Goodpasture disease. Characterization of a single conformational epitope as the target of pathogenic autoantibodies.

Goodpasture disease is a prototype autoimmune disease characterized by the formation of autoantibodies against the heterotrimeric basement membrane collagen type IV, which causes a rapidly progressive glomerulonephritis. The pathogenic antibody response is directed to the non-collagenous (NC1) domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1), but not to the homologous region of the alpha1(IV)NC1. To identify the conformation-dependent immunodominant epitope on the alpha3(IV)NC1, a variety of recombinant NC1 domains were constructed by replacing single residues of alpha3(IV) with the corresponding amino acids from the nonreactive alpha1(IV) chain. Replacement mutations were identified that completely destroyed the Goodpasture epitope in the alpha3(IV) chain. Based on the identification of these critical positions, the epitope was finally reconstructed within the frame of the alpha1(IV) chain. The substitution of nine discontinuous positions in the alpha1(IV)NC1 with amino acid residues from the alpha3 chain resulted in a recombinant construct that was recognized by all patients' sera (n = 20) but by none of the sera from healthy controls (n = 10). This provides, for the first time, the molecular characterization of a single immunodominant conformational epitope recognized by pathogenic autoantibodies in a human autoimmune disease, representing the basis for the development of new epitope-specific strategies in the treatment of Goodpasture disease.     

Point mutations of single amino acids abolish ability of alpha3 NC1 domain to elicit experimental autoimmune glomerulonephritis in rats

We previously showed concordance between Goodpasture syndrome antibody binding and production of experimental glomerulonephritis using human chimeric proteins. We now examine a more limited amino-terminal region of alpha3(IV) non-collagenous domain (NC1) and the impact of single amino acid (AA) mutations of this region on glomerulonephritis induction. Rats were immunized with collagenase-solubilized glomerular basement membrane (csGBM), D3, an alpha1(IV)NC1 chimeric protein with 69 AA of alpha3(IV)NC1 (binds Goodpasture sera), D4, the D3 construct shortened by 4 AA (non-binding), P9, P10, single AA mutants (non-binding), and S2, alpha1(IV)NC1 with 9 AA of alpha3(IV)NC1 (binding). All rats immunized with csGBM and S2 and 50% of D3 rats developed glomerulonephritis. csGBM rats had intense GBM-bound IgG deposits, but S2 and D3 rats had minimal deposits. None of the D4, P9, or P10 rats developed glomerulonephritis. Lymphocytes from nephritic rats proliferated with csGBM, S2, and D3, but not with D4, P9, or P10. Discrete segments of alpha3(IV)NC1 within the alpha1(IV)NC1 backbone can induce glomerulonephritis. Single AA mutations within that epitope render the antigen unresponsive to Goodpasture sera and incapable of inducing glomerulonephritis. These studies support the concordance of glomerulonephritis inductivity and Goodpasture serum binding. Further, they define a critical limited AA sequence within alpha3(IV)NC1 of nine or fewer AA, which confers nephritogenicity to the nonnephritogenic alpha1(IV)NC1 without in vivo antibody binding. This region may be a T-cell epitope responsible for induction of glomerulonephritis in this model in rats and Goodpasture syndrome in man.     

Reactive oxygen species expose cryptic epitopes associated with autoimmune goodpasture syndrome

Goodpasture syndrome is an autoimmune disease of the kidneys and lungs mediated by antibodies and T-cells directed to cryptic epitopes hidden within basement membrane hexamers rich in alpha3 non-collagenous globular (NC1) domains of type IV collagen. These epitopes are normally invisible to the immune system, but this privilege can be obviated by chemical modification. Endogenous drivers of immune activation consequent to the loss of privilege have long been suspected. We have examined the ability of reactive oxygen species (ROS) to expose Goodpasture epitopes buried within NC1 hexamers obtained from renal glomeruli abundant in alpha3(IV) NC1 domains. For some hexameric epitopes, like the Goodpasture epitopes, exposure to ROS specifically enhanced recognition by Goodpasture antibodies in a sequential and time-dependent fashion; control binding of epitopes to alpha3(IV) alloantibodies from renal transplant recipients with Alport syndrome was decreased, whereas epitope binding to heterologous antibodies recognizing all alpha3 NC1 epitopes remained the same. Inhibitors of hydrogen peroxide and hydroxyl radical scavengers were capable of attenuating the effects of ROS in cells and kidney by 30-50%, respectively, thereby keeping the Goodpasture epitopes largely concealed when compared with a 70% maximum inhibition by iron chelators. Hydrogen peroxide administration to rodents was sufficient to expose Goodpasture epitope in vivo and initiate autoantibody production. Our findings collectively suggest that ROS can alter the hexameric structure of type IV collagen to expose or destroy selectively immunologic epitopes embedded in basement membrane. The reasons for autoimmunity in Goodpasture syndrome may lie in an age-dependent deterioration in inhibitor function modulating oxidative damage to structural molecules. ROS therefore may play an important role in shaping post-translational epitope diversity or neoantigen formation in organ tissues.     

Goodpasture autoantibodies unmask cryptic epitopes by selectively dissociating autoantigen complexes lacking structural reinforcement: novel mechanisms for immune privilege and autoimmune pathogenesis

Rapidly progressive glomerulonephritis in Goodpasture disease is mediated by autoantibodies binding to the non-collagenous NC1 domain of alpha3(IV) collagen in the glomerular basement membrane. Goodpasture epitopes in the native autoantigen are cryptic (sequestered) within the NC1 hexamers of the alpha3alpha4alpha5(IV) collagen network. The biochemical mechanism for crypticity and exposure for autoantibody binding is not known. We now report that crypticity is a feature of the quaternary structure of two distinct subsets of alpha3alpha4alpha5(IV) NC1 hexamers: autoantibody-reactive M-hexamers containing only monomer subunits and autoantibody-impenetrable D-hexamers composed of both dimer and monomer subunits. Goodpasture antibodies only breach the quaternary structure of M-hexamers, unmasking the cryptic epitopes, whereas D-hexamers are resistant to autoantibodies under native conditions. The epitopes of D-hexamers are structurally sequestered by dimer reinforcement of the quaternary complex, which represents a new molecular solution for conferring immunologic privilege to a potential autoantigen. Dissociation of non-reinforced M-alpha3alpha4alpha5(IV) hexamers by Goodpasture antibodies is a novel mechanism whereby pathogenic autoantibodies gain access to cryptic B cell epitopes. These findings provide fundamental new insights into immune privilege and the molecular mechanisms underlying the pathogenesis of human autoimmune Goodpasture disease.     

Goodpasture syndrome. Localization of the epitope for the autoantibodies to the carboxyl-terminal region of the alpha 3(IV) chain of basement membrane collagen.

The autoantibodies of patients with Goodpasture syndrome are primarily targeted to the noncollagenous (NC1) domain of the alpha 3(IV) chain of basement membrane collagen (Saus, J., Wieslander, J., Langeveld, J. P. M., Quinones, S., and Hudson, B. G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, the location of the Goodpasture epitope in human alpha 3NC1 was determined, and its structure was partially characterized. This was achieved by identification of regions of alpha 3NC1 which are candidates for the epitope and which are structurally unique among the five known homologous NC1 domains (alpha 1-alpha 5); amino acids that are critical for Goodpasture antibody binding, by selective chemical modifications; and regions that are critical for Goodpasture antibody binding, by synthesis of 12 alpha 3NC1 peptides and measurement of their antibody binding capacity. The carboxyl-terminal region, residues 198-233, was identified as the most likely region for the epitope. By experiment, lysine and cysteine were identified as critical amino acids for antibody binding. Three synthetic peptides were found to inhibit Goodpasture antibody binding to alpha 3NC1 markedly: a 36-mer (residues 198-233), a 12-mer (residues 222-233), and a 5-mer (residues 229-233). Together, these results strongly indicate that the Goodpasture epitope is localized to the carboxyl-terminal region of alpha 3NC1, encompassing residues 198-233 as the primary antibody interaction site and that its structure is discontinuous. These findings provide a conceptual framework for future studies to elucidate a more complete epitope structure by sequential replacement of residues encompassing the epitope using cDNA expression products and peptides synthesized chemically.     

Quaternary organization of the goodpasture autoantigen,the alpha 3(IV) collagen chain. Sequestration of two cryptic autoepitopes by intrapromoter interactions with the alpha4 and alpha5 NC1 domains

Goodpasture's (GP) disease is caused by autoantibodies that target the alpha3(IV) collagen chain in the glomerular basement membrane (GBM). Goodpasture autoantibodies bind two conformational epitopes (E(A) and E(B)) located within the non-collagenous (NC1) domain of this chain, which are sequestered within the NC1 hexamer of the type IV collagen network containing the alpha3(IV), alpha4(IV), and alpha5(IV) chains. In this study, the quaternary organization of these chains and the molecular basis for the sequestration of the epitopes were investigated. This was accomplished by physicochemical and immunochemical characterization of the NC1 hexamers using chain-specific antibodies. The hexamers were found to have a molecular composition of (alpha3)(2)(alpha4)(2)(alpha5)(2) and to contain cross-linked alpha3-alpha5 heterodimers and alpha4-alpha4 homodimers. Together with association studies of individual NC1 domains, these findings indicate that the alpha3, alpha4, and alpha5 chains occur together in the same triple-helical protomer. In the GBM, this protomer dimerizes through NC1-NC1 domain interactions such that the alpha3, alpha4, and alpha5 chains of one protomer connect with the alpha5, alpha4, and alpha3 chains of the opposite protomer, respectively. The immunodominant Goodpasture autoepitope, located within the E(A) region, is sequestered within the alpha3alpha4alpha5 protomer near the triple-helical junction, at the interface between the alpha3NC1 and alpha5NC1 domains, whereas the E(B) epitope is sequestered at the interface between the alpha3NC1 and alpha4NC1 domains. The results also reveal the network distribution of the six chains of collagen IV in the renal glomerulus and provide a molecular explanation for the absence of the alpha3, alpha4, alpha5, and alpha6 chains in Alport syndrome.     

The goodpasture autoantigen. Identification of multiple cryptic epitopes on the NC1 domain of the alpha3(IV) collagen chain

Goodpasture (GP) disease is an autoimmune disorder in which autoantibodies against the alpha3(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 alpha3(IV) chain as residues 17-31 (E(A)) and 127-141 (E(B)) (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 E(A) and/or the E(B) region. Four subpopulations of GP antibodies with distinct epitope specificity for the alpha3(IV)NC1 domain were thus separated and characterized. They were designated GP(A), GP(B), GP(AB), and GP(X), to reflect their reactivity with E(A) only, E(B) only, both regions, and neither, respectively. Hence, regions E(A) and E(B) encompass critical amino acids that constitute three distinct epitopes for GP(A), GP(B), and GP(AB) antibodies, respectively, whereas the epitope for GP(X) antibodies is located in a different unknown region. The GP(A) antibodies were consistently immunodominant, accounting for 60-65% of the total immunoreactivity to alpha3(IV)NC1; thus, they probably play a major role in pathogenesis. Regions E(A) and E(B) 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 alpha3(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.     

Conformational diversity of the Goodpasture antigen, the noncollagenous-1 domain of the alpha3 chain of collagen IV

The noncollagenous-1 domain of the alpha3 chain of collagen IV networks of basement membranes is the target of an antibody-mediated inflammatory response in Goodpasture autoimmune disease. This domain when excised from basement membranes by bacterial collagenase digestion exists in two molecular forms, M(H) and M(L), that differ in cleavage site and mobility in SDS-PAGE. In the present study, M(H) and M(L) were shown to also differ with respect to epitope exposure, susceptibility to endoprotease digestion, and redox states of specific cystene residues, as determined by MS. Moreover, M(H) and M(L) assemble to form different quaternary structures, critically influencing pathogenic epitope(s) exposure and autoantibody binding. Collectively, our findings reveal that M(H) and M(L) are conformational isomers stabilized by a distinct disulfide bond connectivity, and coexist in basement membranes. The hitherto unrecognized conformational diversification of the Goodpasture autoantigen may be of relevance in pathogenesis.     

The goodpasture autoantigen. Mapping the major conformational epitope(s) of alpha3(IV) collagen to residues 17-31 and 127-141 of the NC1 domain

The Goodpasture (GP) autoantigen has been identified as the alpha3(IV) collagen chain, one of six homologous chains designated alpha1-alpha6 that comprise type IV collagen (Hudson, B. G., Reeders, S. T., and Tryggvason, K. (1993) J. Biol. Chem. 268, 26033-26036). In this study, chimeric proteins were used to map the location of the major conformational, disulfide bond-dependent GP autoepitope(s) that has been previously localized to the noncollagenous (NC1) domain of alpha3(IV) chain. Fourteen alpha1/alpha3 NC1 chimeras were constructed by substituting one or more short sequences of alpha3(IV)NC1 at the corresponding positions in the non-immunoreactive alpha1(IV)NC1 domain and expressed in mammalian cells for proper folding. The interaction between the chimeras and eight GP sera was assessed by both direct and inhibition enzyme-linked immunosorbent assay. Two chimeras, C2 containing residues 17-31 of alpha3(IV)NC1 and C6 containing residues 127-141 of alpha3(IV)NC1, bound autoantibodies, as did combination chimeras containing these regions. The epitope(s) that encompasses these sequences is immunodominant, showing strong reactivity with all GP sera and accounting for 50-90% of the autoantibody reactivity toward alpha3(IV)NC1. The conformational nature of the epitope(s) in the C2 and C6 chimeras was established by reduction of the disulfide bonds and by PEPSCAN analysis of overlapping 12-mer peptides derived from alpha1- and alpha3(IV)NC1 sequences. The amino acid sequences 17-31 and 127-141 in alpha3(IV)NC1 have thus been shown to contain the critical residues of one or two disulfide bond-dependent conformational autoepitopes that bind GP autoantibodies.     

A role for collagen IV cross-links in conferring immune privilege to the Goodpasture autoantigen: structural basis for the crypticity of B cell epitopes

The detailed structural basis for the cryptic nature (crypticity) of a B cell epitope harbored by an autoantigen is unknown. Because the immune system may be ignorant of the existence of such "cryptic" epitopes, their exposure could be an important feature in autoimmunity. Here we investigated the structural basis for the crypticity of the epitopes of the Goodpasture autoantigen, the alpha3alpha4alpha5 noncollagenous-1 (NC1) hexamer, a globular domain that connects two triple-helical molecules of the alpha3alpha4alpha5 collagen IV network. The NC1 hexamer occurs in two isoforms as follows: the M-isoform composed of monomer subunits in which the epitopes are accessible to autoantibodies, and the D-isoform composed of both monomer and dimer subunits in which the epitopes are cryptic. The D-isoform was characterized with respect to quaternary structure, as revealed by mass spectrometry of dimer subunits, homology modeling, and molecular dynamics simulation. The results revealed that the D-isoform contains two kinds of cross-links as follows: S-hydroxylysyl-methionine and S-lysyl-methionine cross-links, which stabilize the alpha3alpha5-heterodimers and alpha4alpha4-homodimers, respectively. Construction and analysis of a three-dimensional model of the D-isoform of the alpha3alpha4alpha5 NC1 hexamer revealed that crypticity is a consequence of the following: (a) sequestration of key residues between neighboring subunits that are stabilized by domain-swapping interactions, and (b) by cross-linking of subunits at the trimer-trimer interface, which stabilizes the structural integrity of the NC1 hexamer and protects against binding of autoantibodies. The sequestrated epitopes and cross-linked subunits represent a novel structural mechanism for conferring immune privilege at the level of quaternary structure. Perturbation of the quaternary structure may be a key factor in the etiology of Goodpasture disease.     

Identification of an epitope within human integrin alpha 6 subunit for the binding of autoantibody and its role in basement membrane separation in oral pemphigoid

Oral pemphigoid (OP) is a rare chronic autoimmune disease characterized by blisters and erosive lesions in the oral mucosa. We identified an epitope for the binding of OP autoantibodies within the integrin alpha6 subunit, by cloning four overlapping fragments (A, B, C, and D). Immunoperoxidase studies demonstrated that all of the fragments were present in the oral mucosa. Sera of 20 patients with active OP were studied. All sera bound to integrin alpha6 in DU145 cell lysate by immunoprecipitation and immunoblot assay. The same sera bound only to fragment A and its subfragment A2 on an immunoblot assay. The specificity of the binding was further characterized by blocking and cross-absorption studies. A 14-aa synthetic peptide A2.1, within fragment A2, bound to all the test sera. The sera in this study bound to only one epitope. Controls were sera samples from 10 healthy volunteers and 40 patients with other variants of mucous membrane pemphigoid and mAb GoH3 and BQ16 to integrin alpha6. Control sera did not bind to the full-length integrin alpha6 subunit nor any of the cloned fragments. The OP patient sera and immunoaffinity-purified OP sera, rabbit antisera against fragments A and A2, and mAb GoH3 produced basement membrane separation of oral mucosa in organ culture. This study identifies a peptide within the extracellular domain of integrin alpha6 molecule, to which Abs in the sera from patients with OP bind, and which may play an important role in the pathogenesis of OP.     

Properties of bovine nephritogenic antigen that induces anti-GBM nephritis in rats and its similarity to the Goodpasture antigen.

The nephritogenic antigen that induces antiglomerular basement membrane antibody-induced glomerulonephritis (anti-GBM nephritis) in rats was isolated from collagenase-solubilized bovine renal basement membranes. Purification was achieved using antibody-coupled affinity columns which were originally used for the purification of trypsin-solubilized nephritogenic antigen (Sado et al. 1984a). The nephritogenic antigen was a heteropolymer composed of P2 (Mr 28 kDa) and P3 (Mr 30 kDa) polypeptides as monomers and their dimers in sodium-dodecyl-sulfate (SDS) polyacrylamide gel electrophoresis. The P3 polypeptide was considered to be the nephritogenic epitope, since a fraction composed of the P2 polypeptide alone was not nephritogenic. The properties of the nephritogenic epitope were the same as those of the Goodpasture epitope (M2*), which is a noncollagenous domain of the alpha 3 chain of type IV collagen (Butkowski et al. 1985; Saus et al. 1988), indicating that the nephritogenic antigen is the same as the Goodpasture antigen.     

Hydrophobic amino acid residues are critical for the immunodominant epitope of the Goodpasture autoantigen. A molecular basis for the cryptic nature of the epitope

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 GP(A) autoepitope is encompassed by residues 17-31 (the E(A) region) within the noncollagenous (NC1) domain of the alpha 3(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 E(A) region, but is fully accessible in the hexamer complex. In this study, the identity of residues that compose the GP(A) autoepitope was determined, and the molecular basis of its cryptic nature was explored. This was achieved using site-directed mutagenesis to exchange the alpha3(IV) residues in the E(A) region with the corresponding residues of the homologous but non-immunoreactive alpha1(IV) NC1 domain and then comparing the reactivity of the mutated chimeras with GP(A) and Mab3 antibodies. It was shown that three hydrophobic residues (Ala(18), Ile(19), and Val(27)) and Pro(28) are critical for the GP(A) autoepitope, whereas two hydrophilic residues (Ser(21) and Ser(31)) along with Pro(28) are critical for the Mab3 epitope. These results suggest that the cryptic nature of the GP(A) autoepitope is the result of quaternary interactions of the alpha 3, alpha 4, and alpha 5 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.     

Exon/intron structure of the human alpha 3(IV) gene encompassing the Goodpasture antigen (alpha 3(IV)NC1). Identification of a potentially antigenic region at the triple helix/NC1 domain junction.

The Goodpasture antigen has been identified as the non-collagenous (NC1) domain of alpha 3(IV), a novel collagen IV chain (Saus, J., Wieslander, J., Langeveld, J., Quinones, S., and Hudson, B.G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, the exon/intron structure and sequence for 285 amino acids of human alpha 3(IV), comprising 53 amino acids of the triple-helical domain and the complete NC1 domain (232 amino acids), were determined. Based on the comparison of the amino acid sequences of the alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) NC1 domains, a phylogenetic tree was constructed which indicates that alpha 2(IV) was the first chain to evolve, followed by alpha 3(IV), and then by alpha 1(IV) and alpha 5(IV). The exon/intron structure of these domains is consistent with this evolution model. In addition, it appears that alpha 3(IV) changed most after diverging from the parental gene. Analysis of its primary structure reveals that, at the junction between the triple-helical and NC1 domains, there exists a previously unrecognized, highly hydrophilic region (GLKGKRGDSGSPATWTTR) which is unique to the human alpha 3(IV) chain, containing a cell adhesion motif (RGD) as an integral part of a sequence (KRGDSGSP) conforming to a number of protein kinase recognition sites. Based on primary structure data, we outline new aspects to be explored concerning the molecular basis of collagen IV function and Goodpasture syndrome.     

Comparative analysis of the noncollagenous NC1 domain of type IV collagen: identification of structural features important for assembly, function, and pathogenesis.

Type IV collagen alpha1-alpha6 chains have important roles in the assembly of basement membranes and are implicated in the pathogenesis of Goodpasture syndrome, an autoimmune disorder, and Alport syndrome, a hereditary renal disease. We report comparative sequence analyses and structural predictions of the noncollagenous C-terminal globular NC1 domain (28 sequences). The inferred tree verified that type IV collagen sequences fall into two groups, alpha1-like and alpha2-like, and suggested that vertebrate alpha3/alpha4 sequences evolved before alpha1/alpha2 and alpha5/alpha6. About one fifth of NC1 residues were identified to confer either the alpha1 or alpha2 group-specificity. These residues accumulate opposite charge in subdomain B of alpha1 (positive) and alpha2 (negative) sequences and may play a role in the stoichiometric chain selection upon type IV collagen assembly. Neural network secondary structure prediction on multiple aligned sequences revealed a subdomain core structure consisting of six hydrophobic beta-strands and one short alpha-helix with a significant hydrophobic moment. The existence of opposite charges in the alpha-helices may carry implications for intersubdomain interactions. The results provide a rationale for defining the epitope that binds Goodpasture autoantibodies and a framework for understanding how certain NC1 mutations may lead to Alport syndrome. A search algorithm, based entirely on amino acid properties, yielded a possible similarity of NC1 to tissue inhibitor of metalloproteinases (TIMP) and prompted an investigation of a possible functional relationship. The results indicate that NC1 preparations decrease the activity of matrix metalloproteinases 2 and 3 (MMP-2, MMP-3) toward a peptide substrate, though not to [14C]-gelatin. We suggest that an ancestral NC1 may have been incorporated into type IV collagen as an evolutionarily mobile domain carrying proteinase inhibitor function.     

Properties of the globular domain of type IV collagen and its relationship to the Goodpasture antigen

The globular domain of type IV collagen from bovine glomerular basement membrane was solubilized by collagenase digestion. Components of this domain include several monomer-size and structurally related dimer-size polypeptides. The monomer-size polypeptides were resolved into three fractions (M1, M2, and M3) with slightly different mobilities upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (nonreduced Mr = 24,500-28,300). Chemical and immunochemical studies indicate that each is a distinct component. M2 is reactive with antibodies from patients with Goodpasture syndrome. The molecular weight by sedimentation equilibrium was 32,000 for M2 and 28,000 for M1. The dimers were characterized as two classes, D1 and D2. D1 consists of two sets of nonreactive components (D1a-d and D1a,c) whereas D2 contains one set of four components (D2a-d), each of which is reactive with Goodpasture sera. Chemical and immunochemical studies indicate that a monomer-dimer relationship exists between M1 and D1 and between M2 and D2. The origin of M3 remains undetermined. Rabbit antibodies to type IV collagen alpha chains react with M1 and M2, and antibodies to M1 and M2 react with type IV collagen alpha chains, which provides additional evidence for the localization of the Goodpasture antigen to one of the chains of type IV collagen.     

Identification of Critical Residues of Linear B Cell Epitope on Goodpasture Autoantigen

Background

The autoantigen of anti-glomerular basement membrane (GBM) disease has been identified as the non-collagenous domain 1 of α3 chain of type IV collagen, α3(IV)NC1. Our previous study revealed a peptide on α3(IV)NC1 as a major linear epitope for B cells and potentially nephrogenic, designated as P14 (α3129-150). This peptide has also been proven to be the epitope of auto-reactive T cells in anti-GBM patients. This study was aimed to further characterize the critical motif of P14.

Methods

16 patients with anti-GBM disease and positive anti-P14 antibodies were enrolled. A set of truncated and alanine substituted peptides derived from P14 were synthesized. Circulating antibodies against the peptides were detected by enzyme linked immunosorbent assay (ELISA).

Results

We found that all sera with anti-P14 antibodies reacted with the 13-mer sequence in the C-terminus of P14 (P14c) exclusively. The level of antibodies against P14 was highly correlated with the level of antibodies against P14c (r=0.970, P<0.001). P14c was the core immunogenic region and the amino acid sequence (ISLWKGFSFIMFT) was highly hydrophobic. Each amino acid residue in P14c was sequentially replaced by alanine. Three residues of glycine142, phenylalanine143, and phenylalanine145 were identified crucial for antibody binding based on the remarkable decline (P<0.001) of antibody reaction after each residue replacement.

Conclusions

We defined GFxF (α3142, 143,145) as the critical motif of P14. It may provide some clues for understanding the etiology of anti-GBM disease.     

Characterization of a candidate Borrelia burgdorferi beta3-chain integrin ligand identified using a phage display library

The spirochaetal agents of Lyme disease, Borrelia burgdorferi (sensu lato) bind to integrins alphaIIbbeta3, alphavbeta3 and alpha5beta1 in purified form and on the surfaces of human cells. Using a phage display library of B. burgdorferi (sensu stricto) DNA, a candidate ligand for beta3-chain integrins was identified. The native B. burgdorferi protein, termed p66, is known to be recognized by human Lyme disease patient sera and to be expressed on the surface of the spirochaete. We show here that recombinant p66 binds specifically to beta3-chain integrins and inhibits attachment of intact B. burgdorferi to the same integrins. When expressed on the surface of Escherichia coli, this protein increases the attachment of E. coli to a transfected cell line that expresses alphavbeta3, but not to the parental cell line, which expresses no beta3-chain integrins. Localization of p66 on the surface of B. burgdorferi, the ability of recombinant forms of the protein to bind to beta3-chain integrins and the fact that p66 and B. burgdorferi bind to beta3-chain integrins in a mutually exclusive manner make p66 an attractive candidate bacterial ligand for integrins alphaIIbbeta3 and alphavbeta3.     

Fragmentation of proteins in cartilage treated with interleukin-1: specific cleavage of type IX collagen by matrix metalloproteinase 13 releases the NC4 domain

Degradation of bovine nasal cartilage induced by interleukin-1 (IL-1) was used to study catabolic events in the tissue over 16 days. Culture medium was fractionated by two-dimensional electrophoresis (isoelectric focusing and SDS-PAGE). Identification of components by peptide mass fingerprinting revealed released fragments representing the NC4 domain of the type IX collagen alpha1 chain at days 12 and 16. A novel peptide antibody against a near N-terminal epitope of the NC4 domain confirmed the finding and indicated the presence of one of the fragments already at day 9. Mass spectrometric analysis of the two most abundant fragments revealed that the smallest one contained almost the entire NC4 domain cleaved between arginine 258 and isoleucine 259 in the sequence -ETCNELPAR258-COOH NH2-ITP-. A larger fragment contained the NC4 domain and the major part of the COL3 domain with a cleavage site between glycine 400 and threonine 401 in COL3 (-RGPPGPPGPPGPSG400-COOH NH2-TIG-). The presence of multiple collagen alpha1 (IX) N-terminal sequences demonstrates that the released molecules were cleaved at sites very close to the original N terminus either prior to or due to IL-1 treatment. Matrix metalloproteinase 13 (MMP-13) is active and cleaves fibromodulin in the time interval studied. Cartilage explants treated with MMP-13 were shown to release collagen alpha1 (IX) fragments with the same sizes and with the same cleavage sites as those obtained upon IL-1 treatment. These data describe cleavage by an MMP-13 activity toward non-collagenous and triple helix domains. These potentially important degradation events precede the major loss of type II collagen.