From the Departments of Biochemistry and Molecular
Biology and ¶ Pediatrics, University of Kansas Medical Center,
Kansas City, Kansas 66160
Received for publication, October 2, 2000, and in revised form, November 17, 2000
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ABSTRACT |
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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 GPA
autoepitope is encompassed by residues 17-31 (the EA
region) within the noncollagenous (NC1) domain of the Goodpasture (GP)1
disease is defined by rapidly progressive glomerulonephritis,
with or without lung hemorrhage, which is caused by autoantibodies
targeted to type IV collagen of the glomerular and alveolar basement
membranes. Left untreated, the disease is potentially lethal. If
diagnosed early, GP patients can be treated by immunosuppression and
plasma exchange to remove the toxic autoantibodies. This therapy has
side effects, including a weakening of the natural defenses; thus, a
more specific therapy is highly desirable. A detailed knowledge of the
epitope would facilitate the development of therapies that selectively
remove, neutralize, or prevent synthesis of the pathogenic
autoantibodies and provide a foundation for studies to determine the
etiology of the disease.
The GP autoantigen is the 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
EA region, but is fully accessible in the hexamer complex. In this study, the identity of residues that compose the
GPA autoepitope was determined, and the molecular basis of
its cryptic nature was explored. This was achieved using site-directed
mutagenesis to exchange the
3(IV) residues in the EA
region with the corresponding residues of the homologous but
non-immunoreactive
1(IV) NC1 domain and then comparing the
reactivity of the mutated chimeras with GPA and Mab3
antibodies. It was shown that three hydrophobic residues (Ala18, Ile19, and Val27) and
Pro28 are critical for the GPA autoepitope,
whereas two hydrophilic residues (Ser21 and
Ser31) along with Pro28 are critical for the
Mab3 epitope. These results suggest that the cryptic nature of the
GPA autoepitope is the result of quaternary interactions of
the
3,
4, and
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 chain of type IV collagen (1, 2), one of
the six chains (
1-
6) that compose type IV collagen (3). The GP
autoepitopes are conformational and reside within the 232-residue long
noncollagenous (NC1) domain at the C-terminus of the
3(IV) chain.
Three chains of type IV collagen assemble into triple-helical protomers
(molecule) that further interact with each other at the amino and
carboxyl termini to form supramolecular networks. In the glomerular
basement membrane, which is the main target of autoantibodies, the
3(IV) chain associates with the
4(IV) and
5(IV) chains to form
a cross-linked
3·
4·
5(IV) network (4). The GP epitopes are
cryptic in the
3·
4·
5 NC1 hexamer complex formed by the
interaction of two triple-helical protomers through the NC1 domains
(Fig. 1, top). As a result,
the epitopes are inaccessible for binding of autoantibodies unless the
hexamer dissociates (5-7). Unmasking of previously hidden GP epitopes is thought to be of fundamental importance for understanding the etiology and pathogenesis of GP disease.
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Fig. 1.
Model representing the location within the
native NC1 hexamer complex of the cryptic GP autoepitopes and of the
exposed Mab3 epitope of the 3(IV) NC1
domain. In the type IV collagen networks found in vivo,
two triple-helical collagen protomers interact through their
carboxyl-terminal ends, forming an NC1 hexamer complex
(top). Two conformational GP epitopes, designated
GPA and GPB, have been localized to the
EA and EB regions of the
3(IV) NC1 domain.
These regions jointly form the epitope for Mab3. The GP
epitopes (diagonal lines) are cryptic in the NC1 hexamer
complex and inaccessible for binding of autoantibody (shown for
GPA antibodies; right), but they are exposed
upon dissociation of the hexamer into subunits, allowing binding of the
autoantibody. In contrast, the Mab3 epitope (solid black) is
accessible in both the hexamer and dissociated (monomer) form of the
3(IV)NC1 domain (left). Hence, in the NC1 hexamer
complex, the EA and EB regions contain certain
inaccessible residues that are critical for the GP epitopes and other
accessible residues that are critical for the Mab3 epitope (7).
Two conformational GP autoepitopes have recently been mapped to two
regions of the 3(IV) NC1 domain, designated EA and
EB, by homolog-scanning mutagenesis using chimeric
1/
3 NC1 domains in which the non-immunoreactive
1 NC1 domain
was used as a scaffold for exchanging short homologous
1 sequences
with
3 NC1 segments to ensure correct folding of the epitope. The
immunodominant autoepitope, designated GPA (7), has been
localized to the N-terminal third of the
3(IV) NC1 domain (8, 9) and
specifically to residues 17-31, designated the EA region
(7, 10, 11). Autoantibodies specific for the EA region,
designated GPA, are believed to play an important role in
the pathogenesis of GP disease because they are the predominant
subpopulation (~60-65%) in all sera and have high affinity for
autoantigen (7). Moreover, high titers of GPA antibodies
are correlated with an unfavorable disease outcome (9). A second
autoepitope, designated GPB (7), was also identified in the
central portion of the
3(IV) NC1 domain (9) and was further mapped
to residues 127-141, designated the EB region (7, 10).
In contrast to the cryptic GP autoepitopes, the epitope for Mab3 monoclonal antibody, which is also localized to the EA and EB regions, is fully accessible even in the NC1 hexamer (7). These observations led to the hypothesis that only certain amino acid residues of the EA and EB regions constitute the GPA and GPB autoepitopes and that their cryptic nature in the NC1 hexamer complex is a result of either direct interactions with or close proximity to other NC1 domains in the hexamer, which prevents the access of autoantibodies. Moreover, these critical residues must be distinctly different from those that constitute the Mab3 epitope, which are accessible on the surface of the hexamer complex.
The aim of this study was to identify which residues compose the
GPA autoepitope and to explore the molecular basis of its cryptic nature. This was accomplished using homolog-scanning
mutagenesis to change the 3(IV)-specific residues within the
EA region of an
1/
3 chimera, one at a time, to the
corresponding residues from the homologous but non-immunoreactive
1(IV) NC1 domain. The reactivity of mutated NC1 domains with
GPA and Mab3 antibodies was then compared. It was shown
that three hydrophobic residues (Ala18, Ile19,
and Val27) and Pro28 are critical for the
GPA autoepitope, whereas two hydrophilic residues
(Ser21 and Ser31) along with Pro28
are critical for the Mab3 epitope. These results suggest that the
cryptic nature of the GPA autoepitope is the result of
quaternary interactions among the
3(IV),
4(IV), and
5(IV) NC1
domains of the
3·
4·
5 hexamer complex that bury the one or
more hydrophobic residues. These findings provide critical information
for understanding the etiology and pathogenesis of the disease as well
as for designing drugs that would mimic the epitope and thus block the
binding of GP autoantibodies to autoantigen in vivo.
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EXPERIMENTAL PROCEDURES |
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Site-directed Mutagenesis and Expression of Mutated NC1
Chimeras--
Eight chimeric constructs with individual 3(IV) to
1(IV) amino acid substitutions in the EA epitope region,
designated M1-M8 (see Fig. 2), were constructed using the
GeneEditorTM in vitro site-directed mutagenesis
system (Promega, Madison, WI) according to the manufacturer's
protocol. Substitutions from
3(IV) to
1(IV) amino acids were
introduced by specific primers (Table I).
The previously described C2·6 construct (10), containing the
GP epitope regions EA and EB from the
3(IV)
NC1 domain substituted into the
1(IV) NC1 domain scaffolding, was
used as a template. The clones thus obtained were digested with
NheI and SacII restriction enzymes (New England
Biolabs Inc., Beverly, MA) and subcloned into the pRcX expression
vector (10, 12), which contains the BM-40 signal peptide and the FLAG
peptide (DYKDDDDK) sequences downstream of the cytomegalovirus
promoter. All constructs were sequenced to confirm the
substitutions.
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Human embryonic kidney 293 cells (ATCC 1573 CRL) were transfected with ~5 µg of plasmid DNA using the calcium phosphate precipitation method, and G418-resistant cells were screened for expression of recombinant protein from the culture medium by Western blotting with anti-FLAG monoclonal antibody M2 (Sigma) as previously described (10). Recombinant proteins were purified by affinity chromatography on an anti-FLAG M2 affinity column (Sigma) according to the manufacturer's instructions. The concentration of the purified NC1 domains and chimeras was determined spectrophotometrically using 1.6 A280 = 1 mg/ml (10).
Monoclonal Antibodies--
Several monoclonal antibodies to the
3(IV) NC1 domain were used, the epitopes of which have been
previously localized (7). Mab3 was purchased from Wieslab AB (Lund,
Sweden). M3/1, raised against an amino-terminal synthetic peptide of
the human
3(IV) NC1 domain, and monoclonal antibody 175, raised
against randomly folded recombinant human
3(IV) NC1 domain expressed
in Escherichia coli, were previously described (13).
GP Patient Sera--
The serum or the plasmapheresis fluid from
13 patients diagnosed with Goodpasture disease (GP1-13) was used. The
titer of GP antibodies was measured by ELISA in microtiter plates
coated with 3(IV) NC1 domain (100 ng/well); and for further
analyses, sera were appropriately diluted to yield approximately equal
reactivity. The relative autoantibody reactivity to the EA
and EB regions was measured by ELISA using the C2 and C6
chimeras, respectively (10). GP sera that reacted predominantly with
EA (GPA autoantibodies) were used without
further purification. GP sera that showed significant reactivity
against the EB region required further purification by
absorption of GPB antibodies to a column with immobilized
C6 as previously described (7). The unbound fraction, consisting of
GPA antibodies, was used in the subsequent immunoassays for mapping the epitope location. The bound GPB fraction was
eluted from the C6 affinity column with 3 M guanidinium
chloride and used as a control to establish the correct folding of the
mutated chimeras.
Western Blots-- The proteins (300 ng), separated by SDS-10% polyacrylamide gel electrophoresis under nonreducing conditions, were transferred onto nitrocellulose membranes and immunoblotted with anti-FLAG and Mab3 monoclonal antibodies as well as with GP sera as previously described (10).
Direct and Inhibition Immunoassays--
The 1(IV) NC1 domain
and chimera C6 (negative controls), chimera C2·6 and the
3(IV) NC1
domain (positive controls), and mutated chimeras M1-M8 were coated in
duplicates onto MaxisorpTM ELISA plates (Nunc) at
100 ng/well in 50 mM carbonate buffer, pH 9.6. Binding of
GP autoantibodies or Mab3 was determined by direct ELISA as previously
described (7). Detection was performed with alkaline
phosphatase-conjugated secondary antibodies followed by
p-nitrophenol phosphate, and color development was monitored at 410 nm with a Dynatech MR4000 plate reader. Each serum was analyzed
at least four times, with the median of the results being taken as the
serum's representative value. For inhibition ELISA, the GP sera or
purified GP antibody fractions were incubated overnight at room
temperature with various amounts of recombinant NC1 domains or chimeras
prior to addition to plates coated with the C2·6 chimera.
Statistical Analysis--
The relative optical density of the
mutated protein chimeras with respect to the binding of GP
autoantibodies was calculated as the ratio of the absorption upon ELISA
of each mutated chimera to that of the template C2·6 chimera. Results
are reported as the mean ± S.D. of the serum's median values.
Statistics were calculated using the SPSS software package (Version
9.0). The overall significance of differences in relative binding for
M1-M8 was analyzed by repeated measures-analysis of variance. Reduced binding to individual chimeras, relative to the template C2·6 chimera, was tested by one-sample t tests. Possible serum
subgroups based on nonparallel binding profiles for M1-M8 were
revealed by hierarchical cluster analysis and followed up with
independent-sample t tests and Pearson correlations to
identify the specific chimeras responsible for significantly divergent
profiles. All statistical tests were two-tailed. Significance was
inferred when p was <0.05.
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RESULTS |
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Homolog-scanning Mutagenesis of the Immunodominant GP Epitope
Region EA--
A 15-residue region of 3(IV) NC1,
designated EA (residues 17-31), was sufficient to confer
reactivity for the immunodominant population of GP autoantibodies
(GPA) when substituted into the non-immunoreactive scaffold
of the
1(IV) NC1 domain (7, 10). Thus, the EA region
encompasses the autoepitope for GPA antibodies. The
EA region also contains residues that constitute the
epitope for the Mab3 monoclonal antibody, along with other critical
residues from the EB region (7). Although GPA
and Mab3 antibodies bind to the same EA region, their
respective epitopes are distinct because of dissimilar accessibility
for antibody binding in the NC1 hexamer complex; this suggests that the
EA region comprises buried (or sterically hindered)
residues that compose the GPA autoepitope as well as
surface-exposed residues that compose the Mab3 epitope (7). Here,
homolog-scanning mutagenesis was used to identify which of the eight
3-specific residues within the EA region constitute the
epitopes for GPA and Mab3 antibodies.
The C2·6 chimera, which contains the EA and
EB regions of the 3(IV) NC1 domain in a scaffold of the
1(IV) NC1 domain (Fig. 2,
top), was used as a template for mutagenesis. The
EB region was included in the chimera because it, along
with the EA region, is required for binding of the Mab3
antibody (7). Moreover, the EB region encompasses residues
that constitute the conformational epitope for GPB
autoantibodies. Thus, the binding of GPB antibodies can
serve as a control for assessment of overall conformation of proteins
mutated in the EA region (see below). In this study, each
of the eight
3-specific residues of the EA region was
substituted with the corresponding
1 residue. The mutated chimeras,
designated M1-M8 (Fig. 2, bottom), were then analyzed for
their ability to bind GPA and Mab3 antibodies as well as
the control GPB antibodies.
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Expression and Correct Folding of the M1-M8 Mutated
Chimeras--
The mutated chimeras were expressed in human embryonic
kidney 293 cells. After purification from the culture medium by
affinity chromatography on an anti-FLAG column, the M1-M8 mutants
appeared as a single band on SDS-polyacrylamide gel at the predicted
molecular mass of ~25 kDa (Fig.
3A) and showed reactivity with
anti-FLAG antibodies on Western blots (Fig. 3B). All mutated
chimeras reacted with GPB antibodies as well as or better
than the template C2·6 chimera (Fig. 3C), indicating that
the mutations did not cause protein misfolding. Moreover, certain
chimeras reacted with GPA antibodies, but not with Mab3
antibodies and vice versa (as described in detail below), further
establishing that the loss of reactivity after mutagenesis was due to
removal of a critical binding site and not to protein misfolding. The
epitopes of GPA, GPB, and Mab3 antibodies are
conformational and present only in correctly folded protein, as shown
by the contrast between the recombinant 3(IV) NC1 domain expressed
in human embryonic kidney 293 cells, which is correctly folded, and
that expressed in E. coli, which is misfolded (10). The
misfolded protein did not react with GPA, GPB,
and Mab3 antibodies, although it reacted with monoclonal antibodies that recognize linear epitopes, M3/1 and 175 (Fig.
3D).
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Immunoreactivity of Mutants M1-M8 with GPA
Autoantibodies--
To determine which amino acids of the
EA region are critical for the GPA autoepitope,
the reactivity of the M1-M8 chimeras with sera from 13 GP patients was
analyzed by ELISA (Fig. 4). To allow
comparison of data across multiple experiments, the reactivity of the
mutants was expressed relative to that of the template C2·6 chimera,
which constitutes the positive control because it contains the complete
EA regions of 3 along with the EB region. Chimera C6, containing the EB but not the EA
region of the
3(IV) NC1 domain, was used as a negative control. At
least four measurements were performed for each serum. To exclude the
interference of EB in the binding studies,
EB-depleted GP sera and GP sera with little or no
EB reactivity were used.
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For the overall group of 13 sera (Fig. 4A), the binding
profile for mutated chimeras was characterized by large reductions for
M2 (57%; p < 0.001), M6 (44%; p < 0.001), and M7 (67%; p < 0.001) and a small reduction
in binding for M1 (16%; p = 0.035). The notably large
variability in binding associated with M3 (Fig. 4, A and
B) suggested potentially important differences among the
binding profiles of individual sera. This was confirmed by a cluster
analysis, which revealed two independent serum groups (p = 0.008). The eight sera in group A (Fig.
4C) had large reductions in reactivity with the M2, M6, and
M7 mutants (by 56, 57, and 58%, respectively; p < 0.001). Corrected for reactivity of the negative control (the C6
chimera), the respective reductions with M2, M6, and M7 were 83, 84, and 85%. In contrast, the five sera in group B (Fig. 4D)
had large reductions in reactivity with mutants M2 (60%), M3 (70%),
and M7 (81%) (p < 0.001), along with small reductions
with M1 (30%) and M6 (23%) (p < 0.05). Corrected for the reactivity of the negative control, in group B, the respective reductions with M2, M3, and M7 were 75, 85, and 96%. Group A differed significantly from group B regarding M3, M6, and M7 (p < 0.01). That M3 and M6 were singularly responsible for distinguishing the serum subgroups was shown by the large negative correlation between
M3 and M6 in the group of 13 sera (r = 0.766;
p = 0.002) (Fig. 4B). The reduced reactivity
of M2, M3, M6, and M7 was confirmed by inhibition ELISA (Fig.
5). These findings indicate that
Ala18, Ile19, Val27, and
Pro28 constitute the critical residues of the
GPA autoepitope.
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Immunoreactivity of Mutants M1-M8 with Mab3--
To determine
which amino acids in the EA region are critical for the
Mab3 epitope, the reactivity of the M1-M8 mutants with Mab3 was
determined by Western blotting and ELISA (Fig.
6). Three of the eight mutants, M4, M7,
and M8, showed no reactivity by Western blotting and significantly
decreased reactivity by ELISA (by 97, 85, and 62%, respectively,
relative to the reactivity of the C2·6 chimera; p < 0.005). This indicates that Ser21, Pro28, and
Ser31 are critical residues for the Mab3 epitope. With the
exception of Pro28, this subset of amino acids is different
from that composing the GPA autoepitope, confirming the
supposition that the Mab3 and GPA epitopes are different,
but colocalized within the 15-residue EA region. Consistent
with the location of the Mab3 epitope on the surface of the NC1
hexamer, the two serine residues important for the binding of Mab3 are
hydrophilic and thus predicted to be solvent-accessible in folded
proteins.
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This result also provided additional evidence that the mutated chimeras
were correctly folded, all showing reactivity with GPB and
either GPA or Mab3 antibodies, which bind to conformational epitopes. Mutation of Pro18 abolished the interaction with
both Mab3 and GPA, suggesting that this proline may be
shared by both epitopes. Alternatively, Pro18 could play a
structural role, being required for the correct conformation of the
EA region within the 3(IV) NC1 domain. The mutation P28I
did not seem to affect the overall secondary structure of the NC1
domain because the far-UV circular dichroism spectrum of the M7 mutant
was similar to that of the parent C2·6 chimera and to that of
the recombinant
3(IV) NC1 domain (data not shown). Moreover, if the
P28I mutation had any effect on the conformation, this would be limited
to the EA region, as binding of GPB
autoantibodies to a neighboring EB epitope was not affected.
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DISCUSSION |
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The inaccessibility of the GPA autoepitope,
contrasting with the full accessibility of the Mab3 epitope, afforded
an experimental strategy to identify critical residues of these
conformational epitopes and to explore the molecular basis of the
cryptic nature of the GPA autoepitope. To this end, the
15-residue EA region of the 3(IV) NC1 domain was mutated
at eight
3-specific residues, and the resulting chimeras (M1-M8)
were assessed for their ability to bind GPA and Mab3
antibodies. The M2 (A18D), M3 (I19D), M6 (V27K), and M7 (P28I) chimeras
had greatly decreased binding to GPA antibodies, whereas
the M4 (S21Q), M7 (P28I), and M8 (S31H) chimeras had greatly decreased
binding to Mab3 antibodies. The loss of binding of GPA
antibodies to one set of chimeras and of Mab3 to another, along with
binding of GPB antibodies to all chimeras, indicated that
the overall conformation of the chimeras did not differ from that of
the control (template) C2·6 chimera. Hence, a decrease in antibody
binding to a specific chimera was due to removal of a critical epitope
residue rather than to misfolding and/or mispairing of disulfide bonds.
Thus, the binding profiles indicate that Ala18 and
Pro28, along with either Ile19 in certain GP
patients or Val27 in other GP patients, are critical for
the GPA autoepitope, whereas Ser21,
Ser31, and Pro28 are critical for the Mab3
epitope, as depicted in Fig. 7.
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Interestingly, three of the four GPA residues
(Ala18, Ile19, and Val27) are
hydrophobic (14, 15) and have a high propensity to be buried (16). This
suggests that the cryptic nature of the GPA autoepitope is
because one or more GPA residues participate in hydrophobic
interactions with other NC1 domains in the hexamer complex, thus
burying the epitope and rendering it inaccessible for binding of
autoantibodies (Fig. 7). In contrast, the Mab3 epitope is accessible
because it contains critical residues that have a high propensity to be
located on the hexamer surface (16): two hydrophilic serine residues
and one proline residue, which is most often found in -turns and
other loops on the protein surface. Interestingly, Pro28 is
critical for both GPA and Mab3 epitopes; thus, it may be
important in defining the conformation of the EA region in
the
3(IV) NC1 domain.
The hydrophobic character of the GPA residues represents a
departure from the conventional features of epitopes, which consist predominately of charged or polar residues and tend to be located in
flexible turns or loops on protein surfaces (17, 18). Based on these
features, numerous empirical methods have been developed that
successfully predict the major antigenic sites of native proteins (19).
The 3(IV) NC1 sequence was analyzed with the program Epiplot, which
calculates and plots flexibility, hydrophilicity, and antigenicity
profiles using 13 different scales, chosen as those yielding the best
predictions on proteins whose antigenic structures are known (20). None
of these methods predicted the location of the GPA
autoepitope or the Mab3 epitope, which reside within the amphipathic
EA region of the
3(IV) NC1 domain, as illustrated by the
popular Hopp-Woods hydrophilicity plot in Fig. 8 (upper) (21). Thus, other
factors must govern the immunogenicity of the EA region,
eliciting the production of autoantibodies to GPA residues
on one hand and of antibodies to Mab3 on the other.
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In particular, the lack of immune tolerance to the GPA epitope may be explained by its cryptic nature, which renders it an immunologically privileged site. To avoid autoreactivity, the B cell clones directed to self-antigens are edited out early in development, establishing self-tolerance. Because the GPA epitope is buried in the NC1 hexamer complex under normal physiological conditions, it is sequestered from the immune system. Therefore, if pathogenic factors induce hexamer dissociation, the newly exposed GPA residues would then be perceived as "foreign" by the immune system, eliciting an autoimmune response. What factors trigger this process in vivo remain unclear. Hydrocarbons or viral infections have been suggested as causative agents (22). A recent study provides evidence that reactive oxygen species may act as the physiological mediator for epitope exposure (23).
Certain of the GPA residues identified herein differ from
those found in two recent studies by Wieslander and co-workers (11, 24), who did not identify the overall hydrophobic character of the
epitope. The first study qualitatively investigated the role of 14 3(IV) NC1 domain residues, including six of the eight residues in
the EA region (11). Similar to our findings,
Ile19 and Pro28, but not Thr17,
were found important for binding. However, Ala18 was
reported to be not essential, and the role of Glu24 and
Val27 was not addressed. In addition, Ser21 and
Ser31 were reported to be critical for binding GP
antibodies, whereas we found these residues to be important only for
the binding of the Mab3 antibody. Since a quantitative data analysis
was not reported in that study, it is difficult to directly compare
their findings with ours. A very recent paper from the same group (24) quantitatively analyzed the role of four
3 residues in the
EA region and found Val27 to be critical for GP
antibody binding (similar to our findings), whereas Thr17,
Ala18, and Glu24 had a moderate effect. Some of
the discrepancies may be explained by the different
1/
3 chimeras
used as template for the homolog-scanning mutagenesis. Their template
chimera contained only the EA region, whereas ours
contained both the EA and EB regions of the
3(IV) NC1 domain. Because the EA and EB
regions are in close proximity in the natively folded
3(IV) NC1
domain, mutants M1-M8 used in our work are likely to reproduce the
native GP epitopes more closely. In addition, our study relied on a
mutagenesis strategy that allowed (a) a verification of the
conformation of the mutants by using three different antibodies that
recognize conformational epitopes and (b) a comparison
between the binding of GPA and Mab3 antibodies, a
priori inferred to bind to different residues in the
EA region.
The identification of the critical residues of the GPA
epitope allows the determination of the structural features that
selectively target GPA antibodies to the 3(IV) NC1
domain, among the six homologous NC1 domains of type IV collagen. As
revealed by the comparison in Fig. 8 (lower), all four
GPA residues (Ala18, Ile19,
Val27, and Pro28) occur at the respective
position in the
3 sequence only. Intriguingly, three of the
GPA residues are hydrophobic in the
3 sequence, but the
homologous
1 residues are hydrophilic, charged residues (Asp, Asp,
and Lys, respectively). Analysis of data from experimentally determined
antigenic sites on proteins has revealed that hydrophobic residues are
more likely to be a part of antigenic sites, if they occur on the
surface of a protein (25), as GPA residues do in the
3(IV) NC1 monomer. No other chain besides
3(IV) had a
constellation of three hydrophobic residues at positions 18, 19, and
27. Moreover, Pro28 occurs only in
3, but not in any of
the other five NC1 domains. Therefore, three hydrophobic residues at
positions 18, 19, and 27, together with a proline at position 28, in a
distinct conformation distinguish
3 among the six NC1 domains,
conferring binding of GPA antibodies selectively to the
3(IV) NC1 domain.
The EA region of the 3(IV) NC1 domain emerges as a prime
candidate for a molecular recognition site that specifies the
chain-specific assembly of the
3·
4·
5 network of type IV
collagen. Recently, we showed that the NC1 monomers contain recognition
sequences for selection of chains and protomers that are sufficient to
encode the specificity of assembly of the
1·
2 and
3·
4·
5(IV) networks of the glomerular basement membrane
(26), but their identity is unknown. That the EA region is
a site of interaction between
3 and the other NC1 domains in the
3·
4·
5 hexamer is deduced from its cryptic nature of the
GPA epitope. That the EA region may also be
responsible for the specificity of interaction is suggested by the high
sequence divergence of this region among the six NC1 domains (27). The
EA region is also distinguished by the highest number of
non-conservative amino acid substitutions. Hence, the pattern of
hydrophobic and hydrophilic residues within the EA region
is unique for each of the six NC1 domains and is likely to confer
chain-specific conformations and interactions. In particular, the
EA region of all six NC1 domains may contribute to the
discriminatory interactions that result in specific assembly of
chain-specific networks of type IV collagen.
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FOOTNOTES |
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* This work was supported by Grant DK18381 from the National Institutes of Health (to B. G. H.); by Grant 9920539Z from the American Heart Association, Kansas Affiliate (to D.-B. B.); and by the David Flinchbaugh Memorial Fund.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.
§ The first two authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
913-588-7008; Fax: 913-588-7035; E-mail: bhudson@kumc.edu.
Published, JBC Papers in Press, November 29, 2000, DOI 10.1074/jbc.M008956200
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ABBREVIATIONS |
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The abbreviations used are: GP, Goodpasture; ELISA, enzyme-linked immunosorbent assay.
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REFERENCES |
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1. |
Butkowski, R. J.,
Langeveld, J. P.,
Wieslander, J.,
Hamilton, J.,
and Hudson, B. G.
(1987)
J. Biol. Chem.
262,
7874-7877 |
2. |
Saus, J.,
Wieslander, J.,
Langeveld, J. P.,
Quinones, S.,
and Hudson, B. G.
(1988)
J. Biol. Chem.
263,
13374-13380 |
3. |
Hudson, B. G.,
Reeders, S. T.,
and Tryggvason, K.
(1993)
J. Biol. Chem.
268,
26033-26036 |
4. |
Gunwar, S.,
Ballester, F.,
Noelken, M. E.,
Sado, Y.,
Ninomiya, Y.,
and Hudson, B. G.
(1998)
J. Biol. Chem.
273,
8767-8775 |
5. |
Wieslander, J.,
Langeveld, J.,
Butkowski, R.,
Jodlowski, M.,
Noelken, M.,
and Hudson, B. G.
(1985)
J. Biol. Chem.
260,
8564-8570 |
6. |
Kalluri, R.,
Sun, M. J.,
Hudson, B. G.,
and Neilson, E. G.
(1996)
J. Biol. Chem.
271,
9062-9068 |
7. |
Borza, D.-B.,
Netzer, K. O.,
Leinonen, A.,
Todd, P.,
Cervera, J.,
Saus, J.,
and Hudson, B. G.
(2000)
J. Biol. Chem.
275,
6030-6037 |
8. | 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] |
9. | Hellmark, T., Segelmark, M., Unger, C., Burkhardt, H., Saus, J., and Wieslander, J. (1999) Kidney Int. 55, 936-944[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Netzer, K. O.,
Leinonen, A.,
Boutaud, A.,
Borza, D.-B.,
Todd, P.,
Gunwar, S.,
Langeveld, J. P.,
and Hudson, B. G.
(1999)
J. Biol. Chem.
274,
11267-11274 |
11. |
Hellmark, T.,
Burkhardt, H.,
and Wieslander, J.
(1999)
J. Biol. Chem.
274,
25862-25868 |
12. | Leinonen, A., Netzer, K. O., Boutaud, A., Gunwar, S., and Hudson, B. G. (1999) Kidney Int. 55, 926-935[CrossRef][Medline] [Order article via Infotrieve] |
13. | 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] |
14. | Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132[Medline] [Order article via Infotrieve] |
15. | Rose, G. D., Geselowitz, A. R., Lesser, G. J., Lee, R. H., and Zehfus, M. H. (1985) Science 229, 834-838[Medline] [Order article via Infotrieve] |
16. | Lesser, G. J., and Rose, G. D. (1990) Proteins Struct. Funct. Genet. 8, 6-13[Medline] [Order article via Infotrieve] |
17. | Barlow, D. J., Edwards, M. S., and Thornton, J. M. (1986) Nature 322, 747-748[Medline] [Order article via Infotrieve] |
18. | Hopp, T. P. (1993) Pept. Res. 6, 183-190[Medline] [Order article via Infotrieve] |
19. | Stern, P. S. (1991) Trends Biotechnol. 9, 163-169[Medline] [Order article via Infotrieve] |
20. | Menendez-Arias, L., and Rodriguez, R. (1990) Comput. Appl. Biosci. 6, 101-105[Abstract] |
21. | Hopp, T. P., and Woods, K. R. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 3824-3828[Abstract] |
22. | Wilson, C. B., Borza, D.-B., and Hudson, B. G. (2000) in The Molecular Pathology of Autoimmune Diseases (Theofilopoulos, A. N. , and Bona, A. C., eds), 2nd Ed. , pp. 981-1010, Gordon and Breach Science Publishers, Inc./Harwood Academic Publishers, Newark, NJ |
23. |
Kalluri, R.,
Cantley, L. G.,
Kerjaschki, D.,
and Neilson, E. G.
(2000)
J. Biol. Chem.
275,
20027-20032 |
24. |
Gunnarsson, A.,
Hellmark, T.,
and Wieslander, J.
(2000)
J. Biol. Chem.
275,
30844-30848 |
25. | Kolaskar, A. S., and Tongaonkar, P. C. (1990) FEBS Lett. 276, 172-174[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Boutaud, A.,
Borza, D.-B.,
Bondar, O.,
Gunwar, S.,
Netzer, K. O.,
Singh, N.,
Ninomiya, Y.,
Sado, Y.,
Noelken, M. E.,
and Hudson, B. G.
(2000)
J. Biol. Chem.
275,
30716-30724 |
27. |
Netzer, K. O.,
Suzuki, K.,
Itoh, Y.,
Hudson, B. G.,
and Khalifah, R. G.
(1998)
Protein Sci.
7,
1340-1351 |