The Goodpasture Autoantigen
MAPPING THE MAJOR CONFORMATIONAL EPITOPE(S) OF alpha 3(IV) COLLAGEN TO RESIDUES 17-31 AND 127-141 OF THE NC1 DOMAIN*

Kai-Olaf NetzerDagger , Anu Leinonen, Ariel Boutaud, Dorin-Bogdan Borza, Parvin Todd, Sripad Gunwar, Jan P. M. Langeveld§, and Billy G. Hudson

From the Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160 and the § Institute for Animal Science and Health (ID-DLO), P. O. Box 65, 8200 AB Lelystad, The Netherlands

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Goodpasture (GP) autoantigen has been identified as the alpha 3(IV) collagen chain, one of six homologous chains designated alpha 1-alpha 6 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 alpha 3(IV) chain. Fourteen alpha 1/alpha 3 NC1 chimeras were constructed by substituting one or more short sequences of alpha 3(IV)NC1 at the corresponding positions in the non-immunoreactive alpha 1(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 alpha 3(IV)NC1 and C6 containing residues 127-141 of alpha 3(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 alpha 3(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 alpha 1- and alpha 3(IV)NC1 sequences. The amino acid sequences 17-31 and 127-141 in alpha 3(IV)NC1 have thus been shown to contain the critical residues of one or two disulfide bond-dependent conformational autoepitopes that bind GP autoantibodies.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Goodpasture (GP)1 autoimmune disease is characterized by pulmonary hemorrhage and/or rapidly progressing glomerulonephritis (1). Tissue injury is mediated by anti-basement membrane antibodies that bind alveolar and glomerular basement membranes. The target autoantigen of basement membranes has been identified as the alpha 3(IV) collagen chain, one of six homologous chains designated alpha 1-alpha 6 that comprise type IV collagen (2). In the glomerular basement membrane, the alpha 3(IV) chain exists in a supramolecular network along with the alpha 4(IV) and alpha 5(IV) chains (3). The alpha 3(IV) chain is composed of a long collagenous domain of 1410 amino acids and a non-collagenous (NC1) domain of 232 residues at the carboxyl terminus (4).

The GP autoepitope(s) has been localized to the NC1 domain of the alpha 3(IV) chain (5, 6). Antibodies that bind to the NC1 domain of other alpha (IV) chains may be found in some Goodpasture patients (7, 8), but they only account for about 10% of autoreactivity (9). The autoepitope(s) in the alpha 3(IV)NC1 domain appears to be conformational, because reduction of disulfide bonds abolishes most of the binding (9-11). The identification of the precise amino acid residues that constitute this epitope(s) is important for understanding the etiology and pathogenesis of the GP disease and for the development of diagnostic and therapeutic agents. Several groups have attempted to map the location of the autoepitope(s) by using short linear peptides (9, 11-14) or by site-directed mutagenesis of the alpha 3(IV)NC1 domain expressed in Escherichia coli (15). Although linear sequences have been identified that bind GP antibodies, these findings are at variance with each other. Moreover, prior studies have not addressed whether these linear sequences constitute the major conformational, disulfide bond-dependent epitope(s).

The aim of this study was to identify the alpha 3(IV)NC1 amino acid sequences that form the thus far elusive conformational GP epitope(s). To circumvent the limitations of previous approaches, we pursued an epitope mapping strategy based on chimeric proteins. This approach has been specifically developed and successfully used to map conformational epitopes (16) or autoepitopes (17). We hypothesized that the alpha 3(IV)NC1 regions most likely to form the autoepitope(s) are those most divergent from the other homologous alpha (IV) chains. A series of chimeric alpha 1/alpha 3(IV)NC1 domains were constructed in which these candidate alpha 3(IV)NC1 sequences replaced the corresponding sequences in the non-immunoreactive alpha 1(IV)NC1. The chimeras were expressed in mammalian cells for correct protein folding and disulfide bond formation. We report that two specific sequences, alpha 3(IV)NC1 residues 17-31 and 127-141, contain the critical residues of one or two disulfide bond-dependent conformational GP autoepitopes within the alpha 3(IV)NC1 domain.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

cDNA Manipulation and Chimera Construction-- A suitable vector (Fig. 1) for the expression of recombinant proteins was based on pRc/AC7, a derivative of pRc/CMV (Invitrogen) that contained an expression cassette consisting of the BM-40 5'-untranslated region, BM-40 signal peptide, and an alpha 3 type VI collagen insert (18). By using a two-step inverse PCR with the appropriate primers (Table I), the original insert was replaced by a FLAGTM recognition sequence (Eastman Kodak Co.), and additional restriction sites (NheI, ClaI, HpaI, and SacII) were introduced further downstream. The resulting vector (pRc-X) was used for the expression of the chimeras (Fig. 1, middle). After cleavage of the signal peptide, secreted proteins would contain at the amino terminus a 14-residue fusion sequence (APLADYKDDDDKLA) that included the FLAG peptide (underlined) used for affinity purification.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 1.   Expression of recombinant NC1 domains and chimeric constructs. Bottom, schematic of pRc/CMV. Key elements of the vector are shown. Middle, expression cassette and multiple cloning sites inserted between CMV promoter (PCMV) and bovine growth hormone polyadenylation signal (BGH pA). The position of the translation start codon (ATG) is indicated. Top, schematic representation of the alpha 1(IV)NC1 insert cloned between NheI and SacII sites. The position of the stop codon is shown at the right (TAA). The principle of inverse PCR for the insertion of alpha 3(IV)NC1 sequences is illustrated in the dashed circle (see "Experimental Procedures").

                              
View this table:
[in this window]
[in a new window]
 
Table I
The PCR primer sequences and the restriction enzymes used for cloning of the recombinant proteins

The cDNA for the human alpha 1(IV)NC1 domain was amplified from a human kidney cDNA library (Marathon-ReadyTM, CLONTECH) by PCR using Klentaq polymerase (CLONTECH) and subcloned into pCRTMII vector by using a TA cloning kit (Invitrogen). The inserts with the correct sequence were subcloned into pRc-X. The resulting pRc/falpha 1 construct was subsequently used for the construction of alpha 1/alpha 3 chimeras (Fig. 2). Unless otherwise indicated, Pfu polymerase (Stratagene) was used in the PCR reactions for its low error rates. Restriction enzymes and ligase were purchased from New England Biolabs. The correct sequence of each construct was verified by sequencing.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 2.   Schematic illustration of the alpha 1/alpha 3(IV)NC1 chimeric constructs. At the positions indicated by filled circles, sequences of amino acids from alpha 3(IV)NC1 replaced those in the alpha 1(IV)NC1 (open circles). The disulfide bonds are represented as short lines closing the loops. The arrow indicates the junction between the collagenous and NC1 domains.

The principle of the inverse PCR approach that was used for chimeras C2, C3, C5, and C6 is depicted inside the dashed circle in Fig. 1 (top). The primers (Table I) were designed in a back-to-back orientation, each containing (in 3' to 5' order) residues complementary to the alpha 1(IV)NC1 template, residues complementary to a part of the replacement alpha 3(IV)NC1 sequence, and the recognition site of the inward-cutting BbsI restriction enzyme (GAAGAC(N)2/6). PCR yielded 6.3-kilobase pair amplicons that comprised the whole vector and insert. Digestion with BbsI removed the recognition site and created complementary ends inside the inserted alpha 3(IV) sequence, and then ligation produced a circular expression vector containing a chimeric alpha 1/alpha 3(IV)NC1 insert with no extraneous sequence.

Construction of C1 and C4 chimeras was based on a regular PCR strategy using pRC/falpha 1 as a template and introducing alpha 3 sequences by primers at the 5' and 3' ends of the NC1 insert, respectively. The PCR products were digested with restriction enzymes (Table I) and subcloned into the pRc-X vector for expression. The construction of C7 chimera followed a similar scheme, requiring C1 as template. In order to construct chimera C8, two collagenous Gly-X-Y triplets of the alpha 1(IV) chain had first to be added to the 5' end of the alpha 1(IV)NC1 sequence. An alpha 1(IV)NC1 + Gly-X-Y insert was amplified from a pRc/falpha 1 template, digested with NheI/PpuMI, and ligated into a C3·4 vector preparation cut with the same enzymes. Inverse PCR with this template generated chimera C8.

Six combination chimeras were also constructed as follows: C1·2, C1·4, C2·6, C3·5, C1·2·5, and C7·8. C1·2 chimera was generated using primers for the C1 construct and C2 as template, digested with NheI and ClaI, and then subcloned into the pRc-X vector. The remaining chimeras were generated by subcloning the chimeric insert region of one chimera into a vector preparation of another chimera digested with the same restriction enzymes. C1·4 required subcloning of a NheI/PpuMI C1 insert fragment into the C4 vector; likewise, C2·6 required an ApaI C6 insert in the C2 vector; C3·5 required a PpuMI/SacII C3 insert in the C5 vector; C1·2·5 required an ApaI C5 insert in the C1·2 vector; and C7·8 required a PpuMI/SacII C8 insert in the C7 vector.

Protein Expression and Purification-- Recombinant alpha 1/alpha 3 chimeras were expressed in human embryonic kidney 293 cells (ATCC 1573-CRL) grown in Dulbecco's modified Eagle's medium/F-12 medium (Sigma) supplemented with 5% fetal bovine serum (Sigma) and 50 µg/ml ascorbic acid phosphate magnesium salt (Wako). Five to ten µg of plasmid DNA were transfected by the calcium phosphate co-precipitation method (19) into 70% confluent 293 cells. After 2 days, transfected cells were selected with 250 µg/ml G418 (Life Technologies, Inc.). Resistant cells were screened for expression of recombinant protein by Western blot using an anti-FLAG monoclonal antibody (M2, Kodak) and expanded for quantitative expression. The medium was collected from subconfluent cultures every 48 h, and the recombinant proteins were purified by affinity chromatography on anti-FLAG M2 affinity columns (Kodak) according to the manufacturer's instructions. Protein solutions were concentrated by ultrafiltration (Amicon) and stored at -70 °C. The concentration of recombinant protein solutions was measured spectrophotometrically at 280 nm. An average extinction coefficient A of 1.6 congruent  1 mg/ml was calculated from the amino acid composition of the six human alpha (IV)NC1 domains (20).

Recombinant alpha 3(IV)NC1 expressed in kidney 293 cells and E. coli was prepared as described (21, 22). Native human alpha 3(IV)NC1 was isolated from glomerular basement membrane (23). Human kidneys unsuitable for transplantation were obtained from Midwest Organ Bank, Kansas City, KS.

Sera-- The plasmapheresis fluid or sera from eight patients diagnosed with Goodpasture syndrome (GP1-8) were used. The titer of GP autoantibodies was measured by direct ELISA in plates (Nunc) coated with alpha 3(IV)NC1 (100 ng/well). Relative to the GP1 serum previously described (24), GP1-4 had about the same titer, GP5-6 had a titer about 10-fold lower, and GP7-8 had about 80-fold lower.

Western Blots-- SDS-polyacrylamide gel electrophoresis (25) was performed in 4-20% gradient gels, under non-reducing conditions, using 500 ng of protein per lane. For immunoblotting, the proteins (200 ng of protein/lane) were transferred to nitrocellulose membranes, reacted with GP sera (1:100) and alkaline phosphatase-conjugated goat anti-human IgG (1:1000), then stained with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium.

Direct and Inhibition Immunoassays-- MaxiSorpTM polystyrene microtiter plates (Nunc, Denmark) were coated overnight at room temperature with antigen (50-200 ng/well, as shown) in 50 mM carbonate buffer, pH 9.6, and then blocked with casein or BSA. In some experiments, the antigen was reduced prior to coating by treatment with 10% beta -mercaptoethanol for 5 min at 100 °C. GP sera and normal human sera (negative controls) were diluted in the incubation buffer (2% casein or 2 mg/ml BSA and 0.05% Tween 20 in Tris-buffered saline). Alkaline phosphatase-conjugated goat anti-human IgG (1:2000) was used as secondary antibody. p-Nitrophenol phosphate (1 mg/ml in 1 M diethanolamine buffer, pH 9.8, containing 0.5 mM zinc chloride) was used as substrate, and the development of color was monitored at 410 nm in a Dynatech MR4000 plate reader. For inhibition ELISA, the GP sera were incubated overnight at room temperature with various amounts of recombinant alpha (IV)NC1 domains or chimeras prior to addition to plates coated with alpha 3(IV)NC1. The results shown are the averages of duplicate determinations.

PEPSCAN Analysis-- Mapping of linear epitopes was performed using the "PEPSCAN" method (26). A complete set of solid phase overlapping 12-mer peptides was synthesized onto polyethylene pins following the published sequences of NC1 domains of alpha 1(IV) (GenBankTM accession number P02462) and alpha 3(IV) collagen (GenBankTM accession number X80031). The immunoscreening of these peptides was performed by ELISA. The pins were incubated for 1 h with GP serum (diluted 1:50) and then washed three times. The bound antibody was detected by the reaction with peroxidase-labeled secondary antibody for 30 min, followed by color development with 2,2'-azinobis-3-ethylbenzthiazolinesulfonic acid for another 30 min.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Design and Expression of alpha 1/alpha 3(IV)NC1 Chimeras-- In this study, alpha 1/alpha 3 chimeras were used to identify the conformational epitope(s) of the GP autoantigen. This strategy relied on the high sequence homology between the NC1 domains of alpha 1(IV) and alpha 3(IV) collagen (71% sequence identity and six conserved disulfide bonds), which very likely adopt similar tertiary structures (27). In the chimeras, alpha 1(IV)NC1 acted as an inert "carrier" and provided a three-dimensional scaffold for the substituted alpha 3(IV) sequences.

Since GP sera react preferentially with alpha 3(IV)NC1, but not the other alpha (IV)NC1 domains, the autoepitope(s) must contain amino acids specific to alpha 3(IV)NC1. Our recent comparative analysis of the sequences of alpha (IV)NC1 domains has now permitted the identification of six putative locations of the epitope(s) as short regions (less than 15 residues) in alpha 3(IV)NC1 that are most divergent from other alpha (IV)NC1 domains and that are also predicted to be accessible to solvent (27). Accordingly, six chimeric NC1 domains (C1-C6) were constructed in which these alpha 3(IV)NC1 sequences replaced the corresponding amino acids within the alpha 1(IV)NC1 domain (Fig. 2). Five combination chimeras (C1·2, C1·4, C2·6, C3·5, C1·2·5) were also constructed to allow identification of non-contiguous GP epitopes.

To analyze two previously proposed GP epitopes (11, 15, 28) using this approach, three additional chimeras (C7, C8, and C7·8) were constructed. The 26 amino-terminal amino acids of C7 chimera, which included four collagenous Gly-X-Y triplets, were from the alpha 3(IV) sequence. C8 contained the 36 carboxyl-terminal residues of alpha 3(IV)NC1 and, in addition, had two alpha 1(IV) Gly-X-Y triplets at the amino terminus. The additional Gly-X-Y sequences, also present in the native collagenase-digested alpha 1- and alpha 3(IV)NC1 domains, were incorporated in the C7 and C8 chimeras to emulate the proteins previously used to map the autoepitope (15).

The recombinant chimeric proteins were expressed in the human embryonic kidney 293 cells and isolated from the culture medium as monomers with an apparent molecular mass of about 25-30 kDa by SDS-polyacrylamide gel electrophoresis (Fig. 3a). Unlike expression in E. coli (22), expression in the human kidney 293 cells yields properly folded recombinant NC1 domains that are indistinguishable by FT-IR or immunoassays from those prepared from native sources.2 This cell line has been successfully used to express other proteins with native folding, including basement membrane proteins nidogen (29) and laminin (30).


View larger version (72K):
[in this window]
[in a new window]
 
Fig. 3.   Western blot analysis of the alpha 1/alpha 3(IV) chimeras with GP sera. SDS-polyacrylamide gel electrophoresis was performed in 4-20% gradient gels under non-reducing conditions, and the proteins were stained with Coomassie Blue (a). The molecular mass markers (MW std) indicated by arrowheads were 60, 40, 30, 20, and 15 kDa. Western blotting was performed using GP1 (b), GP2 (c), and GP6 (d) sera at 1:100 dilution.

Immunoreactivity of alpha 1/alpha 3 Chimeras with GP Sera-- The reactivity of the chimeric constructs with GP sera was analyzed by Western blotting as well as by direct and inhibition ELISA. The pattern of autoantibody binding obtained in Western blots with three sera show remarkable similarities (Fig. 3, b-d). Only two chimeras, C2 and C6 (containing residues 17-31 and 127-141 of alpha 3(IV)NC1, respectively), reacted strongly and consistently with GP antibodies, as did combination chimeras containing one or both these regions (C1·2, C2·6, C1·2·5). Some sera showed weak reactivity with other chimeras, but this appears to be due to the cross-reactivity with the alpha 1 backbone, because it was accompanied by comparable binding to alpha 1(IV)NC1. Remarkably, neither C7 nor C8 chimeras bound autoantibodies.

To confirm these findings, the binding of eight GP sera to immobilized chimeras was assessed in direct ELISA (Fig. 4). Sera were diluted proportionally to their titers to allow visualization of the specificity of the low titer sera side by side with the high titer sera. In general, the pattern of reactivity observed in the Western blots was also apparent in the ELISA. All sera reacted strongly with C2 chimera (which averaged 71% of the maximal reactivity, obtained with alpha 3(IV)NC1), C1·2 (47%), C2·6 (70%), and C1·2·5 (64%). There was more variation in the reactivity toward C6 chimera (31% of the reactivity of alpha 3(IV)NC1), which bound significantly only five out of eight sera. All but one low titer serum (GP7) bound more to C2 than to C6 chimera. Sera that showed cross-reactivity with alpha 1(IV)NC1 bound all chimeras, producing a higher background.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4.   Direct ELISA of 14 alpha 1/alpha 3(IV) chimeras with GP sera. Proteins were coated onto plastic plates at 100 ng/well. Recombinant alpha 1-, alpha 2-, and alpha 3(IV)NC1 domains were used as controls. Eight GP sera were diluted in the incubation buffer proportionally to their titers (1:1000 for the reference serum GP1). Two normal sera were used as negative controls at 1:50 dilution. The average ELISA reading of the normal sera (0.09 ± 0.04 A410) was subtracted from the values obtained with GP sera.

The relative reactivity of any given serum toward recombinant proteins was not influenced by the dilution of the serum. This was apparent in the titration curves shown in Fig. 5, which yielded parallel lines for various immobilized proteins. Similar results were obtained with the other sera. All sera had the highest reactivity toward alpha 3(IV)NC1, which was closely followed by C2·6 and C2 chimeras (less than a 2-fold difference in titers). The C6 chimera titers of the sera were more variable, between 2- and 10-fold lower than the alpha 3(IV)NC1 titers, but always higher than those of alpha 1(IV)NC1 and alpha 2(IV)NC1 controls.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5.   Titration of GP1 serum binding to alpha 1/alpha 3(IV) chimeras. The wells were coated with 100 ng of antigen: C2 (open circles), C6 (open triangles), and C2·6 (open squares) chimeras, and controls alpha 1(IV) (filled circles), alpha 2(IV) (filled triangles), and alpha 3(IV) (filled squares) NC1 domains. The binding to immobilized proteins of serial dilutions of the serum was measured by direct ELISA.

Immunodominance of Antibodies Binding to C2 and C6 Chimeras-- It is well established that adsorption of proteins to plastic may cause denaturation, so that the antibody binding measured in direct ELISA may actually be to the denatured antigen. To rule out such artifacts, the interaction between the GP antibodies and antigen was studied in solution by inhibition ELISA in the presence of soluble chimeras and control alpha (IV)NC1 domains. The inhibition curves were determined for three GP sera and were found to be similar. Typical data for one serum are shown in Fig. 6 (top panel). The inhibitory capacity of the chimeras and the control proteins followed the same order as found in direct ELISA, alpha 3(IV)NC1 C2·6 > C2 > C6 > alpha 1(IV)NC1, consistent with the results obtained with the latter technique. The effect of the chimeras was saturable, leveling off at the highest concentration used, where it produced 42-67% inhibition, a significant proportion of the autoantibody reactivity.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 6.   Inhibition ELISA of GP antibodies binding to alpha 3(IV)NC1 domain. Top, inhibition of GP1 serum by soluble alpha 1/alpha 3 chimeras. The GP1 serum, diluted 1:100, was incubated overnight with C2 (open circles), C6 (open triangles), and C2·6 (open squares) chimeras at various concentrations before the immunoassay. Recombinant alpha 1(IV)NC1 (filled circles), alpha 2(IV)NC1 (filled triangles), and alpha 3(IV)NC1 (filled squares) domains were used as controls. Bottom, comparison of eight GP sera by inhibition ELISA using alpha 1/alpha 3 chimeras and alpha (IV)NC1 domains. Sera, diluted as described in Fig. 4, were incubated overnight with 10 µg/ml antigen. ELISA was performed in plates coated with 50 ng/well alpha 3(IV)NC1. Individual data for the eight sera are represented by symbols, and their average is shown as a horizontal line.

The alpha 3(IV)NC1 domain could completely inhibit autoantibody binding and had an I50 (the concentration of competitor at which half-maximal inhibition is achieved) of 0.27 ± 0.03 µg/ml (about 11 nM), in good agreement with the previously reported values of 0.5 (31) and 0.8 µg/ml (9). At high concentrations, alpha 1(IV)NC1 but not alpha 2(IV)NC1 inhibited autoantibody binding to alpha 3(IV)NC1 by about 24%. This effect can be attributed to cross-reactivity, since alpha 3(IV)NC1 is more similar to the alpha 1- and alpha 5(IV)NC1 domains than to alpha 2-, alpha 4-, or alpha 6(IV)NC1 domains (27). An I50 value could not be reliably calculated for chimeras because the inhibition curves they produced could not be fitted adequately to a simple inhibition model. Visual examination of these curves revealed a bi-phasic behavior. The steep inhibition below 2 µg/ml (Fig. 6, top panel) is probably due to the specific alpha 3(IV) sequence in the chimeras, whereas the shallower portion of the curves at higher chimera concentrations, which parallels the alpha 1(IV)NC1 inhibition curve, is likely caused by cross-reactivity with the alpha 1(IV)NC1 scaffolding.

To quantitate the binding specificity of eight GP sera, an inhibition ELISA was performed at a fixed concentration of soluble antigen, 10 µg/ml (Fig. 6, bottom panel). This concentration was chosen to minimize cross-reactivity with alpha 1(IV), while giving almost complete inhibition with alpha 3(IV)NC1. Inhibition with C2·6 was 65 ± 13%, compared with 85 ± 7% for control alpha 3(IV)NC1, demonstrating that this chimera contains the immunodominant autoepitope(s) of alpha 3(IV)NC1. C2·6 chimera had a stronger effect than either C2 (46 ± 8%) or C6 chimeras (23 ± 18%). This indicates that the alpha 3(IV)NC1 residues 17-31 (hereafter referred to as EA) and 127-141 (hereafter referred to as EB) form either two separate epitopes or a single, more complete one, but it appears to rule out significant cross-reactivity between the two homologous sequences.

The data were further analyzed to estimate the fraction of autoreactivity that could be attributed specifically to the alpha 3(IV)NC1 sequences in the chimeras. For each serum, the inhibition produced by the alpha 1(IV)NC1 domain (which averaged 7 ± 4%) was subtracted from the total inhibition given by the chimeras to correct for the cross-reactivity due to the common scaffold, and then the results were normalized to the effect produced by alpha 3(IV)NC1 (Table II). The effect of EA (present in C2 chimera) was strong and consistent (on average 47%, ranging between 27 and 64%) and predominated in seven out of eight sera. In contrast, EB (present in C6 chimera) produced variable inhibition with different sera (on average 18%, ranging between 3 and 56%) and was predominant only in GP7. Together, as in C2·6 chimera, these sequences accounted for most inhibition of GP sera (on average 68%, ranging between 52 and 88%). Only a small fraction of GP reactivity toward alpha 3(IV)NC1 (on average 23%, ranging between 6 and 38%) could not be accounted for by EA, EB, or by cross-reactivity with alpha 1(IV)NC1.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Relative immunoreactivity against various alpha 3(IV)NC1 regions in eight GP sera

Conformational Nature of the Epitope(s)-- It has been previously shown that the reduction of disulfide bonds in alpha 3(IV)NC1 impairs its ability to react with GP antibodies, indicative of a conformational epitope (9-11). Quantitation of this effect with the eight sera used in the present work showed that only 6 ± 5% of the original immunoreactivity remains after reduction of alpha 3(IV)NC1. To evaluate whether EA and EB form a linear or a conformational epitope, the GP reactivity of the alpha 1/alpha 3 chimeras was measured before and after reduction. As in alpha 3(IV)NC1, the reduction of disulfide bonds also abolished binding of autoantibodies to the C2, C6, and C2·6 chimeras and even to alpha 1(IV)NC1 (Fig. 7). Less than 10% of the original immunoreactivity remained in the reduced proteins, although they had the same or higher reactivity with monoclonal antibodies that do not require a conformational epitope, such as anti-FLAG (data not shown). Overall, these results demonstrate that only a small proportion of GP antibodies can recognize linear epitopes and that EA and EB belong to one or two conformational GP epitopes that are disulfide bond-dependent.


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of reduction on the GP reactivity of alpha 1/alpha 3 chimeras. The microtiter plates were coated with 200 ng/well native (hashed bars) or reduced (black bars) antigen in coating buffer. The proteins were reduced by treatment with 10% beta -mercaptoethanol prior to immobilization. Recombinant alpha 1- and alpha 3(IV)NC1 domains were also used as controls. ELISA was performed using GP1, GP2, and GP6 sera at 1:100 dilution.

Comparison of Chimera-based Epitope Mapping with Previous Approaches-- To compare the chimera-based epitope mapping strategy with approaches using linear peptides (9, 11, 13, 14) and to evaluate whether the latter identify linear or conformational GP epitopes, a peptide scanning analysis was performed. A valid comparison between the chimera-based and peptide-based strategies required using the same GP sera. Two complete sets of overlapping 12-mers based on the alpha 1(IV)- and alpha 3(IV)NC1 sequences (Fig. 8) were therefore synthesized and analyzed by the PEPSCAN procedure (26). A previous report using 20-mer peptides to map the GP epitope has indicated nonspecific binding of GP sera to homologous alpha 1- and alpha 3(IV)NC1 peptides (9).


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 8.   PEPSCAN analysis of the alpha 1(IV) and alpha 3(IV)NC1 domains. The reactivity to alpha 3- and alpha 1-derived 12-mer peptides is shown by thick and thin lines, respectively. GP1 (middle), GP2 (top), and GP5 (bottom) sera were used for immunoscreening at 1:50 dilution. The horizontal dotted lines are drawn two standard deviations above the median reading (continuous lines). The positions of EA and EB regions (residues 17-31 and 127-141 of alpha 3(IV)NC1) are also shown.

The PEPSCAN results demonstrated lack of strong specific binding and a high background, presumably due to nonspecific binding. Both alpha 1- and alpha 3(IV)NC1 sequences produced a number of peaks higher than two standard deviations above the median. However, the most reactive peptides (above three standard deviations) varied among the three GP sera tested. The most significant PEPSCAN peak was produced by peptides overlapping residues 94-110 of alpha 3(IV)NC1 with the GP2 serum. Much weaker reactivity was recorded in this region with the other two sera. This region corresponds to the C5 chimera that did not interact with GP sera in direct ELISA (Fig. 4), perhaps due the conformations of the 12-mer peptides on the pin being different from those adopted by the same amino acids in the NC1 domain. Some isolated alpha 3-derived peptides that overlapped the EA region produced peaks in PEPSCAN. However, the interactions were not strong enough to allow unambiguous identification of these residues as part of a GP autoepitope.

Two epitopes previously found by mutagenesis of alpha 3(IV)NC1 expressed in E. coli (15) were not observed in C7 and C8 chimeras, made in eukaryotic cells in the present work. Recombinant alpha 3(IV)NC1 expressed in E. coli was found about four times less reactive than the native human protein (Fig. 9), in agreement with earlier reports (22). In contrast, the recombinant alpha 3(IV)NC1 used in this work, expressed in 293 kidney cells, was found as reactive as native human alpha 3(IV)NC1 (Fig. 9). This suggests a folding difference whereby the full complement of conformational epitopes is not assembled in the E. coli-made protein. It is also possible that mutagenesis to alanine of residues from E. coli alpha 3(IV)NC1 may have caused the reported loss of GP immunoreactivity by affecting the overall structure of the protein and not the epitope itself.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 9.   Comparison of alpha 3(IV)NC1 expressed in "293" kidney cells and E. coli. Microtiter plates were coated with 100 ng of native human alpha 3(IV)NC1 purified from glomerular basement membrane (N) and recombinant alpha 3(IV)NC1 expressed in 293 kidney cell (EK) and E. coli (PK), respectively. BSA-coated wells were used as a negative control. Immunoreactivity with GP1 serum was measured by direct ELISA.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, a new strategy based on chimeric proteins was employed to map regions within alpha 3(IV)NC1 that constitute the conformational epitope(s) for GP autoantibodies. This novel approach has two methodological improvements over previous work. Unlike in peptide-based epitope mapping, short alpha 3(IV)NC1 candidate regions (<15 residues) were grafted onto an inert alpha 1(IV)NC1 framework and expressed in mammalian cells to ensure native folding. The resulting chimeras were assayed for "gain-of-function," i.e. capacity to bind autoantibodies, in contrast with previous site-directed mutagenesis studies (15) that relied on a "loss-of-function" of the protein expressed in E. coli. The results from 14 different chimeras revealed two previously unidentified regions, designated EA and EB (residues 17-31 and 127-141 of alpha 3(IV)NC1, respectively), that strongly bound autoantibodies from eight GP patients. Together, EA and EB accounted for 50-90% (on average 68%) of autoreactivity to alpha 3(IV)NC1.

Among the six candidate regions evaluated in this study, regions EA and EB clearly exhibited a distinct capacity to bind GP antibodies by Western blots, direct ELISA, and inhibition ELISA. The six regions were selected based on the following: (a) autoantibodies preferentially bind the alpha 3(IV)NC1 domain but not the other five homologous NC1 domains of type IV collagen; (b) therefore, regions of substantial sequence divergence between alpha 3(IV)NC1 and the other NC1 domains confer antibody binding to the former. The four regions that were found non-reactive (i.e. those substituted in C1, C3, C4, and C5 chimeras) further distinguish EA and EB as the primary regions for the GP epitope. It is significant that the EA and EB regions are homologous (47% sequence identity) and are located at corresponding positions in the two homologous NC1 subdomains (27), but they are noncontiguous. EA and EB could represent two separate and distinct epitopes or a single epitope EAB, in which EA and EB are held in close proximity to each other by the disulfide bonds. In either case, the complete epitope(s) probably includes additional residues from other regions, less critical for binding. So far, the x-ray crystallographic structures of other protein-antibody complexes have revealed noncontiguous epitopes of 15-22 amino acids that belong to several surface loops (32, 33).

Our results demonstrate that regions EA and EB reproduce very well the authentic GP epitopes in the alpha 3(IV)NC1 domain. Most remarkably, EA and EB form conformational epitopes that require intact disulfide bonds to bind GP antibodies, as demonstrated by loss of GP immunoreactivity of the C2, C6, and C2·6 chimeras upon reduction (Fig. 7). The majority of GP autoantibodies appears to recognize conformational epitopes in alpha 3(IV)NC1 (9-11), but epitope mapping studies have not addressed until now the nature of the epitopes found (see below). Further demonstrating the good mimicry of the original epitope(s), the chimeras produced significant inhibition of GP sera at concentrations in the range of 10-8 M, comparable with alpha 3(IV)NC1 domain. In contrast, linear alpha 3(IV)NC1 peptides produced a comparable effect in inhibition ELISA only at concentrations 100-1000-fold higher (11, 14).

The EA and EB regions have not been previously identified by peptide-based epitope mapping (9, 11-14). As shown here, this was due to the inability of peptide scanning procedures to reliably identify the conformational GP epitope(s). An intrinsic tendency of peptide-based methods to identify sequential epitopes has already been noted (34). Thus, the alpha 1(IV)NC1 framework of the chimeras is instrumental for adoption of the native conformation by EA and EB, and in addition, it may contribute auxiliary residues for binding. It is likely that the previous reports have largely identified linear GP epitopes, which constitute a minority (about 5% of the reactivity against alpha 3(IV)NC1). Furthermore, various linear sequences were found reactive in different studies, suggesting heterogeneity of the linear epitopes. In contrast, the chimera-based approach has successfully identified the critical regions of one or two immunodominant, conformational GP epitope(s) that were consistently recognized by all autoimmune sera used in this work.3

Region EA clearly represents an immunodominant epitope. It was recognized strongly and consistently by all sera analyzed, whereas EB reacted significantly (>10%) with only half of the sera. This may be due to the higher divergence of EA (eight distinct amino acids) compared with EB (five distinct amino acids). The existence of an immunodominant epitope explains the considerable cross-inhibition between GP sera from different patients or between GP sera and certain monoclonal antibodies (13, 31, 35). EA and EB may well be the counterpart of the shared structural determinants on the GP antibodies, found by using an anti-idiotype antibody against anti-alpha 3(IV) IgG (36).

In summary, two specific homologous sequences in alpha 3(IV)NC1 have been identified for the first time to be part of one or two disulfide bond-dependent, conformational and immunodominant GP autoepitopes. This finding provides new knowledge to investigate further the pathogenesis of GP disease. It has recently been shown that alpha 3(IV)NC1 but not alpha 1(IV)NC1 can induce experimental GP disease in mice (21). A very important question, relevant for the identification of the nephritogenic epitope(s) in alpha 3(IV)NC1, is whether any of the alpha 1/alpha 3 chimeras can induce experimental GP syndrome. In myasthenia gravis, another autoimmune disease, the immunodominant epitope on the acetylcholine receptor (known as "MIR" or main immunogenic region) was also pathogenic (37). By providing a highly specific target, the new identification of an immunodominant GP epitope should be useful for the development of more specific therapeutic approaches, such as use of vaccines to induce tolerance or the manipulation of the idiotype network.

    ACKNOWLEDGEMENTS

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

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK 18381 (to B. G. H.) and grants from the Fritz Thyssen Stiftung and the American Heart Association, Kansas Affiliate, Grant KS-94-F-5 (to K.-O. N.) A preliminary report of this work has been presented at the 30th Annual Meeting of the American Society of Nephrology (Netzer K.-O., Gunwar, S., Boutaud, A., Leinonen, A. and Hudson, B.G. (1997) J. Am. Soc. Nephrol. 8, 462 (abstr.)).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Medizinische Klinik I, Kliniken Köln-Merheim, Lehrkrankenhaus der Universität zu Köln, Köln, Germany.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160. E-mail: bhudson{at}kume.edu.

2 A. Boutaud, S. Gunwar, N. Singh, K.-O. Netzer, Y. Sado, Y. Ninomiya, M. E. Noelken, and B. G. Hudson, manuscript in preparation.

3 A report published at the time of submission of this study (38) also used alpha 1/alpha 3 chimeras, but larger regions of alpha 3(IV)NC1 were swapped (the amino-terminal 54 amino acids, the carboxyl-terminal 63 amino acids, and the intervening 115 amino acids, respectively). The conformational nature of the epitopes identified was not explored. Our more fine analysis is in full agreement with the broader conclusions of that study, namely that the amino-terminal third of alpha 3(IV)NC1 accounts for most immunoreactivity with GP sera.

    ABBREVIATIONS

The abbreviations used are: GP, Goodpasture; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; NC1, the noncollagenous domain of type IV collagen; PCR, polymerase-chain reaction; CMV, cytomegalovirus; EA and EB, alpha 3(IV)NC1 residues 17-31 and 127-141, respectively.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Wilson, C., and Dixon, F. (1986) in The Kidney (Berner, B., and Rector, F., eds), 3rd Ed., pp. 800-889, W. B. Saunders Co., Philadelphia
  2. Hudson, B. G., Reeders, S. T., and Tryggvason, K. (1993) J. Biol. Chem. 268, 26033-26036[Free Full Text]
  3. Gunwar, S., Ballester, F., Noelken, M. E., Sado, Y., Ninomiya, Y., and Hudson, B. G. (1998) J. Biol. Chem. 273, 8767-8775[Abstract/Free Full Text]
  4. Mariyama, M., Leinonen, A., Mochizuki, T., Tryggvason, K., and Reeders, S. T. (1994) J. Biol. Chem. 269, 23013-23017[Abstract/Free Full Text]
  5. Butkowski, R. J., Langeveld, J. P., Wieslander, J., Hamilton, J., and Hudson, B. G. (1987) J. Biol. Chem. 262, 7874-7877[Abstract/Free Full Text]
  6. Saus, J., Wieslander, J., Langeveld, J. P., Quinones, S., and Hudson, B. G. (1988) J. Biol. Chem. 263, 13374-13380[Abstract/Free Full Text]
  7. Kalluri, R., Wilson, C. B., Weber, M., Gunwar, S., Chonko, A. M., Neilson, E. G., and Hudson, B. G. (1995) J. Am. Soc. Nephrol. 6, 1178-1185[Abstract]
  8. Dehan, P., Weber, M., Zhang, X., Reeders, S. T., Foidart, J. M., and Tryggvason, K. (1996) Nephrol. Dial. Transplant. 11, 2215-2222[Abstract]
  9. Hellmark, T., Brunmark, C., Trojnar, J., and Wieslander, J. (1996) Clin. Exp. Immunol. 105, 504-510[Medline] [Order article via Infotrieve]
  10. Wieslander, J., Bygren, P., and Heinegard, D. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1544-1548[Abstract]
  11. Kalluri, R., Gunwar, S., Reeders, S. T., Morrison, K. C., Mariyama, M., Ebner, K. E., Noelken, M. E., and Hudson, B. G. (1991) J. Biol. Chem. 266, 24018-24024[Abstract/Free Full Text]
  12. Kefalides, N. A., Ohno, N., Wilson, C. B., Fillit, H., Zabriski, J., and Rosenbloom, J. (1993) Kidney Int. 43, 94-100[Medline] [Order article via Infotrieve]
  13. Levy, J. B., Turner, A. N., George, A. J., and Pusey, C. D. (1996) Clin. Exp. Immunol. 106, 79-85[Medline] [Order article via Infotrieve]
  14. Levy, J. B., Coulthart, A., and Pusey, C. D. (1997) J. Am. Soc. Nephrol. 8, 1698-1705[Abstract]
  15. Kalluri, R., Sun, M. J., Hudson, B. G., and Neilson, E. G. (1996) J. Biol. Chem. 271, 9062-9068[Abstract/Free Full Text]
  16. Hsia, R., Beals, T., and Boothroyd, J. C. (1996) Mol. Microbiol. 19, 53-63[Medline] [Order article via Infotrieve]
  17. Henriksson, E. W., and Pettersson, I. (1997) J. Autoimmun. 10, 559-568[CrossRef][Medline] [Order article via Infotrieve]
  18. Mayer, U., Poschl, E., Nischt, R., Specks, U., Pan, T. C., Chu, M. L., and Timpl, R. (1994) Eur. J. Biochem. 225, 573-580[Abstract]
  19. Sambrook, J. (1989) in Molecular Cloning: A Laboratory Manual (Fritsch, E. F., and Maniatis, T., eds), 2nd Ed., Vol. 3, pp. 16.32-16.40, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  20. Gill, S. C., and von Hippel, P. H. (1989) Anal. Biochem. 182, 319-326[Medline] [Order article via Infotrieve]
  21. Sado, Y., Boutaud, A., Kagawa, M., Naito, I., Ninomiya, Y., and Hudson, B. G. (1998) Kidney Int. 53, 664-671[CrossRef][Medline] [Order article via Infotrieve]
  22. Neilson, E. G., Kalluri, R., Sun, M. J., Gunwar, S., Danoff, T., Mariyama, M., Myers, J. C., Reeders, S. T., and Hudson, B. G. (1993) J. Biol. Chem. 268, 8402-8405[Abstract/Free Full Text]
  23. Wieslander, J., Kataja, M., and Hudson, B. G. (1987) Clin. Exp. Immunol. 69, 332-340[Medline] [Order article via Infotrieve]
  24. Gunwar, S., Ballester, F., Kalluri, R., Timoneda, J., Chonko, A. M., Edwards, S. J., Noelken, M. E., and Hudson, B. G. (1991) J. Biol. Chem. 266, 15318-15324[Abstract/Free Full Text]
  25. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  26. Geysen, H. M., Meloen, R. H., and Barteling, S. J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3998-4002[Abstract]
  27. Netzer, K., Suzuki, K., Itoh, Y., Hudson, B. G., and Khalifah, R. G. (1998) Protein Sci. 7, 1340-1351[Abstract/Free Full Text]
  28. Penades, J. R., Bernal, D., Revert, F., Johansson, C., Fresquet, V. J., Cervera, J., Wieslander, J., Quinones, S., and Saus, J. (1995) Eur. J. Biochem. 229, 754-760[Abstract]
  29. Fox, J. W., Mayer, U., Nischt, R., Aumailley, M., Reinhardt, D., Wiedemann, H., Mann, K., Timpl, R., Krieg, T., Engel, J., and Chu, M. (1991) EMBO J. 10, 3137-3146[Abstract]
  30. Yurchenco, P. D., Quan, Y., Colognato, H., Mathus, T., Harrison, D., Yamada, Y., and O'Rear, J. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10189-10194[Abstract/Free Full Text]
  31. Hellmark, T., Johansson, C., and Wieslander, J. (1994) Kidney Int. 46, 823-829[Medline] [Order article via Infotrieve]
  32. Laver, W. G., Air, G. M., Webster, R. G., and Smith-Gill, S. J. (1990) Cell 61, 553-556[Medline] [Order article via Infotrieve]
  33. Jones, S., and Thornton, J. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13-20[Abstract/Free Full Text]
  34. Schwab, C., Twardek, A., Lo, T. P., Brayer, G. D., and Bosshard, H. R. (1993) Protein Sci. 2, 175-182[Abstract/Free Full Text]
  35. Pusey, C. D., Dash, A., Kershaw, M. J., Morgan, A., Reilly, A., Rees, A. J., and Lockwood, C. M. (1987) Lab. Invest. 56, 23-31[Medline] [Order article via Infotrieve]
  36. Meyers, K. E., Kinniry, P. A., Kalluri, R., Neilson, E. G., and Madaio, M. P. (1998) Kidney Int. 53, 402-407[CrossRef][Medline] [Order article via Infotrieve]
  37. Tzartos, S. J., Cung, M. T., Demange, P., Loutrari, H., Mamalaki, A., Marraud, M., Papadouli, I., Sakarellos, C., and Tsikaris, V. (1991) Mol. Neurobiol. 5, 1-29[Medline] [Order article via Infotrieve]
  38. Ryan, J. J., Mason, P. J., Pusey, C. D., and Turner, N. (1998) Clin. Exp. Immunol. 113, 17-27[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.