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Research Article
Uncovering Cryptic Glycan Markers in Multiple
Sclerosis (MS) and Experimental Autoimmune
Encephalomyelitis (EAE)
Denong Wang,1* Roopa Bhat,2Raymond A. Sobel,3Wei Huang,4Lai-Xi Wang,4
Tomas Olsson,5and Lawrence Steinman2
1Tumor Glycomics Laboratory, SRI International Biosciences Division, Menlo Park,
CA 94025, USA
2Department of Neurology and Neurological Sciences, Stanford University School of Medicine,
Stanford, CA 94305, USA
3Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA
4Institute of Human Virology, Department of Biochemistry & Molecular Biology, University of
Maryland School of Medicine, Baltimore, MD 21201, USA
5Department of Clinical Neuroscience, Karolinska Institute, Stockholm, 171 76, Sweden
Strategy, Management and Health Policy
Enabling
Technology,
Genomics,
Proteomics
Preclinical
Research
Preclinical Development
Toxicology, Formulation
Drug Delivery,
Pharmacokinetics
Clinical Development
Phases I-III
Regulatory, Quality,
Manufacturing
Postmarketing
Phase IV
ABSTRACT Using an integrated antigen microarray approach, we observed epitope-spreading of
autoantibody responses to a variety of antigenic structures in the cerebrospinal fluid (CSF) of patients with
multiple sclerosis (MS) and in the serum of mice with experimental autoimmune encephalomyelitis (EAE).
These included previously described protein- and lipid-based antigenic targets and newly discovered
autoimmunogenic sugar moieties, notably, autoantibodies specific for the oligomannoses in both MS
patient CSF and the sera of mice with EAE. These glycans are often masked by other sugar moieties and
belong to a class of cryptic autoantigens. We further determined that these targets are highly expressed on
multiple cell types in MS and EAE lesions. Co-immunization of SJL/J mice with a Man9-KLH conjugate at
the time of EAE induction elicited highly significant levels of anti-Man9-cluster autoantibodies. Neverthe-
less, this anti-glycan autoantibody response was associated with a significantly reduced clinical severity of
EAE. The potential of these cryptic glycan markers and targeting antibodies for diagnostic and therapeutic
interventions of neurological disorders has yet to be explored. Drug Dev Res 75 : 172–188, 2014. ©
2014 Wiley Periodicals, Inc.
Key words: glycomics; biomarkers; multiple sclerosis; encephalomyelitis; cryptic glycans; cerebrospinal fluid;
autoantibodies
Financial support: NIH grant number R01NS055997 (L. Steinman and D. Wang), U01CA128416 and R56AI108388 (D. Wang),
and R01AI067111 (L-X. Wang), and the Swedish Research Council and the Knut and Alice Wallenberg Foundation (TO).
*Correspondence to: Denong Wang, Tumor Glycomics Laboratory, SRI International Biosciences Division, 333 Ravenswood
Avenue, Menlo Park, CA 94025, USA.
E-mail: denong.wang@sri.com
Received 23 December 2013; Accepted 10 February 2014
Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/ddr.21169
DRUG DEVELOPMENT RESEARCH 75 : 172–188 (2014)
DDR
©2014 Wiley Periodicals, Inc.
INTRODUCTION
Multiple sclerosis (MS) is a complex neurological
disorder in which an adaptive autoimmune response is
thought to target myelin sheath in the central nervous
system (CNS). Pathologically, there is inflammation in
the CNS white matter with the infiltration of multiple
immune cells, including T and B cells, macrophages
and other inflammatory cells. Autoreactive T cells
are believed to be important in the pathogenesis of
MS lesions and in an animal model of MS, experimen-
tal autoimmune encephalomyelitis (EAE) [Lehmann
et al., 1992; McRae et al., 1995; Yu et al., 1996a,
1996b]. Clonal expansion of B cells in brain tissue and
the presence of oligoclonal immunoglobulins in the
CSF of patients with MS are also hallmarks of the
disease [Kabat et al., 1948, 1951; Steinman, 1996;
Genain et al., 1999; Raine et al., 1999; Hueber et al.,
2002].
Evaluation of autoantibodies in autoimmune
diseases has been enabled by development of new
technologies, e.g., microarray-based large-scale auto-
antibody profiling [Robinson et al., 2002, 2003;
Wang et al., 2002; Kanter et al., 2006; Beyer et al.,
2012]. Robinson et al. developed “myelin proteome”
microarrays to profile the evolution of autoantibody
responses in an EAE model [Robinson et al., 2003]
and found that the diversity of autoantibody responses
increased as the disease evolved from acute to chronic
relapsing EAE. Chronic EAE was associated with
extensive intra- and intermolecular epitope spread-
ing of autoreactive B-cell responses. Using human
recombinant protein microarrays to examine auto-
antibody profiles in the CSF of patients with
relapsing-remitting MS (RRMS) led to the identifica-
tion of a panel of proteins as possible differentiators
between RRMS and other neurological diseases
(OND) that warranted further investigation [Beyer
et al., 2012].
Kanter et al. developed lipid arrays to monitor
autoantibody responses against lipids present in the
myelin sheath, including ganglioside, sulfatide, cere-
broside, sphingomyelin, and total brain lipid fractions
[Kanter et al., 2006]. This technology was applied
to monitor lipid-specific antibody responses in CSF
samples from individuals with MS and controls
[Kanter et al., 2006; Ho et al., 2012]. These studies
showed lipid-specific antibodies against sulfatide,
sphingomyelin, and oxidized lipids in the CSF of MS
patients. Sulfatide-specific antibodies were also
detected in SJL/J mice with acute EAE. They fur-
ther demonstrated that immunization of mice with
sulfatide plus myelin peptide produced a more se-
vere disease course of EAE, and administration of
sulfatide-specific antibody exacerbated EAE. Thus,
autoimmune responses to sulfatide and other lipids
are present in individuals with MS and in EAE and
may contribute to the pathogenesis of autoimmune
demyelination.
Carbohydrates represent another class of anti-
genic structures with unique immunological properties
[Wang and Kabat, 1996; Wang, 2004]. In brain, the
myelin sheath and other tissues contain numerous
sugar moieties, either in the form of glycolipids or
as glycoproteins. Expression of brain carbohydrate,
including mannosyl glycans, can be developmentally
and/or differentially regulated [Schachter, 1991; Yuen
et al., 1997; Chai et al., 1999a, 1999b; Michele and
Campbell, 2003; Breloy et al., 2012a, 2012b; Pacharra
et al., 2012]. Some carbohydrates that exist intra-
cellularly as part of the process of protein glycosylation
are masked by other sugar moieties when the mature
glycoproteins are released extracellularly [Brooks et al.,
2002]. Such “cryptic” carbohydrates are, however,
potentially immunogenic [Calarese et al., 2003; Wang
and Lu, 2004; Newsom-Davis et al., 2009; Walker et al.,
2011]. For example, HIV-1 virions [Calarese et al.,
2003; Walker et al., 2011] and certain tumors
[Newsom-Davis et al., 2009; Wang, 2012b; Wang et al.,
2013] abnormally express N-glycan high-mannose
(Man9) clusters. HIV-infected subjects [Trkola et al.,
1996; Walker et al., 2011] and some cancer patients
[Wang, 2012b; Wang et al., 2013] develop anti-
oligomannose autoantibody responses. These observa-
tions prompted us to ask whether MS/EAE-associated
autoantibody responses can also target this class of
cryptic autoantigens.
We have established a versatile bioarray platform
to integrate the protein-, lipid-, and carbohydrate-
based microarrays in order to facilitate identification
of autoimmune targets in autoimmune diseases. In
essence, we have demonstrated a practical approach
to constructing antigen microarrays, with immobiliza-
tion of carbohydrate, lipids/liposomes, and protein
molecules on nitrocellulose-coated micro-glass slides
[Wang et al., 2002, 2005; Wang, 2003; Wang and Lu,
2004]. This bioarray platform was used to display
autoantigens of diverse molecular composition includ-
ing proteins and peptides of myelin sheath, liposomes
of distinct lipid composition, and carbohydrate anti-
gens that display a number of “cryptic” glyco-epitopes
[Wang and Lu, 2004; Newsom-Davis et al., 2009;
Wang, 2012b; Wang et al., 2013]. This bioarray plat-
form supports quantitative measurement of the rela-
tive antibody reactivities (RAR) over a large-panel of
antigens. Thus, it is technically suitable for monitoring
the antigenic epitope-spreading of an autoimmune
response in EAE and MS.
CRYPTIC GLYCAN MARKERS OF MS/EAE 173
Drug Dev. Res.
METHODS AND MATERIALS
Liposome Preparation
Methods of sonication (sonic energy) and extru-
sion (mechanical energy) used to produce liposomes
were as described [Wang et al., 2005]. Two types of
liposomes, homo- and hetero-liposomes, were used in
this study. The former were produced via a single lipid
preparation, e.g., phosphatidylcholine (PTC), cerebro-
side, and sulfatide. The latter contained two different
lipid molecules with PTC as the support to display other
lipid/glycolipid in desired ratios or epitope densities.
For example, the hetero-liposome of sulfatide (Antigen
ID, 30#) is composed of sulfatide and PTC at a ratio of
1:10 (wt/wt), i.e., 0.2 mg sulfatide and 2.0 mg PTC per
ml of liposome suspension in saline. Compositions of all
liposome preparations are given in Table 1.
Printing Protein, Carbohydrate, and
Lipid/Liposome Microarrays
A high-precision microarray robot (PIXSYS
5500C, Cartesian Technologies, Irvine, CA) was used to
spot antigen preparations onto glass slides pre-coated
with nitrocellulose polymer (FAST Slides; Schleicher &
Schuell, Keene, NH) as described [Wang, 2012a]. The
antigen preparations applied include proteins/peptides,
carbohydrates, and liposomes of various composition
(Table 1). Proteins and carbohydrates were dissolved in
phosphate-buffered saline (PBS; pH 7.4) and saline
(0.9% NaCl), respectively. Liposome preparations were
suspended in saline (0.9% NaCl) at the concentrations
specified (Table 1) and were printed in triplicate with
spot sizes of ∼150 μm and at 375 μm intervals, center to
center. The printed microarrays were air-dried and
stored at room temperature (RT) without desiccant
before application.
Staining and Scanning of Microarrays
Immediately before use, the printed microarrays
were rinsed with PBS, pH 7.4, with 0.05% (vol/vol)
Tween 20 and then blocked by incubating the slides in
1% (wt/vol) bovine serum albumin (BSA) in PBS con-
taining 0.05% (wt/vol) NaN3at RT for 30 min. They were
then incubated at RT with antibodies at an indicated
titration in 1% (wt/vol) BSA in PBS containing 0.05%
(wt/vol) NaN3and 0.05% (vol/vol) Tween 20. The sec-
ondary antibodies or streptavidin conjugates applied for
microarray staining are specified in the figure legends.
The stained slides were rinsed five times with PBS with
0.05% (vol/vol) Tween 20, air-dried at RT, and then
scanned for fluorescent signals using a ScanArray5000A
Microarray Scanner (PerkinElmer Life Science, Boston,
MA) following the manufacturer’s manual.
Examination of Presence of Antigens on Array and
Their Antigenic Determinants or Epitopes
It was essential to assess whether the proteins,
synthetic peptides, and carbohydrates were successfully
“printed” and whether desired epitopes or antigenic
determinants were preserved on the chips. A practical
approach was to incubate the printed microarrays with
antibodies, receptors, or lectins known to react with
the printed substance. As illustrated in Figure 1, we
examined preservation of Galanthus nivalis agglutinin
(GNA)-specific glyco-epitopes (Fig. 1A–C) or 2G12-
specific glyco-epitopes (Fig. 1A, B, and D) by staining
the spotted antigen arrays using corresponding probes.
This demonstrated that the GNA-epitopes were pre-
sented by three glyco-conjugates, i.e., Man9-cluster
(4#), M9_2G12-cluster (3#), and Man5–9 RB (1#). In
contrast, 2G12-glyco-epitopes were well-preserved only
by one of the three, i.e., M9_2G12-cluster (3#), in this
microarray substrate.
Microarray Data-Processing, Standardization,
and Statistical Analysis
Fluorescence intensity values for each array spot
and its background were calculated using ScanArray
Express software. SAS Institute’s JMP-Genomics soft-
ware package (http://www.jmp.com/) (Cary, NC) was
used for microarray data standardization and statistical
analysis. In Figure 1, microarray detections are shown
as the mean fluorescent intensities (MFIs) of each
microspot captured by ScanArray 5000A for the arrays
stained with GNA (Fig. 1C) and 2G12 (Fig. 1D),
respectively. Analysis of such microarray raw data in
association with visual inspection of the microarray
image (Fig. 1A), which contained triplicate spots of a
given antigen preparation, provided an evaluation of
reproducibility and variation of this antigen microarray
technology.
In Figures 2B, 3A and B, antigen-specific IgG or
IgM reactivities are shown as microarray scores, which
are the log2 transformed microarray values (mean-
background). Each data point in these figures repre-
sents the mean of corresponding group, i.e., EAE
versus normal controls (Fig. 2B), or MS versus OND
(Fig. 3). The RAR scores specified in Figures 3C and D,
and 4 were defined as the log2 transformed and IQR
standardized microarray values. The IQR function in
JMP-Genomics normalizes array data sets by setting
their interquartile ranges (IQR) to be identical, which
provides internally standardized data sets for further
statistical evaluation of potential biomarkers.
An antigen-by-antigen analysis of variance
(ANOVA) model was applied to obtain statistically
significant differences between groups in comparison.
WANG ET AL.174
Drug Dev. Res.
TABLE 1. Antigen Preparations Used in This Study
Antigen
ID# Antigen name Antgen preparations* Description Source References
1 Man5_9-RB Man 5–9 RB, 0.5 mg/ml Ribonuclease B with Man5–6
GlcNAc2Asn as the main
glycans (pancreas, bovine)
Sigma-Aldrich This report
2 KLH-SH KLH-SH, 0.5 mg/ml Thiolated keyhole limpet
hemocyanin
Present authors Ni et al., Bioconjug Chem.,
17:493–500 (2006)
3 M9(2G12) Man9-Cluster(2G12)-KLH,
0.5 mg/ml
[(Man9GlcNAc2Asn)4]n-KLH Present authors Ni et al., Bioconjug Chem.,
17:493–500 (2006)
4 Man9 Man9-KLH (Man9GlcNAc2Asn)n-KLH,
0.5 mg/ml
Present authors Ni et al., Bioconjug Chem.,
17:493–500 (2006)
5 AGOR AGOR, 0.5 mg/ml Agalacto-orosomucoid From the late Prof. Elvin A.
Kabat (Columbia University)
Wang & Lu, Physiol Genomics
18(2):245–8, (2004)
6 ASOR ASOR, 0.5 mg/ml Asialo-orosomucoid From the late Prof. Elvin A.
Kabat (Columbia University)
Wang & Lu, Physiol Genomics
18(2):245–8, (2004)
7 OR OR, 0.5 mg/ml Orosomucoid (human α1-acid
glycoprotein)
Sigma-Aldrich Wang & Lu, Physiol Genomics
18(2):245–8, (2004)
8 N279 N279, 0.5 mg/ml α(1→6)dextran, NRRL N279 Northern Regional research
laboratory, (Peoria, IL) NRRL
B-2448
Wang, et al., Nat Biotechnol
20(3):275–81, (2002)
9 B1299S B1299S, 0.5 mg/ml α(1→6)dextran, NRRL B-1299S Northern Regional research
laboratory, (Peoria, IL) NRRL
B-2448
Wang, et al., Nat Biotechnol
20(3):275–81, (2002)
10 B1355S B1355S, 0.5 mg/ml α(1→3)(1→6)dextran, NRRL
B-1355S
Northern Regional research
laboratory, (Peoria, IL) NRRL
B-2448
Wang, et al., Nat Biotechnol
20(3):275–81, (2002)
11 B2448 B2448, 0.5 mg/ml Phosphomannan, NRRL
B-2448
Northern Regional research
laboratory, (Peoria, IL) NRRL
B-2448
Kabat et al., J. Exp. Med. 164,
642 (1986)
12 Levan Levan, 0.5 mg/ml Levan (from preparation of
B-512-E dextran)
Commercial Solvents Co. (Terre
Haute, IN)
Kabat et al., J. Exp. Med. 164,
642 (1986)
13 Bacto-Agar Bacto-Agar, 0.5 mg/ml Bacto-agar 20°C extract From the late Prof. Elvin A.
Kabat (Columbia University)
Duckworth & Yaphe,
Carbohydr. Res. 16, 189
(1971)
14 E. coli. 2630 E. coli. LPS 2630,
0.5 mg/ml
Lipopolysaccharides from
Escherichia coli 0111:B4
Sigma-Aldrich This report
15 E. coli. K1 E. coli. K1 CP, 0.5 mg/ml E. coli K1 capsular polysaccha-
ride (CP)
From the late Prof. Elvin A.
Kabat (Columbia University)
Kabat et al., J. Exp. Med. 164,
642 (1986)
16 E. coli. K100 E. coli. K100 CP,
0.5 mg/ml
E. coli K100 capsular polysac-
charide (CP)
From the late Prof. Elvin A.
Kabat (Columbia University)
Kabat et al., J. Exp. Med. 164,
642 (1986)
17 E. coli. K92 E. coli. K92 CP, 0.5 mg/ml E. coli K92 capsular polysac-
charide
From the late Prof. Elvin A.
Kabat (Columbia University)
Kabat et al., J. Exp. Med. 164,
642 (1986)
18 E. coli. LPS 5014 E. coli. LPS 5014,
0.5 mg/ml
Lipopolysaccharides (rough
strains) from Escherichia coli
J5 (Rc mutant)
Sigma-Aldrich This report
19 S. dysenterine
type 1
S. dysenterine type I O-SP,
0.5 mg/ml
Shigella dysenteriae Type 1
O-specific polysaccharide
From Drs. John Robbins and
Rachel Schneerson (NICHD,
NIH)
Pozsgay, et al., Proc Natl Acad
Sci USA 104:14478–14482
(2007)
20 S.typhi LPS S.typhi LPS7261,
0.5 mg/ml
Lipopolysaccharides from Sal-
monella enterica serotype
typhimurium L7261
Sigma-Aldrich This report
21 Cardiolipin/PTC_
1/100 4 M
Cardiolipin/PTC, 0.02 mg/
2 mg/ml, 4°C, 4 Months
Cardiolipin (C1649) Sigma Chemical Co., St. Louis,
MO
This report
22 PTC_D1 PTC 2.0 mg/ml, 4°C, 24 h L-α-Phosphatidylcholine (Egg,
Chicken)
Avanti Polar lipids, Alabaster,
AL
This report
23 PTC_4 M PTC 2.0 mg/ml, 4°C, 4
Months
L-α-Phosphatidylcholine (Egg,
Chicken)
Avanti Polar lipids, Alabaster,
AL
This report
24 PTC_12 M PTC 2.0 mg/ml, 4°C, 12
Months
L-α-Phosphatidylcholine (Egg,
Chicken)
Avanti Polar lipids, Alabaster,
AL
This report
25 PTC_14 M PTC 2.0 mg/ml, 4°C, 14
Months
L-α-Phosphatidylcholine (Egg,
Chicken)
Avanti Polar lipids, Alabaster,
AL
This report
26 Ganglioside/
PTC_1/10
Ganglioside/PTC, 0.2 mg/
2 mg/ml, 4°C, 24 h
Total Ganglioside Extract
(Brain, Porcine-Ammnium
Salt)
Avanti Polar lipids, Alabaster,
AL
This report
27 Erythrosphingosine/
PTC_1/10
Erythrosphingosine/PTC,
0.2 mg/2.0 mg/ml, 4°C,
24 h
D-erythro- Sphingosine (Egg,
Chicken)
Avanti Polar lipids, Alabaster,
AL
This report
28 Phytosphingosine/
PTC_1/10
Phytosphingosine/PTC,
0.2 mg/2.0 mg/ml, 4°C,
24 h
Phytosphingosine
4-Hydroxysphinganine
(Saccharomyces cervisiae)
Avanti Polar lipids, Alabaster,
AL
This report
CRYPTIC GLYCAN MARKERS OF MS/EAE 175
Drug Dev. Res.
Results are graphically presented as a volcano plot
[Zink et al., 2013] for a global comparison of all RAR
scores between the groups in comparison (Fig. 4A) and
as One-way analysis scatterplots for selected targets
(Fig. 4B and C). In the volcano plot, each dot repre-
sents a statistically weighted and quantified difference
between MS and OND groups. The x-axis is the nor-
malized difference (log2 scale) and the y-axis is −log10
TABLE 1. Continued
Antigen
ID# Antigen name Antgen preparations* Description Source References
29 Glucocerebroside/
PTC_1/10
Glucocerebroside/PTC, 0.2 mg/
2 mg/ml, 4°C, 24 h
Glucocerebrosides (Soy, 98%) Avanti Polar lipids, Alabaster,
AL
This report
30 Sulfatide/PTC_1/10 Sulfatide/PTC, 0.2 mg/2.0 mg/
ml, 4°C, 24 h
Sufatides (Cerebroside sulfates)
(Brain, Porcine)
Avanti Polar lipids, Alabaster,
AL
This report
31 Ceremide/
PTC_1/10
Ceremide/PTC, 0.2 mg/2.0 mg/
ml, 4°C, 24 h
Ceramide (Brain, Porcine) Avanti Polar lipids, Alabaster,
AL
This report
32 Cerebroside/
PTC_1/10
Cerebroside/PTC, 0.2 mg/
2.0 mg/ml, 4°C, 24 h
Total Cerebrosides (Brain,
Porcine)
Avanti Polar lipids, Alabaster,
AL
This report
33 DMPS/PTC_1/10 DMPS/PTC, 0.2 mg/2.0 mg/ml,
4°C, 24 h
1,2-Dimyristoyl-sn-Glycero—
3—[phospho-L—
serine](Sodium Salt) (DMPS)
Avanti Polar lipids, Alabaster,
AL
This report
34 Cardiolipin/PTC-
1/20 D1
Cardiolipin/PTC, 0.1 mg/2 mg/
ml, 4°C, 24 h
Cardiolipin-Disodium Salt
(heart, bovin)
Sigma Chemical Co., St. Louis,
MO
This report
35 Cardiolipin/PTC-
1/5_D1
Cardiolipin/PTC, 0.4 mg/2 mg/
ml, 4°C, 24 h
Cardiolipin-Disodium Salt
(heart, bovin)
Sigma Chemical Co., St. Louis,
MO
This report
36 Cardiolipin/PTC-
1/20 4 M
Cardiolipin/PTC, 0.1 mg/2 mg/
ml, 4°C, 4 Months
Cardiolipin-Disodium Salt
(heart, bovin)
Sigma Chemical Co., St. Louis,
MO
This report
37 Cardiolipin/PTC-
1/5 4 M
Cardiolipin/PTC, 0.4 mg/2 mg/
ml, 4°C, 4 Months
Cardiolipin-Disodium Salt
(heart, bovin)
Sigma Chemical Co., St. Louis,
MO
This report
38 GM1/PTC_1/100 GM1/PTC, 0.02 mg/2 mg/ml,
4°C, 24 h
GM1 Ganglioside-Sodium Salt
(Brain, Ovine)
Avanti Polar lipids, Alabaster,
AL
This report
39 GM1/PTC_1/10 GM1/PTC, 0.2 mg/2 mg/ml,
4°C, 14 Months
GM1 Ganglioside-Sodium Salt
(Brain, Ovine)
Avanti Polar lipids, Alabaster,
AL
This report
40 αGal-T2/PTC_1/20 Galαl,3Galβ1-*HDPE/PTC,
0.04 mg/2 mg/ml, 4°C, 12
Months
Galαl,3Galβ1-HDPE (αGal-
terminal di-saccharides)
V-LABS, INC, Louisiana, US This report
41 Lactosamine/PTC Galβl,4GlcNACβ1-HDPE/PTC,
4°C, 12 Months
Galβl,4GlcNACβ1-HDPE (Type
II chain unit)
V-LABS, INC, Louisiana, US This report
42 αGal-T3/PTC Galαl,3Galβl,
4GlcNACβ1-HDPE/PTC,
4°C, 12 Months
Galαl,3Galβl,4GlcNACβl-
(αGal-terminal
di-saccharides)-HDPE
V-LABS, INC, Louisiana, US This report
43 Ceremide Ceremide, 2.0 mg/ml Ceramide (Brain, Porcine) Avanti Polar lipids, Alabaster,
AL
This report
44 Sulfatide Sulfatide, 2.0 mg/ml Sufatides (Cerebroside sulfates),
(Brain, Porcine)
Avanti Polar lipids, Alabaster,
AL
This report
45 Ganglioside Ganglioside, 2.0 mg/ml Total Ganglioside-Ammonium
Salt, (Brain, Porcine)
Avanti Polar lipids, Alabaster,
AL
This report
46 DIMPS DIMPS, 2 mg/ml 1,2-Dimyristoyl-sn-Glycero–3–
[phospho-L–serine](Sodium
Salt) (DMPS)
Avanti Polar lipids, Alabaster,
AL
This report
47 MBP1–11 MBP1–11, 1 mg/ml A synthetic peptide of human
myelin basic protein(MBP),
N-terminal amino acid 1–11
Present authors Robinson et al., Nat. Med.,
8:295–301 (2002)
48 hMBP hMBP, 0.5 mg/ml in 10 mM
HCl
Myelin Basic Protein,
iodination grade (Brain,
Human)
ACCURATE CHEMICAL & SCI-
ENTIFIC CORPORATION,
WESTBURY, NY
Robinson et al., Nat. Med.,
8:295–301 (2002)
49 mMBP mMBP, 0.5 mg/ml Myelin Basic Protein (Brain,
Murine)
ACCURATE CHEMICAL & SCI-
ENTIFIC CORPORATION,
WESTBURY, NY
Robinson et al., Nat. Med.,
8:295–301 (2002)
50 MOG M0G, 1 mg/ml Myelin Oligodendrocyte
Glycoprotein (Brain, Human)
ACCURATE CHEMICAL & SCI-
ENTIFIC CORPORATION,
WESTBURY, NY
Robinson et al., Nat. Med.,
8:295–301 (2002)
51 PLP139–150 PLP139–150, 1 mg/ml A synthetic peptide of human
Myelin Proteolipid Protein
(PLP), Amino acid residue
139–150
Present authors Robinson et al., Nat. Med.,
8:295–301 (2002)
52 Man9-BSA Man9-BSA (Man9GlcNAc2Asn)n-BSA Present authors Wang, et al., Drug Dev. Res.
74:65–80, (2013)
*The antigen concentrations for initial microarray spotting were listed. The subsequent dilutions of each antigens were specified in Figures 1–3.
**HDPE: Neoglycolipids-1,2-di-O-hexadecyl-sn-glycero-3—phosphoethanolamine.
WANG ET AL.176
Drug Dev. Res.
(P-value) for the difference. Spots above the red dashed
line represent signatures that differ significantly
between the groups. For ANOVA scatterplots in
Figures 2C, and 4B and C, each data point represents
the mean of triplicate determinations. A Nominal
Logistic Fit model was applied to assess the perfor-
mance of a serum marker, e.g., M9_2G12_IgG
(Fig. 4B, upper panel), as a classifier for differential
diagnosis between MS and OND.
Enzyme-Linked Immunosorbent Assay (ELISA)
and Immunohistochemistry
ELISA assays were performed as described [Wang
et al., 2002, 2013]. In brief, ELISA microtiter plates
(NUNC, MaxiSorp, Thermo Scientific, Rochester,
NY) coated with corresponding antigens, either Man9-
BSA or PLP139–151 at 10 μg/ml, were incubated with
pre-titrated mouse serum (1:500). The captured
Fig. 1. A carbohydrate microarray analysis of oligomannosyl antigens for expression of the 2G12-like and GNA-like glyco-epitopes. The
mannose-cluster-containing microarrays were stained with 2G12 (5 μg/ml) and a biotinylated GNA (1.0 μg/ml), respectively. The former was
revealed by Alexa647-tagged Goat anti-human IgG-Fc-specific antibodies at 2 μg/ml; then developed with Streptavidin-Cy5 conjugate at 2 μg/ml.
(A) Microarray images stained with either GNA or 2G12. (B) Cluster configuration of the three Man9-conjugates. Microarray detections are shown
as the mean fluorescent intensities (MFIs) of each microspot as captured by the ScanArray 5000A for the arrays stained with GNA (C) and 2G12
(D), respectively. Results were compared using overlay plots of the MFIs of staining signal (circles) versus those of local backgrounds surrounding
the antigen microarrays (crosses)(Cand D). [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]
CRYPTIC GLYCAN MARKERS OF MS/EAE 177
Drug Dev. Res.
Fig. 2. Epitope spreading to a broad range of protein, peptides, lipids, and carbohydrates in the serum of mice with EAE detected by
integrated microarrays. A panel of 51 antigenic structures (Table 1) was spotted in a versatile bioarray substrate in triplicate in 2–4 dilutions
to generate a total of 312 microspots of antigens per array. This platform supports detection of 208 unique antibody signatures, including 104
IgG- and 104 IgM-antibody signatures. (A) Each array was stained with a serum sample at a 1:25 dilution from a mouse with EAE (Right)or
age-matched control SJ/L mouse (Left). The captured IgG (Upper array images) were stained with an anti-IgG antibody conjugated with Cy5
at 2 μg/ml and the captured IgM in the same array (Bottom) was revealed by an R-PE-tagged anti-IgM secondary antibody at 2 μg/ml. Array
locations of myelin proteins (PLP, MOG and MBP), lipids/liposomes (sulfatide, ceramide and DMPS), and high-mannose-clusters (Man9) are
boxed. (B), the levels of IgG and IgM antibodies are shown as mean Ig scores of each group and presented in separate overlay plots. Ig values
of the EAE group (n= 10) are marked as circles and the controls (n= 6) as crosses. *Microarray scores: Antigen-specific IgG or IgM reactivities
are shown as microarray scores, the log2 transformed (mean-background) values of microarray detection. (C) Results of the statistical analysis
of the four probes, including two lipid antigens, PTC and sulfatide and two mannose-clusters, M9_2G12 and Man9. EAE mice examined in
this experiment were induced by immunizing SJL/J mice with myelin PLP139–151 as described [Kanter et al., 2006]. [Color figure can be viewed
in the online issue which is available at wileyonlinelibrary.com]
WANG ET AL.178
Drug Dev. Res.
antigen-specific IgG antibodies were revealed with a
tagged Goat anti-mouse IgG second antibody. For lectin
immunohistochemistry, biotinylated lectin prepara-
tions were applied to stain tissue sections. The tissue-
bound lectins were developed with Streptavidin-HRP
conjugate and DAB substrate. Lectins were pre-titrated
and applied at 10 μg/ml in this study. A nonparametric
statistical test (Wilcoxon rank-sum method) was used to
calculate the significance of differences among compara-
tive groups (Fig. 5).
Fig. 3. Integrated lipid/carbohydrate arrays recognize globally elevated antigen-specific antibodies in the CSF of MS patients as compared with
those detected in the OND CSF. CSF samples from 11 MS (10 RRMS and 1 SPMS) and 9 OND subjects were characterized using microarrays spotted
with 12 lipid and 20 carbohydrate antigens. Each preparation was spotted in triplicate at 2–4 dilutions. As illustrated in the overlay plots (A–D),
this microarray supports detection of 126 unique antibody signatures. These included 63 IgG (Aand C) and 63 IgM (Band D) signatures. Anti-
human IgG or IgM secondary antibodies were used to reveal the antigen-specific antibodies detected by these microarrays.The captured IgG were
stained with an anti-human IgG antibody conjugated with Cy3 at 2 μg/ml and the captured IgM in the same array revealed by a biotinylated anti-
human IgM secondary antibody at 2 μg/ml and developed with Streptavidin-Cy5 conjugate at 2 μg/ml. Microarray data sets in Aand Bwere
processed as described in legend to Figure 1 and illustrated as microarray scores. In Cand D, microarray data sets were further processed using
JMP-Genomics to produce *RAR scores. The results are presented as overlay plots of the mean microarray scores for each group. The colored need-
les that link the group means of each pair of the scores provide a global comparison of antibody profiles of MS group (circles) and OND (crosses).
*RAR Score: RAR—Relative Antibody Reactivity, the value of log2 transformed and IQR-standardized microarray value (Mean-background). For
each antigen in a given concentration, the mean value of triplicate array detections was calculated.
[Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]
CRYPTIC GLYCAN MARKERS OF MS/EAE 179
Drug Dev. Res.
EAE Induction and Man9-KLH Coimmunization
For induction of EAE, we immunized 8–10-week-
old female SJL mice (Jackson Laboratories, Bar Harbor,
ME) with 100 μgscofPLP
139–151 emulsified in complete
Freund’s adjuvant (CFA) at Day 0 without additional
immunization. For the Man9-KLH co-immunization
group, we applied the same immunization protocol as
the EAE control group except with the inclusion of
100 μg of Man9-KLH in the immunization emulsion
together with PLP139–151 in CFA. We monitored clinical
disease phenomena in the mice daily using the following
scoring system: 0, no disease; 1, limp tail; 2, hindlimb
weakness; 3, hindlimb paralysis; 4, hindlimb and
Fig. 4. MS-associated autoantibodies in CSF samples target high-mannose clusters, the cores of N-glycans. (A) Volcano plot analysis of the RAR
scores of all antibody signatures. In the volcano plot, each point represents a biologically unique feature captured by the microarray, i.e., a
normalized difference for a specific antibody signature (RARMS-RAROND). The x-axis is the normalized difference (log2 scale); the y-axis is −log10
(P-value), which weights the levels of significance of a difference. Points above the dashed line (cutoff level 2.5) represent signatures that differ
significantly between the groups based on the Bonferroni test. Two signatures, M9_2G12_IgG and Man9_IgG, were identified by this critical
statistical test as highly significant markers. In Band C, One-way analysis was performed to compare group means among the selected
carbohydrate antigens listed in each panel. Each point in the panels represents the mean value (RAR score) of triplicate array detection of a
subject. The means are shown as bars and standard deviations as diamonds around the mean value. The comparison circles for Student’s “t”-test
appear to the right of the mean diamonds to illustrate the significance of the differences among the means. By graphically showing the
intersections, these circles allow visual inspection of the significance of differences. The more the circles intersect, the less significant their
difference, and vice versa. [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]
WANG ET AL.180
Drug Dev. Res.
forelimb paralysis; 5, death. Mouse sera were taken at
Day 0 before immunization and weekly after immuniza-
tion for monitoring autoantibodies. Animal experiments
were approved by and performed in compliance with the
guidelines of the Stanford University Institutional
Animal Care and Use Committee.
Patient CSF Samples
All human samples were collected and used under
protocols approved by the Institutional Review Boards
of the Karolinska Institute and Stanford University.
Patient demographics and clinical characteristics,
including 10 RRMS, 1 SPMS, and 9 OND individuals
are listed in Table 2. The levels of total IgG and IgM in
these CSF specimens were measured by ELISA when
this microarray study was performed. The mean values
of total IgG in the MS group was 41.64 ±28.41 μg/ml,
which is significantly higher than those in the OND
group (10.47 ±6.34 μg/ml) (“t” test, P= 0.005). The
levels of total IgM in the MS group (0.83 ±0.04 μg/ml)
were not different from those in the OND group
(0.80 ±0.07 μg/ml) (“t” test, P= 0.218).
RESULTS
Monitoring Autoantibodies that Target
Self-Antigens of Distinct Molecular Structures
In this experiment, a panel of 51 antigens of
diverse molecular structures was spotted in the same
bioarray substrate (Table 1). These were protein and
peptides of myelin sheath (47#–51#,n= 5), lipids/
liposomes (21#–46#,n= 26), and carbohydrate antigens
(1#–20#,n= 20). A panel of glycoconjugates that dis-
played cryptic glyco-epitopes, including high-mannose-
clusters, M9_2G12-cluster (3#) and Man9-cluster (4#),
was included in this array. Using these arrays, we
analyzed serum samples from SJL/J mice immunized
with myelin proteolipid protein peptide (PLP139–151)
Fig. 5. Lectin GNA, PHA-L and SNA histochemistry of human brain tissues, normal (0) and MS (1), and mouse spinal cord EAE lesions (2). CNS
tissues were stained with biotinylated lectins and developed with Streptavidin-HRP conjugate and DAB substrate. Lectins were pre-titrated and
applied at 10 μg/ml for tissue staining. Column A, B, and C were stained with lectin GNA, PHA-L and SNA respectively. Normal: A0, B0, and
C0; MS: A1, B1, and C1; EAE: A2, B2, and C2. The carbohydrates recognized by the lectins are indicated for each column.
CRYPTIC GLYCAN MARKERS OF MS/EAE 181
Drug Dev. Res.
[Kanteret al., 2006] for autoantibodies using a group of
age-matched SJL/J mice as control.
Figure 2A shows representative microarray images
in which the captured antigen-specific IgG (Upper) and
IgM (Bottom) are shown for a mouse with EAE (Right
column) and an age-matched control SJL/J mouse (Left
column). Visual inspection of these microarray images
identified four protein/peptide probes, PLP, myelin
oligodendrocyte glycoprotein (MOG) and two prepara-
tions of myelin basic protein (MBP) (marked in red), that
detected markedly increased autoantibodies of both
IgG and IgM isotypes; two lipid probes, sulfatide and
dimyristoylphosphatidylserine (DMPS, yellow), and two
Man9-clusters (white), captured significant amounts of
IgM, but not IgG, in the sample from this panel of mice
with EAE.
A quantitative comparison of the two groups was
conducted to examine the significance of these obser-
vations. Figure 2B shows the overlay plots of antibody
profiles for the two groups where the levels of IgG and
IgM antibodies are shown as the mean Ig scores of each
group. Antibody values of the EAE group (n= 10) were
colored in red and the controls (n= 6) in blue. The
overall antibody profiles between the two groups
appear similar with the exception of specific probes for
autoantibodies to myelin proteins (MBPs [48#and 49#],
MOG [50#] and PLP [51#]), lipids (sulfatide [44#] and
DMPS [46#]), and carbohydrate markers (M9_2G12-
clusters [3#] and Man9-cluster [4#]).
Figure 2C illustrates results of the one-way
ANOVA of four markers, including PTC (22), sulfatide
(44), M9_2G12-cluster (3#), and Man9-cluster (4#).
Similar to brain sulfatide (44), the two Man9-clusters
(3#and 4#) captured increased amounts of autoanti-
bodies of IgM isotype in EAE group as compared with
the control group (NM). However, there was no differ-
ence between the two groups in induction of IgG
autoantibodies for these markers.
These results demonstrate that the PLP-priming
induced autoantibodies of both IgG and IgM isotypes to
myelin proteins PLP, MOG, and MBP, but selectively
elicited IgM responses to a number of lipid and carbo-
hydrate antigens. The latter include autoantibodies to
brain sulfatide and DMPS previously recognized using
PVDF-immobilized lipid arrays [Kanter et al., 2006; Ho
et al., 2012] and to carbohydrate-based novel markers,
M9_2G12-clusters (3#) or Man9-cluster (4#).
Autoantibodies Targeting High-Mannose-Clusters
are Selectively Enriched in the CSF of MS Patients
We next examined whether the carbohydrate and
lipid that detected antibodies in the EAE model might
also detect autoantibodies in the CSF of MS patients
and OND control subjects. For this purpose, we
created a microarray displaying a panel of 32 carbohy-
drate and lipid probes for a focused investigation of
nonprotein markers. The carbohydrate antigens that
display N-glycan cryptic glyco-epitopes, e.g., AGOR
(5#), ASOR (6#), and mannose clusters (1#,3
#, and 4#)
[Wang, 2004, 2012b; Newsom-Davis et al., 2009; Wang
et al., 2013] were included for microarray spotting
(Figs. 3 and 4).
Figure 3A and B show the overlay plots of anti-
body profiles of the two groups. The colored needles
that link the pairs of group means provide a global
comparison of the antibody profiles between the MS
group (red circles) and the OND group (blue crosses).
This comparison reveals global differences in antibody
profiles between the two groups. Specifically, the
microarray scores of CSF-antibody activities in the MS
group are generally higher than those seen in the CSF
of OND subjects. These include not only anti-lipid anti-
bodies (Right, 26#–44#), as previously reported [Ho
et al., 2012], but also anti-carbohydrate antibodies
(Left, 1#–22#). This observation may reflect the fact that
the total Ig concentrations in the CSF of MS patients
are higher than those in the CSF of OND subjects,
TABLE 2. Patient Demographics and Clinical Characteristics for
Cerebrospinal Fluid Samples
Sample ID Diagnosis* Age Gender
Group 1 Multiple sclerosis (MS) (n= 11)
01-119 SPMS 58 M
01-168 RRMS 42 F
01-182 RRMS 27 F
01-188 RRMS 26 F
01-189 RRMS 56 F
01-190 RRMS 26 F
02-012 RRMS 33 F
02-013 RRMS 31 F
02-014** RRMS 37 F
02-015** RRMS 32 F
02-023 RRMS 45 F
Group 2 Other neurological disease (n=9)
01-180 Tension headache 35 M
01-181 Vertigo 36 F
01-184 Tension headache 58 F
01-183 Vertigo 58 F
01-185 Vertigo 51 M
02-001 Slipped disc S1 30 F
02-010 Spinal stenosis in cervical
spine & chronic pain
43 F
02-016 Sensory symptoms 20 F
02-019 Vestibular neuritis 36 M
*Diagnosis at the time of sampling.
**Immunomodulatory treatment being taken by the patient at the time
of sampling.
SPMS, secondary progressive multiple sclerosis; RRMS, relapsing-
remitting multiple sclerosis;
WANG ET AL.182
Drug Dev. Res.
which is one of the hallmarks of MS [Kabat et al., 1948,
1951; Steinman, 1996; Genain et al., 1999; Raine et al.,
1999; Hueber et al., 2002].
We further examine whether there is any selective
enrichment of antigen-specific antibodies in the CSF
of MS patients. We reasoned that identifying such
antibodies might provide clues to pinpoint key
autoimmunogenic targets of MS. For this purpose, we
introduced an approach to establish RAR scores for
microarray detections and then seek targets that
capture the antibody signal with higher RAR scores in
MS patients. Specifically, we normalized microarray
data sets by setting their IQR to be identical using the
JMP-Genomics software package. This statistical opera-
tion effectively “quenches” the variation seen between
subjects that is due to variable antibody concentrations
in the CSF. The two groups illustrate similar Ig-RAR
profiles for both IgG and IgM antibody activities
(Fig. 3C and D). However, a number of probes show
higher IgG-RAR scores in the MS group than in the
OND controls. These include two Man9-clusters (3#
and 4#), three glucose polysaccharides, dextran N279
(8#), B1299S (9#), and B1355S (10#), and a Bacto-agar
(20°C, extracted) antigen (13#) (Fig. 3C).
We then conducted an ANOVA analysis to identify
antibody signatures that differed between the MS and
OND groups based on a critical Bonferroni test.
Figure 4A shows the results in a volcano plot [Zink et al.,
2013], wherein each point represents a statistically
weighted and quantified difference between the MS and
OND groups. The x-axis is normalized difference in log2
scale (RAROND −RARMS) and the y-axis is −log10 (P-
value) for the difference. Of the 126 antibody signatures
captured in this assay (Fig. 3C and D), only two were
above the cutoff line (–log10 [P-value] = 2.5) as highly
significant differentiators between MS and OND. These
were M9_2G12_IgG and Man9_IgG. Results of one-
way ANOVA of the two Man9 clusters are shown in
Figure 4B. Logistic regression analysis suggests that
these CSF autoantibody signatures are highly significant
in the differential diagnosis between MS and OND pro-
ducing the area under the ROC curve values of 0.92929
and 0.87879, respectively. All other antibody signatures
were weighted below the cutoff line (Fig. 4A), including
four signatures that were variably higher in the MS
group than in the OND group. These were N279_IgG
(P= 0.0825), B1299S_IgG (P= 0.0886), B1355S_IgG
(P= 0.1135), and Bacto_Agar_IgG (P= 0.021).
Mannose-Clusters are Aberrantly Expressed in the
CNS in EAE and MS
Based on the Bonferroni test recognition of the
two high-mannose clusters, we hypothesized that the
mannosyl glycans may be aberrantly expressed in vivo in
MS/EAE subjects with exposure of these “cryptic” sugar
moieties to trigger specific autoantibody responses.
Therefore, we examined MS- and EAE-affected CNS
tissues to determine whether they abnormally expressed
high-mannose cluster-related carbohydrates. We
applied lectins of known specificities to monitor expres-
sion of various N-glycan complex carbohydrates in MS
and normal control brain tissue samples and in spinal
cords of EAE mice (Fig. 5). The following three lectins
were applied: (i) GNA, specific for the mannose-
clusters; (ii) Phaseolus vulgaris-L (PHA-L), specific for
Tri-II and m-II clusters; and (iii) Sambucus nigra I
agglutinin (SNA-I), recognizing α2–6 linked Neu5Ac
residues. The results of this study were: A) GNA: In
normal human cerebral cortex (Fig. 5, A0), only the
cytoplasm of neurons (red arrows) was stained. In an
inflammatory MS lesion (A1), GNA stained a number of
cell types, including cells with the morphological
appearance of macrophages and reactive astrocytes
(small red arrow). A dystrophic axon was also strongly
positive (upper right corner, thick red arrow). In an acute
inflammatory EAE lesion (A2), GNA stained many dys-
trophic axons (e.g., thick red arrow) and macrophage
debris (e.g., thin red arrow). There was no positive
staining in the spinal cords of control mice (data not
shown). B) PHA-L: In normal human cerebral cortex
(B0), PHA-L staining was negative. In an active MS
lesion (B1), there was diffuse weak PHA-L staining. In
the EAE lesion (B2), PHA-L staining was negative. C)
SNA: SNA demonstrated positive staining of capillary
endothelial cells in normal human cerebral cortex (C0)
(red arrows). In the active MS lesion (C1), SNA selec-
tively stained cells that were morphologically consistent
with reactive microglia, (e.g., thick red arrow), as well as
endothelial cells (small red arrows) (C1). In the EAE
lesion (C2), SNA stained macrophage debris and
endothelial cells (red arrows). In normal mice, SNA
staining was limited to endothelial cells (data not shown).
Taken together, we observed that: (i) expression
of high-mannose glycans, as defined by GNA, is
restricted to cortical neurons in normal human brain
tissue but is highly and broadly distributed in multiple
cell types in tissues representing MS and EAE lesions;
(ii) Tri/m-II chains detected by PHA-L were not
exposed to lectin staining in normal brain tissue and
were minimally expressed in active MS lesions; and (iii)
α2-6Neu5Ac epitopes (SNA) that were present in blood
vessels and red blood cells in normal brain tissue were
aberrantly expressed in MS and EAE lesions. Thus, the
repertoires of the aberrant carbohydrates in MS and
EAE were not limited to the high-mannose N-glycan
structures. This is indicated by the altered expression of
the SNA epitopes (α2-6Neu5Ac) in these lesions.
CRYPTIC GLYCAN MARKERS OF MS/EAE 183
Drug Dev. Res.
Induction of Anti-Oligomannose IgG Antibodies by
Man9-KLH is Associated with the Reduced Clinical
Severity of EAE
We next determined whether these carbohydrate
targets could alter the course of EAE when used as
immunogens co-delivered with myelin peptides emul-
sified in CFA. Kanter et al. took this approach to
examine the in vivo effect of a lipid marker, sulfatide,
which was identified as an MS and EAE-associated
autoantigen by a lipid array analysis [Kanter et al.,
2006]. They demonstrated that co-immunization of
mice with sulfatide plus myelin peptide resulted in a
more severe disease course and that the administration
of anti-sulfatide antibody exacerbated EAE. In the
present study, we co-immunized SJL mice with Man9-
KLH together with myelin peptide PLP139–151. If the
high-mannose-clusters were immunogenic, this immu-
nization strategy should elicit antibodies targeting
oligomannose antigens and if the anti-carbohydrate
antibodies were pathogenic, induction of these antibod-
ies should worsen the disease course. This in vivo vali-
dation experiment was conducted twice with similar
outcomes. In the first test, 17 mice were used with 5 for
the Man9-cluster experimental group and 12 for the
PLP/CFA control group; the second test employed 20
mice with 10 for each group (Fig. 6).
As illustrated by the EAE score curves (Fig. 6A),
active immunization by Man9-KLH plus PLP did not
worsen but rather improved the course of EAE,
although it elicited high-titers of anti-PLP IgG antibod-
ies, as well as anti-oligomannose IgG antibodies
(Fig. 6B). Although there was no difference in the titers
of anti-PLP IgG antibodies between the PLP control
Fig. 6. Induction of anti-Man9-Cluster IgG antibodies by co-immunization with mannose-clusters and a pathogenic myelin peptide does not
worsen the course of EAE. (A) Overlay plot of the EAE scores of Man9 group (-Δ-, n= 10) and those of vehicle control group (-■-, n= 10). Man9
group: Co-immunization of SJL mice with Man9-KLH (100μg/mouse) and PLP139–151 (100μg/mouse) emulsified in CFA. Vehicle control group:
immunization with PLP plus CFA without additional immunogen. Each point represents the mean ±s.e.m. A statistical test (Mann–Whitney test)
was used to calculate the significance of differences between the two groups. The time points illustrated significant differences (P <0.05) are
marked * on the graph. (B) Anti-Man9 IgG and (C) anti-PLP IgG responses analysed by ELISA. Serum specimens from the EAE experiment
(Fig. 6A) were titrated for antigen-specific ELISA in a preliminary experiment and applied at 1:500 dilutions for ELISA detection of antigen-
specific IgG. (B) ELISA plates coated with Man9-BSA conjugate at 10 μg/ml for detection of Man9-specific IgG at day 21 post-immunization with
Man9-KLH and PLP; (C) ELISA plates were coated with PLP139–151 at 10 μg/ml for the detection of PLP-specific IgG in the same serum specimen.
Serum antibodies specific for the carrier BSA in day 0 pre-immunization and day 21 post-immunization and pre-existing anti-Man9 and anti-PLP
in day 0 sera were below the detection sensitivity of this ELISA (OD 405 nm = or <0.150). One-way ANOVA was performed to compare the
ELISA results obtained between groups. A nonparametric statistical test (Wilcoxon rank-sum method) was applied to calculate the significance
of differences between comparative groups.
WANG ET AL.184
Drug Dev. Res.
and the Man9-co-immunization groups (Fig. 6C, right
panel), the latter elicited markedly increased anti-
Man9-IgG antibodies as determined by a glycan-
specific ELISA assay (Fig. 6B, left panel).
DISCUSSION
Applying integrated protein, lipid, and carbohy-
drate microarrays, we have characterized the autoanti-
body response in the PLP-induced EAE model in SJL/J
mice. We found that the EAE-associated autoantibody
profile was characterized by dominant IgG antibodies
directed at myelin proteins with a broader spectrum of
IgM antibodies targeting lipid- and carbohydrate-based
autoantigens. IgM antibodies specific for brain sulfatide
(44#) and DMPS (46#) that were previously recognized
by Kanter et al. [2006] were confirmed using this
integrated multicomponent microarrays (Fig. 2). In
addition, we detected anti-carbohydrate autoantibodies
specific for the high-mannose type N-glycans (oligo-
mannoses), M9_2G12-clusters (3#) and Man9-cluster
(4#). Similar to anti-sulfatides, IgM but not IgG anti-
oligomannose antibodies were detected in this panel of
EAE mice (Fig. 2B).
Of note, the anti-oligomannose antibodies
detected in EAE were also selectively enriched in the
CSF of MS patients (Fig. 3). The specificities of the
anti-mannose cluster autoantibodies detected in EAE
were strikingly similar to those in the CSF of MS
patients. Specifically, both EAE-derived and MS-d
erived anti-oligomannose antibodies were similarly reac-
tive with the two Man9-clusters, M9_2G12-clusters (3#)
and Man9-cluster (4#). As illustrated in Figure 1B, the
former is a Man9-tetramer conjugate and the latter a
Man9-monomer conjugate. This binding profile is
similar to lectin GNA (Fig. 1C) but differs from the
binding profile of a human monoclonal antibody 2G12
(Fig. 1D) in our microarray assays. The antibody 2G12 is
well characterized as an unusual, domain-exchanged
IgG1 recognizing the Manα(1,2)Man moieties in dense
cluster configurations of high-mannose type N-glycans
as displayed on the outer domain of HIV gp120
glycoprotein [Calarese et al., 2003]. In our microarrays,
2G12 was highly reactive with the M9_2G12 cluster in
[(Man9)4]n-cluster configuration, but only weakly reac-
tive with Man9-cluster in the (Man9)n-configuration
(Fig. 1D). In contrast, lectin GNA was strongly and
equally reactive with the two oligomannose-conjugates
(Fig. 1C). Thus, the binding specificities of the MS
and EAE-associated anti-oligomannose autoantibodies
overlap with the binding profile of GNA.
We examined whether the GNA-defined man-
nosyl glycan markers were expressed in the CNS. In
normal brain tissues (Fig. 5 A0), GNA selectively
stained the cytoplasmic compartment of human cortical
neurons and was negative in other areas. In contrast,
GNA was highly and broadly reactive with multiple
cell types in MS lesions, including macrophages, reac-
tive astrocytes and dystrophic axons (Fig. 5 A1). Simi-
larly, this lectin stained many dystrophic axons and
macrophage debris in the spinal cord lesions of
mice with EAE (Fig. 5 A2). Thus, the GNA-defined
mannosyl glycan markers were aberrantly expressed in
inflammatory/demyelinating CNS lesions.
The EAE model provided a unique experimental
condition to examine the potential immunogenicity of
high-mannose-clusters in vivo. Specifically, we tested
a Man9-KLH conjugate for its capacity to elicit an
acquired immune response to oligomannose epitopes.
We found that a single sc injection of the PLP/CFA
emulsion containing Man9-KLH elicited highly signifi-
cant anti-oligomannose IgG antibodies (Fig. 6B). Thus,
this high-mannose-cluster-conjugate was immunogenic
in vivo in this experimental condition. This effective
anti-carbohydrate immunization may illuminate further
development of novel vaccination strategy targeting the
glycan markers of viruses [Calarese et al., 2003; Wang
and Lu, 2004; Astronomo et al., 2008] and certain
human cancers [Hakomori, 1985; Newsom-Davis et al.,
2009; Wang, 2012b].
Moreover, we observed that induction of IgG
anti-oligomannose response was associated with signifi-
cantly reduced clinical signs of EAE (Fig. 6), a finding
that is in contrast to the results observed following
administration of sulfatide in the same experimental
setting in which a more severe disease course was
observed [Kanter et al., 2006]. Further investigation
will elucidate the physiological and/or pathological roles
of anti-mannose cluster autoantibodies. Whether
autoantibodies can play “Janus-like” opposing roles as
CD47 does in autoimmune brain inflammation [Han
et al., 2012] is under consideration. For example, the
GNA-like autoantibodies that bind dystrophic axons
and cell debris in MS/EAE lesions (Fig. 5) would be
beneficial if they contributed to effective clearance of
myelin debris that exposed the cryptic glyco-epitopes.
Exploring anti-glycan antibodies as potential
serum biomarkers in differential diagnosis and progno-
sis of neurological disorders is an active research area
[Willison, 2002; Willison and Yuki, 2002; Schwarz et al.,
2003, 2006; Lolli et al., 2005; Brettschneider et al.,
2009]. Notably, human serum IgM antibodies specific
for a number of glucosyl targets have been investigated
for MS diagnosis and prognosis [Schwarz et al., 2003,
2006; Brettschneider et al., 2009]. These include
GAGA4 [Glc(α1,4)Glc(α)], GAGA6 [Glc(α1,6)Glc(α)],
GAGA3 [Glc(α1,3)Glc(α)], and GAGA2 [Glc(α1,2)Glc
(α)]. In the present study, we tested a panel of glucose
CRYPTIC GLYCAN MARKERS OF MS/EAE 185
Drug Dev. Res.
polysaccharides, including dextran preparations of
N279, B1299S, and B1355S [Wang and Kabat, 1996].
Dextran N279 predominantly displayed α(1,6)-internal
chain epitopes, i.e., repetitive units of GAGA6 [Glc(α1,
6)Glc(α)]n as linear chains. B1299S was heavily
branched and displays many terminal nonreducing end
epitopes of [Glc(α1,6)Glc(α)]n. B1355S was character-
ized by α(1,3)α(1,6)linkages, which may present the
GAGA3 [Glc(α1,3)Glc(α)]-moieties as both internal
and terminal epitopes. However, this pilot study was
limited in sample sizes, and it is difficult to conclude
whether antibodies for these polysaccharides were sig-
nificantly enriched in the CSF of MS subjects (Fig. 4C).
In addition to these glucose polysaccharides, we
observed CSF-enrichment of anti-Bacto-agar IgG anti-
bodies in the CSF of MS subjects. The Bacto-agar
preparation (20°C, extracted) is a pyruvylated galatosyl
polysaccharide with [4,6pyDGalβ1]-epitopes, which
are also present in microbial polysaccharides, e.g.,
Klebsiella K30 and K33 [Kabat et al., 1980]. Anti-
Dextran and anti-Bacto-agar antibodies are often
present in the human circulation [Kabat and Berg,
1953; Wang et al., 2002]. Detection of these common
anti-carbohydrate antibodies in both CSF and blood
may have utility in monitoring the functional integrity of
the blood-brain barrier. These findings must be further
validated in a larger cohort of MS and OND subjects.
Shared carbohydrate determinants on microbes and
brain tissue, such as manosyl moieties, represents yet
another pathway where the microbiome and autoimmu-
nity intersect in neuroinflammatory disease states
[Wang et al., 2002; Wang and Lu, 2004; Yuki et al.,
2004; Shahrizaila and Yuki, 2011; Ogawa et al., 2013].
ACKNOWLEDGMENTS
We acknowledge Dr. Russell Wolfinger and the
technical support team of SAS Institute for their
instruction and excellent technical assistance in the
use of JMP and JMP-Genomics software; Dr. Jennifer
L. Kanter, Professors William H. Robinson, Leonore
A. Herzenberg, and Leonard A. Herzenberg for help-
ful discussions, Dr. Jiahong Ni for preparation of
[(Man9)4]n-KLH conjugate, Drs. Rachel Schneerson
and John Robbins for Shigella dysenteriae Type 1
O-specific polysaccharide, and the late Professor Elvin
A. Kabat and his previous students, postdoctoral
fellows, and collaborators for their contributions to a
number of carbohydrate antigens listed in Table 1 that
were used in this study. This work is supported in part
by NIH grants R01NS055997 (L. Steinman and D.
Wang), U01CA128416 and R56AI108388 (D. Wang),
and R01AI067111 (L-X. Wang), and by the Swedish
Research Council and the Knut and Alice Wallenberg
Foundation (TO). The content of this article is solely
the responsibility of the authors and does not necessar-
ily represent the official views of the National Institutes
of Health.
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