney 293 cells and purified as described (8.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: 11267-11274Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 9.Sado Y. Boutaud A. Kagawa M. Naito I. Ninomiya Y. Hudson B.G. Kidney Int. 1998; 53: 664-671Abstract Full Text PDF PubMed Scopus (83) Google Scholar). Glomerular basement membrane (GBM) was obtained from human kidneys not suitable for transplantation (generously provided by Midwest Organ Bank, Kansas City, KS) and digested with collagenase to prepare human NC1 hexamers (10.Wieslander J. Kataja M. Hudson B.G. Clin. Exp. Immunol. 1987; 69: 332-340PubMed Google Scholar). Three of the GP sera used in a previous study (8.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: 11267-11274Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), GP1–3, were available in sufficient amount to allow the purification and subsequent characterization of IgG fractions. IgG was purified from these GP sera by affinity chromatography on protein A-agarose (Amersham Pharmacia Biotech), according to the manufacturer's protocol. ChimerasC2 (containing the EA region; residues 17–31 of α3(IV)NC1 domain), C6 (containing theEB region; residues 127–141 of α3(IV)NC1 domain), and C2·6 (containing bothEA and EB regions), as well as control recombinant human α3(IV)NC1, were immobilized to Affi-Gel-10 (Bio-Rad) at a concentration of about 1 mg/ml gel. The IgG fraction of GP sera (∼2.5–5 mg) was applied to each of these affinity columns (containing approximately 0.5 ml of affinity gel). The columns were sequentially washed with 10 volumes (∼5 ml) of Tris-buffered saline (TBS), pH 7.5, and high salt buffer (1m sodium chloride in TBS). The bound IgG was sequentially eluted with increasing concentrations (four column volumes each of 1.5, 3, and 6 m solutions) of guanidinium hydrochloride (GdmCl) in 10 mm Tris buffer, pH 7.5; no additional GP reactivity was detected in fractions eluted under more stringent conditions, such as 8 m GdmCl or 6 m GdmCl at pH 3.0. GdmCl was removed from the eluted IgG fractions by repeated dilution with TBS followed by concentration in Centricon-10 tubes (Millipore). All IgG fractions were concentrated to the same volume (∼1 ml) for subsequent analysis by ELISA. Bovine serum albumin was added (∼1 mg/ml) as carrier protein to the diluted IgG fractions to prevent loss by adsorption. The procedure was repeated for all three GP sera. An alternative purification scheme was used for quantitation of autoantibody subpopulations and involved sequential passage of GP sera through columns containing immobilized α1(IV)NC1 domain,C6, C2, and α3(IV)NC1 domain; the bound autoantibodies were eluted with 6 m GdmCl and processed as above. The reactivity of the purified fractions against α3(IV)NC1 domain (100 ng/well) was assayed by ELISA and the concentration of GP antibodies in each fraction was read from a standard curve made using serial dilutions of GP IgG. Monoclonal antibodies to NC1 domains of type IV collagen (Mab1 (to α1(IV)NC1; also known as Mab6), Mab3 (to α3(IV)NC1; also known as Mab17) (11.Johansson C. Butkowski R. Wieslander J. Connect. Tissue Res. 1991; 25: 229-241Crossref PubMed Scopus (44) Google Scholar), and Mab5 (to α5(IV)NC1)) were purchased from Wieslab AB (Lund, Sweden). The monoclonal antibody M3/1, raised against an N-terminal synthetic peptide of human α3(IV)NC1 domain, and monoclonal antibodies 175, 178, and 189, raised against randomly folded recombinant human α3(IV)NC1 domain expressed in Escherichia coli, were previously described (12.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: 754-760Crossref PubMed Scopus (17) Google Scholar). For immunoblotting, the proteins (200 ng/lane of recombinant NC1 domains and chimeras, and 1 μg/lane of NC1 hexamers) were separated by SDS-polyacrylamide gel electrophoresis under nonreducing conditions and transferred to nitrocellulose membranes. After the remaining binding sites were blocked with casein, the membranes were incubated with appropriately diluted GP antibody fractions or monoclonal antibodies and then with alkaline phosphatase-labeled antibodies to human or mouse IgG, and the blots were developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. Direct and inhibition ELISA were performed as described (8.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: 11267-11274Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The antigens (50–100 ng of chimeras or NC1 domains for direct ELISA, 50 ng of recombinant human α3(IV)NC1 domain for inhibition ELISA) were coated onto plastic plates overnight, at room temperature, in 100 μl of carbonate buffer, pH 9.6. Nonspecific binding sites were blocked with 0.5% bovine serum albumin. Primary and secondary antibodies were diluted in binding buffer (TBS containing 0.2% bovine serum albumin and 0.05% Tween-20). Alkaline phosphatase-labeled antibodies to mouse IgG and human IgG (Sigma) were chosen so that they exhibited no cross-reactivity toward IgG from the other species. The substrate used was 1 mg/ml p-nitrophenol phosphate in 1 mdiethanolamine, pH 9.8, containing 0.5 mm zinc chloride, and color development was monitored at 410 nm with a Dynatech MR4000 plate reader. The results shown were the average of duplicate determinations that differed by less than 10%. A competition assay between GP autoantibodies and monoclonal antibodies (13.Hellmark T. Johansson C. Wieslander J. Kidney Int. 1994; 46: 823-829Abstract Full Text PDF PubMed Scopus (79) Google Scholar) was performed with modifications. Wells coated with α3(IV)NC1 domain (50 ng/well) were preincubated for 30 min with saturating concentrations of monoclonal antibodies Mab3, 175, and M3/1 (1/10–1/25 dilution of cell culture supernatants, as determined by titration); then, GP autoantibodies were added for 1 h, and the amount of human IgG bound was measured using anti-human IgG that did not cross-react with mouse IgG. The results were normalized relative to the binding of GP antibodies in the absence of monoclonal antibody competitor, which was assigned a value of 100%. Alternatively, the overlap of the epitopes was assessed in a two-antibody sandwich assay (14.Harlow E. Lane D. Maniatis T. Fritsch E.F. Sambrook J. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 579-583Google Scholar). Monoclonal antibodies purified on protein A-agarose in the presence of high salt (15.Harlow E. Lane D. Maniatis T. Fritsch E.F. Sambrook J. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 311Google Scholar) were immobilized overnight onto plastic plates (500 ng in 50 μl of binding buffer). After blocking the nonspecific binding sites with bovine serum albumin, the plates were incubated with antigen (250 ng α3(IV)NC1 domain in 50 μl of binding buffer) for 1 h, washed three times, and then incubated with affinity-purified GP autoantibodies for 1 h. Binding of the second antibody layer was detected using alkaline phosphatase-conjugate goat antibodies to human IgG. To correct for differences in the titers of GP antibody fractions, their reactivity toward α3(IV)NC1 domain captured by the monoclonal was normalized to their reactivity to α3(IV)NC1 domain coated directly onto plates by passive adsorption (50 ng/well). To study the sequestration of the GP epitopes within NC1 hexamers isolated from human GBM, an ELISA previously used for bovine hexamers (16.Kalluri R. Sun M.J. Hudson B.G. Neilson E.G. J. Biol. Chem. 1996; 271: 9062-9068Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) was used with the following modifications. Native or dissociated GBM hexamers (300 ng/well) were covalently coupled toN-hydroxysuccinimide-activated plates (Costar) in phosphate-buffered saline, pH 7.4. The native hexamers were dissociated by heating with 6 m GdmCl at 60 °C for 30–60 min or at 90 °C for 15 min and coupled to the plates in the presence of 3m GdmCl to prevent reassociation. Immunoprecipitation experiments were performed in parallel with native and dissociated (as above) NC1 hexamers from human GBM (2 μg), to which equal amounts of antibodies were added in 0.5 ml of TBS, pH 7.5. The following antibodies were used: GP-IgG from two patients, affinity-purified on the α3(IV)NC1 column; posttransplantation Alport alloantibodies eluted from a kidney biopsy, previously shown to bind α3(IV)NC1 (17.Hudson B.G. Kalluri R. Gunwar S. Weber M. Ballester F. Hudson J.K. Noelken M.E. Sarras M. Richardson W.R. Saus J. et al.Kidney Int. 1992; 42: 179-187Abstract Full Text PDF PubMed Scopus (85) Google Scholar); and the monoclonal antibody Mab3. To minimize reassociation of dissociated NC1 hexamer subunits (monomers and dimers), the hexamers in 6 m GdmCl were diluted directly (20–30-fold) in the antibody solution, as described previously (18.Wieslander J. Langeveld J. Butkowski R. Jodlowski M. Noelken M. Hudson B.G. J. Biol. Chem. 1985; 260: 8564-8570Abstract Full Text PDF PubMed Google Scholar). The reactions were allowed to proceed for 30–60 min at room temperature, and then the immune complexes were precipitated with protein G-agarose by gentle rocking for several hours at 4 °C, dissolved in sample buffer (15 min at 90 °C), separated by SDS-polyacrylamide gel electrophoresis, and analyzed by Western blotting with monoclonal antibodies to α3(IV)NC1 (Mab3) and α5(IV)NC1 (Mab5). Previous epitope mapping of GP autoantigen using a panel of α1/α3 chimeric NC1 domains has demonstrated that GP sera bind only chimerasC2 (containing the EA region of α3(IV)NC1) and C6 (containing theEB region), as well as the composite chimeraC2·6 (8.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: 11267-11274Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). To determine whether EA and EB make up a single or two distinct epitopes, GP sera were fractionated by affinity chromatography using the chimeras and α3(IV)NC1 domain as the matrix. Elution of bound autoantibodies was performed stepwise, with increasing concentrations of GdmCl, to separate IgG fractions with different affinities for the solid phase. The specificity of each fraction was determined by ELISA (Fig. 1). The C2-Affi-Gel column retained the antibodies reacting with the EA region of the α3(IV)NC1 domain. This column bound between half and two-thirds of the total reactivity against the α3(IV)NC1 domain in the three sera analyzed (Fig. 1,a–c). The specificity of the C2-bound autoantibodies fell into two distinct patterns. Those eluted at the 3–6 m concentration of GdmCl were specific for theEA epitope, reacting only with the C2chimera, and were therefore designatedGPA . 2In the nomenclature of the affinity-purified autoantibody fractions, the subscript was chosen to designate their reactivity toward the EA andEB regions. In contrast, the antibodies eluted at 1.5 m GdmCl showed additional reactivity to the C6 chimera and were therefore designated GPAB . A detailed examination of the properties of affinity-purified antibody fractions (see Figs. Figure 2, Figure 3, Figure 4below) further confirmed that GPA andGPAB populations are distinct.Figure 2Epitope specificity of GP autoantibody subpopulations. The reactivity of affinity-purified autoantibody subpopulations GPA (a),GPB (b), GPAB (c), and GPX (d) toward α1/α3 chimeric NC1 domains C1–C7 was assessed by ELISA (left panel, 100 ng of protein/well). The chimeras contained the following α3(IV)NC1 residues: 1–6 (C1), 17–31 (C2), 197–208 (C3), 218–232 (C4), 90–104 (C5), 127–141 (C6), and 1–14 plus four Gly-X-Ytriplets (C7). Chimeras showing reactivity in ELISA (C2, C6, and C2·6) were further analyzed by Western blotting (right panel, 200 ng of protein/lane). Recombinant α1(IV)NC1 and α3(IV)NC1 domains were used as negative and positive controls, respectively. NC1 hexamers isolated from human GBM were also used as positive control in Western blot (1 μg/lane).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The analysis of the fractions not bound to theC2-Affi-Gel revealed the presence of two additional populations of GP autoantibodies with distinct epitope specificity. In two of the three sera analyzed (GP-1 and GP-3), the nonbound fraction reacted with the C6 chimera but not with the C2chimera. Thus, these antibodies recognized specifically theEB region and were designatedGPB . The nonbound fraction of the third serum (GP-2) did not react with either C2 or C6chimeras but only with the α3(IV)NC1 domain and was therefore designated GPX . In contrast to the C2 column, much less autoantibody (5–20% of the total reactivity against α3(IV)NC1) bound to theC6 column. The C6 column retained the antibodies reacting with the EB region of the α3(IV)NC1 domain, i.e. the GPB andGPAB populations described above. The elution profiles for all three sera were similar; one example is shown in Fig.1 d. All eluted fractions reacted with both C2 andC6 chimeras, indicating the overlap of theGPB and GPAB fractions. The autoantibody fraction not bound to the C6column contained the GPA antibody subpopulation, which accounted for most reactivity with the C2 chimera (75–90%). The C2·6 column retained antibodies to bothEA and EB regions,i.e. the GPA ,GPB , and GPAB populations. In general, antibodies to EA bound to this column more strongly than those to EB and required higher concentration of GdmCl for elution (an example is shown in Fig. 1 e). In all three sera, about 25–35% of GP autoantibodies passed through the C2·6 column unbound; this fraction consisted of GPX autoantibodies. Affinity columns containing immobilized α3(IV)NC1 and α1(IV)NC1 domains were used as positive and negative controls, respectively. Most GP autoantibodies (90–95%) bound to the α3(IV)NC1-Affi-Gel, as shown in Fig. 1 f. As with the C2·6 column, autoantibodies reacting with C2 chimera required higher concentrations of GdmCl for elution and thus appeared to bind the α3(IV)NC1 domain stronger than those reacting with C6chimera. Only one serum (GP-3) contained antibodies that reacted with α1(IV)NC1. These antibodies were isolated on a column of immobilized α1(IV)NC1 domain, analyzed by direct and inhibition ELISA, and found to be very similar (if not identical) to theGPAB fraction of the same serum. The four populations of GP autoantibodies isolated by affinity chromatography were further characterized for specificity, abundance, and relative affinity for autoantigen (see Figs. Figure 2, Figure 3, Figure 4).GPA autoantibodies were the major population in all three GP sera, accounting for 61, 60, and 66% of the reactivity to α3(IV)NC1. They were purified from the C2 column, after elution of GPAB antibodies with 1.5m GdmCl. Alternatively, they were isolated in larger amounts by a two-step procedure: GP IgG was first passed through aC6-Affi-Gel column to remove GPAB andGPB fractions and then applied to aC2-Affi-Gel column, which retains GPA but not GPX autoantibodies. TheGPA fraction was specific for C2chimera and exhibited no cross-reactivity for other chimeras (Fig.2 a). In inhibition ELISA,C2 chimera was the only potent inhibitor of theGPA subpopulation, besides the positive control α3(IV)NC1 (Fig. 3 a). GPB autoantibodies were found in two out of the three sera analyzed, GP-1 and GP-3, and accounted for 10 and 6% of the reactivity to α3(IV)NC1. They were isolated from the C6column after previous passage of GP IgG through the C2column to remove GPA andGPAB fractions. In Western blots and direct ELISA, GPB autoantibodies were specific forC6 chimera and exhibited no cross-reactivity withC2 or other chimeras (Fig. 2 b). In inhibition ELISA, this fraction was inhibited only by the positive control, α3(IV)NC1 domain, and weakly by the C6 chimera (Fig.3 b). That C6 is about 50-fold weaker inhibitor of than α3(IV)NC1 may indicate that C6 does not encompass the full epitope for GPB antibodies. Moreover, thatGPB antibodies react well with C6immobilized on a solid phase (in direct ELISA and Western blot) suggests that these antibodies have sufficiently high avidity forC6 (due to multivalent binding) despite lower affinity. GPAB autoantibodies were separated from the C2 column by elution with 1.5 m GdmCl. They were present in all three sera but in relatively small amounts: 10, 6, and 12%. In Western blots and direct ELISA, this subpopulation reacted with bothC2 and C6 chimeras, and to a smaller extent with α1(IV)NC1 and the other chimeras (Fig. 2 c). In inhibition ELISA, the GPAB population showed preference either for the EA epitope, as in GP-2 serum, or for the EB epitope, as in GP-3 serum (Fig.3 c), as indicated by the difference (more than 10-fold) in the inhibitory potency of C2 and C6 chimeras, respectively. GPX autoantibodies constituted the second most abundant fraction in all sera analyzed, accounting for 20, 35, and 17% of the reactivity to α3(IV)NC1. They bound neither C2,C6, nor any other α1/α3 chimeras (Fig. 2 d) and were only inhibited by α3(IV)NC1 (Fig. 3 d). Based solely on these data, it could not be ascertained whether theGPX antibodies represented a homogeneous subpopulation or a mixture. The relative affinity of the autoantibody populations for α3(IV)NC1 was measured by inhibition ELISA (Fig.4), and their affinity was expressed as IC50, the concentration of soluble α3(IV)NC1 required to inhibit antibody binding to immobilized α3(IV)NC1 by 50%.GPA antibodies had IC50 values ranging between 0.015 and 0.3 μg/ml (0.6–12 nm). Due to relatively large amounts of antibodies required in this assay, the less abundant fractions GPB andGPAB were assayed together, as eluted from theC6 column in the 3 m GdmCl fraction. Their IC50 values ranged between 0.021 and 0.107 μg/ml (0.84–4.28 nm). GPX autoantibodies consistently had the lowest affinity for α3(IV)NC1 (range, 0.675–9.03 μg/ml, or 27–360 nm), at least 15-fold lower that other fractions from the same serum. Thus, the relative affinities of GPA and GPB antibodies were similar, and both were much higher than those ofGPX autoantibodies. The distinction, proximity, and accessibility of the multiple epitopes that bind the GP autoantibody populations were also investigated using five different monoclonal antibodies that were against the α3(IV)NC1 domain. First, the epitopes for the monoclonal antibodies were determined by ELISA (Fig. 5, top panel) and Western blot (not shown), using α1/α3 chimeras. Monoclonal antibody M3/1 reacted exclusively with C7, the chimera containing the 14 N-terminal amino acids of α3(IV)NC1 domain that were used as a synthetic peptide to prepare the antibody (12.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: 754-760Crossref PubMed Scopus (17) Google Scholar). Monoclonal antibodies 175, 178, and 189 reacted with only C5 chimera, indicating that their epitope is encompassed by residues 90–104 of the α3(IV)NC1 domain. Monoclonal antibody Mab3 reacted exclusively with chimera C2·6, but not with chimeras C2 orC6. Hence, regions EA andEB together, encompassing residues 17–31 and 127–141 (see above), form the epitope for Mab3. That Mab3 recognizedEA and EB regions jointly demonstrated the close proximity of these noncontiguous sites within α3(IV)NC1. Moreover, these results demonstrate that among the five monoclonal antibodies, Mab3 binds to the same region asGPA , GPB , andGPAB antibodies. Like GP antibodies, Mab3 also recognizes a conformational epitope, because its reactivity was completely abolished by the reduction of disulfide bonds of α3(IV)NC1 domain or C2·6 chimera (data not shown). In contrast, the epitopes for monoclonal antibodies 175, 178, 189, and M3/1 are linear because their reactivity with α3(IV)NC1 domain was relatively unaffected by reduction. The location of the epitopes of monoclonal antibodies and their relationship with the GP epitopes in regions EA and EB are shown in Fig. 5, bottom panel. The finding that EA andEB regions together encompass the epitope for Mab3 provided an independent strategy to determine the location of GP epitopes relative to those of Mab3 and other monoclonal antibodies to α3(IV)NC1 by competition experiments. Mab3 produced the highest inhibition of all GP subpopulations (50.7 ± 6.1%), compared with other monoclonal antibodies in competition ELISA (Fig.6, top panel). This must be caused by the close proximity and/or the overlap between the epitope of Mab3 and those for GPA ,GPB , and GPAB antibodies. Moreover, GPX antibodies were also inhibited significantly by Mab3, suggesting that their epitope must be in relatively close to the EA andEB regions. Although inhibition produced by the control monoclonal antibodies 175 (20 ± 4.8%) and M3/1 (33.7 ± 3.9%) was always lower than that produced with Mab3, it could not be ascertained whether this was due to proximity of the epitopes or mere steric hindrance. To distinguish between these possibilities, a two-antibody sandwich assay (14.Harlow E. Lane D. Maniatis T. Fritsch E.F. Sambrook J. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 579-583Google Scholar) was performed. The α3(IV)NC1 antigen was first bound to a layer of immobilized monoclonal antibody, and then its ability to further react with GP antibodies—and thus form an antibody-antigen-antibody “sandwich”—was measured by ELISA. As found by direct competition, GP antibodies could not bind α3(IV)NC1 simultaneously with Mab3, indicating that their epitopes are identical or in close proximity. Nevertheless, GP antibodies bound to α3(IV)NC1 captured by Mabs 175, 178, 189, and M3/1, indicating that the epitopes of these monoclonal antibodies were relatively far away from the GP epitopes. To better understand how GP autoantibodies bind to GBM in vivo, the pathogenic event that leads to glomerulonephritis, we next studied the reactivity of purified GP antibodies with the autoantigen in the native hexameric form. In basement membranes, α3(IV)NC1 domain occurs as a hexameric complex, which represents the end-to-end connection between two triple helical collagen molecules. Our previous studies of isolated NC1 hexamers of bovine origin revealed that the GP epitopes are sequestered and do not bind antibodies, unless the hexamer is dissociated into subunits by 6 m GdmCl or low pH (18.Wieslander J. Langeveld J. Butkowski R. Jodlowski M. Noelken M. Hudson B.G. J. Biol. Chem. 1985; 260: 8564-8570Abstract Full Text PDF PubMed Google Scholar). Later, this cryptic property was also demonstrated for human GBM hexamers (19.Weber M. Meyer zum Buschenfelde K.H. Kohler H. Clin. Exp. Immunol. 1988; 74: 289-294PubMed Google Scholar). However, identification of several subpopulations of GP antibodies raised the possibility that they may differ in their reactivity to native human GBM, not all being cryptic. Here, we investigated the relative accessibility of individual GP epitopes in the human NC1 hexamer complex by ELISA, as described previously (16.Kalluri R. Sun M.J. Hudson B.G. Neilson E.G. J. Biol. Chem. 1996; 271: 9062-9068Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), and by immunoprecipitation. Sequestration of GP epitopes within the hexamers was manifested in ELISA as a significant increase (3–4-fold) in the reactivity of GP sera with human GBM hexamers after dissociation (Fig.7, top panel), which contrasted with a decrease in the reactivity of control antibodies Mab3 and Alport alloantibodies (see below). GP autoantibody populations were individually assayed and found to react better with dissociated NC1 hexamers (Fig. 7, bottom panel), the relative reactivity of dissociated GBM hexamers ranging between 225 and 850% (on average, 398 ± 181%). Therefore, all GP epitopes are cryptic. The increased reactivity must have been caused by hexamer dissociation and not by denaturation of its subunits, because the treatment with GdmCl of α3(IV)NC1 monomers only produced a slight decrease (to 86 ± 10%) in their reactivity with GP antibodies. Moreover, treatment with GdmCl of native hexamers in the presence of reducing agents (such as dithiothreitol) strongly diminished their reactivity with GP antibodies (data not shown), consistent with the conformational nature of GP epitopes. Unlike GP antibodies, several control antibodies bound less to dissociated human GBM hexamers (20–50% of the reactivity of native hexamers), thus ruling out the possibility that hexamer reactivity was increased nonspecifically by the treatment with GdmCl (e.g.due to increased coating efficiency). These controls were antibodies of which the epitopes are known to be accessible in bovine GBM hexamers. Among these, posttransplantation Alport alloantibodies were used because they bind to the same α3·α4·α5 network of type IV collagen network as GP antibodies (20.Gunwar S. Ballester F. Noelken M.E. Sado Y. Ninomiya Y. Hudson B.G. J. Biol. Chem. 1998; 273: 8767-8775Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) and also produce anti-GBM glomerulonephritis. Mab3 and Mab1 are also known to react with intact NC1 hexamers, having been used to isolate hexamers containing α3(IV)NC1 and α1(IV)NC1, respectively, by affinity chromatography (21.Johansson C. Butkowski R. Wieslander J. J. Biol. Chem. 1992; 267: 24533-24537Abstract Full Text PDF PubMed Google Scholar, 22.Kahsai T.Z. Enders G.C. Gunwar S. Brunmark C. Wieslander J. Kalluri R. Zhou J. Noelken M.E. Hudson B.G. J. Biol. Chem. 1997; 272: 17023-17032Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Interestingly, the Mab3 epitope was readily accessible in the NC1 hexamer, in contrast with the GP epitopes, although both are located in the EA and EB regions, comprising 15 amino acids each. To address the nature of the small but measurable reactivity of native GBM hexamers in ELISA, the interaction between GBM hexamers and GP antibodies was further studied by immunoprecipitation. Because in this assay, binding occurred in solution, possible artifacts caused by immobilization of hexamers to the ELISA plates were excluded. GP antibodies from two different patients (affinity-purified on α3(IV)NC1) and control antibodies (Mab3 and Alport alloantibodies) were allowed to interact with both native and dissociated GBM hexamers under native-like conditions, and then the immune complexes were precipitated with protein G and analyzed by Western blot (Fig.8) for the presence of α3(IV)NC1 (top panel)—precipitated directly by antibodies—and α5(IV)NC1 (bottom panel)—co-precipitated only when associated with the α3(IV)NC1 domain. Whole GP IgG was used because much larger amounts of antibody were required in this assay compared with ELISA, and all GP populations were shown to be cryptic. When incubated with native hexamers, GP antibodies precipitated only α3(IV) monomers, but no α3(IV) dimers, although both were present in the original GBM hexamers (cf. Fig. 8, right lane). However, α3(IV)NC1 dimers were precipitated by GP antibodies from dissociated GBM hexamers (along with an increased amount α3(IV)NC1), showing that the inertness of dimers is not an intrinsic property but must be caused by sequestration of their GP epitopes within the hexamer configuration. Unlike GP antibodies, monoclonal antibody Mab3 and Alport alloantibodies precipitated both α3(IV)NC1 monomers and dimers without requiring prior dissociation of GBM hexamers. The control antibodies also co-precipitated the α5(IV)NC1 domain in both monomer and dimer forms, demonstrating binding to whole NC1 hexamers and thus the accessibility of their epitopes. In contrast, even though α3(IV)NC1 monomers were precipitated by GP antibodies from native GBM hexamers, they were not accompanied by α5(IV) monomers, indicating that GP antibodies bind only to dissociated α3(IV) monomers, but not to intact hexamers. Therefore, GP epitopes on the monomers are also cryptic and sequestered within the NC1 hexamers. Because these monomers were not present in the GBM hexamer preparation (as established by analytical gel-filtration), they must have been extracted from the native hexamer during the incubation with GP antibodies—either passively, due to a dynamic equilibrium between hexamer and its subunits, or actively, by the effect of antibodies on the hexamers. Overall, immunoprecipitation demonstrated that GP antibodies did not bind to intact GBM hexamers, although they reacted with α3(IV)NC1 monomers that were dislodged from the hexamer under nondenaturing conditions and with α3(IV)NC1 dimers after hexamer dissociation in vitro. Overall, our results demonstrate that GP autoantibodies are heterogeneous, consisting of a mixture of populations that recognize multiple epitopes on α3(IV)NC1 domain. Four distinct subpopulations, designated GPA ,GPB , GPAB , andGPX , were isolated by affinity chromatography using NC1 chimeras and characterized in this work.GPA and GPB antibodies were specific for the EA (residues 17–31) andEB (residues 127–141) regions of α3(IV)NC1, respectively. Hence, regions EA andEB encompass critical amino acid residues that constitute two distinct epitopes. GPAB subpopulation recognized both EA andEB regions individually, but not as a single entity. The cross-reactivity of GPAB antibodies suggests that their epitopes include amino acids conserved between the homologous EA and EB regions and therefore are distinct from the epitopes of the specificGPA and GPB subpopulations. GPX antibodies did not recognize either the EA or EB region, indicating the existence of one or more additional epitopes at a different location. Although EA andEB encompassed distinct epitopes for GP antibodies, they were both required for binding of Mab3 monoclonal antibody. That EA or EB together form a single epitope for Mab3 established the close proximity of these two regions in the native structure of the α3(IV)NC1 domain. Of several monoclonal antibodies recognizing different α3(IV)NC1 epitopes, only Mab3 competed effectively with GP autoantibodies, providing an independent confirmation that their epitopes map to the same regions." @default.
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• W2034215619 title "The Goodpasture Autoantigen" @default.
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