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Profiling of Alopecia Areata Autoantigens
Based on Protein Microarray Technology*
Angelika Lueking,
a,b,c
Otmar Huber,
b,d,e
Christopher Wirths,
d
Kirsten Schulte,
a
Karola M. Stieler,
f
Ulrike Blume-Peytavi,
f
Axel Kowald,
g
Karin Hensel-Wiegel,
d
Rudolf Tauber,
d
Hans Lehrach,
g
Helmut E. Meyer,
a,h
and Dolores J. Cahill
a,h,i,j
Protein biochips have a great potential in future parallel
processing of complex samples as a research tool and in
diagnostics. For the generation of protein biochips, highly
automated technologies have been developed for cDNA
expression library production, high throughput protein ex-
pression, large scale analysis of proteins, and protein
microarray generation. Using this technology, we present
here a strategy to identify potential autoantigens involved
in the pathogenesis of alopecia areata, an often chronic
disease leading to the rapid loss of scalp hair. Only little is
known about the putative autoantigen(s) involved in this
process. By combining protein microarray technology
with the use of large cDNA expression libraries, we pro-
filed the autoantibody repertoire of sera from alopecia
areata patients against a human protein array consisting
of 37,200 redundant, recombinant human proteins. The
data sets obtained from incubations with patient sera
were compared with control sera from clinically healthy
persons and to background incubations with anti-human
IgG antibodies. From these results, a smaller protein sub-
set was generated and subjected to qualitative and quan-
titative validation on highly sensitive protein microarrays
to identify novel alopecia areata-associated autoantigens.
Eight autoantigens were identified by protein chip tech-
nology and were successfully confirmed by Western blot
analysis. These autoantigens were arrayed on protein mi-
croarrays to generate a disease-associated protein chip.
To confirm the specificity of the results obtained, sera
from patients with psoriasis or hand and foot eczema as
well as skin allergy were additionally examined on the
disease-associated protein chip. By using alopecia areata
as a model for an autoimmune disease, our investigations
show that the protein microarray technology has potential
for the identification and evaluation of autoantigens as
well as in diagnosis such as to differentiate alopecia
areata from other skin diseases. Molecular & Cellular
Proteomics 4:1382–1390, 2005.
Autoimmune diseases affect 5% of the world population,
and our understanding and treatment of human autoimmune
diseases has to be improved (1). Often the current diagnostic
tools are limited because there is no assay to detect the quality
of the patients’ response to drugs. Thus, in addition to improved
diagnostics the discrimination between drug responder and
drug non-responder before the onset of therapy might be a goal
for future strategies in the treatment of autoimmune diseases.
A characteristic feature of many autoimmune diseases is
the production of autoantibodies (2). Although the pathogenic
role for most of the autoantibodies in various autoimmune
diseases is not clear, the identification of the autoantigens
that are targeted by the autoantibodies during the immune
response may present an important tool for diagnosis, classifi-
cation, and prognosis. Additionally profiling the autoantibody
repertoire may help to elucidate the pathophysiology of auto-
immunity, enabling novel treatments such as antigen-tolerating
therapy (3, 4). Protein microarrays have been used for detection
and validation of autoantibodies in biological fluids (5, 6).
Alopecia areata is an autoimmune disease of the hair folli-
cles that affects between 2 and 4% of patients seen in der-
matological practice (7) resulting in rapid loss of hair, often
featured by nail dystrophy. The loss may reverse completely,
become chronic, or progress to loss of all scalp hair (alopecia
totalis) and all body hair (alopecia universalis). It is diagnosed
by the clinical finding of a patchy hairless area with some
dystrophic hairs called “exclamation point” hairs at the border
of the patch. It may be confirmed by characteristic histological
findings of a dense peri- and intrabulbar inflammatory infiltrate
of anagen hair follicles like a bee swarm. The disease is
characterized by a frequently relapsing course with good
response to systemic immunosuppression (corticosteroids) or
topical immunomodulation with diphencyprone (8).
Progress has been achieved over the past 5–10 years in the
characterization of hair follicle antigens targeted by antibodies
in alopecia areata (9). Candidate autoantigens that have been
identified include the 44/46-kDa hair-specific keratin and tri-
chohyalin. Moreover there is evidence that anti-hair follicle
antibodies are modulated during the disease process and can
From the
a
Medical Proteome Center, Ruhr-University Bochum, Uni
-
versita¨ tsstrasse 150, 44780 Bochum, Germany,
d
Institute of Clinical
Chemistry and Pathobiochemistry, Charite´ -Campus Benjamin Frank-
lin, Hindenburgdamm 30, 12200 Berlin, Germany,
f
Center of Experi
-
mental and Applied Cutaneous Physiology, Department of Dermatol-
ogy, Charite´ -Campus Mitte, Schumannstr. 20-21, 10117 Berlin,
Germany,
g
Max Planck Institute for Molecular Genetics, Ihnestrasse
73, 14195 Berlin, Germany,
h
PROT@GEN, Otto-Hahn-Str. 15, 44227
Dortmund, Germany, and
i
Centre for Human Proteomics, Royal Col
-
lege of Surgeons in Ireland, Dublin 2, Ireland
Received, January 26, 2005, and in revised form, May 10, 2005
Published, MCP Papers in Press, June 6, 2005, DOI 10.1074/
mcp.T500004-MCP200
Technology
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.1382 Molecular & Cellular Proteomics 4.9
This paper is available on line at http://www.mcponline.org
occur before clinically evident hair loss (9). In addition, differ-
entiating anagen keratinocytes are an important structure in
the autoimmune etiology of alopecia both in autoimmune
endocrine syndrome type I-associated alopecia areata and at
least in a subgroup of patients with alopecia areata unrelated
to autoimmune polyendocrine syndrome type I (10).
There is evidence for a polygenic inheritance, and diverse
candidate genes have so far been identified by case-control
and family-based studies: HLA class II genes (11), interleu-
kin-1 cluster genes (12, 13), and polymorphism in the inter-
feron-induced p78 protein MxA, which is highly expressed in
anagen hair bulbs of alopecia areata lesions (14).
In this study, we combined protein microarray technology
with the concept of an arrayed expression library enabling
direct connection from the protein product of an individual
expression clone to its corresponding cDNA sequence infor-
mation (for reviews, see Refs. 15–17). Using robot technology,
a human fetal brain cDNA expression library (hEx1) was
picked into microtiter plates, and high density protein arrays
were produced on filter membranes followed by in situ expres-
sion (18). In an initial analysis of this library, more than 66% of
the clones contain inserts in the correct reading frame. 64% of
these clones comprise full-length proteins (5, 19). Based on an
oligonucleotide fingerprinting analysis, ⬃13,000 different genes
have been determined in this expression library indicating a high
variance of presented proteins. Therefore, this library seems to
be highly suitable for unbiased screening assays.
Screening protein arrays with body fluids from large numbers
of patients with an autoimmune disease would allow the iden-
tification of potentially novel autoantigens as well as potentially
assist the prognosis, diagnosis, and subtyping of autoimmune
diseases based on the presence of specific autoantibodies. In
diagnostic applications denatured proteins are preferentially
used due to their higher stability during storage. Because our
study has the aim to develop a disease-associated protein chip
suitable for diagnosis, we have used denatured proteins and
focused on autoantibodies recognizing linear epitopes. Pep-
tides, which present linear epitopes, have been successfully
used to profile the autoantibody repertoire (6, 20, 21).
Here we used alopecia areata as a model for a T-cell-
mediated autoimmune disease and profiled the autoantibody
repertoire of alopecia areata patients. Following identification of
putative autoantigens, we extended the recently described ap-
proach by arraying the identified putative autoantigens on pro-
tein microarrays to generate a disease-associated protein chip
to specify and differentiate between these candidate autoanti-
gens. To further confirm the specificity of the results obtained,
sera from patients with psoriasis or hand and foot eczema were
additionally analyzed on the disease-associated protein chip.
EXPERIMENTAL PROCEDURES
Patient Data—24 patients with alopecia areata (12 alopecia areata
circumscripta, two alopecia areata of diffuse type, seven alopecia
areata subtotalis or universalis, one alopecia areata ophiasis type,
two alopecia areata data not available) in an active stage without any
systemic or topical therapy were randomly chosen for participation in
this investigation. The patient control group consisted of patients with
normal hair growth and no personal or family history of alopecia
areata. The disease control group consists of sera that were collected
from patients with hand and foot or atopic eczema (n ⫽ 5) and
psoriasis (n ⫽ 4) at a mean age of 38 years.
The alopecia areata patients, of which 7 of 24 were male (29%) and
17 of 24 were female (71%) at a mean age of 35 years in male and 38
years in female, agreed to donate a blood sample after having re-
ceived written information and signed the informed consent. 2 of 7
(30%) of the male population had atopic diathesis, whereas 8 of 18
(47%) of the female population had atopy. Autoimmune parameters
(antinuclear antibodies (ANA)
1
and autoantibodies against TSH recep-
tor (TRAK)) were analyzed in addition. The patient population showed
in total in 9 of 24 (38%) a positive antinuclear antibody level (22% in
male and 16% in female), and in 6 of 24 patients (25%) antibodies
against thyroid were found to be positive. All patients showed IgG
levels in the normal range. One patient also was diagnosed to suffer
from vitiligo. This study was accredited by the Charite´ ethics com-
mission. Blood was collected from alopecia areata patients in BD
Biosciences Vacutainer systems. After centrifugation at 3000 rpm/min
serum was distributed and analyzed in the central clinical chemistry
unit at the Institute of Clinical Chemistry and Pathobiochemistry,
Charite´ Campus Benjamin Franklin for the following parameters: IgG,
IgE, ANA, autoantibodies against thyroid peroxidase (anti-TPO), and
TRAK. IgG was quantified on a Behring NephelometerII® by a mech-
anized nephelometric assay (Dade Behring). IgE was determined on a
UniCap 100® analyzer by a mechanized fluorescence enzyme immu-
noassay (Sweden Diagnostics). ANA was determined manually by
indirect autoimmunofluorescence microscopy. Anti-TPO was ana-
lyzed on a Kryptor® analyzer with an automated immunofluorescence
assay from BRAHMS Diagnostica using time-resolved amplified cryp-
tate emission technology, TRAK was quantified with a luminescence
receptor assay with a coated tube system based on the anti-TSH
receptor autoantibody-mediated inhibition of binding of labeled TSH
to the TSH receptor.
Expression Vector and Bacterial Strain—A protein expression sub-
set of a cDNA library (hEx1) from human fetal brain cloned in the
protein expression vector pQE30NST (GenBank
TM
accession number
AF074376) and transformed into the Escherichia coli strain SCS1
(Stratagene) (18), which consisted of 37,200 clones, was used for
autoantibody profiling.
Sequence Analysis of Expression Clones—cDNA inserts were
PCR-amplified and tag-sequenced as described previously (19). The
sequences were searched against public databases (National Center
for Biotechnology Information (NCBI)) (22).
Serum Profiling on High Density Protein Arrays—High density pro-
tein arrays of the protein expression set of the hEx1 library were
obtained from the German Resource Center for Genome Research
(RZPD) and prepared as described previously (18). For serum profil-
ing, the filters were blocked in 3% (w/v) nonfat, dry milk powder in
TBST (TBS, 0.1% (v/v) Tween 20) for 2 h, washed twice in TBST, and
subsequently incubated with serum pools, each containing four pa-
tient sera adjusted to identical IgG levels (10
g/ml) and diluted 1:20
in 2% (w/v) BSA, TBST for 16 h. Following three 30-min TBST washes
1
The abbreviations used are: ANA, antinuclear antibodies; TRAK,
autoantibodies against TSH receptor; TSH, thyroid-stimulating hor-
mone; anti-TPO, autoantibodies against thyroid peroxidase; AP, al-
kaline phosphatase; CV, coefficient of variation; FGFR3, fibroblast
growth factor receptor 3; DCM, dilated cardiomyopathy; EPF, en-
demic pemphigus foliaceus; SLE, systemic lupus erythematosus; E2,
ubiquitin carrier protein; DC, disease control.
Profiling of Alopecia Areata Autoantigens
Molecular & Cellular Proteomics 4.9 1383
and subsequent incubation with the secondary antibody (either
mouse anti-human IgG, Sigma, 1:5000 dilution or mouse anti-human
IgG3, Sigma, 1:400 dilution as appropriate) in 2% (w/v) BSA, TBST,
the filters were washed three times for 30 min in TBST. This was
followed by incubation with the tertiary antibody (rabbit anti-mouse
IgG alkaline phosphatase (AP)-conjugated, Sigma, 1:5000 dilution) in
2% (w/v) BSA, TBST. Subsequently the filters were washed three
times in TBST-T (TBST, 1% Triton X-100) for 20 min each followed by
a 10-min wash in TBS and a further wash for 10 min in AP buffer (1 m
M
MgCl
2
, 0.1 M Tris, pH 9.5) and subsequent incubation in 25 mM
Attophos (JBL Scientific) in AP buffer for 5 min. The filters were
illuminated with long wave UV light, and the images were taken using
a high resolution charge-coupled device detection system (Fuji). Im-
age analysis was performed with VisualGrid (GPC Biotech, Munich,
Germany).
Protein Expression and Purification in High Throughput—Each pro-
tein was expressed in 1-ml cultures in deep well microtiter plates. The
proteins were extracted from each culture (23) and purified as de-
scribed previously (19).
Generation of Protein Microarrays—FAST slides (Schleicher &
Schuell) were placed in a Q-Array System (Genetix, New Milton, UK)
equipped with humidity control (65%), and 16 or 24 blunt-ended
stainless steel print tips with a tip diameter of 150
m were used to
generate the protein arrays. All protein antigens were spotted in
duplicate onto two fields in three different concentrations (undiluted
and 1:5 and 1:10 diluted). For most of the proteins, spotting of the
undiluted purified proteins leads to immobilization of 15 fmol of pro-
tein/spot on the microarray (undiluted: 15 fmol; 1:5: 3 fmol; 1:10: 1.5
fmol, respectively). Each protein microarray included several control
spots, such as human or mouse serum IgG in three concentrations
(human IgG: 1:1000, 1:2500, and 1:5000; mouse anti-human IgG:
1:1000, 1:2500, and 1:5000).
Serum Profiling on Protein Microarrays—After spotting, the protein
chips were blocked in 2% (w/v) BSA, TBST, 0.1% (v/v) Tween 20 at
room temperature, and the serum was added in a 1:100 dilution in 2%
(w/v) BSA, TBST. The protein chips were incubated in a humidified
atmosphere for 16 h at 4 °C. Following three 30-min TBST washes
and subsequent incubation with the secondary antibody (mouse anti-
human IgG, Sigma, 1:5000 dilution) in 2% (w/v) BSA, TBST, the
protein chips were washed three times for 20 min in TBST. This was
followed by incubation with the tertiary antibody (rabbit anti-mouse
IgG-Cy3) in 2% (w/v) BSA, TBST. Subsequently the chips were
washed three times each in TBST for 20 min. All secondary and
tertiary antibody incubation steps were performed for1hatroom
temperature and carried out in a volume of 200
l underneath a cover
slide in the dark. These protein chips were imaged using a confocal
microarray reader (ScanArray 4000, PerkinElmer Life Science), and im-
age analysis was performed using ScanArray Express (PerkinElmer Life
Sciences). One protein microarray from each batch of the spotted chips
was incubated with an antibody directed against the amino-terminal
RGS His
6
tag of the immobilized recombinant protein. The resulting
intensities, which reflect the corresponding protein concentration, were
used for normalization by dividing each intensity value from the serum
incubation by the corresponding RGS His
6
intensity value.
Western Immunoblot Analyses—For Western blot analyses, pro-
teins were purified under denaturing conditions from 300 ml of bac-
terial cultures grown at 37 °C. Expression of proteins fused with a His
6
tag was induced in the cultures with 1 mM isopropyl

-D-thiogalacto-
pyranoside at an A
578
of 0.6 –0.7. After 4 h cells were pelleted,
resuspended in 10 ml of buffer B (8
M urea, 0.1 M NaH
2
PO
4
,10mM
Tris/HCl, pH 8.0) and incubated at 4 °C for 1 h. Poly-Prep® chroma-
tography columns (Bio-Rad) containing 0.5 ml nickel-nitrilotriacetic
acid-agarose (Qiagen) were equilibrated twice with 10 ml of buffer B.
After centrifugation, 10 ml of the cell lysate were loaded onto the
columns, which were subsequently washed three times with 10 ml of
buffer C (8
M urea, 0.1 M NaH
2
PO
4
,10mM Tris/HCl, pH 6.3). Elution
was performed with 5 ml of buffer E (8
M urea, 0.1 M NaH
2
PO
4
,10mM
Tris/HCl, pH 4.5).
100 ng of each protein were separated by SDS-PAGE and trans-
ferred to PVDF membrane (PolyScreen
®
, PerkinElmer Life Sciences)
by semidry blotting. The membrane was blocked in TST buffer (10 m
M
Tris/HCl, pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween 20) for1hand
incubated for 1 h with serum adjusted to 10
g/ml IgG in TST
(alopecia areata patients serum and control patients serum) or diluted
1:1000 in TST (control human serum). After three washings in TST, the
membranes were incubated for 30 min with goat anti-human IgG-
horseradish peroxidase antibody (Dianova) diluted 1:10,000 in TST
and subsequently washed in TST. Chemoluminescence detection
was performed with a FujiFilm LAS-1000 imager by scanning mem-
branes treated with Lumi-Light® Western blotting substrate (Roche
Applied Science).
Bioinformatic Analysis—Following the image analysis the mean
intensity (median background subtracted) was determined for each
protein feature.
To determine putative biomarkers, the average value of the protein
duplicates or quadruplicates was calculated (average
protein
of patient
serum
n
or control serum
n
value) followed by the determination of the
quality of the screen by calculating the coefficient of variation of the
control antibodies, human IgG and mouse IgG, respectively. These
values were also used for interchip normalization. To determine and
consider differences of protein chip production, each chip from a
screen was normalized against the RGS His
6
epitope tag intensity,
which reflects the corresponding protein concentration. The mean
value of all average intensities of the control serum group (aver-
age
protein
of control serum
n
values) was calculated that is called the
“average
protein X
control” value. Dividing the average value of each
individual patient serum (named average
protein X
patient serum
n
value) by the “average
protein X
control value” results in the so-called
factor F. We suggested F values above 2 for disease-significant
autoantigens.
RESULTS AND DISCUSSION
Profiling the Antibody Repertoire of Alopecia Areata Pa-
tients—In this study we screened a human high density pro-
tein array containing 37,200 redundant, recombinant proteins
derived from a human fetal brain expression library with sera
obtained from 20 alopecia areata patients. We generated five
serum pools, each containing four patient sera, and incubated
these serum pools with the high density protein filters. The
use of five serum pools instead of higher numbers of individ-
ual samples reduced the requirements of materials, e.g. the
high density protein arrays, as well as of time and labor.
The data sets obtained from screenings of the five serum
pools were compared with data sets obtained from screen-
ings of 11 individual control sera from clinically healthy per-
sons and to background incubations with anti-human IgG.
From these data, proteins reacting with antibodies present in
alopecia areata patient serum pools were identified. When we
considered proteins that were detected by antibodies from
two or more of the five patient pools but not by antibodies
from the control sera, we identified a subset of 23 proteins.
5⬘-tag sequencing of these clones was performed, and their
sequences were used for BLAST searches against the public
databases including GenBank
TM
and Unigene (22).
Profiling of Alopecia Areata Autoantigens
1384 Molecular & Cellular Proteomics 4.9
Validation of Putative Autoantigens on Protein Microar-
rays—The 23 recombinant proteins selected above were ex-
pressed from the bacterial clones and purified, in parallel,
under denaturing conditions. This corresponded to the state
of the proteins immobilized on the PVDF protein array. In
addition, 10 randomly chosen proteins that were only de-
tected once from the pooled serum screenings were included.
This strategy allowed evaluation of the relevance of the data
obtained by screening the patient serum pools. In addition,
two proteins representing stathmin or a fragment thereof,
which is an antigen of a naturally occurring autoantibody (24),
were included to ascertain the quality of the sera used. Fol-
lowing high throughput expression and purification, recombi-
nant proteins were used in three concentrations for the gen-
eration of protein microarrays. To enable inter- and intrachip
image analysis as well as process and batch control, human
IgG (in the form of human serum) and mouse IgG (in the form
of mouse anti-human IgG) were included in the chip design.
The protein microarrays were incubated separately with
sera from 24 alopecia areata patients including the 20 patient
sera examined in the initial high density protein filter screen-
ing. In addition, the 11 control sera used in the initial filter
screening were also incubated separately on the protein mi-
croarrays. To determine the quality of the serum screening,
the coefficient of variation (CV) of the process control anti-
gens, namely human IgG and mouse IgG, was determined for
each incubated microarray, and the average CV was then
calculated. The analysis of both human IgG and mouse IgG
had a low CV (1:1000, 1:2500, and 1:5000 dilutions: 9, 22, and
19% for human IgG and 15, 25, and 33% for the mouse IgG,
respectively) indicating a comparable quality between patient
and control serum incubations.
By bioinformatic analysis, significant differences between
patient and control sera were determined for 10 proteins
(Table I). Only two of these proteins were not in the same
reading frame as the RGS His
6
epitope tag. Sequence
searches and comparisons using the amino acid sequences
obtained for the reading frames of these two proteins did not
allow the identification of a putative autoantigen. This indi-
cates that our presented strategy has a strong bias to identify
real existing proteins as potential biomarkers. As seen in
Table I, these proteins were all detected by two or more serum
pools, whereas proteins only detected by one serum pool
were not regarded as significant. However, we found several
differences in the percentages between pools and microarray
results. These differences may result from the lower numbers
of pools, which result in less precise percentages when com-
pared with the higher number of percentages in the microar-
ray experiments. In addition, the experiments based on mi-
croarrays are more sensitive and precise because they work
for example with purified and concentrated proteins.
To further confirm the presence of autoantibodies against
the putative autoantigens identified in the pool screenings,
these proteins were expressed and purified in larger scale and
analyzed on Western blots with patient sera. Autoantibodies
against each of the antigens (except the FGFR3 protein) iden-
tified in the screen were detectable in the Western blot anal-
yses as shown for some examples (Fig. 1B). Serum from
patient P1 gave high overall background and did not allow
detection of autoantigen bands. Comparison of the results
obtained with high density arrays and Western blots, how-
ever, did not show complete correlation of detection of the
different putative autoantigens (also compare Fig. 3, A and C,
for alopecia areata patient and Fig. 3, B and D, for disease
control). This could be explained by the presence of serum
autoantibodies reacting with epitopes on the microarrays that
may have partially refolded, but these antibodies no longer
detect the antigen following SDS-PAGE and Western blotting
as can be seen for the FGFR3 protein. Otherwise epitopes
have to be completely unfolded (e.g. by SDS) to be detected.
TABLE I
Analysis of putative alopecia areata specific autoantigens
The column “Hits in pools” shows the numbers of pools detecting the corresponding autoantigens, whereas the column “Hits in Microarrays”
shows the numbers of single sera detecting the corresponding autoantigen. The column “Factor Ø” describes the average detection levels
obtained from the microarray experiments with patient sera compared to microarray experiments obtained from control sera. The column
“ORF” gives information about a correct open reading frame of the antigen detected. EGF, epidermal growth factor.
Clone ID
MPMGp800 . . .
Accession no.
(GenBank
TM
)
Gene name
Hits in pools
(percentage)
Hits in
microarrays
(percentage)
Factor
Ø
ORF
K18585 AV652428 cDNA clone GLCDAC05 4 (80%) 12 (50%) 2 ⫹
M10510 XM031401 EGF-like domain, multiple 3 (EGFL3) 3 (60%) 11 (46%) 2 ⫺
J01523 AK022755 cDNA FLJ12693 fis, clone NT2RP1000324 2 (40%) 19 (79%) 5 ⫹
O22528 NM004436 Endosulfine
␣
(ENSA) 2 (40%) 15 (63%) 5 ⫹
K24594 NM003134 Signal recognition particle subunit 14 2 (40%) 12 (50%) 3 ⫹
D04547 NM022965 FGFR3 2 (40%) 18 (75%) 6 ⫹
O05529 M91670 Keratinocyte ubiquitin carrier protein (E2-EPF) 2 (40%) 17 (71%) 5 ⫹
M17541 BC006318 Erythrocyte membrane protein band 4.9 (dematin) 2 (40%) 20 (83%) 5 ⫹
B20572 NM007029 Neuron-specific growth-associated protein (SCG10) 2 (40%) 12 (50%) 11 ⫹
F11552 NM006769 LMO4 2 (40%) 18 (75%) 17 ⫺
Profiling of Alopecia Areata Autoantigens
Molecular & Cellular Proteomics 4.9 1385
In a previous study, the IgG- and IgG3-specific antibody
repertoire of dilated cardiomyopathy (DCM) patients was pro-
filed (25). In this study, 10 single sera were screened against
the human protein filter array containing 37,200 human pro-
teins, and 48 IgG-specific and 32 IgG3-specific autoantigens
were identified. The quantitative validation using highly sensitive
protein microarrays leads to the confirmation of 10 IgG- and 6
IgG3-specific autoantigens. In the alopecia areata study, we
used five serum pools, each containing four different sera from
alopecia areata patients, and we identified only 23 putative
autoantigens on the human protein filter array. When compared
with the DCM study, the number of identified putative autoan-
tigens is remarkably lower. However, at least eight of these
proteins with in-frame amino acid sequences were confirmed
using both highly sensitive protein microarrays and Western
blots leading to similar results when compared with our previ-
ous DCM study. This indicates that the use of pools of sera,
instead of single serum incubations, is a reliable method for the
first screening step on the human filter arrays. We cannot rule
out that by using serum pools additional putative autoantigens
will/may remain unidentified. However, we expect that the most
predominately existing putative autoantigens will be detected
by this method. For the development of a disease-associated
protein chip, we chose just those proteins that were correctly
expressed (correct reading frame with respect to the RGS His
6
epitope) and confirmed by Western blotting.
Development of a Disease-associated Protein Microarray
Suitable for Diagnosis of Alopecia Areata Patients—Eight au-
toantigens were identified by protein chip technology and
successfully confirmed by Western blot analysis. These au-
toantigens were arrayed on protein microarrays to generate a
disease-associated protein chip that may be suitable for fast
diagnosis. Two control proteins representing natural autoan-
tigens (24) were included as well as human and mouse IgG
allowing process control and determination of serum quality
(Fig. 2A).
The protein microarrays were incubated with a selection of
20 of the currently present 24 patient sera. These incubations
were compared with incubations with 10 new collected con-
trol sera. To further confirm the specificity of the results ob-
tained, sera from patients with psoriasis or hand and foot
eczema as well as skin allergy (so-called disease control sera)
FIG.1.Representative Western blot analysis of the putative autoantigens with sera from alopecia areata patients. A, Coomassie-
stained input gel showing the putative autoantigen proteins used for Western blot analysis. B, control blot using the secondary anti-human IgG
peroxidase antibody. C–E, Western blot analysis with patient sera P12, P16, and P17. F and G, Western blots incubated with sera from control
persons without obvious skin disease. H and I, analysis of sera from patients with psoriasis (H) and neurodermatitis (I) (also see Fig. 3D, DC2
and DC5). A–E, lane 1, SCG 10; lane 2, EGI-like domain, multiple 3 in wrong reading frame; lane 3, GLCDAC05; lane 4,
␣
-endosulfine; lane 5,
NOL8; lane 6, FGFR3; lane 7, dematin; lane 8, signal recognition particle subunit 14; lane 9, EPF autoantigen; lane 10 human IgG loading
reference. F–I, loading as in A–E with EGI-like domain, multiple 3 omitted.
Profiling of Alopecia Areata Autoantigens
1386 Molecular & Cellular Proteomics 4.9
were additionally analyzed on the disease-associated protein
chip (Fig. 2B). The whole experiment was performed in two
independent batches of arrays.
The analysis of the human IgG as well as the mouse IgG
showed a low CV (batch 1: 32, 19, and 26%, batch 2: 8, 9, and
24% for human IgG; batch 1: 27, 31, and 17%, batch 2: 8, 7,
and 34% for the mouse IgG, respectively) indicating good
reproducibility between patient, control, and disease control
serum incubations.
When compared with the group of control sera (using av-
erage intensity values of each protein and protein concentra-
tion), 18 patient sera could be characterized by the presence
of autoantibodies against two or more of the chosen biomar-
ker proteins (average, 3.4 recognized autoantigens). One pa-
tient showed the presence of autoantibody to one of the
selected proteins, and in another patient, serum autoantibod-
ies against the chosen biomarkers were completely missing.
In Western blot analyses, however, autoantigens in the serum
of this patient could be detected suggesting that this serum
was perhaps altered either during transportation or storage.
Consequently using the disease-associated protein chip, we
could successfully determine 90% (relevance criteria, ⬎2 au-
toantigens detected) of the alopecia areata patients having
this disease (Fig. 3A). Comparison of sera from patients with
other skin diseases (psoriasis, neurodermatitis, and hand-foot
eczema) designated as disease control (DC) group with the
control group showed that the disease control sera recog-
nized 1.8 autoantigens on average. In detail, no disease con-
trol serum recognized more than three potential autoantigens.
Three (of seven) disease control sera recognized three au-
toantigens, two disease control sera reacted against two pro-
teins, and two disease control sera had no autoantibodies
against the chosen biomarker panel (Fig. 3B). This indicates
that there are antibodies in disease control sera directed
against the putative alopecia areata-specific autoantigens.
Especially the NOL8 antigen appears to be a more general
autoantigen in skin diseases. Similar results were obtained by
Robinson et al. (6) who have shown that several autoantigens
such as Ro52 or histone H2A were recognized by more than
one autoimmune disease (Sjo¨ gren syndrome, SLE, and mixed
connective tissue disease) (6).To address this issue, we sug-
gest the analysis of more patient samples of alopecia areata
and other dermatological diseases as a control, which may
lead to the identification of certain protein panels character-
FIG.2. Development of a disease-associated protein chip suitable for diagnosis of alopecia areata patients. Fields 1– 8, putative
autoantigens (1, cDNA clone GLCDAC05; 2, cDNA FLJ12693 fis, clone NT2RP1000324NOL8; 3,
␣
-endosulfine; 4, signal recognition particle
subunit 14; 5, FGFR3; 6, endemic pemphigus foliaceus autoantigen; 7, dematin; 8, SCG10); fields 9 and 10, natural occurring autoantigens
(control proteins; stathmin); field 11, human IgG; field 12, mouse IgG (process control proteins). All proteins in fields 1–12 were spotted in
quadruplicates in three different concentrations. In addition, 96 randomly selected proteins are spotted that serve as targets for unspecific
cross-reaction in case of highly concentrated autoantibodies in the serum. A, protein chip incubated with an anti-RGS His
6
antibody to ensure
quality of the chip. B, diagnostic fields incubated with different samples.
Profiling of Alopecia Areata Autoantigens
Molecular & Cellular Proteomics 4.9 1387
istic for each disease. Alternatively the relevance criteria
should be chosen more stringently. However, this also re-
quires an improvement of the disease-associated protein
chip. We suggest that a protein biochip leading to an average
detection of about five autoantigens may result in a better
discrimination of related diseases. A further selection of pu-
tative autoantigens can be achieved using for example an-
other type of expression library such as a T-cell-specific or a
tissue-specific expression library.
Functional Relation of Putative Autoantigens—We identified
eight putative autoantigens by profiling the autoantibody rep-
ertoire of alopecia areata patients (Table I). We detected pro-
teins with no clear pathophysiological role except FGFR3,
which reveals a strong relation to hair diseases. Below we
discuss the role of some of the identified proteins.
FGFR3 shows homology to epidermal growth factor re-
ceptor, which plays an important role in the control of hair
cycle progression. Expression of FGFR3 was strongly de-
tected in the superbasal layers and the inner layers of hair
follicles and in wounded skin. Using RNA in situ hybridiza-
tion analysis, FGFR3 RNA was detected in precuticle cells in
the periphery of the hair bulb. However, the function of
FIG.3.Identification of alopecia areata patients by detection of two or more autoantigens. A, comparison of each patient’s signal
intensity to the average intensity of the control group. Autoantigens that show a double or higher intensity are marked with a red circle. B,
comparison of each disease control signal intensity to the average intensity of the control group. Autoantigens that show a double or higher
intensity are marked with a red circle. C, detection of putative autoantigens on Western blots with patient sera. Specific signals for autoantigens
were detected on a FujiFilm LAS-1000 imager after background deduction relative to human IgG input. The figure summarizes the results of
three independent blots. D, Western blot analysis of the putative autoantigens with sera from disease patients. Detection was performed as
described in C. Protein 1, SCG 10; 2, GLCDAC05; 3,
␣
-endosulfine; 4, NOL8; 5, FGFR3; 6, dematin; 7, signal recognition particle subunit 14;
8, EPF autoantigen.
Profiling of Alopecia Areata Autoantigens
1388 Molecular & Cellular Proteomics 4.9
FGFR3 in the autoimmune disease alopecia areata is not
clear.
The endemic pemphigus foliaceus (EPF) autoantigen origi-
nally was identified by screening a keratinocyte expression
library with autoantibodies from a patient with the blistering
skin disease EPF. The isolated cDNA encodes a member of
the ubiquitin carrier proteins (E2). Interestingly this autoanti-
gen is generated by a translational reading frameshift from the
same cDNA (26, 27). The relevance of this autoantigen in the
pathogenesis of EPF or of other autoimmune diseases is
currently unknown.
Dematin is an actin-binding and bundling protein of the
erythrocyte membrane skeleton. SCG10-related proteins
have a function in microtubule destabilization. The functional
block by phosphorylation further supports the importance of
the SCG10 family proteins in neuronal cytoskeletal regulation,
particularly as to microtubule dynamics.
However, it is unclear whether the identified potential au-
toantigens are functionally related to the autoimmune dis-
ease. When comparing various putative autoantigens specific
for other autoimmune diseases, such as rheumatoid arthritis,
only some autoantigens are described with known functional
significance in the disease. For other diseases no functional
significance was found for these autoantigens (28). Interest-
ingly, however, some of those autoantigens with unknown
function in relation to the autoimmune disease have still been
used as biomarkers of the corresponding disease (29).
In an another approach, we have profiled the autoantibody
repertoire of an SLE mouse model on a mouse TH
1
cDNA
expression library and identified for example the mouse lectin
and galactose-binding protein soluble 3 protein as a putative
autoantigen (30). When compared with the human SLE, au-
toantibodies against the human lectin and galactoside-bind-
ing soluble 3 (galectin-3) have been described already, indi-
cating a certain homology between mouse models to human
diseases (31). This suggests that applying our strategy to the
research of autoimmune diseases leads to valuable data.
* This work was supported in part by the Bundesministerium fu¨r
Bildung und Forschung (BMBF) Grant 0811870 (BioFuture) and the
Max-Planck-Gesellschaft. The costs of publication of this article were
defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
b
Both authors contributed equally to this work.
c
To whom correspondence and requests for materials should be
addressed: Protagen, Otto-Hahn-Str. 15, 44227 Dortmund, Germany.
Tel.: 49-0-231-9742-6300; Fax: 49-0-231-9742-6301; E-mail:
angelika.lueking@protagen.de.
e
Supported by the Sonnenfeld-Stiftung and from a grant from the
FCI (Funds of the Chemical Industry).
j
Supported by Science Foundation Ireland Award 02/CE1/B141.
REFERENCES
1. Davidson, A., and Diamond, B. (2001) Autoimmune diseases. N. Engl.
J. Med. 345, 340 –350
2. von Mu¨ hlen, C. A., and Tan, E. M. (1995) Autoantibodies in the diagnosis of
systemic rheumatic diseases. Semin. Arthritis Rheum. 24, 323–358
3. Robinson, W. H., Fontoura, P., Lee, B. J., de Vegvar, H. E., Tom, J., Pedotti,
R., DiGennaro, C. D., Mitchell, D. J., Fong, D., Ho, P. P., Ruiz, P. J.,
Maverakis, E., Stevens, D. B., Bernard, C. C., Martin, R., Kuchroo, V. K.,
van Noort, J. M., Genain, C. P., Amor, S., Olsson, T., Utz, P. J., Garren,
H., and Steinman, L. (2003) Protein microarrays guide tolerizing DNA
vaccine treatment of autoimmune encephalomyelitis. Nat. Biotechnol.
21, 1033–1039
4. Brocke, S., Gijbels, K., Allegretta, M., Ferber, I., Piercy, C., Blankenstein, T.,
Martin, R., Utz, U., Karin, N., Mitchell, D., Veromaa, T., Waisman, A.,
Gaur, A., Conlon, P., Ling, N., Fairchild, P. J., Wraith, D. C., O’Garra, A.,
Fathman, C. G., and Steinman, L. (1996) Treatment of experimental
encephalomyelitis with a peptide analogue of myelin basic protein. Na-
ture 379, 343–346
5. Lueking, A., Possling, A., Huber, O., Beveridge, A., Horn, M., Eickhoff, H.,
Schuchardt, J., Lehrach, H., and Cahill, D. J. (2003) A non-redundant
human protein chip for antibody screening and serum profiling. Mol. Cell.
Proteomics 2, 1342–1349
6. Robinson, W. H., DiGennaro, C., Hueber, W., Haab, B. B., Kamachi, M.,
Dean, E. J., Fournel, S., Fong, D., Genovese, M. C., de Vegvar, H. E.,
Skriner, K., Hirschberg, D. L., Morris, R. I., Muller, S., Pruijn, G. J., van
Venrooij, W. J., Smolen, J. S., Brown, P. O., Steinman, L., and Utz, P. J.
(2002) Autoantigen microarrays for multiplex characterization of autoan-
tibody responses. Nat. Med. 8, 295–301
7. Price, V. H. (1991) Alopecia areata: clinical aspects. J. Investig. Dermatol.
96, 68S.
8. Freyschmidt-Paul, P., Happle, R., and Hoffmann, R. (2003) Alopecia areata.
Clinical aspects, pathogenesis and rational therapy of a T-cell-induced
autoimmune disease. Hautarzt 54, 713–722
9. Tobin, D. J. (2003) Characterization of hair follicle antigens targeted by the
anti-hair follicle immune response. J. Investig. Dermatol. Symp. Proc. 8,
176 –181
10. Hedstrand, H., Perheentupa, J., Ekwall, O., Gustafsson, J., Michaelsson,
G., Husebye, E., Rorsman, F., and Kampe, O. (1999) Antibodies against
hair follicles are associated with alopecia totalis in autoimmune polyen-
docrine syndrome type I. J. Investig. Dermatol. 113, 1054 –1058
11. Messenger, A. G., and McDonagh, A. J. (2000) Alopecia areata: aetiology
and pathogenesis, in Hair and Its Disorders (Camacho, F. M., Randall,
V. A., and Price, V. H., eds) pp. 177–186, Martin Dunitz, London
12. Tazi-Ahnini, R., Cox, A., McDonagh, A. J., Nicklin, M. J., di Giovine, F. S.,
Timms, J. M., Messenger, A. G., Dimitropoulou, P., Duff, G. W., and Cork,
M. J. (2002) Genetic analysis of the interleukin-1 receptor antagonist and
its homologue IL-1L1 in alopecia areata: strong severity association and
possible gene interaction. Eur. J. Immunogenet. 29, 25–30
13. Tarlow, J. K., Clay, F. E., Cork, M. J., Blakemore, A. I., McDonagh, A. J.,
Messenger, A. G., and Duff, G. W. (1994) Severity of alopecia areata is
associated with a polymorphism in the interleukin-1 receptor antagonist
gene. J. Investig. Dermatol. 103, 387–390
14. Freyschmidt-Paul, P., Happle, R., and Hoffmann, R. (2004) Alopecia areata
in animal models: insights in pathogenesis and therapy in a T-cell me-
diated autoimmune disorder. J. Dtsch. Dermatol. Ges. 4, 260 –273
15. Cahill, D. J., and Nordhoff, E. (2003) Protein arrays and their role in pro-
teomics. Adv. Biochem. Eng. Biotechnol. 83, 177–187
16. Lueking, A., Konthur, Z., Eickhoff, H., Bu¨ ssow, K., Lehrach, H., and Cahill,
D. J. (2001) Protein microarrays—a tool for the post-genomic era. Curr.
Genomics 2, 151–159
17. Walter, G., Bu¨ ssow, K., Cahill, D., Lueking, A., and Lehrach, H. (2000)
Protein arrays for gene expression and molecular interaction screening.
Curr. Opin. Microbiol. 3, 298 –302
18. Bu¨ ssow, K., Cahill, D., Nietfeld, W., Bancroft, D., Scherzinger, E., Lehrach,
H., and Walter, G. (1998) A method for global protein expression and
antibody screening on high-density filters of an arrayed cDNA library.
Nucleic Acids Res. 26, 5007–5008
19. Bu¨ ssow, K., Nordhoff, E., Lu¨ bbert, C., Lehrach, H., and Walter, G. (2000) A
human cDNA library for high-throughput protein expression screening.
Genomics 65, 1– 8
20. Quintana, F. J., Hagedorn, P. H., Elizur, G., Merbl, Y., Domany, E., and
Cohen, I. R. (2004) Functional immunomics: microarray analysis of IgG
autoantibody repertoires predicts the future response of mice to induced
diabetes. Proc. Natl. Acad. Sci. U. S. A. 101, Suppl. 2, 14615–14621
Profiling of Alopecia Areata Autoantigens
Molecular & Cellular Proteomics 4.9 1389
21. Quintana, F. J., and Cohen, I. R. (2004) The natural autoantibody repertoire
and autoimmune disease. Biomed. Pharmacother. 58, 276 –281
22. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller,
W., and Lipman, D. J. (1997) Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs. Nucleic Acids Res. 25,
3389 –3402
23. Lueking, A., Horn, M., Eickhoff, H., Bu¨ ssow, K., Lehrach, H., and Walter, G.
(1999) Protein microarrays for gene expression and antibody screening.
Anal. Biochem. 270, 103–111
24. Lacroix-Desmazes, S., Kaveri, S. V., Mouthon, L., Ayouba, A., Malanchere,
E., Coutinho, A., and Kazatchkine, M. D. (1998) Self-reactive antibodies
(natural autoantibodies) in healthy individuals. J. Immunol. Methods 216,
117–137
25. Horn, S., Lueking, A., Murphy, D., Staudt, A., Gutjahr, C., Schulte, K.,
Koenig, A., Landsberger, M., Lehrach, H., Felix, S. B., and Cahill, D.
(2005) Profiling humoral auto-immune repertoire of dilated cardiomyop-
athy (DCM) patients and development of a disease-associated protein
chip. Proteomics, in press
26. Liu, Z., Haas, A. L., Diaz, L. A., Conrad, C. A., and Giudice, G. J. (1996)
Characterization of a novel keratinocyte ubiquitin carrier protein. J. Biol.
Chem. 271, 2817–2822
27. Liu, Z., Dias, L. A., Haas, A. L., and Giudice, G. J. (1992) cDNA cloning of
a novel human ubiquitin carrier protein. J. Biol. Chem. 267, 15829 –15835
28. Ooka, S., Matsui, T., Nishioka, K., and Kato, T. (2003) Autoantibodies to
low-density-lipoprotein-receptor-related protein 2 (LRP2) in systemic au-
toimmune diseases. Arthritis Res. Ther. 5, R174 –R180
29. Corrigall, V. M., and Panayi, G. S. (2002) Autoantigens and immune path-
ways in rheumatoid arthritis. Crit. Rev. Immunol. 22, 281–293
30. Gutjahr, C., Murphy, D., Lueking, A., Koenig, A., Janitz, M., O’Brien, J.,
Korn, B., Horn, S., Lehrach, H., and Cahill, D. J. (2005) Mouse protein
arrays from a T
H
1 cell cDNA library for antibody screening and serum
profiling. Genomics 85, 285–296
31. Lim, Y., Lee, D. Y., Lee, S., Park, S. Y., Kim, J., Cho, B., Lee, H., Kim, H. Y.,
Lee, E., Song, Y. W., and Jeoung, D. I. (2002) Identification of autoanti-
bodies associated with systemic lupus erythematosus. Biochem. Bio-
phys. Res. Commun. 295, 119 –124
Profiling of Alopecia Areata Autoantigens
1390 Molecular & Cellular Proteomics 4.9
