Evaluation of anti α-d-Glc: P -(1→4)-α-d-Glc p (GAGA4) IgM antibodies as a biomarker for multiple sclerosis

Article abstract of DOI:10.1039/c8ra04897e

The correct diagnosis of multiple sclerosis (MS) remains challenging due to the complex pathophysiological and clinical characteristics of the disease. Consequently, there has been immense interest in finding a non-invasive diagnostic test for MS. Recent studies found that serum anti-α-d-Glcp-(1→4)-α-d-Glcp (GAGA4) IgM antibodies were upregulated in MS patients, and this finding led to the development of a commercial diagnostic test (gMS Dx test), although the test has poor selectivity and has not been independently validated. Herein, we developed an enzyme-linked immunosorbent assay (ELISA) to evaluate the use and reliability of several anti-glucose IgM antibodies, including those against GAGA4, as diagnostic biomarkers for MS. In contrast to previous studies, our results show that serum anti-GAGA4 IgM antibody levels are not significantly higher in MS patients, which could potentially explain the poor selectivity of the commercial test.

Full text of DOI:10.1039/c8ra04897e

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Evaluation of anti a-D-Glcp-(1/4)-a-D-Glcp
(GAGA4) IgM antibodies as a biomarker for multiple
Cite this: RSC Adv., 2018, 8, 28086
sclerosis†
Chriselle D. Braganza,ab Kristiana T. Santoso,ab Emma M. Dangerfield,ab Anne C. La
ab
ab
Flamme,bc Mattie S. M. Timmer
and Bridget L. Stocker
*
*
The correct diagnosis of multiple sclerosis (MS) remains challenging due to the complex pathophysiological
and clinical characteristics of the disease. Consequently, there has been immense interest in finding a non-
invasive diagnostic test for MS. Recent studies found that serum anti-a-D-Glcp-(1/4)-a-D-Glcp (GAGA4)
IgM antibodies were upregulated in MS patients, and this finding led to the development of a commercial
diagnostic test (gMS® Dx test), although the test has poor selectivity and has not been independently
validated. Herein, we developed an enzyme-linked immunosorbent assay (ELISA) to evaluate the use and
reliability of several anti-glucose IgM antibodies, including those against GAGA4, as diagnostic
biomarkers for MS. In contrast to previous studies, our results show that serum anti-GAGA4 IgM antibody
levels are not significantly higher in MS patients, which could potentially explain the poor selectivity of
the commercial test.
Received 8th June 2018
Accepted 22nd July 2018
DOI: 10.1039/c8ra04897e
rsc.li/rsc-advances
protein IgM antibodies have been suggested to predict early
relapse in MS patients,8 however in other studies, no such
correlation was observed.9 A multitude of other antibodies have
also been studied for their potential use as biomarkers for MS,
including antibodies against the Epstein Barr nuclear antigen,10
heat shock proteins,11,12 and complement regulators,13,14
although none have been successfully validated in a clinical
setting.7,15
Introduction
Multiple sclerosis (MS) is a chronic autoimmune disease char-
acterized by damage to the protective myelin sheaths that
insulate nerve bers.1 Demyelination of the nerve bers impairs
or prevents the transduction of signals throughout the central
nervous system (CNS) resulting in an array of symptoms such as
fatigue, blurred vision, muscle weakness and cognitive
impairment.2 Symptoms can vary widely among individuals and
consequently, MS diagnosis is oen difficult and requires
a combination of blood tests, magnetic resonance imaging, and
evoked potential tests, which measure the electrical conduction
of nerves.3 Cerebrospinal uid, obtained via lumbar puncture,
can also be analyzed for the presence of oligoclonal IgG bands
that are indicative of inammation in the CNS,4 however this
procedure is non-specic to MS and is difficult to repeat regu-
larly due to its invasive nature.5
Recently, anti-glycan IgM antibodies have been described as
potential biomarkers for MS.16,17 In 2005, Lolli et al. observed
high IgM autoantibody titers against the N-glucosylated peptide
CSF114(Glc) in patients with RRMS.16 It was determined that
a native N-linked b-D-glucopyranose-asparagine moiety was the
minimal epitope required for antibody binding. More recently,
it was observed that puried N-glucosylated glycoproteins from
Haemophilus inuenzae were preferentially recognized by anti-
bodies from a subpopulation of MS patients, thereby providing
the rst example of an N-glucosylated native antigen for anti-
bodies in MS.18 In 2003, Schwarz et al. immobilized a range of
glycans onto a glass chip via a cyanuric 1,8-diamino-3,6-
dioxaoctane linker19 and used this glycan array to test for
serum anti-glycan antibodies.17,19–21 From this work, it was
determined that serum anti-a-glucose (a-Glc) IgM antibodies,
specically those against a-D-Glcp-(1/4)-a-D-Glcp (GAGA4)
(Fig. 1), were signicantly up-regulated in MS patients. These
ndings then led to the development of a commercial blood test
known as the gMS® Dx test, which claims to have a high positive
predictive value for MS and the ability to differentiate MS from
other neurological disorders.17,20,21 However, the test has a low
sensitivity rate of 33.7%,21 and has not been widely validated.
To develop a non-invasive diagnostic tool for MS, recent
research has focused on serum-derived antibodies as potential
biomarkers for the disease.6,7 In some studies, high levels of
anti-myelin oligodendrocyte glycoprotein and anti-myelin basic
aSchool of Chemical and Physical Sciences, Victoria University of Wellington, P. O. Box
600, Wellington 6140, New Zealand
bCentre for Biodiscovery, Victoria University of Wellington, P. O. Box 600, Wellington
6140, New Zealand. E-mail: mattie.timmer@vuw.ac.nz; bridget.stocker@vuw.ac.nz
cMalaghan Institute of Medical Research, Victoria University of Wellington, P. O. Box
7060, Wellington 6242, New Zealand
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra04897e
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Fig. 1 Structure of GAGA4 with the cyanuric linker.19
Moreover, others have identied antibodies against several a- simple triazole-functionalized linker is envisioned to eliminate
glucosyl antigens in healthy donors.22–24
the high levels of non-specic binding observed with more
Given the poor selectivity of the gMS® Dx test, we sought to complex linkers.27
independently validate the potential of a variety of glucosyl
To measure serum anti-glycan IgM levels, we will use an
antigens for the detection of IgM antibodies as diagnostic ELISA protocol that presents the antigens in such a way that
biomarkers for MS. To this end, we synthesized a range of a- their carbohydrate moieties are exposed for antibody binding
and b-glucose–glycoprotein conjugates, which contain native (Fig. 3). This will be achieved by rst coating the plates with the
glycosidic linkages at their reducing ends to avoid potential antigen–BSA conjugates, followed by incubation with serum
non-specic antibody responses. These conjugates were then samples. Incubation with the secondary anti-human IgM anti-
used in an enzyme-linked immunosorbent assay (ELISA) with body that is coupled to horseradish peroxidase will then allow
serum samples from healthy donors and patients with for the transformation of 3,30,5,50-tetramethylbenzidine (TMB)
relapsing-remitting multiple sclerosis (RRMS) to ascertain and thus, determination of the level of bound anti-glycan IgM
whether differences in specic glycan IgM responses were antibodies.
observed between the two patient populations. Our results
indicate that anti-glycan IgM antibody levels vary tremendously
among individuals and as a result, no specic anti-glycan IgM
Synthesis of antigens
It was proposed that glyconconjuates 2–7 could be prepared
response was seen for patients with RRMS compared to healthy
from the corresponding free carboxylic acid 8 via the formation
controls. The ndings of our study provide insight into the poor
of N-hydroxysuccinimide (NHS)-activated ester and subsequent
conjugation to bovine serum albumin (BSA) (Scheme 1).
selectivity observed with the commercial gMS® Dx test.
Carboxylic acid 8, in turn, should be accessible by way of
a copper catalyzed azide-alkyne Huisgen 1,3-dipolar cycloaddi-
Results and discussion
tion (CuAAC)28 of 4-pentynoic acid (9) with azide-functionalized
In order to compare the level of anti-glycan IgM antibodies in glycan 10, whereby the glycan would be prepared via a glyco-
patients with RRMS and healthy controls (HC), we aim to sylation reaction of suitably protected sugar 11 with 3-azido-
prepare carbohydrate antigens and control glycoprotein conju- propanol (12). Here, the stereochemistry at the anomeric
gates 1–7 (Fig. 2). Here, the free azidopropyl linker-BSA conju- position would be controlled through the use of coupling
gate (1) will be used as a negative control to measure conditions that favor the formation of the theromodynamic
background titers. L-Rha-BSA (2) will be used as the positive product (a-anomer), or through neighboring group participa-
control as previous studies on human anti-glycan antibodies tion (b-anomer). Preparation of the free azidopropyl linker-BSA
revealed that anti-L-rhamnose (L-Rha) antibodies were present conjugate (1) would be achieved via the CuAAC of 3-azidopro-
in high levels in the majority of subjects.22,25,26 The synthesis of panol (12) with 4-pentynoic acid (9) and subsequent protein
a selection of a- and b-linked glucose-containing antigens (3–7) conjugation.
will also be undertaken, including glycoconjugate 4, which
With a suitable synthetic strategy in place, we rst under-
contains the GAGA4-motif previously suggested to be up- took the synthesis of the free azidopropyl linked-BSA conju-
regulated in patients with MS.17 Similarly, synthesis and anal- gate (1) and the positive control Rha-BSA (2) (Scheme 2).
ysis of glycoconjugate 7 containing D-maltotriose will allow for Glycosylation of peracetylated L-rhamnose (13) with 3-azido-
investigations into whether this glycoconjugate can elicit propanol (12) using BF3$Et2O as the activator gave glycoside 14
a similar IgM antibody response to that seen with GAGA4, since as the a-anomer [J1,2 ¼ 1.5 Hz] exclusively (Scheme 2A). The
7 also possesses the GAGA4 moiety at its terminus. Use of the acetyl protecting groups in 14 were subsequently removed
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Fig. 2 Controls and antigens to be used in this study.
under Zemplen conditions29 to give deprotected azidopropyl L- uronium tetrauoroborate (TSTU),26 in the presence of NEt3
Rha glycoside 15. Installation of a carboxylic acid group onto 3- (Scheme 2B). The NHS esters were then cross-linked to BSA26 to
azidopropanol (12) or azidopropyl rhamnopyranoside 15 was yield free azidopropyl linked-BSA conjugate 1 and L-rha-BSA
then achieved via CuAAC28 with 4-pentynoic acid (9) to give the conjugate 2. Analysis of the nal conjugates was then under-
corresponding carboxy-modied triazoles 16a and 16b in good taken using MALDI-TOF spectrometry to determine the
yields of 82% and 77%, respectively. Each derivative was then average molecular weight of the protein aer conjugation to
converted to its corresponding NHS ester using an activated the sugar, which was used to estimate the number of antigens
form of NHS, N,N,N0,N0-tetramethyl-O-(N-succinimidyl) per protein molecule (Fig. S1†). Here, conjugate 1 was
´
Fig. 3 Schematic representation of the ELISA protocol used in this study.
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Scheme 1 Retrosynthetic analysis of glycoconjugate targets 2–7.
observed to contain an average of 42 linker molecules per BSA, (TPABr) (3 equiv.) as mild activators. Here, the glycosylation
and conjugate 2 contained 35 rhamnose molecules per BSA. reactions occurred via the corresponding glycosyl bromide
Having successfully synthesized the azidopropyl linked-BSA intermediates, which underwent in situ anomerisation33 to form
conjugate 1 and the L-Rha-BSA conjugate 2, we then used the desired benzylated glycosides in 85% yields with a 7 : 1 a : b
a similar strategy to prepare the glucose-functionalized anti- ratio. The protected a-glycosides were readily separated from
gens. Synthesis of the a-linked antigens began with the prepa- their b-glycoside counterparts via silica gel ash column chro-
ration of the thioglycosides30 of per-acetylated D-glucose (17a) matography to give the target products 19a [J1,2 ¼ 3.8 Hz] and
and per-acetylated D-maltose (17b), with subsequent deacetyla- 19b [J1,2 ¼ 3.7 Hz] in 68% and 65% isolated yields, respectively.
tion29 and benzylation31 to give the fully protected thioglyco- Carboxylic acid groups were then installed on the benzylated a-
sides 18a and 18b, respectively, in good overall yields (Scheme glycosides through the use of CuAAC reactions with 4-pentynoic
3). To achieve good a-selectivity during the ensuing glycosyla- acid (9), followed by hydrogenolysis using H2 gas and Pd(OH)2
tion reactions with 3-azidopropanol (12), we optimized glyco- and treatment with TSTU to afford the NHS esters. Conjugation
sylation reaction conditions previously reported by Sato et al.,32 of the activated esters to BSA then gave the target a-linked Glc-
and used CuBr2 (3 equiv.) and tetrapropylammonium bromide antigens a-Glc-BSA (3) and GAGA4-BSA (4). MALDI-TOF analysis
Scheme 2 Synthesis of the positive and negative control antigens.
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Scheme 3 Synthesis of a-Glc based antigens 3 and 4.
of glycoconjugates 3 and 4 showed average loadings of 31 and titres against the free azidopropyl linker-BSA conjugate (1) for
25 glycans per BSA, respectively (Fig. S1†). each subject were subtracted from the IgM titres obtained for
To prepare the b-Glc antigens, per-acetylated D-glucose (17a), each glycan. An additional cohort of plasma samples (6 HC and
per-acetylated D-maltose (17b), and peracetylated D-maltotriose 8 MS) were also analysed in addition to the previous sera
(17c) were converted to the corresponding a-glycosyl bromides samples in order to evaluate any possible differences in sample
using HBr-HOAc,34 before undergoing glycosylation with type (Fig. 4B).
acceptor 12 in the presence of AgOTf to selectively produce the
As anticipated, the highest IgM response was observed
b-linked products (Scheme 4). Deacetylation29 then furnished against the positive control Rha (2), in the sera of both HC and
the deprotected b-azidopropyl glycosides 20a–c in good overall RRMS patients (Fig. 4A), and a similar response was observed
yields (38–58%). Here, selective formation of the b-product was when plasma samples were included (Fig. 4B). This result is
conrmed by 1H NMR [J1,2 ¼ 7.9 Hz (20a), 8.0 Hz (20b) and consistent with previous ndings by Huejt et al., where all 106
8.0 Hz (20c)]. Formation of the NHS-activated esters was then tested healthy subjects showed high anti-Rha antibody titres.22
undertaken as described above, with conjugation to BSA High IgM titres were also observed towards b-Glc (5) for most
affording the target antigens b-Glc-BSA (5), GAGB4-BSA (6) and subjects, which is again consistent with previous studies.22,25 No
GAGAB4-BSA (7). Average glycan loadings of 25 glycans per BSA signicant difference was observed between HC and MS
for glycoconjugates 5 and 6, and 21 glycans per BSA for glyco- patients for Rha- or b-Glc-specic IgM levels in serum or
conjugate 7 were determined by MALDI-TOF analysis (Fig. S1†). plasma.
In previous studies by Schwarz et al., it was demonstrated
that anti-a-Glc serum IgM levels were higher in RRMS patients
compared to HC.17 In contrast, we observed no difference
Serum anti-glycan IgM levels
between the anti-a-Glc (3) IgM levels of RRMS patients and HC.
Brettschneider et al. observed elevated levels of anti-GAGA4 IgM
antibodies in RRMS patients and determined that this differ-
ence allowed MS patients to be differentiated from HC and
patients with other neurological diseases.21 These ndings by
Brettschneider et al. formed the basis of the gMS® Dx test for
With the antigen–BSA conjugates in hand, we then determined
serum anti-glycan IgM levels in RRMS patients and HC. To this
end, the sera from 10 HC and 40 RRMS patients were analysed
by ELISA for IgM antibodies specic to each of the carbohydrate
antigens Rha (2), a-Glc (3), GAGA4 (4), b-Glc (5), GAGB4 (6) and
GAGAGB4 (7) (Fig. 4A). To account for non-specic binding, IgM
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Scheme 4 Synthesis of b-Glc based antigens 5–7.
the early detection of MS. We, however, did not observe a decrease in anti-GAGB4 IgM levels in RRMS patients
increased levels of anti-GAGA4 (4) IgM antibodies in RRMS compared to HC, with the difference becoming signicant (p ¼
patients. Instead, a lower level of these antibodies was observed 0.035) when plasma samples were tested. Serum IgM antibody
when compared to HC, with this difference trending towards levels to GAGAGB4 (7) were also measured in order to compare
signicance (p ¼ 0.056) when serum samples were used. When these results to those obtained for GAGA4 (4) since both contain
combined with results from the plasma samples, however, the the same terminal structural motif. We observed anti-GAGAGB4
difference in anti-GAGA4 IgM levels became signicantly lower IgM levels that were even lower than those measured for GAGA4,
(p ¼ 0.040).
To investigate whether the reducing-end glycosidic linkage difference trending towards signicance (p ¼ 0.083).
inuenced IgM levels, GAGB4 (6) was included in our study. Overall, for each of the antigens, the HC group demonstrated
with the IgM titres being lower in RRMS patients and the
Similar to the results obtained for GAGA4 (4), we observed either comparable or higher IgM levels compared to the RRMS
Fig. 4 Serum anti-glycan IgM levels for healthy controls (HC, n ¼ 10) and relapsing-remitting multiple sclerosis (RRMS, n ¼ 40) patients (A);
serum and plasma anti-glycan IgM levels for HC (n ¼ 16) and MS patients (n ¼ 48) (B). Data are expressed as median values and interquartile
ranges, with bars above and below indicating the maximum and minimum DOD values, respectively. *p < 0.05 by Mann–Whitney U Test.
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population. In addition, we observed a reduction in IgM levels
as the length of the carbohydrate scaffold was increased, which
highlights the impact of the carbohydrate chain length on IgM
binding. No signicant upregulation of anti-glycan IgM anti-
body responses was detected for RRMS patients in this study,
which is in contrast to earlier studies by Schwarz et al.17 and
Brettschneider et al.21 Moreover, as illustrated in our work, and
8 T. Berger, P. Rubner, F. Schautzer, R. Egg, H. Ulmer,
I. Mayringer, E. Dilitz, F. Deisenhammer and M. Reindl, N.
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that of others,22 anti-glycan IgM levels vary strongly amongst the 10 K. Munger, L. Levin, E. O'Reilly, K. Falk and A. Ascherio,
human population. Due to this extensive variability in human Mult. Scler. J., 2011, 17, 1185–1193.
antibody levels, it is challenging to use IgM antibodies as 11 S. Chiba, S. Yokota, K. Yonekura, S. Tanaka, H. Furuyama,
biomarkers for disease, and our ndings may go some way in
explaining the poor selectivity of 33.7% observed when using
the gMS® Dx test.21
H. Kubota, N. Fujii and H. Matsumoto, J. Neurol. Sci.,
2006, 241, 39–43.
12 F. J. Quintana, M. F. Farez, V. Viglietta, A. H. Iglesias,
Y. Merbl, G. Izquierdo, M. Lucas, A. S. Basso, S. J. Khoury,
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Conclusion
´
13 C. Pinter, S. Beltrami, D. Caputo, P. Ferrante and A. Clivio, J.
In summary, the results of this study show that the IgM anti-
body response to the simple GAGA4 moiety is not upregulated
in RRMS patients. In contrast, we observed a lower level of anti-
GAGA4 IgM antibodies in RRMS patients, thereby highlighting
the challenge in nding a suitable antibody biomarker for MS.
Thus, there still remains an unmet need for a non-invasive,
early diagnostic tool for MS. Whether a suitable antigen with
a more robust and reproducible antibody response can be
found to meet this need, remains to be seen.
NeuroVirol., 2000, 6, S42–S46.
14 G. Ingram, S. Hakobyan, N. P. Robertson and B. P. Morgan,
Clin. Exp. Immunol., 2009, 155, 128–139.
15 G. Ingram, J. J. Bugert, S. Loveless and N. P. Robertson, Eur.
J. Neurol., 2010, 17, 1386–1389.
16 F. Lolli, B. Mulinacci, A. Carotenuto, B. Bonetti, G. Sabatino,
B. Mazzanti, A. M. D'Ursi, E. Novellino, M. Pazzagli,
L. Lovato, M. C. Alcaro, E. Peroni, M. C. Pozo-Carrero,
F. Nuti, L. Battistini, G. Borsellino, M. Chelli, P. Rovero
and A. M. Papini, Proc. Natl. Acad. Sci. U. S. A., 2005, 102,
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17 M. Schwarz, L. Spector, M. Gortler, O. Weisshaus, L. Glass-
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Conflicts of interest
The authors declare no conict of interest.
18 M. T. C. Walvoort, C. Testa, R. Eilam, R. Aharoni, F. Nuti,
G. Rossi, F. Real-Fernandez, R. Lanzillo, V. Brescia Morra,
F. Lolli, P. Rovero, B. Imperiali and A. M. Papini, Sci. Rep.,
2016, 6, 39430.
19 M. Schwarz, L. Spector, A. Gargir, A. Shtevi, M. Gortler,
R. T. Altstock, A. A. Dukler and N. Dotan, Glycobiology,
2003, 13, 749–754.
20 M. S. Freedman, J. Laks, N. Dotan, R. T. Altstock, A. A. Dukler
and C. J. Sindic, Mult. Scler., 2009, 15, 422–430.
21 J. Brettschneider, T. D. Jaskowski, H. Tumani, S. Abdul,
D. Husebye, H. Seraj, H. R. Hill, E. Fire, L. Spector,
J. Yarden, N. Dotan and J. W. Rose, J. Neuroimmunol., 2009,
217, 95–101.
Acknowledgements
The authors would like to thank NZ Lottery Health Research
(340915/13) and the Health Research Council (Hercus Fellow-
ship, BLS, 2013/33) for nancial support. We are extremely
grateful to Liz Goode, Kathryn Hally, Dr Lisa Johnston and Carl
Beyers for collecting the blood samples used in this study. We
would like to thank Sven Sondhauss for helping with the
MALDI-TOF and Vimal Patel for helping with the ELISA
protocol. We would also like to thank Dr Lisa Woods for assis-
tance with statistical analysis of the data.
22 M. E. Huejt, M. Vuskovic, D. Vasiliu, H. Xu, P. Obukhova,
N. Shilova, A. Tuzikov, O. Galanina, B. Arun, K. Lu and
N. Bovin, Mol. Immunol., 2009, 46, 3037–3049.
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