Citrullination of linear and cyclic altered peptide ligands from myelin basic protein (MBP87-99) epitope elicits a Th1 polarized response by T cells isolated from multiple sclerosis patients: Implications in triggering disease

Article abstract of DOI:10.1021/jm800891n

Derangement of cellular immunity is central in the pathophysiology of multiple sclerosis (MS) and is often manifested by abnormal cytokine production. We investigated cytokine secretion in peripheral blood mononuclear cells (PBMC) of 18 MS patients and 15 controls and correlated cytokine polarization with the nature of antigenic stimulus. We synthesized two novel citrullinated peptides, linear [Cit⁹¹, Ala⁹⁶, Cit⁹⁷]MBP_87-99 and cyclo(87-99)[Cit⁹¹, Ala⁹⁶, Cit⁹⁷]MBP _87-99 that resulted from citrullination of 91,97 Arg residues in antagonists, linear [Arg⁹¹, Ala⁹⁶]MBP_87-99 and cyclo(87-99)[Arg⁹¹, Ala⁹⁶]MBP_87-99 peptides. PBMC from MS patients and controls were cultured with citrullinated peptides, and both peptides caused a Th1 polarization in all MS patients studied. In contrast, culture with noncitrullinated MBP peptides resulted in heterogeneous cytokine secretion that differed between individual patients. Thus, citrullination of self-antigens may potentially trigger disease in susceptible individuals. This finding may open new avenues in drug design of new substances that inhibit citrullination and arrest epitope spreading and worsening of MS.

Full text of DOI:10.1021/jm800891n

7834
J. Med. Chem. 2008, 51, 7834–7842
Citrullination of Linear and Cyclic Altered Peptide Ligands from Myelin Basic Protein
(MBP87-99) Epitope Elicits a Th1 Polarized Response by T Cells Isolated from Multiple
Sclerosis Patients: Implications in Triggering Disease
George Deraos,† Kokona Chatzantoni,‡ Minos-Timotheos Matsoukas,† Theodore Tselios,† Spyros Deraos,† Maria Katsara,†,§
Panagiotis Papathanasopoulos,| Demitrios Vynios,† Vasso Apostolopoulos,*,§, Athanasia Mouzaki,*,‡, and John Matsoukas*,†,
Department of Chemistry, UniVersity of Patras, Patras 26500, Greece, DiVision of Hematology, Department of Internal Medicine, Medical
School, UniVersity of Patras, Patras 26110, Greece, Neurology Clinic, Medical School and UniVersity Hospital, UniVersity of Patras,
Patras 26500, Greece, and Immunology and Vaccine Laboratory, Burnet Institute (Austin Campus), Heidelberg, Victoria 3084, Australia
ReceiVed July 18, 2008
Derangement of cellular immunity is central in the pathophysiology of multiple sclerosis (MS) and is often
manifested by abnormal cytokine production. We investigated cytokine secretion in peripheral blood
mononuclear cells (PBMC) of 18 MS patients and 15 controls and correlated cytokine polarization with the
nature of antigenic stimulus. We synthesized two novel citrullinated peptides, linear [Cit91, Ala96,
Cit97]MBP87-99 and cyclo(87-99)[Cit91, Ala96, Cit97]MBP87-99 that resulted from citrullination of 91,97 Arg
residues in antagonists, linear [Arg91, Ala96]MBP87-99 and cyclo(87-99)[Arg91, Ala96]MBP87-99 peptides.
PBMC from MS patients and controls were cultured with citrullinated peptides, and both peptides caused
a Th1 polarization in all MS patients studied. In contrast, culture with noncitrullinated MBP peptides resulted
in heterogeneous cytokine secretion that differed between individual patients. Thus, citrullination of self-
antigens may potentially trigger disease in susceptible individuals. This finding may open new avenues in
drug design of new substances that inhibit citrullination and arrest epitope spreading and worsening of MS.
Introduction
(mutated peptides) derived from agonist (wild type) peptides
from MBP with the aim to shift T helper cell responses from
the proinflammatory Th1 (IL-2, IFN-γ) to the antiinflammatory
Th2(IL-4,IL-5,orIL-10).7-11 PeptidesintheMHC-peptide-TCR
complex, causing antagonism, have been shown to have fewer
hydrogen bonds between peptide side chains to the comple-
mentarity-determining region 3 loops of the TCR by analysis
of cocrystals.12 A loss of hydrogen bond contact can cause wild
type, agonist, or even superagonist (hyperstimulatory APL)
peptides to become antagonists.12 Similarly, structures of APL
from vesicular stomatitis virus and human immunodeficiency
virus in complex with MHC and/or TCR demonstrated that very
minor conformational changes in the peptide side chain resulted
in profound biological effects.13,14 It has been demonstrated that
APLs derived from guinea pig MBP74-85, and human MBP83-99
and MBP87-99 epitopes have antagonistic effects by preventing
experimental autoimmune encephalomyelitis (EAE) and divert-
ing Th1 (IL-2, IFN-γ) cytokines toward Th2/Th3 (IL-10). Two
APL derived from MBP83-99 epitope have entered into human
clinical trials with varying responses and side effects.15-18 We
previously reported that a linear APL of the wild type MBP87-99
with mutations at Lys91 (principle TCR contact residue) and
Pro96 (secondary TCR contact residue) resulting in [Arg91,
Ala96]MBP87-99 (P6) was able to protect Lewis rats from
development of EAE when co-injected with the encephalitogenic
MBP74-85 peptide.19,20 In addition, head-to-tail cyclization of
Multiple sclerosis (MSa) is a chronic disorder of the central
nervous system that is predominantly characterized by local T
cell and macrophage infiltration, leading to demyelination and
loss of neurologic function.1 MS is an autoimmune disease
triggered by CD4+ T cells, although autoantibodies cannot be
excluded. More recently, T helper (Th)17 cells have been shown
to be involved in MS.2 Antigens within the myelin sheath, such
as myelin basic protein (MBP), proteolipid protein, and myelin
oligodendrocyte glycoprotein,3,4 have been described as the main
constituents of which self-reactive CD4+ T cells and autoan-
tibodies are directed against.
Altered peptide ligands (APL) are usually defined as ana-
logues derived from the native, wild type peptide, which carry
amino acid mutations at T cell receptor (TCR) contact residues.5
Engagement of these APL by TCR usually impairs or drastically
alters normal T cell function while retaining major histocom-
patibility complex (MHC) binding. Some of these APL (also
known as mutant or antagonist peptides) are able to specifically
antagonize and/or inhibit T cell activation induced by the wild
type immunogenic (agonist) peptide.6 Interest into the thera-
peutic management of MS has involved peptide analogues
* To whom correspondence should be addressed. For V.A.: phone, +613-
9287-0666; fax, +613-9287-0600; e-mail, vasso@burnet.edu.au. For A.M.:
phone/fax, +30-2610-969123; e-mail, mouzaki@med.upatras.gr. For J.M.:
phone/fax, +30-2610-997180; e-mail, imats@chemistry.upatras.gr.
†
P6-cyclo(87-99)[Arg91, Ala96]MBP87-99 (P7) peptide8 com-
Department of Chemistry, University of Patras.
Department of Internal Medicine, University of Patras.
Burnet Institute (Austin Campus).
Medical School and University Hospital, University of Patras.
21,22
‡
pletelyblockedthedevelopmentofEAEinducedbyMBP74-85
.
§
Furthermore, peripheral blood mononuclear cells (PBMC) from
controls and MS patients cultured with P6 and P7 significantly
increased the Th2/Th1 cytokine ratio.23
|
These authors contributed equally.
a Abbreviations: MS, multiple sclerosis; Th, T helper; MBP, myelin basic
protein; APL, altered peptide ligand; TCR, T cell receptor; MHC, major
histocompatibility complex; EAE, experimental autoimmune encephalo-
myelitis; PBMC, peripheral blood mononuclear cells; PAD, peptidylarginine
deiminase; Cit, citrulline; TLC, thin layer chromatography; MD, molecular
dynamics; SD, steepest descent; CG, conjugate gradient.
Numerous studies have implicated the role of citrullination
of self-peptides in the pathogenesis of autoimmune diseases24
including MS.25-27 Citrullination is the conversion of Arg to
citrulline (Cit), resulting in the positive charge of Arg side chain
10.1021/jm800891n CCC: $40.75
2008 American Chemical Society
Published on Web 11/19/2008

Citrullinated MBP87-99 Peptides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7835
Scheme 1. Conversion Reaction of Peptidyl-Arginine to Peptidyl-Citrulline by PAD II Enzyme
Table 1. Peptide Analogues Synthesized and Used in This Study
compound
description
amino acid sequence
wild type
P1
P2
P3
P4
P5
P6
P7
cP6
cP7
linear MBP87-99
linear MBP72-85
linear APL MBP72-85
negative control
cyclic MBP72-85
ValHisPhePheLys91AsnIleValThrPro96ArgThrPro
Glu-Lys-Ser-Glu-Arg-Ser-Glu-Asp-Glu-Asn-Pro-Val
Glu-Lys-Ser-Glu-Arg-Ser-Glu-Ala-Glu-Asn-Pro-Val
cyclo[(D-F)LLReKDap]
Glu-Lys-Ser-Glu-Arg-Ser-Glu-Asp-Glu-Asn-Pro-Val-NH2
Glu-Lys-Ser-Glu-Arg-Ser-Glu-Ala-Glu-Asn-Pro-Val-NH2
ValHisPhePheArg91AsnIleValThrAla96ArgThrPro
ValHisPhePheArg91AsnIleValThrAla96ArgThrPro
ValHisPhePheCit91AsnIleValThrAla96Cit97ThrPro
cyclo(87-99)ValHisPhePheCit91AsnIleValThrAla96Cit97ThrPro
cyclic APL MBP72-85
linear APL, [Arg91, Ala96]MBP87-99
cyclo-APL, cyclo(87-99)[Arg91, Ala96]MBP87-99
linear [Cit91, Ala96, Cit97]MBP87-99
cyclo(87-99)[Cit91, Ala96, Cit97]MBP87-99
Scheme 2. Conversion Reaction of Cyclo(87-99)[Arg91, Ala96]MBP87-99 (P7) by PAD II Enzyme
into neutral (Scheme 1). This transformation is referred to as
deimination, an enzymatic reaction catalyzed by peptidylarginine
deiminases (PAD). Citrullination results in a reduced positive
charge of the protein, lowering its affinity to the negatively
charged myelin phosphatidylserine residues which creates a
noncompact structure of MBP sequence that is readily degrad-
able by cathepsin D. Cathepsin D cleaves Phe-Phe peptide
bonds, most of which are located in the internal regions of the
protein, not exposed to solvent.25,26 Therefore, citrullination of
MBP results in a breakdown of the protein, leading to new
citrullinated peptide epitopes that could trigger an immune
response. In fact a charged isomer of MBP that is citrullinated
and referred to as MBP-C8 has been shown to be recognized
preferential response to MBP newly formed citrullinated pep-
tides may be present and could contribute to disease.
Herein, we describe the conversion of linear and cyclic
MBP87-99 APLs, P6 and P7, respectively, to their citrullinated
analogues cP6 ([Cit91, Ala96, Cit97]MBP87-99
) and cP7
(cyclo(87-99)[Cit91, Ala96, Cit97]MBP87-99) and their effects on
cytokine stimulation by PBMC isolated from MS patients and
control subjects. It is demonstrated that the APLs P6 and P7 in
their citrullinated forms (cP6 and cP7) result in the polarization
of Th1 cytokines in MS patients PBMC, hence indicating that
citrullination of MBP peptides may trigger disease. Furthermore,
molecular modeling was used to gain insights into the actions
noted by the citrullinated peptides.
by CD4+ T cells from MS patients and control subjects.28
T
Results
cell lines from MS patients also responded with higher frequency
and sensitivity to MBP-C8.27 Thus, given the high amount of
citrullinated MBP protein in MS patients’ brain tissue, a
Peptide Synthesis and Citrullination. The peptides used in
this study are shown in Table 1. The native (wild type) linear

7836 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Deraos et al.
Figure 1. (A) HPLC analysis of linear [Arg91, Ala96]MBP87-99 (P6) before treatment with enzyme PAD II and (B) after treatment with enzyme
PAD II (cP6). (C) Cyclo(87-99)[Arg91, Ala96]MBP87-99 (P7) after treatment with enzyme PAD II (cP7). The citrullinated analogues (cP6 and cP7)
are eluted later than noncitrullinated P6 and P7. Final purity HPLC analysis of (D) cP6 and (E) cP7. Column used was Nucleosil C18, 250 mm
× 4.6 mm, 5 µm. TRcit was 15.67 min (cP6) and 17.65 min (cP7). Elution conditions were as follows: 5% solvent b to 100% solvent b in 32 min;
flow rate of 1 mL/min. Solvent a consisted of TFA in H2O, 0.08% (v/v). Solvent b consisted of TFA in AcN, 0.08% (v/v).
peptide (MBP87-99), linear APL with mutations at position Lys91
and Pro96 to result in [Arg91, Ala96]MBP87-99 (P6) and its
corresponding cyclic peptide, cyclo(87-99)[Arg91, Ala96]-
MBP87-99 (P7), were synthesized in house (Department of
Chemistry, University of Patras), and were >95% pure as
assessed by HPLC. In addition, control peptides (P1, P2, P4,
P5) corresponding to the MBP72-85 epitope, immunodominant
guinea pig peptides, and a negative control cyclic peptide (P3)
were also synthesized with >95% purity.
Ala96, Cit97]MBP87-99 (Scheme 2) by incubation with PAD II.
Arginine modification was detected by thin layer chromatog-
raphy (TLC) and was verified by reversed phase HPLC and
mass spectroscopy. The citrullinated peptide eluted slightly later
than the initial non-enzyme treated peptide (Figure 1). The
converted citrullinated analogues cP6 and cP7 were measured
to be 20.4% and 23.47%, respectively, of the final products.
Citrullinated peptides were further purified and lyophilized, and
the final purity HPLC of cP6 and cP7 (Figure 1D and Figure
1E) showed that peptides were >95% pure, the same level of
purity as the parent peptide.
Peptides P6 and P7were converted to their citrullinated forms
cP6 [Cit91, Ala96, Cit97]MBP87-99 and cP7 cyclo(87-99)[Cit91,

Citrullinated MBP87-99 Peptides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7837
Figure 2. Net change in cytokine production over placebo values (cultures in plain culture media) of PBMC cultures from MS patients and
controls cultured in the presence of 10 pg/mL per 106 cells of control peptides (P1-P5) and wild type peptide. Cytokines are measured in pg/mL
per 106 cells: (A-K) results from individual MS patients. Color code for cytokines is as follows: IL-10 ) green; IL-4 ) blue; IL-2 ) yellow;
IFN-γ ) red. Left-hand side panels show cytokine production by control and patient PBMC cultured in plain culture media for 72 h without the
addition of peptides (placebo values).
Citrullination of APL Results in Th1 Cytokine Polariza-
tion. PBMC from MS patients and control subjects cultured in
the presence of wild type and control peptides (P1-P5) clearly
demonstrate the heterogeneity of the responses of MS patients
PBMC to the peptides (Figure 2). The results shown are grouped
together for control subjects and plotted separately for 11
individual MS patients. It was of interest to note that PBMC
cultured with citrullinated analogues, cP6 and cP7, resulted in
an increase in the secretion of Th1 cytokines IL-2 and IFN-γ
in all MS patients PBMC (n ) 18) (Figure 3). Furthermore,

7838 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Deraos et al.
human MHC class II (HLA-DR2) and the deduced model for
binding to murine MHC class II (H2-I-As).9,10,29-31 In addition,
His88 and Phe89 are in proximity to the cyclic citrullinated
analogue (cP7), much closer to that of its linear counterpart,
cP6. Furthermore, in both models cP6 and cP7, Cit97 points up
toward the TCR (Figure 4C); thus, it is quite possible that Cit97
may make novel contacts with the TCR, leading to T cell
stimulation. Likewise, Cit91 could also make novel contacts with
the TCR leading to altered responses. Furthermore, Figure 5
shows the superimposition of His88, Phe89, and Phe90 counter-
parts of linear (cP6) and cyclic (cP7) analogues, with the same
region obtained from that of the X-ray crystal structure of the
native MBP83-99 peptide (PDB code 1ymm).32
Discussion
Posttranslational modifications (citrullination, phosphorylation,
deamidation, deimination, methylation, glycosylation) are common
biological processes that alter specific parts of a protein after
synthesis. Most proteins undergo some form of posttranslational
modification, and almost all amino acids can be altered by one or
more of these processes. The modified protein thus contains new
or rare amino acids or new specific side groups that can have critical
influence on the structure and function of the protein. Furthermore,
degradation of the protein results in several new peptide fragments
that may elicit immune responses. The conversion of Arg to
citrulline33 may result in the induction of an altered immune
response compared to the native noncitrullinated counterpart. Such
responses have been implicated in the pathogenesis of autoimmune/
inflammatory diseases, such as MS and rheumatoid arthritis.25,26,34,35
In rheumatoid arthritis detection of antibodies against cyclic
citrullinated peptides (anti-CPP antibodies) is an indicator and
diagnosis of the disease.36,37 In addition, T cells have been shown
to be stimulated to citrullinated hen egg-white lysozyme peptides
bound to MHC.38 Furthermore, citrullinated peptides bind with
higher affinity to HLA-DR4 and lead to activation of CD4+ T
cells in HLA-DR4-IE transgenic mice.39 In the adult human brain
15-20% of MBP is citrullinated, whereas in the brain of MS
patients the amount of citrullinated MBP is about 3-fold higher
(45-60%).40 CD4+ T cells from MS patients recognize citrulli-
nated MBP (MBP-C8)28 and respond with higher sensitivity.27 An
increased proportion of citrullinated MBP (MBP-C8) is present in
the brains of MS patients. Isolation of MBP-C8 from guinea pig
brains was shown to be encephalitogenic in Lewis rats. MBP-C8
was also able to reinduce EAE in Lewis rats that had been
previously recovered from unmodified MBP induced EAE.41
Different MBP charged isomers (C1-C8) isolated from normal
and MS white matter tissue indicated that deimination was found
at R43, R49, R54, R65, R97, R122, R130, and R162. The citrullinated
residues varied between MS samples, and partial deimination at
several residues was common.42 Other reports also suggest that
R25, R33, R159, and R170 are also citrullinated.40,43,44 Hence, R97
studied herein has been found to be citrullinated in MS patients,
and the cP6 and cP7 studied herein reflect the natural state of
citrullinated arginine residues. Given the high amount of citrulli-
nated MBP protein in MS patients brain tissue and areas of spinal
cord,45 it is highly likely that preferential T cell responses to MBP
newly formed citrullinated peptides may be present and could
contribute to disease.
Figure 3. Net change in cytokine production over placebo values of
PBMC cultures of MS patients and control subjects cultured in the
presence of 10 pg/mL per 106 cells of peptide analogues P6, P7, cP6,
cP7, and wild type. Grouped results from n ) 15 controls and n ) 18
MS patients are shown ( standard deviation.
the increase in Th1 cytokines was accompanied by a decrease
in the Th2 cytokine, IL-10, creating a clear Th1 polarization.
In contrast, culture with the noncitrullinated linear APL peptide
P6 resulted in a moderate induction of IL-10 and a moderate
reduction of IL-2 and IFN-γ, demonstrating a Th2 dominance,
as previously demonstrated.23 Moreover, culture with the
noncitrullinated cyclic peptide P7 resulted in a reduction in IL-2
cytokine and induction of IFN-γ and IL-10, demonstrating a
mixed Th1/Th2 response (Figure 3). Finally, culture with the
wild type native peptide, MBP83-99, resulted in a reduction of
IL-10 cytokine, as expected. There was no effect in the secretion
of the Th2 cytokine IL-4 by any of the peptides tested. By
contrast, culture of PBMC from control donors (n ) 15) with
peptides P6, P7, cP6, and cP7 resulted in the stimulation of
IFN-γ and IL-10 cytokines whereas wild type peptide stimulated
only the production of IL-10. Similar to MS PBMC, peptides
P6, P7, cP6, and cP7 had no effect on IL-4 production by
control PBMC.
Structural Insights of cP6 and cP7 Peptides. In order to
gain insights into the structures of cP6 and cP7 peptides in their
free state used in this study, molecular dynamics (MD)
simulations and minimization protocols were used. The six
lowest energy frames for cP6 (Figure 4A) and cP7 (Figure 4B)
peptides and the global energy minimum structures (Figure 4C)
demonstrate some differences and similarities. The linear
analogue (cP6) is extended compared to its cyclic form (cP7).
Hence, the substitution of Arg91 and Arg97 to citrullines in its
linear conformation allows a more extended conformation
compared to the linear noncitrullinated analog (P6). The primary
TCR contact amino acids His88, Phe89, Cit91 in the linear
analogue (cP6) point up toward the TCR, and the major MHC
anchor amino acid Phe90 points down into the MHC. This is in
line with the crystal structure of MBP83-99 in complex with
We previously noted that double mutations of the linear wild
type peptide MBP87-99 at positions 91 and 96 with Arg and
Ala, respectively, P6, suppressed guinea pig MBP72-85 induced
EAE in Lewis rats. Head-to-tail cyclization of linear APL P6
led to cyclic P7, which retained its antagonistic effect in an
EAE model. In addition, P6 and P7 have been shown to bind

Citrullinated MBP87-99 Peptides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7839
Figure 4. Representative lowest energy conformations of (A) linear [Cit91, Ala96, Cit97]MBP87-99 (cP6) and (B) cyclo(87-99)[Cit91, Ala96,
Cit97]MBP87-99 (cP7) after MD simulations and minimizations with SD and CG algorithms. (C) Global energy minima for citrullinated cP6 andcP7
peptides after MD simulations.
to HLA-DR4, increase the Th2/Th1 cytokine ratio of PBMC
suspected importance of citrullination as a major mechanism
in triggering autoimmune/inflammatory diseases such as MS.
In particular, culture of PBMC from MS patients with citrul-
linated analogues cP6 and cP7 increased the secretion of IL-2
and IFN-γ and simultaneously decreased the secretion of IL-
10, characteristic of Th1 polarization. On the contrary, the
noncitrullinated linear APL peptide P6 induced the production
of IL-10 and reduced the production of IL-2 and IFN-γ
characteristic of Th2 polarization. Cyclic APL peptide P7,
however, stimulated IFN-γ, but simultaneously IL-10 was also
induced. These findings indicate that citrullination of APL
(antagonistic) peptides from MBP87-99 immunodominant pep-
tide, leading to Th1 dominance, may have implications in
exacerbations or triggering disease. It would be of interest to
study the effects of the citrullinated peptides on the numbers
and function of T regulatory cell populations48 from MS patients.
from MS patients, and were able to suppress the proliferation
23
of a CD4+ T cell clone specific to wild type MBP87-99
.
Changing cytokine secretion patterns is an important approach
in regulating disease. In MS patients, the majority of MBP87-99
specific CD4+ T cells, isolated during active disease, secrete
Th1 proinflammatory cytokines whereas during remission the
cytokine profile shifts to Th2/Th3 antiinflammatory cytokines.46
Hence, the induction of Th2 responses during active disease
should reverse disease. In addition, the role of IL-10 in the
induction and function of natural and antigen induced regulatory
T cells in autoimmune diseases has been demonstrated.47
Cytokine secretion investigations were included in the evaluation
study herein of linear and cyclic APL (P6 and P7) and were
compared to their citrullinated converted analogues (cP6 and
cP7). The results of the present study strongly support the long

7840 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Deraos et al.
with each patient’s PBMC responding differently when cultured
with each peptide. Molecular modeling suggested that both Cit91
and Cit97 residues point up toward the TCR, thus implicating
the possible interaction with the CDR3 loops of the TCR and
inducing an altered cytokine response. This study provides direct
experimental evidence that citrullination is essential for antigenic
recongition of self-epitopes by MS T cells and may open new
avenues in drug design of citrullination inhibitors to suppress
disease.
Experimental Procedures
1. Synthesis of Linear and Cyclic Analogues. Control peptides
P1-P5 (Table 1) were synthesized and used in this study. In
addition, the native (wild type) linear peptide (MBP87-99) and
its mutant analogues, linear [Arg91, Ala96]MBP87-99 (P6) and
cyclo(87-99)[Arg91, Ala96]MBP87-99 (P7), were synthesized as
previously described.19,21,22 Briefly, the syntheses of linear
peptides were carried out in solid phase using the Fmoc/tBu
methodology, utilizing the 2-chlorotrityl chloride resin. For the
total synthesis Na-Fmoc protected L-amino acids were used. The
protected final products were further used for the synthesis of
the cyclic analogues. To a mixture of the linear protected peptide
in dry DMF and 2,4,6-collidine was added 1-hydroxy-7-
azabenzotriazol. The solution was then added dropwise to a
solution of O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium
tetrafluoroborate in dry dimethylformamide for 2 h and stirred
for 4 h. The reaction was followed by the ninhydrin test on TLC.
The solvent was removed from the reaction mixture under
reduced pressure, affording a light-yellow oily residue. The
cyclic protected peptides were precipitated from H2O and were
dried in vacuo for 12 h. The protected cyclic peptides were
treated with the deprotection mixture DCM/TFA/ethanedithiol/
anisole/H2O (32/65/2/1) for 4 h at room temperature. The
resulting solution was concentrated under vacuum to a small
volume. The final free cyclic peptides were precipitated as a
light-yellow amorphous solid by the addition of diethyl ether,
filtered, and dried in vacuo for 12 h. The purity of peptides were
>80%. The final crude products were further purified using RP-
HPLC, and the resultant peptide purities were >95% (Figure
1). All peptides used in this study are in Table 1.
2. Citrullination of Linear and Cyclic Analogues Using
Peptidylarginine Deiminase. Linear [Arg91, Ala96]MBP87-99 (P6)
and cyclo(87-99)[Arg91, Ala96]MBP87-99 (P2) peptides were treated
with PAD II (Sigma Chemical Co.) to substitute all arginine residues
with citrullines.33,53 Usually, an amount of 4.9 mg of peptide (about
267 nmol for P6 and 318 nmol for P7) was dissolved in 0.1 mL of
10 mM HEPES buffer, pH 7.6, containing 5 mM CaCl2 and 2 mM
dithiothreitol. This was followed by the addition of 2 units of PAD
II, and the mixture was incubated at 52 °C for 48 h. The enzyme
treatment was stopped by heating at 100 °C for 5 min, and the
solutions were clarified by centrifugation at 14000g for 5 min. Arg
modifications were assessed by TLC, using 5% p-dimethylami-
nobenzaldehyde in HCl as developer, where citrulline residues
produced a yellow color. Arg modifications were also verified by
reversed phase HPLC. Modified peptides were further purified/
lyophilized, and the resultant peptide purities were >95% (Figure
1). P6 in its citrullinated form is designated cP6 (linear [Cit91, Ala96,
Cit97]MBP87-99), and P7 in its citrullinated form is designated as
cP7 (cyclo(87-99)[Cit91, Ala96, Cit97]MBP87-99). All peptides used
are shown in Table 1.
Figure 5. Superimposition of His88, Phe89, and Phe90 obtained from
the X-ray structure of MBP83-99 peptide with the lowest energy
conformations of linear [Cit91, Ala96, Cit97]MBP87-99 (cP6) and
cyclo(87-99)[Cit91, Ala96, Cit97]MBP87-99 (cP7) analogues.
Furthermore, these studies may open new avenues in drug design
of citrullination inhibitors.
Moreover, previous conformations of the cyclic APL P749
revealed that the proximity of residues at positions 91 and 97
were not favorable. Charge repulsion between Arg91 and Arg97
due to the positive charges at these position, seemed to account
for the opposite orientations of Arg91 and Arg97 side chains. A
ꢀ II turn present in the region 92-96 is a conformational feature
in P7.50 This conformational characteristic is responsible for a
more compact conformation of the APL, which may be
responsible for its altered effect. This conformational pharma-
cophore model defines the MHC (Phe90) anchor and TCR (His88,
Phe89) contact sites and the overall volume of the target
molecules resulting in weak interactions with the TCR and,
hence, possibly its antagonistic outcomes.49,51,52 In order to gain
insights of the structures of their citrullinated analogues cP6
and cP7 in possibly inducing disease, MD simulations of cP6
and cP7 were performed. The primary TCR contact amino acids
His88, Phe89, Cit91 in the linear analogue (cP6) point up toward
the TCR with Phe90 pointing down into the MHC. Of interest,
in both models cP6 and cP7, Cit91 and Cit97 point up toward
the TCR, thus implicating that both these noncanonical amino
acids may play a role in the activation of T cells leading to
altered or enhanced responses. Furthermore, it is clear that there
is overlap of His88, Phe89, and Phe90 residues between the models
and with that of the crystal structure in complex with MHC, in
particular cyclo(87-99)[Cit91, Ala96, Cit97]MBP87-99 (cP7) with
the native MBP83-99 peptide.
Conclusion
We synthesized citrullinated analogues of a linear and cyclic
APL from MBP87-99. The results presented here demonstrate
that culture of PBMC from MS patients with citrullinated
MBP87-99 APL results in a strong and uniform Th1 polarization.
In contrast, culture with noncitrullinated MBP87-99 or other MBP
peptides resulted in heterogeneous cytokine secretion patterns,
Table 2. Demographic Details of MS Patients and Control Subjectsa
subjects
MS patients, n ) 18 12F, 6M 35.1 (17-66)
controls, n ) 15 8F, 7M 28.5 (20-55)
sex
age
type of disease phase of disease
treatment
no rx (n ) 12), IFN-ꢀ (n ) 6) 2.58 (1-6.5)
N/A N/A
EDSS
disease duration
RR
acute
N/A
5.01 (0-19)
N/A
N/A
a Age (years), duration of disease (years), and EDSS scores are given as median with (range); n, sample size; F, female; M, male; RR, remitting-relapsing
MS; no rx, no treatment; IFN-ꢀ, under continuous prophylactic therapy with IFN-ꢀ (IFN-ꢀ1ꢀ, Betaferon, Schering A.G., 0.25 mg subcutaneously every
second day or IFN-ꢀ1R, Avonex, Biogen Inc., 3 µg intramuscularly once a week or Rebif, Serono A.G., 44 µg subcutaneously every second day). N/A, not
applicable.

Citrullinated MBP87-99 Peptides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7841
3. Biological Evaluation. 3.1. Study Subjects. Eighteen adult
patients with definite MS of the remitting relapsing type (RR-MS)
were studied. The patients were treated and followed up in the
Neurology Clinic of Patras University Hospital (PUH). All patients
were in the acute phase of the disease at the time of blood sample
collection and did not suffer from any additional ailment. The blood
samples were drawn before administration of treatment. A control
group consisted of 15 adult healthy volunteers. An amount of 10
mL of heparinized venous blood was drawn from MS patients and
control subjects once. Demographic details of all study subjects
are shown in Table 2. Informed consent was obtained from each
participating patient. PUH abides by the Helsinki declaration on
ethical principles for medical research involving human subjects.
3.2. Cell Cultures and Measurement of Cytokines. PBMC
were isolated from MS patients and control subjects and cultured
for 72 h in culture medium as described previously in the presence
or absence of peptides (wild type, P6, P7, cP6, or cP7) at 10 pg/
mL of culture per 106 cells.21 This stimulation peptide dose was
chosen after titration of peptides from 1 pg/mL to 1 µg/mL and
found to be the most effective (not shown). At the end of the culture
period culture supernatants were collected by centrifugation at
12 000 rpm at 4 °C for 1 min and stored at -30 °C. ELISA assays
for human IL-2, IFN-γ, IL-4, and IL-10 were performed according
to the manufacturers instructions (Diaclone, Besanc¸on, France).
4. Molecular Modeling. All computer calculations were per-
formed on Pentium IV 2.14 GHz and Silicon Graphics O2
workstations using Discovery Studio, version 1.7, Quanta 2005
molecular modeling systems by Accelrys Software Inc. (San Diego,
CA). The representative conformers of linear and cyclic analogues
were generated using backbone unrestrained molecular dynamics
simulation. Thus, populations of the various conformers that
represent local minima at the potential energy surface were
identified.
4.1. Generating the Initial Conformations. The starting
conformations of linear [Cit91, Ala96, Cit97]MBP87-99 (cP6) and
cyclo(87-99)[Cit91, Ala96, Cit97]MBP87-99 (cP7) peptide analogues
were extendedly built, consisting of L-amino acids, on the CHARMm
force field.54 The dielectric constant was set to 45, as DMSO, which
is a polar, aprotic solvent that simulates better the receptor
environment during all the experiments. The generalized Born
solvent model with a simple switching55 was used, as well as the
SHAKE algorithm, which serves bond geometry constraints during
the MD simulations.56 First, the structures of linear [Cit91, Ala96,
Cit97]MBP87-99 (cP6) and cyclo(87-99)[Cit91, Ala96, Cit97]MBP87-99
(cP7) peptides in their free state were energy-minimized using
steepest descent (SD) and conjugate gradient (CG) algorithms, with
10 000 steps and rmsd of 0.001 Å as energy convergence criterion.
4.2. Molecular Dynamics (MD) Simulations. MD runs were
performed using the CHARMm force field as follows: Heating,
from 0 to 300 K gradually, and equilibration were set with a time
step of 0.002 ps while the time step of production was 0.002 ps
for a total time of 1 ns. Parameters on saving the resulting
frequencies were set in such a way in order to extract 500
conformations for each molecule. Six local energy minimum
conformations were extracted from each molecule according to the
potential energy surface. After the dynamics simulations, minimiza-
tion protocols were performed on the chosen structures as follows:
SD and CG algorithms, with 10 000 steps using an rmsd of 0.001
Å as the energy convergence criterion.
References
(1) Katsara, M.; Matsoukas, J.; Deraos, G.; Apostolopoulos, V. Towards
immunotherapeutic drugs and vaccines against multiple sclerosis. Acta
Biochim Biophys Sin. 2008, 40, 636–642.
(2) Holmoy, T.; Hestvik, A. L. Multiple sclerosis: immunopathogenesis
and controversies in defining the cause. Curr. Opin. Infect. Dis. 2008,
21, 271–278.
(3) Polman, C. H.; Uitdehaag, B. M. New and emerging treatment options
for multiple sclerosis. Lancet Neurol. 2003, 2, 563–566.
(4) Steinman, L. Multiple sclerosis: a coordinated immunological attack
against myelin in the central nervous system. Cell 1996, 85, 299–
302.
(5) Evavold, B. D.; Sloan-Lancaster, J.; Allen, P. M. Tickling the TCR:
selective T-cell functions stimulated by altered peptide ligands.
Immunol. Today 1993, 14, 602–609.
(6) Evavold, B. D.; Allen, P. M. Separation of IL-4 production from Th
cell proliferation by an altered T cell receptor ligand. Science 1991,
252, 1308–1310.
(7) Hedegaard, C. J.; Krakauer, M.; Bendtzen, K.; Lund, H.; Sellebjerg,
F.; Nielsen, C. H. T helper cell type 1 (Th1), Th2 and Th17 responses
to myelin basic protein and disease activity in multiple sclerosis.
Immunology 2008, 125, 161–169.
(8) Katsara, M.; Deraos, G.; Tselios, T.; Matsoukas, J.; Apostolopoulos,
V. Design of novel cyclic altered peptide ligands of myelin basic
protein MBP83-99 that modulate immune responses in SJL/J mice.
J. Med. Chem. 2008, 51, 3971–3978.
(9) Katsara, M.; Yuriev, E.; Ramsland, P. A.; Deraos, G.; Tselios, T.;
Matsoukas, J.; Apostolopoulos, V. A double mutation of MBP(83-
99) peptide induces IL-4 responses and antagonizes IFN-gamma
responses. J. Neuroimmunol. 2008, 200, 77–89.
(10) Katsara, M.; Yuriev, E.; Ramsland, P. A.; Deraos, G.; Tselios, T.;
Matsoukas, J.; Apostolopoulos, V. Mannosylation of mutated MBP83-
99 peptides diverts immune responses from Th1 to Th2. Mol. Immunol.
2008, 45, 3661–3670.
(11) Mouzaki, A.; Tselios, T.; Papathanassopoulos, P.; Matsoukas, I.;
Chatzantoni, K. Immunotherapy for multiple sclerosis: basic insights
for new clinical strategies. Curr. NeuroVasc. Res. 2004, 1, 325–340.
(12) Degano, M.; Garcia, K. C.; Apostolopoulos, V.; Rudolph, M. G.;
Teyton, L.; Wilson, I. A. A functional hot spot for antigen recognition
in a superagonist TCR/MHC complex. Immunity 2000, 12, 251–261.
(13) Kalergis, A. M.; Nathenson, S. G. Altered peptide ligand-mediated
TCR antagonism can be modulated by a change in a single amino
acid residue within the CDR3 beta of an MHC class I-restricted TCR.
J. Immunol. 2000, 165, 280–285.
(14) Thomson, C. T.; Kalergis, A. M.; Sacchettini, J. C.; Nathenson, S. G.
A structural difference limited to one residue of the antigenic peptide
can profoundly alter the biological outcome of the TCR-peptide/MHC
class I interaction. J. Immunol. 2001, 166, 3994–3997.
(15) Bielekova, B.; Goodwin, B.; Richert, N.; Cortese, I.; Kondo, T.; Afshar,
G.; Gran, B.; Eaton, J.; Antel, J.; Frank, J. A.; McFarland, H. F.;
Martin, R. Encephalitogenic potential of the myelin basic protein
peptide (amino acids 83-99) in multiple sclerosis: results of a phase
II clinical trial with an altered peptide ligand. Nat. Med. 2000, 6, 1167–
1175.
(16) Crowe, P. D.; Qin, Y.; Conlon, P. J.; Antel, J. P. NBI-5788, an altered
MBP83-99 peptide, induces a T-helper 2-like immune response in
multiple sclerosis patients. Ann. Neurol. 2000, 48, 758–765.
(17) Kappos, L.; Comi, G.; Panitch, H.; Oger, J.; Antel, J.; Conlon, P.;
Steinman, L. Induction of a non-encephalitogenic type 2 T helper-
cell autoimmune response in multiple sclerosis after administration
of an altered peptide ligand in a placebo-controlled, randomized phase
II trial. The Altered Peptide Ligand in Relapsing MS Study Group.
Nat. Med. 2000, 6, 1176–1182.
(18) Kim, H. J.; Antel, J. P.; Duquette, P.; Alleva, D. G.; Conlon, P. J.;
Bar-Or, A. Persistence of immune responses to altered and native
myelin antigens in patients with multiple sclerosis treated with altered
peptide ligand. Clin. Immunol. 2002, 104, 105–114.
(19) Tselios, T.; Daliani, I.; Deraos, S.; Thymianou, S.; Matsoukas, E.;
Troganis, A.; Gerothanassis, I.; Mouzaki, A.; Mavromoustakos, T.;
Probert, L.; Matsoukas, J. Treatment of experimental allergic en-
cephalomyelitis (EAE) by a rationally designed cyclic analogue of
myelin basic protein (MBP) epitope 72-85. Bioorg. Med. Chem. Lett.
2000, 10, 2713–2717.
(20) Tselios, T.; Probert, L.; Daliani, I.; Matsoukas, E.; Troganis, A.;
Gerothanassis, I. P.; Mavromoustakos, T.; Moore, G. J.; Matsoukas,
J. M. Design and synthesis of a potent cyclic analogue of the myelin
basic protein epitope MBP72-85: importance of the Ala81 carboxyl
group and of a cyclic conformation for induction of experimental
allergic encephalomyelitis. J. Med. Chem. 1999, 42, 1170–1177.
(21) Tselios, T.; Apostolopoulos, V.; Daliani, I.; Deraos, S.; Grdadolnik,
S.; Mavromoustakos, T.; Melachrinou, M.; Thymianou, S.; Probert,
Acknowledgment. This work was supported by grants from
the Ministry of Development Secreteriat of Research and
Technology of Greece (HRAKLEITOS, EPAN, PENED) and
the University of Patras (Karatheodoris). M.K. was also sup-
ported by a Du Pre grant from MSIF, and V.A. was supported
by an NH&MRC career development award (Grant No.
223316).

7842 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Deraos et al.
L.; Mouzaki, A.; Matsoukas, J. Antagonistic effects of human cyclic
MBP(87-99) altered peptide ligands in experimental allergic en-
cephalomyelitis and human T-cell proliferation. J. Med. Chem. 2002,
45, 275–283.
(39) Hill, J. A.; Southwood, S.; Sette, A.; Jevnikar, A. M.; Bell, D. A.;
Cairns, E. Cutting edge: the conversion of arginine to citrulline allows
for a high-affinity peptide interaction with the rheumatoid arthritis-
associated HLA-DRB1*0401 MHC class II molecule. J. Immunol.
2003, 171, 538–541.
(40) Moscarello, M. A.; Mastronardi, F. G.; Wood, D. D. The role of
citrullinated proteins suggests a novel mechanism in the pathogenesis
of multiple sclerosis. Neurochem. Res. 2007, 32, 251–256.
(41) Cao, L.; Sun, D.; Whitaker, J. N. Citrullinated myelin basic protein
induces experimental autoimmune encephalomyelitis in Lewis rats
through a diverse T cell repertoire. J. Neuroimmunol. 1998, 88, 21–
29.
(42) Kim, J. K.; Mastronardi, F. G.; Wood, D. D.; Lubman, D. M.; Zand,
R.; Moscarello, M. A. Multiple sclerosis: an important role for post-
translational modifications of myelin basic protein in pathogenesis.
Mol. Cell. Proteomics 2003, 2, 453–462.
(43) Harauz, G.; Musse, A. A. A tale of two citrullinessstructural and
functional aspects of myelin basic protein deimination in health and
disease. Neurochem. Res. 2007, 32, 137–158.
(44) Wood, D. D.; Moscarello, M. A. The isolation, characterization, and
lipid-aggregating properties of a citrulline containing myelin basic
protein. J. Biol. Chem. 1989, 264, 5121–5127.
(45) Raijmakers, R.; Vogelzangs, J.; Croxford, J. L.; Wesseling, P.; van
Venrooij, W. J.; Pruijn, G. J. Citrullination of central nervous system
proteins during the development of experimental autoimmune en-
cephalomyelitis. J. Comp. Neurol. 2005, 486, 243–253.
(46) Correale, J.; Gilmore, W.; McMillan, M.; Li, S.; McCarthy, K.; Le,
T.; Weiner, L. P. Patterns of cytokine secretion by autoreactive
proteolipid protein-specific T cell clones during the course of multiple
sclerosis. J. Immunol. 1995, 154, 2959–2968.
(47) Wraith, D. C. Role of interleukin-10 in the induction and function of
natural and antigen-induced regulatory T cells. J. Autoimmun. 2003,
20, 273–275.
(22) Tselios, T.; Daliani, I.; Probert, L.; Deraos, S.; Matsoukas, E.; Roy,
S.; Pires, J.; Moore, G.; Matsoukas, J. Treatment of experimental
allergic encephalomyelitis (EAE) induced by guinea pig myelin basic
protein epitope 72-85 with a human MBP(87-99) analogue and
effects of cyclic peptides. Bioorg. Med. Chem. 2000, 8, 1903–1909.
(23) Matsoukas, J.; Apostolopoulos, V.; Kalbacher, H.; Papini, A. M.;
Tselios, T.; Chatzantoni, K.; Biagioli, T.; Lolli, F.; Deraos, S.;
Papathanassopoulos, P.; Troganis, A.; Mantzourani, E.; Mavromousta-
kos, T.; Mouzaki, A. Design and synthesis of a novel potent myelin
basic protein epitope 87-99 cyclic analogue: enhanced stability and
biological properties of mimics render them a potentially new class
of immunomodulators. J. Med. Chem. 2005, 48, 1470–1480.
(24) Matsuo, K.; Xiang, Y.; Nakamura, H.; Masuko, K.; Yudoh, K.; Noyori,
K.; Nishioka, K.; Saito, T.; Kato, T. Identification of novel citrullinated
autoantigens of synovium in rheumatoid arthritis using a proteomic
approach. Arthritis Res. Ther. 2006, 8, R175.
(25) Pritzker, L. B.; Joshi, S.; Gowan, J. J.; Harauz, G.; Moscarello, M. A.
Deimination of myelin basic protein. 1. Effect of deimination of arginyl
residues of myelin basic protein on its structure and susceptibility to
digestion by cathepsin D. Biochemistry 2000, 39, 5374–5381.
(26) Pritzker, L. B.; Joshi, S.; Harauz, G.; Moscarello, M. A. Deimination
of myelin basic protein. 2. Effect of methylation of MBP on its
deimination by peptidylarginine deiminase. Biochemistry 2000, 39,
5382–5388.
(27) Tranquill, L. R.; Cao, L.; Ling, N. C.; Kalbacher, H.; Martin, R. M.;
Whitaker, J. N. Enhanced T cell responsiveness to citrulline-containing
myelin basic protein in multiple sclerosis patients. Mult. Scler. 2000,
6, 220–225.
(28) Martin, R.; Whitaker, J. N.; Rhame, L.; Goodin, R. R.; McFarland,
H. F. Citrulline-containing myelin basic protein is recognized by T-cell
lines derived from multiple sclerosis patients and healthy individuals.
Neurology 1994, 44, 123–129.
(48) Scalzo, K.; Plebanski, M.; Apostolopoulos, V. Regulatory T-cells:
immunomodulators in health and disease. Curr. Top. Med. Chem. 2006,
6, 1759–1768.
(49) Spyranti, Z.; Dalkas, G. A.; Spyroulias, G. A.; Mantzourani, E. D.;
Mavromoustakos, T.; Friligou, I.; Matsoukas, J. M.; Tselios, T. V.
Putative bioactive conformations of amide linked cyclic myelin basic
protein peptide analogues associated with experimental autoimmune
encephalomyelitis. J. Med. Chem. 2007, 50, 6039–6047.
(50) Mantzourani, E. D.; Tselios, T. V.; Grdadolnik, S. G.; Brancale, A.;
Platts, J. A.; Matsoukas, J. M.; Mavromoustakos, T. M. A putative
bioactive conformation for the altered peptide ligand of myelin basic
protein and inhibitor of experimental autoimmune encephalomyelitis
[Arg91, Ala96] MBP87-99. J. Mol. Graphics Modell. 2006, 25, 17–
29.
(51) Mantzourani, E. D.; Platts, J. A.; Brancale, A.; Mavromoustakos, T. M.;
Tselios, T. V. Molecular dynamics at the receptor level of immun-
odominant myelin basic protein epitope 87-99 implicated in multiple
sclerosis and its antagonists altered peptide ligands: triggering of
immune response. J. Mol. Graphics Modell. 2007, 26, 471–481.
(52) Mantzourani, E. D.; Tselios, T. V.; Grdadolnik, S. G.; Platts, J. A.;
Brancale, A.; Deraos, G. N.; Matsoukas, J. M.; Mavromoustakos, T. M.
Comparison of proposed putative active conformations of myelin basic
protein epitope 87-99 linear altered peptide ligands by spectroscopic
and modelling studies: the role of positions 91 and 96 in T-cell receptor
activation. J. Med. Chem. 2006, 49, 6683–6691.
(53) Rothnagel, J. A.; Rogers, G. E. Citrulline in proteins from the
enzymatic deimination of arginine residues. Methods Enzymol. 1984,
107, 624–631.
(54) Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.;
Swaminathan, S.; Karplus, M. CHARMM: a program for macromo-
lecular energy, minimization, and dynamics calculations. J. Comput.
Chem. 1983, 4, 187–217.
(55) Chen, J.; Im, W.; Brooks, C. L. Balancing solvation and intramolecular
interactions: toward a consistent generalized Born force field. J. Am.
Chem. Soc. 2006, 128, 3728–3736.
(56) Ryckaert, J.-P.; Ciccotti, G.; Berendsen, H. J. C. Numerical integration
of the Cartesian equations of motion of a system with constraints:
molecular dynamics of n-alkanes. J. Comput. Phys. 1977, 23, 327–341.
(29) Gauthier, L.; Smith, K. J.; Pyrdol, J.; Kalandadze, A.; Strominger,
J. L.; Wiley, D. C.; Wucherpfennig, K. W. Expression and crystal-
lization of the complex of HLA-DR2 (DRA, DRB1*1501) and an
immunodominant peptide of human myelin basic protein. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 11828–11833.
(30) Smith, K. J.; Pyrdol, J.; Gauthier, L.; Wiley, D. C.; Wucherpfennig,
K. W. Crystal structure of HLA-DR2 (DRA*0101, DRB1*1501)
complexed with a peptide from human myelin basic protein. J. Exp.
Med. 1998, 188, 1511–1520.
(31) Wucherpfennig, K. W.; Hafler, D. A.; Strominger, J. L. Structure of
human T-cell receptors specific for an immunodominant myelin basic
protein peptide: positioning of T-cell receptors on HLA-DR2/peptide
complexes. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 8896–8900.
(32) Hahn, M.; Nicholson, M. J.; Pyrdol, J.; Wucherpfennig, K. W.
Unconventional topology of self peptide-major histocompatibility
complex binding by a human autoimmune T cell receptor. Nat.
Immunol. 2005, 6, 490–496.
(33) Fearon, W. R. The carbamido diacetyl reaction: a test for citrulline.
Biochem. J. 1939, 33, 902–907.
(34) Boggs, J. M.; Rangaraj, G.; Koshy, K. M.; Ackerley, C.; Wood, D. D.;
Moscarello, M. A. Highly deiminated isoform of myelin basic protein
from multiple sclerosis brain causes fragmentation of lipid vesicles.
J. Neurosci. Res. 1999, 57, 529–535.
(35) Gyorgy, B.; Toth, E.; Tarcsa, E.; Falus, A.; Buzas, E. I. Citrullination:
a posttranslational modification in health and disease. Int. J. Biochem.
Cell Biol. 2006, 38, 1662–1677.
(36) Girelli, F.; Foschi, F. G.; Bedeschi, E.; Calderoni, V.; Stefanini, G. F.;
Martinelli, M. G. Is anti cyclic citrullinated peptide a useful laboratory
test for the diagnosis of rheumatoid arthritis? Eur. Ann. Allergy Clin.
Immunol. 2004, 36, 127–130.
(37) Lee, A. N.; Beck, C. E.; Hall, M. Rheumatoid factor and anti-CCP
autoantibodies in rheumatoid arthritis: a review. Clin. Lab. Sci. 2008,
21, 15–18.
(38) Ireland, J.; Herzog, J.; Unanue, E. R. Cutting edge: unique T cells
that recognize citrullinated peptides are a feature of protein immuniza-
tion. J. Immunol. 2006, 177, 1421–1425.
JM800891N