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J. Biol. Chem., Vol. 275, Issue 40, 30844-30848, October 6, 2000
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,, and
From the Department of Nephrology, Lund University
Hospital, S-22185 Lund, Sweden and § Wieslab AB, S-22370
Lund, Sweden
Received for publication, May 31, 2000, and in revised form, July 12, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Goodpasture disease fulfils all criteria for a
classical autoimmune disease, where autoantibodies targeted against the
non-collagenous domain of the Goodpasture disease is known and characterized as a classic
autoimmune disease. The disease is B cell and antibody mediated, with
autoantibodies directed against proteins in the glomerular basement
membrane and lung alveoli. When bound to self-structures in the
kidney and lung, the antibodies initiate an inflammatory destruction of
tissue by recruitment of complement leading to rapidly progressive
glomerulonephritis often accompanied with severe and life threatening
lung hemorrhage. The major self-epitope is located on the Trying to map an epitope for a specific autoimmune disorder is very
difficult, since in most cases autoantibodies against a variety of
epitopes on the target structure are formed. For Goodpasture syndrome a
limited epitope distribution was indicated when binding of
autoantibodies to collagen IV was successfully blocked by one single
monoclonal antibody (5, 6). In our first attempt to map the Goodpasture
epitope we used linear synthetic peptides of the To avoid the problem with linear peptides and mal-folded recombinants
expressed in bacterial systems, new mapping strategies has been
initiated by several groups, where recombinant collagen IV is expressed
in eukaryotic cell lines. By substitution of amino acid residues in the
The principal goal for this study was to investigate if a reactive
epitope could be created in the Patients and Sera--
The sera of 20 Goodpasture patients with
biopsy-proven anti-glomerular basement membrane nephritis were obtained
from the serum bank at the Department of Nephrology, Lund University
Hospital. All patients showed crescentic glomerulonephritis with linear deposits of IgG at direct immunofluorescence. Seven of the patients had
in addition overt lung hemorrhage. The mean age was 49 years ranging
from 18 to 78 years, 10 males and 10 females. The mean serum creatinin
at time of diagnosis was 920. Twelve of the patients were maintained on
dialysis, four had died and four survived with native functional
kidneys after 6 months of follow-up. Sera from five healthy blood
donors were used as controls.
Antibodies--
The alkaline phosphatase-conjugated antibodies,
goat anti-human IgG (Fc part) and goat anti-rabbit IgG, were purchased
from Sigma and the rabbit anti-mouse IgG from Dako, Glostrup, Denmark.
The polyclonal rabbit anti-collagen X antibodies were manufactured as
sera at the laboratory by immunizing a rabbit with keyhole limpet
hemocyanin-conjugated peptide (GYP GAK GER GSP GSD GKP GYP GKP
GLD GC-(KLH)). It was diluted 1/200. The monoclonal antibody against the 6 × His was purchased from Serotec (MCA1396, Serotec, Kidlington, Great Britain) and diluted 1/2000.
Expression Vector--
In this study the expression vector
pCEP4-BM40-HisEK was a kind gift from Dr. A. Aspberg, Cell and
Molecular Biology, Lund University.1 The vector is
based on the pCEP4 vector (Invitrogen, Leek, The Netherlands) and adds
a BM40 signal peptide followed by a hexahistidine tag and an
enterokinase D cleavage site N-terminal of the recombinant protein.
Construction of DNA for Expression--
The basic DNA construct
used in this study is a cDNA coding for the human Cell Culturing and Transfection--
HEK-293 EBNA cells were
cultured in 90-mm cell culture plates (TPP, Trasadingen,
Switzerland) in a Dulbecco's modified Eagle's medium with 10%
fetal calf serum (Life Technologies, Inc., Paisley, Great Britain). For
every construct 8 × 105 HEK-293 EBNA cells were
transfected with 20 µg of plasmid using an electroporator (Bio-Rad),
with the electrical settings 200 V, 960 microfarads in a 0.4-cm cuvette.
After electroporation the cells were seeded on new plates, and after
reaching confluence, about 48 h after transfection, selection with
hygromycin was initiated. Transfected, hygromycin-resistant cells were
cultured until a desired number of plates had reach confluence then
collection of supernatants were initiated. During harvesting, the
transfected cells were kept in fetal calf serum-free Dulbecco's
modified Eagle's medium supplemented with 50 mg/liter ascorbate
(17). The expected size of the recombinant protein is approximately 31 kDa.
Purification of Recombinant Protein--
500 ml of serum-free
conditioned medium were precipitated by adding ammonium sulfate to 50%
saturation and then solved in 20 mM NaPi
buffer, pH 7.8, with 250 mM NaCl and applied to 3 ml of
ProBindd resin (Invitrogen) equilibrated in the same
buffer. The column was then washed with a 20 mM
NaPi buffer, pH 6.0, with 500 mM NaCl and
eluted in a 20 mM NaPi buffer, pH 6.0, 500 mM NaCl, 500 mM imidazol. The eluted
samples were concentrated and dialyzed against a phosphate-buffered
saline buffer. The purified recombinant proteins were analyzed by
silver stained SDS-polyacrylamide gel electrophoresis gels and by
immunoblotting using antibodies against the type X collagen domain and
antibodies against the His tag. Protein concentrations were determined
using the BCA (Pierce) method and by measuring the absorbance at 280 nm.
Enzyme-linked Immunosorbent Assay
(ELISA)2--
The microtiter plates
(Nunc Immunoplate, Roskilde, Denmark) were coated overnight at
4 °C with 0.025 µg/well of purified recombinant protein in coating
buffer (50 mM Na2CO3, 0.05%
NaN3, pH 9.6). The plates were washed three times with 0.15 M NaCl, 0.05% (v/v) Tween 20 and then incubated for 1 h at room temperature with 100 µl/well of sera diluted 1/100 in
phosphate-buffered saline-bovine serum albumin (1.5 mM KH2PO4, 8 mM
Na2HPO4, 0.12 M NaCl, 2.5 mM KCl, 0.05% (w/v) NaN3 containing 0.2%
(w/v) bovine serum albumin, pH 7.3). After three new washes the plates
were incubated with alkaline phosphatase-conjugated goat anti-human IgG
for an additional hour. The amount of bound antibodies was detected by
the use of p-nitrophenyl phosphate (Sigma) (1 mg/ml) in
substrate buffer (1 M diethanolamine, 0.5 mM
MgCl2, pH 9.8), as substrate. Color development was
measured spectrophotometrically at 405 nm after 1-h incubation at room
temperature. All assays were run in duplicate.
Inhibition ELISA--
The inhibition ELISA was performed in the
same way as described above, with the exception of the preceding
overnight preincubation at 4 °C of patient sera with recombinant
protein in concentrations from 10 to 0.0001 µg/ml. The plates
were coated with recombinant
To find the optimal concentration of patient sera for inhibition, two
dilution series were made for each of the six patient sera used in the
inhibition ELISA. One of the dilution series was preincubated with
recombinant SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting--
A
10% NuPAGE gel from Novex (San Diego, CA) was run in a MOPS
buffer system according to the supplier's recommendations. The gels
were either silver-stained or transferred to a polyvinylidene difluoride membrane using a semidry blotting procedure (18). The
antibodies were diluted as described above, and the membranes were
treated as described elsewhere (6).
In a previous study we were able to recreate an immunoreactive
epitope in the non-reactive 
3-chain of collagen IV initiates an
inflammatory destruction of the basement membrane in kidney glomeruli
and lung alveoli. This leads to a rapidly progressive
glomerulonephritis and severe pulmonary hemorrhage. Previous studies
have indicated a limited epitope for the toxic antibodies in the
N-terminal part of the non-collagenous domain. The epitope has been
partially characterized by recreating the epitope in the non-reactive
1-chain by exchanging nine residues to the corresponding ones of
3. In this study we have investigated to what extent each of these
amino acids contribute to the antibody binding in different patient sera. The results show that seven of the nine substitutions are enough
to get an epitope that is recognized equally well as the native
3-chain by all sera from 20 clinically verified Goodpasture patients. Furthermore, the patient sera reactivity against the different recombinant chains used in the study are very similar, with
some minor exceptions, strongly supporting a highly defined and
restricted epitope. We are convinced that the restriction of the
epitope is of significant importance for the understanding of the
etiology of the disease. Thereby also making every step on the way to
characterization of the epitope a crucial step on the way to specific
therapy for the disease.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3-NC1
domain of collagen IV. Collagen IV 3-chain has a limited
distribution in the body and is only found in a few specialized
basement membranes including the glomerular and alveolar basement
membranes, thus explaining the specific organ involvement in
Goodpasture disease. Goodpasture disease is indeed an antibody mediated
disease as proven by the transfer of disease to monkeys by
injection of kidney bound antibodies from Goodpasture patients (1) and
the therapeutic effect of treating patients with plasma exchange and
immunosuppressive drugs to reduce the amount of circulating antibodies
(2). The Goodpasture epitope is a conformational epitope, which is
indicated by the loss of reactivity to autoantibodies when the tertiary
protein structure is disrupted by reduction of disulfide bonds (3). The
epitope is also known as a cryptotope, i.e. the epitope is
hidden in the native protein structure and is fully exposed first when
the protein is partially denatured (4).
3(IV) NC1 domain to
block the binding of autoantibodies to collagen IV (7). With this
method we were unable to map any epitope on the 3(IV) NC1 domain
although for one patient an epitope on the 1(IV) NC1 domain was
found. In a study by Kalluri et al. (8), using linear
peptides, they suggested the C-terminal part of the 3 (IV) NC1
domain to comprise the Goodpasture epitope. However this study as well
as ours suffered from the disadvantage of using linear peptides to
characterize a conformational epitope and the results have not been confirmed.
3(IV) NC1 against the corresponding ones from the homologous but
non-reactive 1(IV) chain, and expressing the constructs in an
eukaryotic expression system correctly folded proteins with intact
conformational epitopes are produced. All these studies have emphasized
the N-terminal part of the 3 (IV) NC1 as the principal epitope
region (9-11). Furthermore, we found that only autoantibodies against
the N-terminal third of the 3(IV) NC1 domain are pathologically
significant (12). Following studies have revealed a small region within
the N-terminal part of the 3 NC1 as the major target for the
circulating antibodies. This epitope has then been recreated by
substitution of a few a.a. residues in the non-reactive 1-chain to
the corresponding ones from 3 (13-15). In our hands, all 20 patients in the cohort recognized this epitope (15).
1(IV) NC1 with fewer than the nine
substitutions, previously reported, and to what extent each of these
amino acids contribute to the antibody binding in different patient sera.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1(IV) NC1.
This was fused to cDNA coding for a stretch of around 100 base
pairs of the triple helical part of collagen X, expressing the epitope
for the anti-type X collagen antibody described above. Furthermore, in
this construct called S1, five point mutations have been introduced
causing substitutions of amino acids in five positions (P5-P9) in the
1(IV) NC1 to the corresponding amino acids of 3(IV) NC1 (Fig.
1b). Substitutions of these
five amino acids have in a previous study been shown to be of vital
importance for epitope recognition (15), although the substitution of
these five amino acids was not enough to make the non-reactive 1
reactive. In the same study an immunoreactive epitope was created by
the introduction of four additional amino acid substitutions, in
positions P1-P4. Since the aim of this study is to investigate if a
reactive epitope can be created with fewer than these four additional
substitutions, P1-P4, we made recombinants with all possible
combinations of the four additional substitutions, in all 14 constructs. For the introduction of mutations in the 14 different
constructs, the overlap extension polymerase chain reaction
technique (16) was applied using the primers 1 and 2 and 3-13 in Table
I and the S1 construct as template (Fig. 1b). The 14 constructs were besides the five common substitutions substituted in
one or more of the positions P1-P4. The constructs were named
according to which positions that were substituted, e.g. a
construct substituted in positions 1 and 3 is called R13 for
"recombinant substituted in positions 1 and 3." The protein-coding cDNAs were then inserted in the pCEP4-BM40-HisEK vector (Fig. 1a).
View larger version (21K):
[in a new window]
Fig. 1.
a, a schematic drawing of the cDNAs
inserted into the expression vector (pCEP4-BM40-HisEK). This construct
result in an expressed protein containing a 6 × His tag followed
by a 30-amino acid stretch of type X collagen and the NC1 domain from
type IV collagen. b, in the basic construct, S1, amino acids
in positions P5-P9 are substituted from the 1 ones (upper
row) to the corresponding ones of 3(IV) NC1 (lower
row). The positions for substitutions in the 1 protein sequence
are indicated by arrows and numbers. For example,
the R12 construct is substituted in positions P1 and P2 plus
P5-P9.
3-NC1 (0.025 µg/well).
3(IV) NC1 (2 ng/well), and the analogous series was
preincubated with an equal volume of phosphate-buffered saline-bovine
serum albumin buffer. In this way two "dilution curves" were
achieved, where the absorbance for the curve produced from the dilution
series afflicted by inhibition from the 3(IV) NC1 declined earlier
and reached background absorbance earlier than the non-inhibited
dilution series. The serum dilution that gave the largest difference,
in percentages, between the inhibited and non-inhibited dilution series
was used in the inhibition ELISA assays for the different recombinants.
Antibodies bound to the coat were detected with alkaline
phosphatase-conjugated secondary antibodies as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1(IV) NC1 domain by substitution of
single amino acids to the corresponding ones of 3 (15). In that
study two principle constructs were made, one with five amino acid
substitutions in positions known to be critical for epitope recognition
(P5-P9), called the S1 construct, and one with four additional
substitutions in non-conserved positions in the same region (P1-P4),
called S2. It was shown that the five substitution recombinants only
reacted weak with the antibodies, whereas the nine-substitution
recombinant showed immunoreactivity to all tested sera. In this present
study, we have investigated what impact each of these amino acids has
on the affinity of autoantibodies from different patients. Thereby
further characterizing the immune response in Goodpasture disease to
this major epitope. To achieve this, all possible combinations of the
four additional substitutions were made in a total of 14 new
recombinants. The S1 construct was used as template, and then
subsequent substitutions were introduced with site-directed mutagenesis
using the primers listed in Table I.
Primers used to introduce amino acid substitutions in the collagen IV
1NC1 domain
Initial Screening of Reactivity--
Initial tests of the 14 recombinants' immunoreactivity against all 20 patient sera were
performed using ELISA and inhibition ELISA. Of the 14 tested
recombinants only five were reactive with the patient sera, one of the
seven substitutions (R12) and four of the eight
substitutions (R123, R124, R134, and R234). The other recombinants did not show reactivity above the reactivity against the
recombinant 1(IV) NC1 (data not shown) and therefore not further
evaluated. The reactive recombinants R12, R123, R134, and R234 were
carefully analyzed again to establish their reactivity compared with
the nine-substitution recombinant (S2), recombinant 3, and
recombinant 1. In Fig. 2, the
immunoblotting results are shown.
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ELISA--
The recombinants were first analyzed using direct ELISA
(Fig. 3). With this assay, we found no
significant difference in reactivity for the R12 recombinant compared
with the S2 protein. In line with these results, none of the eight
substitution recombinants had a significantly lower reactivity
than the 3 recombinant. Although it is noticeable that recombinant
R134 that lack substitution 2 has a significantly lower reactivity than
R12, probably indicating a greater importance for substitution 2. In
fact, two of the samples did not react with this R134 recombinant, and
another six of them showed low reactivity.
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Inhibition ELISA--
In contrast to the direct ELISA, the
inhibition ELISA revealed differences in affinity to the different
recombinants. The highest inhibitory effect was found using recombinant
3, S2, or R12, while all the eight substitution recombinants showed
a significantly lower inhibitory capacity than the seven-substitution R12 recombinant. The inhibition curves from one of the samples are
shown in Fig. 4a, and the mean
normalized inhibition values for all samples are shown in Fig.
4b.
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In conclusion, the ELISA studies have shown that the seven-substitution
recombinant R12 is as reactive as the native 3 and the nine
substitution recombinant S2 described previously (15).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Circulating autoantibodies against different parts of the 3(IV)
chain, as well as antibodies against other (IV) chains, are detected
in serum from patients with Goodpasture disease (6, 19). However, the
major epitope region is found to be the N-terminal part of the 3(IV)
NC1 (9, 11), and only antibodies against this part of the molecule
correlate with disease progression (12, 15). In this study we have
further narrowed down and characterized the molecular properties of
this epitope recognized by the pathogenic autoantibodies. A recombinant
protein comprised of the 1(IV) NC1 domain with seven amino acids
substituted to the corresponding ones from the 3 chain was
constructed. This recombinant protein, R12, was recognized by the
autoantibodies from all patients with Goodpasture disease, in both
direct ELISA and in inhibition ELISA, to the same degree as recombinant
3. These results support the previous findings where we and others
have localized the major epitope to the same region (13-15).
The very limited region recognized by all Goodpasture sera indicates a similar immunization and maturation process in all patients. Interestingly, an overlapping T-cell epitope is found by Phelps and co-workers (20, 21). However, if this process is initiated by a foreign immunogen, i.e. molecular mimicry, or if it is a self-immunization with degraded type IV collagen, is yet unknown.
As shown in Fig. 5a, the
epitope is localized to a small loop in the secondary sequence. The
epitope seems to be dependent on correct folding of this loop,
indicated both by the fact that disruption of the disulfide bridges (4,
7) as well as changes of the charge of certain residues disturb the
immunoreactivity. This is shown by the substitutions in positions 3 and
4, of which one, but not both, of the resulting amino acids must have a
negative charge for the epitope to be fully recognized. In R124 both
residues are negatively charged, and in R123 both residues are
uncharged, and in both, the changes result in a loss of affinity. The
preservation of the positively charged lysine in position P2, seen in
the S1 and R134 recombinants (Fig. 5b), dramatically reduce
affinity to the recombinants. Surprisingly, the R12, as well as the S2, R123, and R124, actually reacted stronger than the recombinant 3(IV)
in the direct ELISA. This could possibly be explained by a less rigid
structure in the 1(IV) background that results in a more accessible
epitope. This theory is supported by the fact that this difference in
reactivity did not appear when the recombinants were analyzed using
inhibition ELISA. An alternative explanation for the higher reactivity
could be that the produced recombinant, e.g. R12, displays
an epitope more similar to a hypothetical mimicry structure than the
native 3(IV) NC1 does.
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Although all samples recognize one very limited area on the 3(IV)
NC1, there are some differences in recognition pattern between the
different samples. As discussed above the effect of charge changes
within the loop have different effect on antibodies from different sera
(especially the R134). Furthermore the relative amount of antibodies
against the epitope defined by the R12 constructs varies from serum to
serum, ranging from 65 to 95%.
We believe that this study of the Goodpasture epitope adds new and
important data that will help us to understand the underlying immunological mechanisms in Goodpasture disease in particular, but also
for autoimmune diseases in general. By using the R12 recombinant
protein in an assay instead of the complete 3(IV) NC1, a more
specific diagnostic test could be developed that could distinguish
between pathogenic antibodies and harmless autoantibodies.
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ACKNOWLEDGEMENTS |
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We thank Lena Gunnarsson for skillful technical assistance and Dr. Anders Aspberg, CMB, Lund University, for providing the expression vector.
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FOOTNOTES |
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* This work was supported by Swedish Medical Research Council Grant 16X-09487, the Lundberg Foundation, the Greta och Johan Koch's Foundation, the Tegger's Foundation, and by a grant from the "Network for Inflammation Research" funded by the Swedish Foundation for Strategic Research.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.
¶ To whom correspondence should be addressed: Wieslab AB, S-22370 Lund, Sweden. Tel.: 46-46-2862840; Fax: 46-46-140890; E-mail: jw@wieslab.se.
Published, JBC Papers in Press, July 14, 2000, DOI 10.1074/jbc.M004717200
1 A. Aspberg, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviation used is: ELISA, enzyme-linked immunosorbent assay.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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| 6. | Hellmark, T., Johansson, C., and Wieslander, J. (1994) Kidney Int. 46, 823-829 |
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 B. G. Hudson, K. Tryggvason, M. Sundaramoorthy, and E. G. Neilson Alport's Syndrome, Goodpasture's Syndrome, and Type IV Collagen N. Engl. J. Med., June 19, 2003; 348(25): 2543 - 2556. [Full Text] [PDF]
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M. David, D.-B. Borza, A. Leinonen, J. M. Belmont, and B. G. Hudson Hydrophobic Amino Acid Residues Are Critical for the Immunodominant Epitope of the Goodpasture Autoantigen. A MOLECULAR BASIS FOR THE CRYPTIC NATURE OF THE EPITOPE J. Biol. Chem., February 23, 2001; 276(9): 6370 - 6377. [Abstract] [Full Text] [PDF]
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