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
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ABSTRACT |
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The Goodpasture (GP) autoantigen has been
identified as the 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 The GP autoepitope(s) has been localized to the NC1 domain of the
The aim of this study was to identify the 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
The cDNA for the human
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
Construction of C1 and C4 chimeras was based on a
regular PCR strategy using pRC/f
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
Recombinant 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 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% 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 Design and Expression of
Since GP sera react preferentially with
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
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).
Immunoreactivity of
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
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 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
The
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
The data were further analyzed to estimate the fraction of
autoreactivity that could be attributed specifically to the Conformational Nature of the Epitope(s)--
It has been
previously shown that the reduction of disulfide bonds in 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
The PEPSCAN results demonstrated lack of strong specific binding and a
high background, presumably due to nonspecific binding. Both
Two epitopes previously found by mutagenesis of In the present study, a new strategy based on chimeric proteins
was employed to map regions within 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
Our results demonstrate that regions EA and
EB reproduce very well the authentic GP epitopes
in the 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 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- In summary, two specific homologous sequences in 3(IV) collagen chain, one of six homologous chains
designated
1-
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
3(IV) chain.
Fourteen
1/
3 NC1 chimeras were constructed by substituting one or
more short sequences of
3(IV)NC1 at the corresponding positions in
the non-immunoreactive
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
3(IV)NC1 and C6 containing residues 127-141 of
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
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
1- and
3(IV)NC1 sequences. The amino acid sequences 17-31 and 127-141 in
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
3(IV) collagen chain, one of six homologous
chains designated
1-
6 that comprise type IV collagen (2). In the
glomerular basement membrane, the
3(IV) chain exists in a
supramolecular network along with the
4(IV) and
5(IV) chains (3).
The
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).
3(IV) chain (5, 6). Antibodies that bind to the NC1 domain of other
(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
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
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).
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
3(IV)NC1 regions most likely to form the autoepitope(s) are
those most divergent from the other homologous
(IV) chains. A series
of chimeric
1/
3(IV)NC1 domains were constructed in which these
candidate
3(IV)NC1 sequences replaced the corresponding sequences in
the non-immunoreactive
1(IV)NC1. The chimeras were expressed in
mammalian cells for correct protein folding and disulfide bond
formation. We report that two specific sequences,
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
3(IV)NC1 domain.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
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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 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
3(IV)NC1 sequences is illustrated in the dashed
circle (see "Experimental Procedures").
The PCR primer sequences and the restriction enzymes used for cloning
of the recombinant proteins
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/f
1 construct
was subsequently used for the construction of
1/
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.
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Fig. 2.
Schematic illustration of the
1/
3(IV)NC1 chimeric
constructs. At the positions indicated by filled
circles, sequences of amino acids from
3(IV)NC1 replaced those
in the
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.
1(IV)NC1 template, residues complementary to a part of the
replacement
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
3(IV) sequence, and
then ligation produced a circular expression vector containing a
chimeric
1/
3(IV)NC1 insert with no extraneous sequence.
1 as a template and introducing
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
1(IV) chain had first to be added to the 5' end of the
1(IV)NC1
sequence. An
1(IV)NC1 + Gly-X-Y insert was amplified from
a pRc/f
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.
1/
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
1 mg/ml was calculated from the amino acid
composition of the six human
(IV)NC1 domains (20).
3(IV)NC1 expressed in kidney 293 cells and E. coli was prepared as described (21, 22). Native human
3(IV)NC1 was isolated from glomerular basement membrane (23). Human kidneys unsuitable for transplantation were obtained from Midwest Organ Bank,
Kansas City, KS.
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.
-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
(IV)NC1 domains or chimeras prior to addition to plates coated with
3(IV)NC1. The results shown are the averages of duplicate determinations.
1(IV)
(GenBankTM accession number P02462) and
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
1/
3(IV)NC1 Chimeras--
In this
study,
1/
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
1(IV) and
3(IV) collagen (71% sequence identity and six
conserved disulfide bonds), which very likely adopt similar tertiary
structures (27). In the chimeras,
1(IV)NC1 acted as an inert
"carrier" and provided a three-dimensional scaffold for the
substituted
3(IV) sequences.
3(IV)NC1, but not the other
(IV)NC1 domains, the autoepitope(s) must contain amino acids
specific to
3(IV)NC1. Our recent comparative analysis of the
sequences of
(IV)NC1 domains has now permitted the identification of
six putative locations of the epitope(s) as short regions (less than 15 residues) in
3(IV)NC1 that are most divergent from other
(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
3(IV)NC1 sequences replaced the corresponding amino acids within the
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.
3(IV) sequence.
C8 contained the 36 carboxyl-terminal residues of
3(IV)NC1 and, in addition, had two
1(IV) Gly-X-Y
triplets at the amino terminus. The additional Gly-X-Y
sequences, also present in the native collagenase-digested
1- and
3(IV)NC1 domains, were incorporated in the C7 and
C8 chimeras to emulate the proteins previously used to map
the autoepitope (15).
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Fig. 3.
Western blot analysis of the
1/
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.
1/
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
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
1 backbone, because it was accompanied by comparable binding to
1(IV)NC1. Remarkably, neither C7 nor C8
chimeras bound autoantibodies.
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
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
1(IV)NC1 bound all chimeras, producing a higher background.
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Fig. 4.
Direct ELISA of 14 1/
3(IV) chimeras with GP
sera. Proteins were coated onto plastic plates at 100 ng/well.
Recombinant
1-,
2-, and
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.
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
3(IV)NC1
titers, but always higher than those of
1(IV)NC1 and
2(IV)NC1
controls.
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Fig. 5.
Titration of GP1 serum binding to
1/
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
1(IV) (filled circles),
2(IV) (filled
triangles), and
3(IV) (filled squares) NC1 domains.
The binding to immobilized proteins of serial dilutions of the serum
was measured by direct ELISA.
(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,
3(IV)NC1 > C2·6 > C2 > C6 >
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.
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Fig. 6.
Inhibition ELISA of GP antibodies binding
to 3(IV)NC1 domain. Top,
inhibition of GP1 serum by soluble
1/
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
1(IV)NC1
(filled circles),
2(IV)NC1 (filled triangles),
and
3(IV)NC1 (filled squares) domains were used as
controls. Bottom, comparison of eight GP sera by inhibition
ELISA using
1/
3 chimeras and
(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
3(IV)NC1.
Individual data for the eight sera are represented by
symbols, and their average is shown as a horizontal
line.
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,
1(IV)NC1 but not
2(IV)NC1 inhibited autoantibody
binding to
3(IV)NC1 by about 24%. This effect can be attributed to
cross-reactivity, since
3(IV)NC1 is more similar to the
1- and
5(IV)NC1 domains than to
2-,
4-, or
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
3(IV) sequence in the chimeras, whereas the shallower portion of the
curves at higher chimera concentrations, which parallels the
1(IV)NC1 inhibition curve, is likely caused by cross-reactivity with
the
1(IV)NC1 scaffolding.
1(IV), while giving almost
complete inhibition with
3(IV)NC1. Inhibition with C2·6 was 65 ± 13%, compared with 85 ± 7% for control
3(IV)NC1, demonstrating that this chimera contains the
immunodominant autoepitope(s) of
3(IV)NC1. C2·6 chimera
had a stronger effect than either C2 (46 ± 8%) or
C6 chimeras (23 ± 18%). This indicates that the
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.
3(IV)NC1 sequences in the chimeras. For each serum, the inhibition produced by
the
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
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
3(IV)NC1 (on average
23%, ranging between 6 and 38%) could not be accounted for by
EA, EB, or by
cross-reactivity with
1(IV)NC1.
Relative immunoreactivity against various 3(IV)NC1 regions in eight
GP sera
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
3(IV)NC1.
To evaluate whether EA and
EB form a linear or a conformational epitope,
the GP reactivity of the
1/
3 chimeras was measured before and
after reduction. As in
3(IV)NC1, the reduction of disulfide bonds
also abolished binding of autoantibodies to the C2,
C6, and C2·6 chimeras and even to
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.
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Fig. 7.
Effect of reduction on the GP reactivity
of 1/
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%
-mercaptoethanol prior to immobilization. Recombinant
1- and
3(IV)NC1 domains were also used as controls. ELISA was performed
using GP1, GP2, and GP6 sera at 1:100 dilution.
1(IV)-
and
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
1- and
3(IV)NC1 peptides (9).
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Fig. 8.
PEPSCAN analysis of the
1(IV) and
3(IV)NC1
domains. The reactivity to
3- and
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
3(IV)NC1) are also
shown.
1- and
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
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
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.
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
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
3(IV)NC1 used in this work,
expressed in 293 kidney cells, was found as reactive as native human
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
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.
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Fig. 9.
Comparison of
3(IV)NC1 expressed in "293" kidney cells and
E. coli. Microtiter plates were coated with 100 ng of native human
3(IV)NC1 purified from glomerular basement
membrane (N) and recombinant
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
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
3(IV)NC1 candidate regions
(<15 residues) were grafted onto an inert
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
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
3(IV)NC1.
3(IV)NC1 domain but not the other five homologous NC1 domains of
type IV collagen; (b) therefore, regions of substantial
sequence divergence between
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).
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
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
3(IV)NC1
domain. In contrast, linear
3(IV)NC1 peptides produced a comparable
effect in inhibition ELISA only at concentrations 100-1000-fold higher
(11, 14).
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
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
3(IV) IgG
(36).
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
3(IV)NC1 but not
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
3(IV)NC1, is whether any of the
1/
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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 1/
3 chimeras, but larger regions of
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
3(IV)NC1 accounts for most immunoreactivity with GP sera.
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ABBREVIATIONS |
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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, 3(IV)NC1
residues 17-31 and 127-141, respectively.
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REFERENCES |
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