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Molecular Properties of the Goodpasture Epitope

2000; Elsevier BV; Volume: 275; Issue: 40 Linguagem: Inglês

10.1074/jbc.m004717200

1083-351X

Andreas Gunnarsson, Thomas Hellmark, Jörgen Wieslander,

Galectins and Cancer Biology

Goodpasture disease fulfils all criteria for a classical autoimmune disease, where autoantibodies targeted against the non-collagenous domain of the α3-chain of collagen IV initiates an inflammatory destruction of the basement membrane in kidney glomeruli and lung alveoli. This leads to a rapidly progressive glomerulonephritis and severe pulmonary hemorrhage. Previous studies have indicated a limited epitope for the toxic antibodies in the N-terminal part of the non-collagenous domain. The epitope has been partially characterized by recreating the epitope in the non-reactive α1-chain by exchanging nine residues to the corresponding ones of α3. In this study we have investigated to what extent each of these amino acids contribute to the antibody binding in different patient sera. The results show that seven of the nine substitutions are enough to get an epitope that is recognized equally well as the native α3-chain by all sera from 20 clinically verified Goodpasture patients. Furthermore, the patient sera reactivity against the different recombinant chains used in the study are very similar, with some minor exceptions, strongly supporting a highly defined and restricted epitope. We are convinced that the restriction of the epitope is of significant importance for the understanding of the etiology of the disease. Thereby also making every step on the way to characterization of the epitope a crucial step on the way to specific therapy for the disease. Goodpasture disease fulfils all criteria for a classical autoimmune disease, where autoantibodies targeted against the non-collagenous domain of the α3-chain of collagen IV initiates an inflammatory destruction of the basement membrane in kidney glomeruli and lung alveoli. This leads to a rapidly progressive glomerulonephritis and severe pulmonary hemorrhage. Previous studies have indicated a limited epitope for the toxic antibodies in the N-terminal part of the non-collagenous domain. The epitope has been partially characterized by recreating the epitope in the non-reactive α1-chain by exchanging nine residues to the corresponding ones of α3. In this study we have investigated to what extent each of these amino acids contribute to the antibody binding in different patient sera. The results show that seven of the nine substitutions are enough to get an epitope that is recognized equally well as the native α3-chain by all sera from 20 clinically verified Goodpasture patients. Furthermore, the patient sera reactivity against the different recombinant chains used in the study are very similar, with some minor exceptions, strongly supporting a highly defined and restricted epitope. We are convinced that the restriction of the epitope is of significant importance for the understanding of the etiology of the disease. Thereby also making every step on the way to characterization of the epitope a crucial step on the way to specific therapy for the disease. enzyme-linked immunosorbent assay Goodpasture disease is known and characterized as a classic autoimmune disease. The disease is B cell and antibody mediated, with autoantibodies directed against proteins in the glomerular basement membrane and lung alveoli. When bound to self-structures in the kidney and lung, the antibodies initiate an inflammatory destruction of tissue by recruitment of complement leading to rapidly progressive glomerulonephritis often accompanied with severe and life threatening lung hemorrhage. The major self-epitope is located on the α3-NC1 domain of collagen IV. Collagen IV α3-chain has a limited distribution in the body and is only found in a few specialized basement membranes including the glomerular and alveolar basement membranes, thus explaining the specific organ involvement in Goodpasture disease. Goodpasture disease is indeed an antibody mediated disease as proven by the transfer of disease to monkeys by injection of kidney bound antibodies from Goodpasture patients (1Lerner R.A. Glassock R.J. Dixon F.J. J. Exp. Med. 1967; 126: 989-1004Crossref PubMed Scopus (529) Google Scholar) and the therapeutic effect of treating patients with plasma exchange and immunosuppressive drugs to reduce the amount of circulating antibodies (2Turner N. Lockwood C.M. Rees A.J. Schrier R.W. Gottschalk C.W. Diseases of the Kidney. Little Brown and Co., Boston1993: 1865-1894Google Scholar). The Goodpasture epitope is a conformational epitope, which is indicated by the loss of reactivity to autoantibodies when the tertiary protein structure is disrupted by reduction of disulfide bonds (3Wieslander J. Bygren P. Heinegard D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1544-1548Crossref PubMed Google Scholar). The epitope is also known as a cryptotope, i.e. the epitope is hidden in the native protein structure and is fully exposed first when the protein is partially denatured (4Wieslander 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).Trying to map an epitope for a specific autoimmune disorder is very difficult, since in most cases autoantibodies against a variety of epitopes on the target structure are formed. For Goodpasture syndrome a limited epitope distribution was indicated when binding of autoantibodies to collagen IV was successfully blocked by one single monoclonal antibody (5Pusey C.D. Dash A. Kershaw M.J. Morgan A. Reilly A. Rees A.J. Lockwood C.M. Lab. Invest. 1987; 56: 23-31PubMed Google Scholar, 6Hellmark T. Johansson C. Wieslander J. Kidney Int. 1994; 46: 823-829Abstract Full Text PDF PubMed Scopus (78) Google Scholar). In our first attempt to map the Goodpasture epitope we used linear synthetic peptides of the α3(IV) NC1 domain to block the binding of autoantibodies to collagen IV (7Hellmark T. Brunmark C. Trojnar J. Wieslander J. Clin. Exp. Immunol. 1996; 105: 504-510Crossref PubMed Scopus (24) Google Scholar). With this method we were unable to map any epitope on the α3(IV) NC1 domain although for one patient an epitope on the α1(IV) NC1 domain was found. In a study by Kalluri et al. (8Kalluri 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: 24018-24024Abstract Full Text PDF PubMed Google Scholar), using linear peptides, they suggested the C-terminal part of the α3 (IV) NC1 domain to comprise the Goodpasture epitope. However this study as well as ours suffered from the disadvantage of using linear peptides to characterize a conformational epitope and the results have not been confirmed.To avoid the problem with linear peptides and mal-folded recombinants expressed in bacterial systems, new mapping strategies has been initiated by several groups, where recombinant collagen IV is expressed in eukaryotic cell lines. By substitution of amino acid residues in the α3(IV) NC1 against the corresponding ones from the homologous but non-reactive α1(IV) chain, and expressing the constructs in an eukaryotic expression system correctly folded proteins with intact conformational epitopes are produced. All these studies have emphasized the N-terminal part of the α3 (IV) NC1 as the principal epitope region (9Quinones S. Bernal D. Garcia-Sogo M. Elena S.F. Saus J. J. Biol. Chem. 1992; 267: 19780-19784Abstract Full Text PDF PubMed Google Scholar, 10Kalluri R. Sun M.J. Hudson B.G. Neilson E.G. J. Biol. Chem. 1996; 271: 9062-9068Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 11Ryan J.J. Mason P.J. Pusey C.D. Turner N. Clin. Exp. Immunol. 1998; 113: 17-27Crossref PubMed Scopus (36) Google Scholar). Furthermore, we found that only autoantibodies against the N-terminal third of the α3(IV) NC1 domain are pathologically significant (12Hellmark T. Segelmark M. Unger C. Burkhardt H. Saus J. Wieslander J. Kidney Int. 1999; 55: 936-944Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Following studies have revealed a small region within the N-terminal part of the α3 NC1 as the major target for the circulating antibodies. This epitope has then been recreated by substitution of a few a.a. residues in the non-reactive α1-chain to the corresponding ones from α3 (13Netzer 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 (128) Google Scholar, 14Borza D.B. Netzer K.-O. Leinonen A. Todd P. Cervera J. Saus J. Hudson B.G. J. Biol. Chem. 2000; 275: 6030-6037Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 15Hellmark T. Burkhardt H. Wieslander J. J. Biol. Chem. 1999; 274: 25862-25868Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In our hands, all 20 patients in the cohort recognized this epitope (15Hellmark T. Burkhardt H. Wieslander J. J. Biol. Chem. 1999; 274: 25862-25868Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar).The principal goal for this study was to investigate if a reactive epitope could be created in the α1(IV) NC1 with fewer than the nine substitutions, previously reported, and to what extent each of these amino acids contribute to the antibody binding in different patient sera.RESULTSIn a previous study we were able to recreate an immunoreactive epitope in the non-reactive α1(IV) NC1 domain by substitution of single amino acids to the corresponding ones of α3 (15Hellmark T. Burkhardt H. Wieslander J. J. Biol. Chem. 1999; 274: 25862-25868Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In that study two principle constructs were made, one with five amino acid substitutions in positions known to be critical for epitope recognition (P5–P9), called the S1 construct, and one with four additional substitutions in non-conserved positions in the same region (P1–P4), called S2. It was shown that the five substitution recombinants only reacted weak with the antibodies, whereas the nine-substitution recombinant showed immunoreactivity to all tested sera. In this present study, we have investigated what impact each of these amino acids has on the affinity of autoantibodies from different patients. Thereby further characterizing the immune response in Goodpasture disease to this major epitope. To achieve this, all possible combinations of the four additional substitutions were made in a total of 14 new recombinants. The S1 construct was used as template, and then subsequent substitutions were introduced with site-directed mutagenesis using the primers listed in Table I.Initial Screening of ReactivityInitial tests of the 14 recombinants' immunoreactivity against all 20 patient sera were performed using ELISA and inhibition ELISA. Of the 14 tested recombinants only five were reactive with the patient sera, one of the seven substitutions (R12) and four of the eight substitutions (R123, R124, R134, and R234). The other recombinants did not show reactivity above the reactivity against the recombinant α1(IV) NC1 (data not shown) and therefore not further evaluated. The reactive recombinants R12, R123, R134, and R234 were carefully analyzed again to establish their reactivity compared with the nine-substitution recombinant (S2), recombinant α3, and recombinant α1. In Fig. 2, the immunoblotting results are shown.ELISAThe recombinants were first analyzed using direct ELISA (Fig. 3). With this assay, we found no significant difference in reactivity for the R12 recombinant compared with the S2 protein. In line with these results, none of the eight substitution recombinants had a significantly lower reactivity than the α3 recombinant. Although it is noticeable that recombinant R134 that lack substitution 2 has a significantly lower reactivity than R12, probably indicating a greater importance for substitution 2. In fact, two of the samples did not react with this R134 recombinant, and another six of them showed low reactivity.Figure 3Mean absorbance in the direct ELISA where the different recombinants were incubated with sera from 20 biopsy-proven Goodpasture patients. Bars indicate S.D. values.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Inhibition ELISAIn contrast to the direct ELISA, the inhibition ELISA revealed differences in affinity to the different recombinants. The highest inhibitory effect was found using recombinant α3, S2, or R12, while all the eight substitution recombinants showed a significantly lower inhibitory capacity than the seven-substitution R12 recombinant. The inhibition curves from one of the samples are shown in Fig. 4 a, and the mean normalized inhibition values for all samples are shown in Fig.4 b.Figure 4a, one representative Goodpasture serum is inhibited by different amounts of recombinant proteins.Crosses indicate recombinant α1(IV) NC1, boxesrecombinant α3(IV) NC1, triangles the S1 recombinant,closed circles the S2 and the R12 recombinants, open circles the R134 recombinant, and diamonds the R234, R123, and R124 recombinants. b, all recombinants were tested for their ability to inhibit the binding of antibodies from the Goodpasture patients to native α3. Equal amounts of recombinant protein were added, and the greatest inhibition was defined as 100% inhibition and used as reference for determining the inhibition for the other recombinants. Displayed in the figure is the mean inhibition of the sera in percentage for each recombinant. Bars indicate S.D. values.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In conclusion, the ELISA studies have shown that the seven-substitution recombinant R12 is as reactive as the native α3 and the nine substitution recombinant S2 described previously (15Hellmark T. Burkhardt H. Wieslander J. J. Biol. Chem. 1999; 274: 25862-25868Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar).DISCUSSIONCirculating autoantibodies against different parts of the α3(IV) chain, as well as antibodies against other α(IV) chains, are detected in serum from patients with Goodpasture disease (6Hellmark T. Johansson C. Wieslander J. Kidney Int. 1994; 46: 823-829Abstract Full Text PDF PubMed Scopus (78) Google Scholar, 19Neilson E.G. Kalluri R. Sun M.J. Gunwar S. Danoff T. Mariyama M. Maers J.C. Reeders S.T. Hudson B.G. J. Biol. Chem. 1993; 268: 8402-8405Abstract Full Text PDF PubMed Google Scholar). However, the major epitope region is found to be the N-terminal part of the α3(IV) NC1 (9Quinones S. Bernal D. Garcia-Sogo M. Elena S.F. Saus J. J. Biol. Chem. 1992; 267: 19780-19784Abstract Full Text PDF PubMed Google Scholar, 11Ryan J.J. Mason P.J. Pusey C.D. Turner N. Clin. Exp. Immunol. 1998; 113: 17-27Crossref PubMed Scopus (36) Google Scholar), and only antibodies against this part of the molecule correlate with disease progression (12Hellmark T. Segelmark M. Unger C. Burkhardt H. Saus J. Wieslander J. Kidney Int. 1999; 55: 936-944Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 15Hellmark T. Burkhardt H. Wieslander J. J. Biol. Chem. 1999; 274: 25862-25868Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In this study we have further narrowed down and characterized the molecular properties of this epitope recognized by the pathogenic autoantibodies. A recombinant protein comprised of the α1(IV) NC1 domain with seven amino acids substituted to the corresponding ones from the α3 chain was constructed. This recombinant protein, R12, was recognized by the autoantibodies from all patients with Goodpasture disease, in both direct ELISA and in inhibition ELISA, to the same degree as recombinant α3. These results support the previous findings where we and others have localized the major epitope to the same region (13Netzer 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 (128) Google Scholar, 14Borza D.B. Netzer K.-O. Leinonen A. Todd P. Cervera J. Saus J. Hudson B.G. J. Biol. Chem. 2000; 275: 6030-6037Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 15Hellmark T. Burkhardt H. Wieslander J. J. Biol. Chem. 1999; 274: 25862-25868Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar).The very limited region recognized by all Goodpasture sera indicates a similar immunization and maturation process in all patients. Interestingly, an overlapping T-cell epitope is found by Phelps and co-workers (20Phelps R.G. Jones V.L. Coughlan M. Turner A.N. Rees A.J. J. Biol. Chem. 1998; 273: 11440-11447Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 21Phelps R.G. Rees A.J. Kidney Int. 1999; 56: 1638-1653Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). However, if this process is initiated by a foreign immunogen, i.e. molecular mimicry, or if it is a self-immunization with degraded type IV collagen, is yet unknown.As shown in Fig. 5 a, the epitope is localized to a small loop in the secondary sequence. The epitope seems to be dependent on correct folding of this loop, indicated both by the fact that disruption of the disulfide bridges (4Wieslander 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,7Hellmark T. Brunmark C. Trojnar J. Wieslander J. Clin. Exp. Immunol. 1996; 105: 504-510Crossref PubMed Scopus (24) Google Scholar) as well as changes of the charge of certain residues disturb the immunoreactivity. This is shown by the substitutions in positions 3 and 4, of which one, but not both, of the resulting amino acids must have a negative charge for the epitope to be fully recognized. In R124 both residues are negatively charged, and in R123 both residues are uncharged, and in both, the changes result in a loss of affinity. The preservation of the positively charged lysine in position P2, seen in the S1 and R134 recombinants (Fig. 5 b), dramatically reduce affinity to the recombinants. Surprisingly, the R12, as well as the S2, R123, and R124, actually reacted stronger than the recombinant α3(IV) in the direct ELISA. This could possibly be explained by a less rigid structure in the α1(IV) background that results in a more accessible epitope. This theory is supported by the fact that this difference in reactivity did not appear when the recombinants were analyzed using inhibition ELISA. An alternative explanation for the higher reactivity could be that the produced recombinant, e.g. R12, displays an epitope more similar to a hypothetical mimicry structure than the native α3(IV) NC1 does.Figure 5a, a model (22Siebold B. Deutzmann R. Kühn K. Eur. J. Biochem. 1988; 176: 617-624Crossref PubMed Scopus (85) Google Scholar) of the R12 NC1 domain from type IV collagen, where each ball represents one amino acid. The nine amino acid residues P1–P9 are indicated with a dark gray tone. The epitope region is enlarged, and the different residues are indicated with one-letter symbols. Here the mutated residues are indicated with a dark gray tone. The P1–P9 positions are indicated as well as the charge or polarity of these amino acids.b, a lineup showing the amino acid sequence for the analyzed recombinants covering the principal epitope region, including all positions for substitution except P9. The amino acid in the P9 position is common for the α3(IV) NC1 and the recombinants, but differs from the corresponding one of α1(IV) NC1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Although all samples recognize one very limited area on the α3(IV) NC1, there are some differences in recognition pattern between the different samples. As discussed above the effect of charge changes within the loop have different effect on antibodies from different sera (especially the R134). Furthermore the relative amount of antibodies against the epitope defined by the R12 constructs varies from serum to serum, ranging from 65 to 95%.We believe that this study of the Goodpasture epitope adds new and important data that will help us to understand the underlying immunological mechanisms in Goodpasture disease in particular, but also for autoimmune diseases in general. By using the R12 recombinant protein in an assay instead of the complete α3(IV) NC1, a more specific diagnostic test could be developed that could distinguish between pathogenic antibodies and harmless autoantibodies. Goodpasture disease is known and characterized as a classic autoimmune disease. The disease is B cell and antibody mediated, with autoantibodies directed against proteins in the glomerular basement membrane and lung alveoli. When bound to self-structures in the kidney and lung, the antibodies initiate an inflammatory destruction of tissue by recruitment of complement leading to rapidly progressive glomerulonephritis often accompanied with severe and life threatening lung hemorrhage. The major self-epitope is located on the α3-NC1 domain of collagen IV. Collagen IV α3-chain has a limited distribution in the body and is only found in a few specialized basement membranes including the glomerular and alveolar basement membranes, thus explaining the specific organ involvement in Goodpasture disease. Goodpasture disease is indeed an antibody mediated disease as proven by the transfer of disease to monkeys by injection of kidney bound antibodies from Goodpasture patients (1Lerner R.A. Glassock R.J. Dixon F.J. J. Exp. Med. 1967; 126: 989-1004Crossref PubMed Scopus (529) Google Scholar) and the therapeutic effect of treating patients with plasma exchange and immunosuppressive drugs to reduce the amount of circulating antibodies (2Turner N. Lockwood C.M. Rees A.J. Schrier R.W. Gottschalk C.W. Diseases of the Kidney. Little Brown and Co., Boston1993: 1865-1894Google Scholar). The Goodpasture epitope is a conformational epitope, which is indicated by the loss of reactivity to autoantibodies when the tertiary protein structure is disrupted by reduction of disulfide bonds (3Wieslander J. Bygren P. Heinegard D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1544-1548Crossref PubMed Google Scholar). The epitope is also known as a cryptotope, i.e. the epitope is hidden in the native protein structure and is fully exposed first when the protein is partially denatured (4Wieslander 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). Trying to map an epitope for a specific autoimmune disorder is very difficult, since in most cases autoantibodies against a variety of epitopes on the target structure are formed. For Goodpasture syndrome a limited epitope distribution was indicated when binding of autoantibodies to collagen IV was successfully blocked by one single monoclonal antibody (5Pusey C.D. Dash A. Kershaw M.J. Morgan A. Reilly A. Rees A.J. Lockwood C.M. Lab. Invest. 1987; 56: 23-31PubMed Google Scholar, 6Hellmark T. Johansson C. Wieslander J. Kidney Int. 1994; 46: 823-829Abstract Full Text PDF PubMed Scopus (78) Google Scholar). In our first attempt to map the Goodpasture epitope we used linear synthetic peptides of the α3(IV) NC1 domain to block the binding of autoantibodies to collagen IV (7Hellmark T. Brunmark C. Trojnar J. Wieslander J. Clin. Exp. Immunol. 1996; 105: 504-510Crossref PubMed Scopus (24) Google Scholar). With this method we were unable to map any epitope on the α3(IV) NC1 domain although for one patient an epitope on the α1(IV) NC1 domain was found. In a study by Kalluri et al. (8Kalluri 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: 24018-24024Abstract Full Text PDF PubMed Google Scholar), using linear peptides, they suggested the C-terminal part of the α3 (IV) NC1 domain to comprise the Goodpasture epitope. However this study as well as ours suffered from the disadvantage of using linear peptides to characterize a conformational epitope and the results have not been confirmed. To avoid the problem with linear peptides and mal-folded recombinants expressed in bacterial systems, new mapping strategies has been initiated by several groups, where recombinant collagen IV is expressed in eukaryotic cell lines. By substitution of amino acid residues in the α3(IV) NC1 against the corresponding ones from the homologous but non-reactive α1(IV) chain, and expressing the constructs in an eukaryotic expression system correctly folded proteins with intact conformational epitopes are produced. All these studies have emphasized the N-terminal part of the α3 (IV) NC1 as the principal epitope region (9Quinones S. Bernal D. Garcia-Sogo M. Elena S.F. Saus J. J. Biol. Chem. 1992; 267: 19780-19784Abstract Full Text PDF PubMed Google Scholar, 10Kalluri R. Sun M.J. Hudson B.G. Neilson E.G. J. Biol. Chem. 1996; 271: 9062-9068Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 11Ryan J.J. Mason P.J. Pusey C.D. Turner N. Clin. Exp. Immunol. 1998; 113: 17-27Crossref PubMed Scopus (36) Google Scholar). Furthermore, we found that only autoantibodies against the N-terminal third of the α3(IV) NC1 domain are pathologically significant (12Hellmark T. Segelmark M. Unger C. Burkhardt H. Saus J. Wieslander J. Kidney Int. 1999; 55: 936-944Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Following studies have revealed a small region within the N-terminal part of the α3 NC1 as the major target for the circulating antibodies. This epitope has then been recreated by substitution of a few a.a. residues in the non-reactive α1-chain to the corresponding ones from α3 (13Netzer 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 (128) Google Scholar, 14Borza D.B. Netzer K.-O. Leinonen A. Todd P. Cervera J. Saus J. Hudson B.G. J. Biol. Chem. 2000; 275: 6030-6037Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 15Hellmark T. Burkhardt H. Wieslander J. J. Biol. Chem. 1999; 274: 25862-25868Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In our hands, all 20 patients in the cohort recognized this epitope (15Hellmark T. Burkhardt H. Wieslander J. J. Biol. Chem. 1999; 274: 25862-25868Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The principal goal for this study was to investigate if a reactive epitope could be created in the α1(IV) NC1 with fewer than the nine substitutions, previously reported, and to what extent each of these amino acids contribute to the antibody binding in different patient sera. RESULTSIn a previous study we were able to recreate an immunoreactive epitope in the non-reactive α1(IV) NC1 domain by substitution of single amino acids to the corresponding ones of α3 (15Hellmark T. Burkhardt H. Wieslander J. J. Biol. Chem. 1999; 274: 25862-25868Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In that study two principle constructs were made, one with five amino acid substitutions in positions known to be critical for epitope recognition (P5–P9), called the S1 construct, and one with four additional substitutions in non-conserved positions in the same region (P1–P4), called S2. It was shown that the five substitution recombinants only reacted weak with the antibodies, whereas the nine-substitution recombinant showed immunoreactivity to all tested sera. In this present study, we have investigated what impact each of these amino acids has on the affinity of autoantibodies from different patients. Thereby further characterizing the immune response in Goodpasture disease to this major epitope. To achieve this, all possible combinations of the four additional substitutions were made in a total of 14 new recombinants. The S1 construct was used as template, and then subsequent substitutions were introduced with site-directed mutagenesis using the primers listed in Table I.Initial Screening of ReactivityInitial tests of the 14 recombinants' immunoreactivity against all 20 patient sera were performed using ELISA and inhibition ELISA. Of the 14 tested recombinants only five were reactive with the patient sera, one of the seven substitutions (R12) and four of the eight substitutions (R123, R124, R134, and R234). The other recombinants did not show reactivity above the reactivity against the recombinant α1(IV) NC1 (data not shown) and therefore not further evaluated. The reactive recombinants R12, R123, R134, and R234 were carefully analyzed again to establish their reactivity compared with the nine-substitution recombinant (S2), recombinant α3, and recombinant α1. In Fig. 2, the immunoblotting results are shown.ELISAThe recombinants were first analyzed using direct ELISA (Fig. 3). With this assay, we found no significant difference in reactivity for the R12 recombinant compared with the S2 protein. In line with these results, none of the eight substitution recombinants had a significantly lower reactivity than the α3 recombinant. Although it is noticeable that recombinant R134 that lack substitution 2 has a significantly lower reactivity than R12, probably indicating a greater importance for substitution 2. In fact, two of the samples did not react with this R134 recombinant, and another six of them showed low reactivity.Inhibition ELISAIn contrast to the direct ELISA, the inhibition ELISA revealed differences in affinity to the different recombinants. The highest inhibitory effect was found using recombinant α3, S2, or R12, while all the eight substitution recombinants showed a significantly lower inhibitory capacity than the seven-substitution R12 recombinant. The inhibition curves from one of the samples are shown in Fig. 4 a, and the mean normalized inhibition values for all samples are shown in Fig.4 b.Figure 4a, one representative Goodpasture serum is inhibited by different amounts of recombinant proteins.Crosses indicate recombinant α1(IV) NC1, boxesrecombinant α3(IV) NC1, triangles the S1 recombinant,closed circles the S2 and the R12 recombinants, open circles the R134 recombinant, and diamonds the R234, R123, and R124 recombinants. b, all recombinants were tested for their ability to inhibit the binding of antibodies from the Goodpasture patients to native α3. Equal amounts of recombinant protein were added, and the greatest inhibition was defined as 100% inhibition and used as reference for determining the inhibition for the other recombinants. Displayed in the figure is the mean inhibition of the sera in percentage for each recombinant. Bars indicate S.D. values.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In conclusion, the ELISA studies have shown that the seven-substitution recombinant R12 is as reactive as the native α3 and the nine substitution recombinant S2 described previously (15Hellmark T. Burkhardt H. Wieslander J. J. Biol. Chem. 1999; 274: 25862-25868Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In a previous study we were able to recreate an immunoreactive epitope in the non-reactive α1(IV) NC1 domain by substitution of single amino acids to the corresponding ones of α3 (15Hellmark T. Burkhardt H. Wieslander J. J. Biol. Chem. 1999; 274: 25862-25868Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In that study two principle constructs were made, one with five amino acid substitutions in positions known to be critical for epitope recognition (P5–P9), called the S1 construct, and one with four additional substitutions in non-conserved positions in the same region (P1–P4), called S2. It was shown that the five substitution recombinants only reacted weak with the antibodies, whereas the nine-substitution recombinant showed immunoreactivity to all tested sera. In this present study, we have investigated what impact each of these amino acids has on the affinity of autoantibodies from different patients. Thereby further characterizing the immune response in Goodpasture disease to this major epitope. To achieve this, all possible combinations of the four additional substitutions were made in a total of 14 new recombinants. The S1 construct was used as template, and then subsequent substitutions were introduced with site-directed mutagenesis using the primers listed in Table I. Initial Screening of ReactivityInitial tests of the 14 recombinants' immunoreactivity against all 20 patient sera were performed using ELISA and inhibition ELISA. Of the 14 tested recombinants only five were reactive with the patient sera, one of the seven substitutions (R12) and four of the eight substitutions (R123, R124, R134, and R234). The other recombinants did not show reactivity above the reactivity against the recombinant α1(IV) NC1 (data not shown) and therefore not further evaluated. The reactive recombinants R12, R123, R134, and R234 were carefully analyzed again to establish their reactivity compared with the nine-substitution recombinant (S2), recombinant α3, and recombinant α1. In Fig. 2, the immunoblotting results are shown.ELISAThe recombinants were first analyzed using direct ELISA (Fig. 3). With this assay, we found no significant difference in reactivity for the R12 recombinant compared with the S2 protein. In line with these results, none of the eight substitution recombinants had a significantly lower reactivity than the α3 recombinant. Although it is noticeable that recombinant R134 that lack substitution 2 has a significantly lower reactivity than R12, probably indicating a greater importance for substitution 2. In fact, two of the samples did not react with this R134 recombinant, and another six of them showed low reactivity.Inhibition ELISAIn contrast to the direct ELISA, the inhibition ELISA revealed differences in affinity to the different recombinants. The highest inhibitory effect was found using recombinant α3, S2, or R12, while all the eight substitution recombinants showed a significantly lower inhibitory capacity than the seven-substitution R12 recombinant. The inhibition curves from one of the samples are shown in Fig. 4 a, and the mean normalized inhibition values for all samples are shown in Fig.4 b.In conclusion, the ELISA studies have shown that the seven-substitution recombinant R12 is as reactive as the native α3 and the nine substitution recombinant S2 described previously (15Hellmark T. Burkhardt H. Wieslander J. J. Biol. Chem. 1999; 274: 25862-25868Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Initial Screening of ReactivityInitial tests of the 14 recombinants' immunoreactivity against all 20 patient sera were performed using ELISA and inhibition ELISA. Of the 14 tested recombinants only five were reactive with the patient sera, one of the seven substitutions (R12) and four of the eight substitutions (R123, R124, R134, and R234). The other recombinants did not show reactivity above the reactivity against the recombinant α1(IV) NC1 (data not shown) and therefore not further evaluated. The reactive recombinants R12, R123, R134, and R234 were carefully analyzed again to establish their reactivity compared with the nine-substitution recombinant (S2), recombinant α3, and recombinant α1. In Fig. 2, the immunoblotting results are shown.ELISAThe recombinants were first analyzed using direct ELISA (Fig. 3). With this assay, we found no significant difference in reactivity for the R12 recombinant compared with the S2 protein. In line with these results, none of the eight substitution recombinants had a significantly lower reactivity than the α3 recombinant. Although it is noticeable that recombinant R134 that lack substitution 2 has a significantly lower reactivity than R12, probably indicating a greater importance for substitution 2. In fact, two of the samples did not react with this R134 recombinant, and another six of them showed low reactivity.Inhibition ELISAIn contrast to the direct ELISA, the inhibition ELISA revealed differences in affinity to the different recombinants. The highest inhibitory effect was found using recombinant α3, S2, or R12, while all the eight substitution recombinants showed a significantly lower inhibitory capacity than the seven-substitution R12 recombinant. The inhibition curves from one of the samples are shown in Fig. 4 a, and the mean normalized inhibition values for all samples are shown in Fig.4 b.In conclusion, the ELISA studies have shown that the seven-substitution recombinant R12 is as reactive as the native α3 and the nine substitution recombinant S2 described previously (15Hellmark T. Burkhardt H. Wieslander J. J. Biol. Chem. 1999; 274: 25862-25868Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Initial Screening of ReactivityInitial tests of the 14 recombinants' immunoreactivity against all 20 patient sera were performed using ELISA and inhibition ELISA. Of the 14 tested recombinants only five were reactive with the patient sera, one of the seven substitutions (R12) and four of the eight substitutions (R123, R124, R134, and R234). The other recombinants did not show reactivity above the reactivity against the recombinant α1(IV) NC1 (data not shown) and therefore not further evaluated. The reactive recombinants R12, R123, R134, and R234 were carefully analyzed again to establish their reactivity compared with the nine-substitution recombinant (S2), recombinant α3, and recombinant α1. In Fig. 2, the immunoblotting results are shown. Initial tests of the 14 recombinants' immunoreactivity against all 20 patient sera were performed using ELISA and inhibition ELISA. Of the 14 tested recombinants only five were reactive with the patient sera, one of the seven substitutions (R12) and four of the eight substitutions (R123, R124, R134, and R234). The other recombinants did not show reactivity above the reactivity against the recombinant α1(IV) NC1 (data not shown) and therefore not further evaluated. The reactive recombinants R12, R123, R134, and R234 were carefully analyzed again to establish their reactivity compared with the nine-substitution recombinant (S2), recombinant α3, and recombinant α1. In Fig. 2, the immunoblotting results are shown. ELISAThe recombinants were first analyzed using direct ELISA (Fig. 3). With this assay, we found no significant difference in reactivity for the R12 recombinant compared with the S2 protein. In line with these results, none of the eight substitution recombinants had a significantly lower reactivity than the α3 recombinant. Although it is noticeable that recombinant R134 that lack substitution 2 has a significantly lower reactivity than R12, probably indicating a greater importance for substitution 2. In fact, two of the samples did not react with this R134 recombinant, and another six of them showed low reactivity. The recombinants were first analyzed using direct ELISA (Fig. 3). With this assay, we found no significant difference in reactivity for the R12 recombinant compared with the S2 protein. In line with these results, none of the eight substitution recombinants had a significantly lower reactivity than the α3 recombinant. Although it is noticeable that recombinant R134 that lack substitution 2 has a significantly lower reactivity than R12, probably indicating a greater importance for substitution 2. In fact, two of the samples did not react with this R134 recombinant, and another six of them showed low reactivity. Inhibition ELISAIn contrast to the direct ELISA, the inhibition ELISA revealed differences in affinity to the different recombinants. The highest inhibitory effect was found using recombinant α3, S2, or R12, while all the eight substitution recombinants showed a significantly lower inhibitory capacity than the seven-substitution R12 recombinant. The inhibition curves from one of the samples are shown in Fig. 4 a, and the mean normalized inhibition values for all samples are shown in Fig.4 b.In conclusion, the ELISA studies have shown that the seven-substitution recombinant R12 is as reactive as the native α3 and the nine substitution recombinant S2 described previously (15Hellmark T. Burkhardt H. Wieslander J. J. Biol. Chem. 1999; 274: 25862-25868Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In contrast to the direct ELISA, the inhibition ELISA revealed differences in affinity to the different recombinants. The highest inhibitory effect was found using recombinant α3, S2, or R12, while all the eight substitution recombinants showed a significantly lower inhibitory capacity than the seven-substitution R12 recombinant. The inhibition curves from one of the samples are shown in Fig. 4 a, and the mean normalized inhibition values for all samples are shown in Fig.4 b. In conclusion, the ELISA studies have shown that the seven-substitution recombinant R12 is as reactive as the native α3 and the nine substitution recombinant S2 described previously (15Hellmark T. Burkhardt H. Wieslander J. J. Biol. Chem. 1999; 274: 25862-25868Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). DISCUSSIONCirculating autoantibodies against different parts of the α3(IV) chain, as well as antibodies against other α(IV) chains, are detected in serum from patients with Goodpasture disease (6Hellmark T. Johansson C. Wieslander J. Kidney Int. 1994; 46: 823-829Abstract Full Text PDF PubMed Scopus (78) Google Scholar, 19Neilson E.G. Kalluri R. Sun M.J. Gunwar S. Danoff T. Mariyama M. Maers J.C. Reeders S.T. Hudson B.G. J. Biol. Chem. 1993; 268: 8402-8405Abstract Full Text PDF PubMed Google Scholar). However, the major epitope region is found to be the N-terminal part of the α3(IV) NC1 (9Quinones S. Bernal D. Garcia-Sogo M. Elena S.F. Saus J. J. Biol. Chem. 1992; 267: 19780-19784Abstract Full Text PDF PubMed Google Scholar, 11Ryan J.J. Mason P.J. Pusey C.D. Turner N. Clin. Exp. Immunol. 1998; 113: 17-27Crossref PubMed Scopus (36) Google Scholar), and only antibodies against this part of the molecule correlate with disease progression (12Hellmark T. Segelmark M. Unger C. Burkhardt H. Saus J. Wieslander J. Kidney Int. 1999; 55: 936-944Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 15Hellmark T. Burkhardt H. Wieslander J. J. Biol. Chem. 1999; 274: 25862-25868Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In this study we have further narrowed down and characterized the molecular properties of this epitope recognized by the pathogenic autoantibodies. A recombinant protein comprised of the α1(IV) NC1 domain with seven amino acids substituted to the corresponding ones from the α3 chain was constructed. This recombinant protein, R12, was recognized by the autoantibodies from all patients with Goodpasture disease, in both direct ELISA and in inhibition ELISA, to the same degree as recombinant α3. These results support the previous findings where we and others have localized the major epitope to the same region (13Netzer 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 (128) Google Scholar, 14Borza D.B. Netzer K.-O. Leinonen A. Todd P. Cervera J. Saus J. Hudson B.G. J. Biol. Chem. 2000; 275: 6030-6037Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 15Hellmark T. Burkhardt H. Wieslander J. J. Biol. Chem. 1999; 274: 25862-25868Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar).The very limited region recognized by all Goodpasture sera indicates a similar immunization and maturation process in all patients. Interestingly, an overlapping T-cell epitope is found by Phelps and co-workers (20Phelps R.G. Jones V.L. Coughlan M. Turner A.N. Rees A.J. J. Biol. Chem. 1998; 273: 11440-11447Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 21Phelps R.G. Rees A.J. Kidney Int. 1999; 56: 1638-1653Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). However, if this process is initiated by a foreign immunogen, i.e. molecular mimicry, or if it is a self-immunization with degraded type IV collagen, is yet unknown.As shown in Fig. 5 a, the epitope is localized to a small loop in the secondary sequence. The epitope seems to be dependent on correct folding of this loop, indicated both by the fact that disruption of the disulfide bridges (4Wieslander 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,7Hellmark T. Brunmark C. Trojnar J. Wieslander J. Clin. Exp. Immunol. 1996; 105: 504-510Crossref PubMed Scopus (24) Google Scholar) as well as changes of the charge of certain residues disturb the immunoreactivity. This is shown by the substitutions in positions 3 and 4, of which one, but not both, of the resulting amino acids must have a negative charge for the epitope to be fully recognized. In R124 both residues are negatively charged, and in R123 both residues are uncharged, and in both, the changes result in a loss of affinity. The preservation of the positively charged lysine in position P2, seen in the S1 and R134 recombinants (Fig. 5 b), dramatically reduce affinity to the recombinants. Surprisingly, the R12, as well as the S2, R123, and R124, actually reacted stronger than the recombinant α3(IV) in the direct ELISA. This could possibly be explained by a less rigid structure in the α1(IV) background that results in a more accessible epitope. This theory is supported by the fact that this difference in reactivity did not appear when the recombinants were analyzed using inhibition ELISA. An alternative explanation for the higher reactivity could be that the produced recombinant, e.g. R12, displays an epitope more similar to a hypothetical mimicry structure than the native α3(IV) NC1 does.Although all samples recognize one very limited area on the α3(IV) NC1, there are some differences in recognition pattern between the different samples. As discussed above the effect of charge changes within the loop have different effect on antibodies from different sera (especially the R134). Furthermore the relative amount of antibodies against the epitope defined by the R12 constructs varies from serum to serum, ranging from 65 to 95%.We believe that this study of the Goodpasture epitope adds new and important data that will help us to understand the underlying immunological mechanisms in Goodpasture disease in particular, but also for autoimmune diseases in general. By using the R12 recombinant protein in an assay instead of the complete α3(IV) NC1, a more specific diagnostic test could be developed that could distinguish between pathogenic antibodies and harmless autoantibodies. Circulating autoantibodies against different parts of the α3(IV) chain, as well as antibodies against other α(IV) chains, are detected in serum from patients with Goodpasture disease (6Hellmark T. Johansson C. Wieslander J. Kidney Int. 1994; 46: 823-829Abstract Full Text PDF PubMed Scopus (78) Google Scholar, 19Neilson E.G. Kalluri R. Sun M.J. Gunwar S. Danoff T. Mariyama M. Maers J.C. Reeders S.T. Hudson B.G. J. Biol. Chem. 1993; 268: 8402-8405Abstract Full Text PDF PubMed Google Scholar). However, the major epitope region is found to be the N-terminal part of the α3(IV) NC1 (9Quinones S. Bernal D. Garcia-Sogo M. Elena S.F. Saus J. J. Biol. Chem. 1992; 267: 19780-19784Abstract Full Text PDF PubMed Google Scholar, 11Ryan J.J. Mason P.J. Pusey C.D. Turner N. Clin. Exp. Immunol. 1998; 113: 17-27Crossref PubMed Scopus (36) Google Scholar), and only antibodies against this part of the molecule correlate with disease progression (12Hellmark T. Segelmark M. Unger C. Burkhardt H. Saus J. Wieslander J. Kidney Int. 1999; 55: 936-944Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 15Hellmark T. Burkhardt H. Wieslander J. J. Biol. Chem. 1999; 274: 25862-25868Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In this study we have further narrowed down and characterized the molecular properties of this epitope recognized by the pathogenic autoantibodies. A recombinant protein comprised of the α1(IV) NC1 domain with seven amino acids substituted to the corresponding ones from the α3 chain was constructed. This recombinant protein, R12, was recognized by the autoantibodies from all patients with Goodpasture disease, in both direct ELISA and in inhibition ELISA, to the same degree as recombinant α3. These results support the previous findings where we and others have localized the major epitope to the same region (13Netzer 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 (128) Google Scholar, 14Borza D.B. Netzer K.-O. Leinonen A. Todd P. Cervera J. Saus J. Hudson B.G. J. Biol. Chem. 2000; 275: 6030-6037Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 15Hellmark T. Burkhardt H. Wieslander J. J. Biol. Chem. 1999; 274: 25862-25868Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The very limited region recognized by all Goodpasture sera indicates a similar immunization and maturation process in all patients. Interestingly, an overlapping T-cell epitope is found by Phelps and co-workers (20Phelps R.G. Jones V.L. Coughlan M. Turner A.N. Rees A.J. J. Biol. Chem. 1998; 273: 11440-11447Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 21Phelps R.G. Rees A.J. Kidney Int. 1999; 56: 1638-1653Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). However, if this process is initiated by a foreign immunogen, i.e. molecular mimicry, or if it is a self-immunization with degraded type IV collagen, is yet unknown. As shown in Fig. 5 a, the epitope is localized to a small loop in the secondary sequence. The epitope seems to be dependent on correct folding of this loop, indicated both by the fact that disruption of the disulfide bridges (4Wieslander 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,7Hellmark T. Brunmark C. Trojnar J. Wieslander J. Clin. Exp. Immunol. 1996; 105: 504-510Crossref PubMed Scopus (24) Google Scholar) as well as changes of the charge of certain residues disturb the immunoreactivity. This is shown by the substitutions in positions 3 and 4, of which one, but not both, of the resulting amino acids must have a negative charge for the epitope to be fully recognized. In R124 both residues are negatively charged, and in R123 both residues are uncharged, and in both, the changes result in a loss of affinity. The preservation of the positively charged lysine in position P2, seen in the S1 and R134 recombinants (Fig. 5 b), dramatically reduce affinity to the recombinants. Surprisingly, the R12, as well as the S2, R123, and R124, actually reacted stronger than the recombinant α3(IV) in the direct ELISA. This could possibly be explained by a less rigid structure in the α1(IV) background that results in a more accessible epitope. This theory is supported by the fact that this difference in reactivity did not appear when the recombinants were analyzed using inhibition ELISA. An alternative explanation for the higher reactivity could be that the produced recombinant, e.g. R12, displays an epitope more similar to a hypothetical mimicry structure than the native α3(IV) NC1 does. Although all samples recognize one very limited area on the α3(IV) NC1, there are some differences in recognition pattern between the different samples. As discussed above the effect of charge changes within the loop have different effect on antibodies from different sera (especially the R134). Furthermore the relative amount of antibodies against the epitope defined by the R12 constructs varies from serum to serum, ranging from 65 to 95%. We believe that this study of the Goodpasture epitope adds new and important data that will help us to understand the underlying immunological mechanisms in Goodpasture disease in particular, but also for autoimmune diseases in general. By using the R12 recombinant protein in an assay instead of the complete α3(IV) NC1, a more specific diagnostic test could be developed that could distinguish between pathogenic antibodies and harmless autoantibodies. We thank Lena Gunnarsson for skillful technical assistance and Dr. Anders Aspberg, CMB, Lund University, for providing the expression vector.