(Received for publication, September 18, 1995; and in revised form, January 12, 1996)
From the
The family of type IV collagen comprises six chains numbered
1 through
6. The
3(IV) NC1 domain is the primary target
antigen for autoantibodies from patients with anti-basement membrane
disease and Goodpasture syndrome. Earlier peptide studies suggested
that the last 36 amino acids of the
3 NC1 domain probably contains
one recognition site for Goodpasture autoantibodies, and an algorithm
analysis of secondary structure from a later study predicted a second
possible upstream epitope near the triple helix junction. We have used
several analytic approaches to evaluate the likelihood of two
immunologic epitopes for the Goodpasture antigen. In our first set of
studies, peptide antibodies directed against these two putative regions
co-inhibited Goodpasture autoantibodies binding to denatured human
3(IV) NC1 monomer by nearly 80%, with the helix-junction region of
the
3 NC1 domain contributing 26% of the binding sites and the
C-terminal region contributing the remaining 50%. Second, both of these
candidate regions are normally sequestered within the associated
3(IV) NC1 hexamer but become more visible for binding by
anti-peptide antibodies upon their dissociation, a property that is
shared by the Goodpasture autoantibodies. Third, segment deletions of
recombinant
3 NC1 domain further confirmed the presence of two
serologic binding sites. Finally, we looked more closely at the
C-terminal binding region of the
3(IV) NC1 domain. Since the
lysines in that region have been previously advanced as possible
contact sites, we created several substitutions within the C-terminal
epitope of the
3 NC1 domain. Substitution of lysines to alanines
revealed lysines 219 and 229 as essential for antibody binding to this
distal site; no lysines were present in the NC1 part of the helix-NC1
junction region. Substitutions involving arginine and cysteines to
alanines in the same C-terminal region did not produce significant
reductions in antibody binding. In summary, our findings characterize
two Goodpasture epitopes confined to each end of the
3 NC1 domain;
one is lysine-dependent, and the other is not. We propose, as a
hypothetical model, that these two immunologically privileged regions
fold to form an optimal pathogenic structure within the NC1 domain of
the
3 chain. These sites are subsequently concealed by NC1 hexamer
assembly of type IV collagen.
Goodpasture syndrome is an autoimmune disease characterized
typically by rapidly progressive glomerulonephritis and pulmonary
hemorrhage mediated by anti-GBM
autoantibodies(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) .
The principal target for these antibodies is the NC1 domain of the
3(IV) chain of type IV
collagen(1, 3, 12, 13, 14, 15) .
Type IV collagen constitutes the major protein of mammalian basement
membranes(1, 16, 17, 18) . It plays
a role in providing tensile strength and scaffolding for the binding
and alignment of other basement membrane molecules, like laminin,
entactin, proteoglycan, and
fibronectin(1, 19, 20, 21, 22) .
Type IV collagen is composed of six genetically distinct
-chains
(
1-
6)(1, 12, 15, 23, 24, 25, 26, 27, 28, 29, 30, 31) .
The type IV collagen protomer is characterized by three distinct
structural domains: the amino-terminal collagenase-resistant
collagenous 7S domain, the carboxyl-terminal noncollagenous NC1 domain, (
)and the major triple helical region between the terminal
domains(1) . Bacterial collagenase solubilizes assembled type
IV protomers, digesting the triple helix and leaving NC1 hexamers and
7S resistant fragments(3) . Detailed studies of the NC1 domain
of type IV collagen from several tissues indicate that these hexamers
are comprised of dimers (45,000-55,000) and monomers
(24,000-28,000), the ratio of which varies from tissue to
tissue(3, 13, 32) .
The 3(IV) chain
has also been cloned from several different species and localized to
human chromosome 2(24, 33) . The full-length amino
acid sequence of the
3(IV) NC1 domain reveals several structural
similarities to the other chains of type IV collagen as well as some
distinct differences(34) . Some regions of the NC1 domain, for
example, have been implicated in the activation of polymorphonuclear
neutrophils (35) . Other mutations in the
3(IV) chain have
also been detected as polymorphisms in patients with autosomal
recessive Alport syndrome(1, 36) , and in Alport
patients with post-transplant anti-GBM nephritis, the target for their
anti-GBM antibodies is also the
3(IV) NC1
domain(1, 37, 38) . Recently, the human
3(IV) NC1 domain has been expressed in recombinant form and shown
to selectively bind Goodpasture autoantibodies(39) . Using this
recombinant protein and site-specific antibodies, we have further
characterized the Goodpasture antibody epitopes.
Figure 1:
3(IV) NC1 domain and
its mutants expressed in Escherichia coli as fusion proteins
encoded by pDS-MCS. The
3(IV) NC1 domain and all the deletions and
mutant proteins are summarized in the list below the plasmid picture.
All the recombinant proteins were expressed with a six-histidine tag (underlined). The histidine leader in each construct was
extended by one lysine residue created during the reformation of the
5`-BamHI site. The
3 chain of type IV collagen is a
polypeptide of 1670 amino acids(34) . The NC1 domain starts at
residue 1439 and ends at amino acid 1670 (232 amino acids). The present
study was conducted on an E. coli expressed recombinant
fragment of
3(IV) chain starting at amino acid 1427 and ending at
residue 1670. This fragment contains 12 amino acids of the triple
helix-NC1 junction Gly-X-Y sequence and the entire
NC1 domain of the
3(IV) chain. In the present study, the residue
1439 of the
3(IV) chain was designated as amino acid 1, the first
amino acid of the
3(IV) NC1 domain.
Inhibition ELISA was performed as
before (41, 43) with slight modification. The ELISA
plates (NUNC-immuno plates-Maxisorp, InterMed, Denmark) were coated
with 25 ng of denatured human 3(IV) NC1 monomer. The plated were
coated overnight at room temperature. Upon washing and blocking the
plates with bovine serum albumin as described earlier, the plates were
incubated with Goodpasture antibodies (1:1000) containing increasing
amounts of either recombinant
3(IV) NC1 or the mutants. The
increasing concentrations were 2, 4, 6, 8, and 10 µg. The
recombinant stocks are stored in 8 M urea and 125 mM imidazole. The final solution containing the Goodpasture
antibodies contains micromolar amounts of urea to enable optimal
solubilization. The plates were incubated overnight and developed from
this point as described earlier.
For inhibition ELISA using the
3(IV) NC1 site specific antibodies, the dilution curves for each
of the antibodies were determined by direct ELISA. The optimal
dilutions were used in inhibition assay against the Goodpasture
antibodies with denatured human
3(IV) NC1 monomer as the antigen.
Briefly, the plates were coated with 25 ng of denatured human
3(IV) NC1 monomer and followed with either anti-
3-c-36
peptide antibody or anti-
3-n-18 peptide antibody. Subsequently,
1:50 dilution of human Goodpasture antibodies (saturating antibody
concentration for 25 ng of human antigen) were applied and developed
for reading at absorbance 405 nm. In the case of double antibody
inhibition study, the antigen-coated plates were incubated with
anti-
3-c-36 peptide antibody followed by different dilutions of
anti-
3-n-18 peptide antibody. Human Goodpasture antibodies were
applied in the last step before development.
Previous reports have suggested two regions of the 3(IV)
NCI domain as possible candidates for the Goodpasture
epitope(41, 44) . An interaction site for antibody was
initially proposed for the last 36 amino acids of the
3 NC1 domain (41) based on peptide analysis. A year later a structural
algorithm in another study predicted a second interaction site near the
triple helix-NC1 junction of the
3 NC1 domain(2) .
We prepared antibodies
to two regions of the 3 NC1 domain (
3/c-36 and
3/n-18
regions) that may form the putative Goodpasture epitopes. Both of these
antibodies bind 3-4-fold better to the denatured bovine NC1
hexamer than to the associated form (Fig. 2). This effect was
also observed with the Goodpasture antibodies. Control antibodies did
not bind to either form of the antigen. Antibodies generated against
the 160-kDa associated bovine NC1 hexamer revealed similar binding to
both forms of the bovine NC1 hexamer (Fig. 2). As an additional
control, an interesting effect was observed with anti-
3(IV) NC1
antibody obtained from an Alport patient following renal
transplantation. These antibodies are made by some Alport patients in
response to exposure to normal
3(IV) chains in the renal
allograft; patients develop severe anti-GBM nephritis and reject their
transplanted kidneys. The major target for these anti-GBM
alloantibodies is also the
3(IV) NC1
domain(37, 38, 43) . We found that these
alloantibodies, in contrast to the Goodpasture antibody, bind strongly
to the associated form of bovine NC1 hexamer and 4-5-fold less
against denatured bovine NC1 hexamer.
Figure 2:
Cryptic property of type IV collagen NC1
hexamer. The effect of denaturation of the bovine NC1 hexamer into NC1
monomers and dimers in binding to the anti-3(IV) NC1 antibodies
and anti-NC1 hexamer antibodies is presented in this experiment. The
Goodpasture antibodies, anti-
3/c-36 antibody, and anti-
3/n-18
antibody show a 3-4-fold increase in binding upon denaturation of
the NC1 hexamer. The Alport alloantibodies (some patients with Alport
syndrome develop anti-
3(IV) NC1 antibodies upon renal allograft
transplantation) showed the opposite effect. The anti-associated NC1
hexamer antibody shows no change in binding under either condition. The
control antibodies did not reveal any significant binding to either
form of the antigen.
Figure 3:
Inhibition analysis with site-specific
anti-3(IV) NC1 antibodies. Panel A, the plates were
coated with 25 ng of denatured human
3(IV) NC1 hexamer and a
dilution curve was obtained for the anti-
3-C.36 peptide antibody (solid rectangles) and anti-
3-N.18 peptide antibody (solid circles). Panel B, each of the two peptide
antibodies were assayed for their capacity to inhibit Goodpasture
autoantibody binding (GP-1) to the antigen. The dilutions refer to the
different peptide antibody dilutions used in the assay. The rectangles represent the anti-
3-C.36 peptide antibody,
and the circle represents anti-
3-N.18 peptide antibody.
The Goodpasture antibody binding to the human
3(IV) NC1 was
calculated as 100% for these experiments. Panel C, a double
antibody inhibition was performed to address the cumulative effect of
the two
3(IV) NC1-specific peptide antibodies in inhibiting the
Goodpasture antibodies binding to the antigen. The maximal binding
dilution of 1:50 for Goodpasture antibody was used in this experiment
(data not shown).
Figure 4:
Characterization of the deletion and point
mutants of recombinant 3(IV) NC1 domain. The deletions and point
mutations within the
3(IV) NC1 domain were generated as described
under ``Materials and Methods'' using the primers presented
in Table 1. All of the recombinant proteins were expressed in E. coli using the plasmid presented in Fig. 2. All of
the recombinants were analyzed by 15% SDS-PAGE as shown in panels A and B. A small amount of degradation was observed for all
the proteins during the purification procedure. The
3 and
3/n-26/c-36 recombinants contain a second band under the major
band, which corresponds to the predicted size of the protein. One
microgram of protein was loaded in each lane, as established by BCA
protein assay and absorbance at 280 nm. One-tenth of this amount was
further analyzed by immunoblotting and ELISA measurements using
Goodpasture antibodies. Panel C shows the immunoblotting (inset) and ELISA results for the
3,
3/n-26,
3/n-26/c-36, and
3/n-26/c-KK recombinant proteins. GP-1,
GP-2, and GP-3 designate three different circulating antibodies from
Goodpasture patients. Panel D shows the immunoblotting (inset) and ELISA measurements using the same three antibodies
against
3,
3/c-K.206,
3/c-K.213,
3/c-K.219,
3/c-K.229,
3/c-K.230,
3/c-KK,
3/c-KKK, and
3/c-R.223 recombinant proteins. The dilution of Goodpasture
antibodies used was 1:500. All of the recombinant proteins were coated
on the plates in the presence of 6 M guanidine HCl, 50 mM Tris-Cl, pH 7.5.
Chemical modification studies previously suggested that
lysines may be necessary for Goodpasture autoantibody binding in the
C-terminal binding region(41) . Therefore, lysine 229 and
lysine 230 were mutated to alanines along with the deletion of 3
NC1/n-26 region. This mutant (
3/n-26/c.KK) revealed significant
reduction in its binding to Goodpasture antibodies, similar to mutant
3/n-26/c-36 (Fig. 4, panel C). We further analyzed
the role of individual lysines within the region. There are a total of
six lysines present in the
3(IV) NC1 domain, and five of them are
present in the
3 NC1/c36 region. The
3 NC1/n26 does not
contain any lysines in the NC1 part of this region. Although there are
two lysines present in the triple helical part of this region, previous
studies suggest that these may not be involved in the Goodpasture
autoantibody binding(27) . Collagenase digestion of GBM cleaves
the
3(IV) NC1 region at two places, one at the triple helix-NC1
border region and the other at a site nine amino acids (including two
lysines) away from the NC1 region, into the triple helix(27) .
These two forms of
3(IV) NC1 domains bind Goodpasture
autoantibodies strongly(27) . Therefore, the two lysines in the
triple helical region of the
3 NC1/n26 do not seem important in
the Goodpasture autoantibody binding. Hence, in the present study these
lysines were not mutated.
The lysines in the 3 NC1/c36 region
(Lys-206, -213, -219, -229, and -230) were individually substituted in
the
3 NC1/c36 region to alanines. SDS-PAGE analysis was performed
to check for the predicted size (Fig. 4, panel B).
Immunoblotting and ELISA experiments were performed on these mutants
using the same Goodpasture antibodies as in the experiment shown in Fig. 4, panel C. Replacement of lysine 219
(
3/c-K.219) or lysine 229 (
3/c-K.229) resulted in a steep
reduction in binding with Goodpasture antibodies (Fig. 4, panel D). Replacement of lysines 206, 213, and 230 did not
produce any significant change in their binding to Goodpasture
antibodies when compared with recombinant
3(IV) NC1 domain.
Additionally, an arginine substitution (arginine 223) to alanine did
not change in Goodpasture antibodies binding either.
In order to
determine if lysines 219 and 229 have a synergistic effect on binding
to antibody, two new mutants were designed. One mutant involved the
substitution of three lysines (positions 219, 229, and 230) to alanines
(3/c-KKK), and the other mutant involved lysines 229 and 230
(
3/c-KK) also changed to alanines. Both of these mutants showed
similar reduction in binding to antibody as the substitutions in
3/c-K.219 and
3/c-K.229. Therefore, the effects of lysines
219 and 229 seem independent and are not additive, since mutation of
both at the same time (
3/c-KKK) did not show any noticeable
difference in binding as compared with mutants
3/c-K.219 and
3/c-K.229 alone.
Figure 5:
Inhibition of Goodpasture autoantibody
binding to denatured human 3(IV) NC1 domain by deletion and point
mutants of recombinant
3(IV) NC1 domain. Inhibition ELISA was
performed using denatured human
3(IV) NC1 hexamer as the coating
antigen and GP-1 antibody. The figure shows the representative
inhibition curves for six different recombinant proteins. The
recombinant
3(IV) NC1 shows the maximum inhibition at 55%, with
the recombinant
6(IV) NC1 exhibiting less than 10% inhibition. A
summary of the inhibition effects seen for nine different proteins is
presented in Fig. 6. The ELISA plates were coated with 25 ng of
dissociated human
3(IV) NC1 hexamer. Goodpasture antibodies were
used at a dilution of 1:1000.
, recombinant
3;
,
3/n-26;
,
3/n-26/c-36;
,
3/c-K.229;
,
3/c-R.223;
, r
6.
Figure 6:
Relative inhibition of recombinant
3(IV) NC1 mutants. The denatured human NC1 hexamers were further
separated by gel filtration and high pressure liquid chromatography to
yield denatured human
3(IV) NC1 monomer(41) . Recombinant
3(IV) NC1 inhibits Goodpasture antibody binding to denatured human
3(IV) NC1 monomer by 55% at saturating concentration. The
competitive inhibition of antibody binding to denatured human
3(IV) NC1 monomer observed with the recombinant
3(IV) NC1 was
assigned a maximum value of 100%. All the inhibition values observed
for the mutants were expressed relative to the recombinant; the largest
inhibition was seen for mutant
3/n-26/c-36 at 32.7%. r-
6, recombinant
6; r-
3, recombinant
3.
Immunologic epitopes like those comprising the Goodpasture antigen have been studied in a variety of systems. Epitopes for which the complete structures have been elucidated by x-ray crystallography typically contain between 15 and 22 residues contributed from different regions of a target protein(49) . This is consistent with the view that while the primary structures of native epitopes are often discontinuous (49, 50, 51, 52) , two- or three-dimensional folding creates a conformational or multiheaded determinant. A recent study using a similar approach to ours identified two nonlinear domains of apoB100 as key parts of a discontinuous MB47 epitope(53) . In a separate study, two monoclonal anti-islet cell antibodies (MICAs 1 and 3) binding to GAD65 were found dependent on two regions, a middle region comprising amino acids 244-295 and a C-terminal region of 41 amino acids, suggesting a conformational epitope is formed from these two domains(54) . Others have also reported that lysine residues are critical for the binding of the anti-HLA-A2 CREG antibodies to their antigen(47) .
In the present study, we have further
distinguished two important interaction sites for Goodpasture
autoantibody within the 3(IV) NC1 domain using serologic analyses
and recombinant mutagenesis. One site resides among the last 36 amino
acids, with lysines 219 and 229 being particularly necessary, and a
second site lies within the first 26 amino acids of the triple
helix-NC1 junction. These results bring together and solidify two
previous suggestions regarding the determinants of the Goodpasture
epitope (41, 44) and advance the notion that
Goodpasture antibody may recognize an internalized structure.
The
3(IV) NC1 hexamers found in fragments of digested basement type IV
collagen bind Goodpasture autoantibodies to only a limited extent, and
these hexamers do not induce Goodpasture-like syndrome in rabbits (54) ; further denaturation of the
3(IV) NC1 hexamers into
monomer and dimer subunits by denaturants, however, greatly increases
the binding of antibody, and these isolated
3(IV) NC1 dimers
induce Goodpasture disease in rabbits. The poor recognition of hexamer
prior to denaturation suggests that candidate Goodpasture epitopes are
immunologically privileged within the normal hexamer configuration.
This privilege is not extended to binding sites recognized by
anti-
3(IV) alloantibodies in Alport patients with anti-basement
membrane disease following renal transplantation.
Our experiments
with anti-peptide antibodies to the Goodpasture antigen, directed
toward the 3 NC1/n26 and the
3 NC1/c36 regions, further
support the notion of internalized or cryptic epitopes comprising the
Goodpasture autoantigen. Anti-peptide antibodies to both regions bind
weakly to the associated
3(IV) NC1 hexamer but strongly against
hexamer denatured into monomer and dimers. Furthermore, these
antibodies cumulatively inhibit Goodpasture autoantibodies binding to
monomers by 75%. Taken together, these experiments suggest that the
epitopes within these two ends of the
3(IV) NC1 domain are
probably the most important regions. Exposure of such privileged
epitopes by smoke, oxidants, organic solvents, or infection, as a
hypothesis, may modify collagen interactions in basement membranes in situ that facilitate subsequent inflammation and further
opportunity for more antibody accumulation in organ
tissues(48) .
Inferring from the results obtained here, we
propose as a hypothetical model that two immunologically privileged
regions fold to form an optimal antigenic structure within the NC1
domain of the 3 chain. These Goodpasture sites are subsequently
concealed by the normal assembly of type IV collagen into hexamers.