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Hydrophobic Amino Acid Residues Are Critical for the Immunodominant Epitope of the Goodpasture Autoantigen

A MOLECULAR BASIS FOR THE CRYPTIC NATURE OF THE EPITOPE*
Open AccessPublished:March 02, 2001DOI:https://doi.org/10.1074/jbc.M008956200
      Goodpasture (GP) autoimmune disease is caused by autoantibodies to type IV collagen that bind to the glomerular basement membrane, causing rapidly progressing glomerulonephritis. The immunodominant GPAautoepitope is encompassed by residues 17–31 (the EAregion) within the noncollagenous (NC1) domain of the α3(IV) chain. The GP epitope is cryptic in the NC1 hexamer complex that occurs in the type IV collagen network found in tissues and inaccessible to autoantibodies unless the hexamer dissociates. In contrast, the epitope for the Mab3 monoclonal antibody is also located within the EA region, but is fully accessible in the hexamer complex. In this study, the identity of residues that compose the GPA autoepitope was determined, and the molecular basis of its cryptic nature was explored. This was achieved using site-directed mutagenesis to exchange the α3(IV) residues in the EAregion with the corresponding residues of the homologous but non-immunoreactive α1(IV) NC1 domain and then comparing the reactivity of the mutated chimeras with GPA and Mab3 antibodies. It was shown that three hydrophobic residues (Ala18, Ile19, and Val27) and Pro28 are critical for the GPA autoepitope, whereas two hydrophilic residues (Ser21 and Ser31) along with Pro28 are critical for the Mab3 epitope. These results suggest that the cryptic nature of the GPA autoepitope is the result of quaternary interactions of the α3, α4, and α5 NC1 domains of the hexamer complex that bury the one or more hydrophobic residues. These findings provide critical information for understanding the etiology and pathogenesis of the disease as well as for designing drugs that would mimic the epitope and thus block the binding of GP autoantibodies to autoantigen.
      GP
      Goodpasture
      ELISA
      enzyme-linked immunosorbent assay
      Goodpasture (GP)1disease is defined by rapidly progressive glomerulonephritis, with or without lung hemorrhage, which is caused by autoantibodies targeted to type IV collagen of the glomerular and alveolar basement membranes. Left untreated, the disease is potentially lethal. If diagnosed early, GP patients can be treated by immunosuppression and plasma exchange to remove the toxic autoantibodies. This therapy has side effects, including a weakening of the natural defenses; thus, a more specific therapy is highly desirable. A detailed knowledge of the epitope would facilitate the development of therapies that selectively remove, neutralize, or prevent synthesis of the pathogenic autoantibodies and provide a foundation for studies to determine the etiology of the disease.
      The GP autoantigen is the α3 chain of type IV collagen (
      • Butkowski R.J.
      • Langeveld J.P.
      • Wieslander J.
      • Hamilton J.
      • Hudson B.G.
      ,
      • Saus J.
      • Wieslander J.
      • Langeveld J.P.
      • Quinones S.
      • Hudson B.G.
      ), one of the six chains (α1–α6) that compose type IV collagen (
      • Hudson B.G.
      • Reeders S.T.
      • Tryggvason K.
      ). The GP autoepitopes are conformational and reside within the 232-residue long noncollagenous (NC1) domain at the C-terminus of the α3(IV) chain. Three chains of type IV collagen assemble into triple-helical protomers (molecule) that further interact with each other at the amino and carboxyl termini to form supramolecular networks. In the glomerular basement membrane, which is the main target of autoantibodies, the α3(IV) chain associates with the α4(IV) and α5(IV) chains to form a cross-linked α3·α4·α5(IV) network (
      • Gunwar S.
      • Ballester F.
      • Noelken M.E.
      • Sado Y.
      • Ninomiya Y.
      • Hudson B.G.
      ). The GP epitopes are cryptic in the α3·α4·α5 NC1 hexamer complex formed by the interaction of two triple-helical protomers through the NC1 domains (Fig. 1, top). As a result, the epitopes are inaccessible for binding of autoantibodies unless the hexamer dissociates (
      • Wieslander J.
      • Langeveld J.
      • Butkowski R.
      • Jodlowski M.
      • Noelken M.
      • Hudson B.G.
      ,
      • Kalluri R.
      • Sun M.J.
      • Hudson B.G.
      • Neilson E.G.
      ,
      • Borza D.-B.
      • Netzer K.O.
      • Leinonen A.
      • Todd P.
      • Cervera J.
      • Saus J.
      • Hudson B.G.
      ). Unmasking of previously hidden GP epitopes is thought to be of fundamental importance for understanding the etiology and pathogenesis of GP disease.
      Figure thumbnail gr1
      Figure 1Model representing the location within the native NC1 hexamer complex of the cryptic GP autoepitopes and of the exposed Mab3 epitope of the α3(IV) NC1 domain. In the type IV collagen networks found in vivo, two triple-helical collagen protomers interact through their carboxyl-terminal ends, forming an NC1 hexamer complex (top). Two conformational GP epitopes, designated GPA and GPB, have been localized to the EA and EB regions of the α3(IV) NC1 domain. These regions jointly form the epitope for Mab3. The GP epitopes (diagonal lines) are cryptic in the NC1 hexamer complex and inaccessible for binding of autoantibody (shown for GPA antibodies; right), but they are exposed upon dissociation of the hexamer into subunits, allowing binding of the autoantibody. In contrast, the Mab3 epitope (solid black) is accessible in both the hexamer and dissociated (monomer) form of the α3(IV)NC1 domain (left). Hence, in the NC1 hexamer complex, the EA and EB regions contain certain inaccessible residues that are critical for the GP epitopes and other accessible residues that are critical for the Mab3 epitope (
      • Borza D.-B.
      • Netzer K.O.
      • Leinonen A.
      • Todd P.
      • Cervera J.
      • Saus J.
      • Hudson B.G.
      ).
      Two conformational GP autoepitopes have recently been mapped to two regions of the α3(IV) NC1 domain, designated EA and EB, by homolog-scanning mutagenesis using chimeric α1/α3 NC1 domains in which the non-immunoreactive α1 NC1 domain was used as a scaffold for exchanging short homologous α1 sequences with α3 NC1 segments to ensure correct folding of the epitope. The immunodominant autoepitope, designated GPA (
      • Borza D.-B.
      • Netzer K.O.
      • Leinonen A.
      • Todd P.
      • Cervera J.
      • Saus J.
      • Hudson B.G.
      ), has been localized to the N-terminal third of the α3(IV) NC1 domain (
      • Ryan J.J.
      • Mason P.J.
      • Pusey C.D.
      • Turner N.
      ,
      • Hellmark T.
      • Segelmark M.
      • Unger C.
      • Burkhardt H.
      • Saus J.
      • Wieslander J.
      ) and specifically to residues 17–31, designated the EA region (
      • Borza D.-B.
      • Netzer K.O.
      • Leinonen A.
      • Todd P.
      • Cervera J.
      • Saus J.
      • Hudson B.G.
      ,
      • Netzer K.O.
      • Leinonen A.
      • Boutaud A.
      • Borza D.-B.
      • Todd P.
      • Gunwar S.
      • Langeveld J.P.
      • Hudson B.G.
      ,
      • Hellmark T.
      • Burkhardt H.
      • Wieslander J.
      ). Autoantibodies specific for the EA region, designated GPA, are believed to play an important role in the pathogenesis of GP disease because they are the predominant subpopulation (∼60–65%) in all sera and have high affinity for autoantigen (
      • Borza D.-B.
      • Netzer K.O.
      • Leinonen A.
      • Todd P.
      • Cervera J.
      • Saus J.
      • Hudson B.G.
      ). Moreover, high titers of GPA antibodies are correlated with an unfavorable disease outcome (
      • Hellmark T.
      • Segelmark M.
      • Unger C.
      • Burkhardt H.
      • Saus J.
      • Wieslander J.
      ). A second autoepitope, designated GPB (
      • Borza D.-B.
      • Netzer K.O.
      • Leinonen A.
      • Todd P.
      • Cervera J.
      • Saus J.
      • Hudson B.G.
      ), was also identified in the central portion of the α3(IV) NC1 domain (
      • Hellmark T.
      • Segelmark M.
      • Unger C.
      • Burkhardt H.
      • Saus J.
      • Wieslander J.
      ) and was further mapped to residues 127–141, designated the EB region (
      • Borza D.-B.
      • Netzer K.O.
      • Leinonen A.
      • Todd P.
      • Cervera J.
      • Saus J.
      • Hudson B.G.
      ,
      • Netzer K.O.
      • Leinonen A.
      • Boutaud A.
      • Borza D.-B.
      • Todd P.
      • Gunwar S.
      • Langeveld J.P.
      • Hudson B.G.
      ).
      In contrast to the cryptic GP autoepitopes, the epitope for Mab3 monoclonal antibody, which is also localized to the EA and EB regions, is fully accessible even in the NC1 hexamer (
      • Borza D.-B.
      • Netzer K.O.
      • Leinonen A.
      • Todd P.
      • Cervera J.
      • Saus J.
      • Hudson B.G.
      ). These observations led to the hypothesis that only certain amino acid residues of the EA and EB regions constitute the GPA and GPB autoepitopes and that their cryptic nature in the NC1 hexamer complex is a result of either direct interactions with or close proximity to other NC1 domains in the hexamer, which prevents the access of autoantibodies. Moreover, these critical residues must be distinctly different from those that constitute the Mab3 epitope, which are accessible on the surface of the hexamer complex.
      The aim of this study was to identify which residues compose the GPA autoepitope and to explore the molecular basis of its cryptic nature. This was accomplished using homolog-scanning mutagenesis to change the α3(IV)-specific residues within the EA region of an α1/α3 chimera, one at a time, to the corresponding residues from the homologous but non-immunoreactive α1(IV) NC1 domain. The reactivity of mutated NC1 domains with GPA and Mab3 antibodies was then compared. It was shown that three hydrophobic residues (Ala18, Ile19, and Val27) and Pro28 are critical for the GPA autoepitope, whereas two hydrophilic residues (Ser21 and Ser31) along with Pro28are critical for the Mab3 epitope. These results suggest that the cryptic nature of the GPA autoepitope is the result of quaternary interactions among the α3(IV), α4(IV), and α5(IV) NC1 domains of the α3·α4·α5 hexamer complex that bury the one or more hydrophobic residues. These findings provide critical information for understanding the etiology and pathogenesis of the disease as well as for designing drugs that would mimic the epitope and thus block the binding of GP autoantibodies to autoantigen in vivo.

      DISCUSSION

      The inaccessibility of the GPA autoepitope, contrasting with the full accessibility of the Mab3 epitope, afforded an experimental strategy to identify critical residues of these conformational epitopes and to explore the molecular basis of the cryptic nature of the GPA autoepitope. To this end, the 15-residue EA region of the α3(IV) NC1 domain was mutated at eight α3-specific residues, and the resulting chimeras (M1–M8) were assessed for their ability to bind GPA and Mab3 antibodies. The M2 (A18D), M3 (I19D), M6 (V27K), and M7 (P28I) chimeras had greatly decreased binding to GPA antibodies, whereas the M4 (S21Q), M7 (P28I), and M8 (S31H) chimeras had greatly decreased binding to Mab3 antibodies. The loss of binding of GPAantibodies to one set of chimeras and of Mab3 to another, along with binding of GPB antibodies to all chimeras, indicated that the overall conformation of the chimeras did not differ from that of the control (template) C2·6 chimera. Hence, a decrease in antibody binding to a specific chimera was due to removal of a critical epitope residue rather than to misfolding and/or mispairing of disulfide bonds. Thus, the binding profiles indicate that Ala18 and Pro28, along with either Ile19 in certain GP patients or Val27 in other GP patients, are critical for the GPA autoepitope, whereas Ser21, Ser31, and Pro28 are critical for the Mab3 epitope, as depicted in Fig. 7.
      Figure thumbnail gr7
      Figure 7Schematic representation of the GPA autoepitope, highlighting quaternary interactions in the NC1 hexamer as the molecular basis of its cryptic nature. Within the EA region of the α3(IV) NC1 domain, hydrophobic residues Ala18, Ile19, Val27, and Pro28 are critical for the GPA epitope (diagonal lines). One or more of these GPA residues are buried within the NC1 hexamer by hydrophobic interactions with residues of other NC1 domains, rendering the epitope cryptic and inaccessible for antibody binding (left). Upon hexamer dissociation, the buried residues are exposed and become accessible for binding (right). In contrast, hydrophilic residues Ser21 and Ser31as well as Pro28 are critical for the Mab3 epitope (solid black), which also includes some residues from the EB region. These residues are exposed on the surface of the NC1 hexamers and monomers, thus accessible for Mab3 binding. Pro28 is critical for both GPA and Mab3 epitopes, and it is possibly important for the native conformation of the EA region.
      Interestingly, three of the four GPA residues (Ala18, Ile19, and Val27) are hydrophobic (
      • Kyte J.
      • Doolittle R.F.
      ,
      • Rose G.D.
      • Geselowitz A.R.
      • Lesser G.J.
      • Lee R.H.
      • Zehfus M.H.
      ) and have a high propensity to be buried (
      • Lesser G.J.
      • Rose G.D.
      ). This suggests that the cryptic nature of the GPA autoepitope is because one or more GPA residues participate in hydrophobic interactions with other NC1 domains in the hexamer complex, thus burying the epitope and rendering it inaccessible for binding of autoantibodies (Fig. 7). In contrast, the Mab3 epitope is accessible because it contains critical residues that have a high propensity to be located on the hexamer surface (
      • Lesser G.J.
      • Rose G.D.
      ): two hydrophilic serine residues and one proline residue, which is most often found in β-turns and other loops on the protein surface. Interestingly, Pro28 is critical for both GPA and Mab3 epitopes; thus, it may be important in defining the conformation of the EA region in the α3(IV) NC1 domain.
      The hydrophobic character of the GPA residues represents a departure from the conventional features of epitopes, which consist predominately of charged or polar residues and tend to be located in flexible turns or loops on protein surfaces (
      • Barlow D.J.
      • Edwards M.S.
      • Thornton J.M.
      ,
      • Hopp T.P.
      ). Based on these features, numerous empirical methods have been developed that successfully predict the major antigenic sites of native proteins (
      • Stern P.S.
      ). The α3(IV) NC1 sequence was analyzed with the program Epiplot, which calculates and plots flexibility, hydrophilicity, and antigenicity profiles using 13 different scales, chosen as those yielding the best predictions on proteins whose antigenic structures are known (
      • Menendez-Arias L.
      • Rodriguez R.
      ). None of these methods predicted the location of the GPAautoepitope or the Mab3 epitope, which reside within the amphipathic EA region of the α3(IV) NC1 domain, as illustrated by the popular Hopp-Woods hydrophilicity plot in Fig.8 (upper) (
      • Hopp T.P.
      • Woods K.R.
      ). Thus, other factors must govern the immunogenicity of the EA region, eliciting the production of autoantibodies to GPA residues on one hand and of antibodies to Mab3 on the other.
      Figure thumbnail gr8
      Figure 8Structural features of the EAregion. Upper, the hydrophilicity of the α3(IV) NC1 domain was calculated and plotted according to the Hopp-Woods scale, using a window of six residues. The peaks indicate the most hydrophilic regions of a protein, located on the surface and predicted to be the major antigenic sites. The residues that compose the cryptic GPA epitope are located in an amphipathic region. Lower, comparison of the amino acid sequences of the six α(IV) NC1 domains in the EA region. The α3 amino acids critical for binding of GPAautoantibodies are boxed, and those critical for Mab3 binding are encircled. Notice the high sequence divergence among the six chains at these positions, including numerous non-conservative substitutions. The amino acids conserved between α3 and the other chains (shaded) are likely to play a structural role.
      In particular, the lack of immune tolerance to the GPAepitope may be explained by its cryptic nature, which renders it an immunologically privileged site. To avoid autoreactivity, the B cell clones directed to self-antigens are edited out early in development, establishing self-tolerance. Because the GPA epitope is buried in the NC1 hexamer complex under normal physiological conditions, it is sequestered from the immune system. Therefore, if pathogenic factors induce hexamer dissociation, the newly exposed GPA residues would then be perceived as “foreign” by the immune system, eliciting an autoimmune response. What factors trigger this process in vivo remain unclear. Hydrocarbons or viral infections have been suggested as causative agents (
      • Wilson C.B.
      • Borza D.-B.
      • Hudson B.G.
      ). A recent study provides evidence that reactive oxygen species may act as the physiological mediator for epitope exposure (
      • Kalluri R.
      • Cantley L.G.
      • Kerjaschki D.
      • Neilson E.G.
      ).
      Certain of the GPA residues identified herein differ from those found in two recent studies by Wieslander and co-workers (
      • Hellmark T.
      • Burkhardt H.
      • Wieslander J.
      ,
      • Gunnarsson A.
      • Hellmark T.
      • Wieslander J.
      ), who did not identify the overall hydrophobic character of the epitope. The first study qualitatively investigated the role of 14 α3(IV) NC1 domain residues, including six of the eight residues in the EA region (
      • Hellmark T.
      • Burkhardt H.
      • Wieslander J.
      ). Similar to our findings, Ile19 and Pro28, but not Thr17, were found important for binding. However, Ala18 was reported to be not essential, and the role of Glu24 and Val27 was not addressed. In addition, Ser21 and Ser31 were reported to be critical for binding GP antibodies, whereas we found these residues to be important only for the binding of the Mab3 antibody. Since a quantitative data analysis was not reported in that study, it is difficult to directly compare their findings with ours. A very recent paper from the same group (
      • Gunnarsson A.
      • Hellmark T.
      • Wieslander J.
      ) quantitatively analyzed the role of four α3 residues in the EA region and found Val27 to be critical for GP antibody binding (similar to our findings), whereas Thr17, Ala18, and Glu24 had a moderate effect. Some of the discrepancies may be explained by the different α1/α3 chimeras used as template for the homolog-scanning mutagenesis. Their template chimera contained only the EA region, whereas ours contained both the EA and EB regions of the α3(IV) NC1 domain. Because the EA and EBregions are in close proximity in the natively folded α3(IV) NC1 domain, mutants M1–M8 used in our work are likely to reproduce the native GP epitopes more closely. In addition, our study relied on a mutagenesis strategy that allowed (a) a verification of the conformation of the mutants by using three different antibodies that recognize conformational epitopes and (b) a comparison between the binding of GPA and Mab3 antibodies, a priori inferred to bind to different residues in the EA region.
      The identification of the critical residues of the GPAepitope allows the determination of the structural features that selectively target GPA antibodies to the α3(IV) NC1 domain, among the six homologous NC1 domains of type IV collagen. As revealed by the comparison in Fig. 8 (lower), all four GPA residues (Ala18, Ile19, Val27, and Pro28) occur at the respective position in the α3 sequence only. Intriguingly, three of the GPA residues are hydrophobic in the α3 sequence, but the homologous α1 residues are hydrophilic, charged residues (Asp, Asp, and Lys, respectively). Analysis of data from experimentally determined antigenic sites on proteins has revealed that hydrophobic residues are more likely to be a part of antigenic sites, if they occur on the surface of a protein (
      • Kolaskar A.S.
      • Tongaonkar P.C.
      ), as GPA residues do in the α3(IV) NC1 monomer. No other chain besides α3(IV) had a constellation of three hydrophobic residues at positions 18, 19, and 27. Moreover, Pro28 occurs only in α3, but not in any of the other five NC1 domains. Therefore, three hydrophobic residues at positions 18, 19, and 27, together with a proline at position 28, in a distinct conformation distinguish α3 among the six NC1 domains, conferring binding of GPA antibodies selectively to the α3(IV) NC1 domain.
      The EA region of the α3(IV) NC1 domain emerges as a prime candidate for a molecular recognition site that specifies the chain-specific assembly of the α3·α4·α5 network of type IV collagen. Recently, we showed that the NC1 monomers contain recognition sequences for selection of chains and protomers that are sufficient to encode the specificity of assembly of the α1·α2 and α3·α4·α5(IV) networks of the glomerular basement membrane (
      • Boutaud A.
      • Borza D.-B.
      • Bondar O.
      • Gunwar S.
      • Netzer K.O.
      • Singh N.
      • Ninomiya Y.
      • Sado Y.
      • Noelken M.E.
      • Hudson B.G.
      ), but their identity is unknown. That the EA region is a site of interaction between α3 and the other NC1 domains in the α3·α4·α5 hexamer is deduced from its cryptic nature of the GPA epitope. That the EA region may also be responsible for the specificity of interaction is suggested by the high sequence divergence of this region among the six NC1 domains (
      • Netzer K.O.
      • Suzuki K.
      • Itoh Y.
      • Hudson B.G.
      • Khalifah R.G.
      ). The EA region is also distinguished by the highest number of non-conservative amino acid substitutions. Hence, the pattern of hydrophobic and hydrophilic residues within the EA region is unique for each of the six NC1 domains and is likely to confer chain-specific conformations and interactions. In particular, the EA region of all six NC1 domains may contribute to the discriminatory interactions that result in specific assembly of chain-specific networks of type IV collagen.

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