IgG Antibodies to Cyclic Citrullinated Peptides Exhibit Profiles Specific in Terms of IgG Subclasses, Fc-Glycans and a Fab-Peptide Sequence
IgG Antibodies to Cyclic Citrullinated Peptides Exhibit Profiles Specific in Terms of IgG Subclasses, Fc-Glycans and a Fab- Peptide Sequence
Susanna L. Lundstr om * 0 1 2 3
Ca tia Fernandes-Cerqueira 1 2 3
A. Jimmy Ytterberg 0 1 2 3
Elena Ossipova 1 2 3
Aase H. Hensvold 1 2 3
Per-Johan Jakobsson 1 2 3
Vivianne Malmstr om 1 2 3
Anca I. Catrina 1 2 3
Lars Klareskog 1 2 3
Karin Lundberg 1 2 3
Roman A. Zubarev 0 2 3
0 Division of Physiological Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet , Stockholm , Sweden,
1 Rheumatology Unit, Department of Medicine, Karolinska Institutet , Stockholm , Sweden
2 Funding: This study was supported by funds from the Swedish Strategic Research Funds (SSF), from the Swedish Research Council (VR), from the European Research Council (ERC), the EU projects Gums&Joints (contract No. 261460) and Trigger (contract No. 306029) as well as Innovative Medicine Initiative BTCure (115142-2) and EU project FP7-HEALTH-2012-INNOVATION-1 Euro- TEAM (305549-2). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript
3 Editor: Joseph J Barchi, National Cancer Institute at Frederick , United States of America
The Fc-glycan profile of IgG1 anti-citrullinated peptide antibodies (ACPA) in rheumatoid arthritis (RA) patients has recently been reported to be different from non-ACPA IgG1, a phenomenon which likely plays a role in RA pathogenesis. Herein we investigate the Fc-glycosylation pattern of all ACPA-IgG isotypes and simultaneously investigate in detail the IgG protein-chain sequence repertoire. IgG from serum or plasma (S/P, n514) and synovial fluid (SF, n54) from 18 ACPA-positive RA-patients was enriched using Protein G columns followed by ACPA-purification on cyclic citrullinated peptide-2 (CCP2)-coupled columns. Paired
ACPA (anti-CCP2 eluted IgG) and IgG flow through (FT) fractions were analyzed by
LC-MS/MS-proteomics. IgG peptides, isotypes and corresponding
Fc-glycopeptides were quantified and interrogated using uni- and multivariate
statistics. The Fc-glycans from the IgG4 peptide EEQFNSTYR was validated using
protein A column purification. Relative to FT-IgG4, the ACPA-IgG4 Fc-glycan-profile
contained lower amounts (p50.002) of the agalacto and asialylated
corefucosylated biantennary form (FA2) and higher content (p50.001) of sialylated
glycans. Novel differences in the Fc-glycan-profile of ACPA-IgG1 compared to
FT-IgG1 were observed in the distribution of bisected forms (n55, p50.0001,
decrease) and mono-antennnary forms (n53, p50.02, increase). Our study also
confirmed higher abundance of FA2 (p50.002) and lower abundance of
afucosylated forms (n54, p50.001) in ACPA-IgG1 relative to FT-IgG1 as well as
lower content of IgG2 (p50.0000001) and elevated content of IgG4 (p50.004) in
ACPA compared to FT. One l-variable peptide sequence was significantly
increased in ACPA (p50.0001). In conclusion, the Fc-glycan profile of both
ACPAIgG1 and ACPA-IgG4 are distinct. Given that IgG1 and IgG4 have different
Fcreceptor and complement binding affinities, this phenomenon likely affects ACPA
effector- and immune-regulatory functions in an IgG isotype-specific manner. These
findings further highlight the importance of antibody characterization in relation to
functional in vivo and in vitro studies.
Rheumatoid arthritis (RA) is a common chronic autoimmune disease
characterized by joint inflammation and subsequent cartilage and bone
destruction . The presence of anti-citrullinated peptide antibodies (ACPA)
in patients with more severe disease progression and in asymptomatic individuals
years prior to disease onset, suggests that these autoantibodies play an important
role in RA pathogenesis [1, 5, 6]. It has recently been demonstrated that the
IgGFc-region in ACPA has distinct features, both in terms of the distribution of IgG
subclasses and IgG1-Fc-glycosylation pattern . The ACPA-IgG1-Fc region
contains more truncated forms compared to the total IgG1 pool , which
becomes more pronounced following onset of the disease . In addition, IgG1
and IgG4 have been reported to be the predominant subclasses of antibodies that
react with cyclic citrullinated peptides (CCP), citrullinated vimentin and
citrullinated fibrinogen [8, 9]. These features can influence the affinity of IgGs to
Fc-receptors and complement, and thereby modulate their activity of effector
functions and regulatory pathways.
It is possible to use LC-MS/MS proteomics methodologies to investigate
features in the IgG14 repertoire, both on the peptide sequence level and for the
Fc-glycosylation pattern. The protein sequence region containing the N-linked
Fcglycan can be characterized according to IgG subclasses after trypsin digestion that
produces well-known peptides; IgG1: EEQYNSTYR [P01857], IgG2: EEQFNSTFR
[P01859], IgG3: EEQYNSTFR [P01860] and IgG4: EEQFNSTYR [P01861],
(accession numbers correspond to UniProt IDs) [15, 16]. Known polymorphisms
in IgG3 also result in EEQFNSTFR [17, 18]. This variant is frequently found in
individuals of European descent [17, 18].
As shown in Figure 1, the IgG-Fc attached oligosaccharide comprises a core
biantennary heptasaccharide moiety ([A2]; nomenclature is according to Royle
et al. ). If the glycan is mono-antennary, it is referred to as A1 . The first
sugar unit (an N-acetyl-glucosamine) is normally core fucosylated (e.g. FA2, FA1)
. An absence of this fucose may affect antibody effector functions. For
example, the afucosylated IgG1 variant has 50- to 100- fold higher affinity to
FccRIIIa and the increased affinity is associated with an enhanced
antibodydependent cellular cytotoxicity (ADCC) [14, 21]. Similarly, if the biantennary
structure is bisected with an extra N-acetyl-glucosamine (FA2B), it can also
increase the affinity to FccRIII and display more potent ADCC . The outer
glucosamine units can be elongated with galactoses (FA2Gn, n51 or 2) and the
galactoses can be further extended with sialic acids (FA2GnSn, n51 or 2). The
presence of terminal sialic acid on the glycan reduces Fc-receptor affinity 10-fold
and, a-2,6-sialylation on the Fc-glycan can actively suppress inflammation via
binding to the Specific Intercellular adhesion molecule-3-Grabbing Non-integrin
(SIGN) receptors [14, 23].
Some aspects of Fc-glycosylation of IgG and ACPA from RA patients, and in
particular of ACPAs from the IgG1 subclass, have been described previously
[7, 10, 2427]. However, there is no information available that describes the
glycosylation patterns of ACPAs which takes into account both detailed
glycosylation patterns of all IgG subclasses as well as the quantitative distribution
of these subclasses in serum/plasma (S/P) and synovial fluid (SF) of RA patients.
In the present study, we have used one single method, proteomic-type LC-MS/MS
, to simultaneously investigate differences both in the protein/peptide
abundances and in IgG14-Fc glycan structures in ACPA-IgG and in
non-ACPAIgG derived from serum/plasma and synovial fluid samples from ACPA positive
Materials and Methods
Subjects and samples
Serum (n58), plasma (n57) and synovial fluid ([SF], n54) were obtained from
18 anti-CCP2 positive patients diagnosed with RA according to the ACR/EULAR
2010 criteria (Serum/Plasma [S/P]: 2277 years, 6 females; SF: 4269 years, 2
females) (Supplementary Table S1). SF from three of the patients was sampled at
two occasions, with one year between sampling dates. Nine of the patients were
sampled following short symptom duration (0.54 years) and nine of the patients
were sampled following long symptom duration (1647 years), (Table S1) .
Patients were attending the Rheumatology Clinic at Karolinska University
Hospital, and were receiving health care and treatment according to clinical
guidelines and practice. All participants gave their oral consent as specified in the
ethical approval (Case number 2006-476-31/4, Stockholm Regional Ethics
Committee), and in line with Swedish law. Patients consent was documented in
the medical records by the respective treating physician. This was done after the
patient had received information about the study and after approving
participation in the study.
Protein G and CCP2 column purification
ACPA (anti-CCP2 reactive IgGs) were obtained as recently described . Briefly,
SF (1525 mL) samples were treated with hyaluronidase and centrifuged at 3000 g
for 5 min. Supernatant proteins were precipitated with saturated ammonium
sulphate and dialyzed against PBS. Plasma and serum (1020 mL) samples were
centrifuged at 3000 g for 5 min and diluted 1:5 (v/v) in PBS. IgGs from SF and
diluted S/P samples were purified on HiTrap Protein G HP columns (GE
Healthcare, Sweden), according to the manufacturers instructions. Eluted IgGs
were dialyzed against PBS and applied to CCP2 affinity columns (kindly provided
by Euro-Diagnostica, Sweden). ACPAs were eluted using 0.1 M glycineHCl
buffer (pH 2.7) and pH was immediately adjusted to 7.4 with 1 M Tris (pH 9).
The unbound IgG fraction, i.e. the flow through, (FT), was also collected. Both
ACPA and FT fractions were concentrated and buffer exchanged to PBS using
10 kDa Microsep UF Centrifugal Device (Pall Life Science, USA). Recovery degree
and purity of total ACPAs were assessed by measuring anti-CCP2 reactivity
(Immunoscan CCPlus assay, Euro-Diagnostica, Sweden) and by SDS-PAGE/
Coomassie Brilliant Blue staining.
Protein A column purification
Protein A HP Spin Trap affinity columns (GE Healthcare, Sweden) were used in
order to separate IgG3 from IgG1, IgG2 and IgG4 in both ACPA and FT samples
previously purified from plasma, sera and SF. Due to limited sample material,
pooled samples were used as well as individual samples from subjects 9, 11, 15 and
16 (Table S1). ACPA and FT samples were loaded on pre-equilibrated (20 mM
sodium phosphate buffer, pH 7.4) protein A columns and incubated for 4 min
with gentle mixing. IgG3 was collected by 30 s centrifugation at 75 g and the
columns were washed two times with equilibration buffer (via centrifugation, 30 s
at 75 g). Subsequently, IgG1, IgG2 and IgG4 were jointly collected by eluting
protein A columns with 0.1 M Glycin-HCL, pH 2.7. Eluates were collected in
tubes containing pH neutralizing buffer (1 M Tris-HCL, pH 9). Samples were
directly buffer-exchanged to PBS using 10 kDa Nanosep UF Centrifugal Device
(Pall Life Science, Port Washington, NY).
Sample preparations and liquid chromatography - mass
Prior to LC-MS/MS analysis, IgG samples (5 mg/sample) were digested by trypsin
as previously described (29, 30). Briefly, samples were reduced with 20 mM
dithiothreitol for 30 min at 56C and alkylated with 66 mM iodoacetamide for
30 min in darkness. Trypsin was added (1:50; enzyme:protein) and digestion was
performed at 37C overnight. Peptides were desalted using C18 StageTip (Thermo
Fisher Scientific, Waltham, MA), dried using SpeedVac and resuspended in 0.1%
formic acid and 0.5% acetonitrile solution. Samples were kept at 10C and
injected on the column in 5 mL aliquots containing 0.3 mg of digest.
Glycosidase treatment was performed over night at 37C on trypsin digest using
2.5 mU of 1) a(23,6,8,9)-Sialidase A and 2) a(23,6,8,9)-Sialidase A and b(1
3,4)-Galactosidase (ProZyme, Hayward, CA) dissolved in 10 mL of the
corresponding X10 buffers provided by the manufacturer.
Samples were analyzed in triplicates in a randomized order using reversed phase
liquid chromatography (LC) system (Easy-nLC, Proxeon, Thermo Fisher
Scientific) connected to a hybrid LTQ Orbitrap Velos ETD mass spectrometer
(Thermo Fisher Scientific, Waltham, MA) operating in positive ion mode. The
survey mass spectrum covering the range of m/z 3002000 was obtained with a
resolution of 60,000 at m/z 400. Following each MS scan, top five most abundant
precursor ions were selected for MS/MS with collision induced dissociation (CID)
and electron transfer dissociation (ETD). The instrument was calibrated externally
using internal lock mass calibration on m/z 429.088735 and 445.120025.
LCseparation of the peptides and glycopeptides were performed on a 10 cm long
fused silica tip column (SilicaTips New Objective Inc.) packed in house with 3 mm
C18-AQ ReproSil-Pur (Dr. Maisch GmbH, Germany). The chromatographic
separation was achieved using a water (A) and acetonitrile (B) solvent system both
containing 0.1% formic acid. The gradient was set up as following: 3235% (B) in
35 min, 36295% (B) in 5 min, 95% (B) for 8 min and 3% (B) for 10 min. The
flow rate was set at 300 nl/min.
Protein identification and quantification
The MS/MS spectra were extracted from.raw files into.mgf files using in-house written
RAW_to_MGF software . Mascot (Matrix Science) search engine v.2.3.02 was
used for protein identification with a concatenated version of the SwissProt protein
sequence database (April, 2013, 20242 entries). Peptide mass error tolerance was set at
10 ppm, MS/MS fragment mass accuracy at 0.5 Da and tryptic digestion was set with a
maximum of two missed cleavages. Carbamidomethylation of cysteine was used as a
fixed modification, while the variable modifications were asparagine and glutamine
deamidation, methionine oxidation as well as N-glycosylation
(HexNAc[m]dHex[n]Hex[o]; m, n and o are the number of N-acetyl-hexoseamines,
deoxyhexoses and hexoses, respectively). Peptide and protein quantification was
performed with in-house written software Quanti .
Glycopeptide identification and quantification
IgG-glycopeptide amino acid sequences and glycoforms were characterized as
previously described . Briefly, IgG Fc-glycopeptides were identified in LC-MS/
MS datasets by their characteristic retention times and accurate monoisotopic
masses (within ,10 ppm from the theoretical values) of doubly and triply
charged ions (IgG1: EEQYNSTYR, IgG2 or IgG3 [IgG2/3]: EEQFNSTFR and IgG4
(or IgG3) [IgG4/(3)]: EEQFNSTYR (or EEQYNSTFR)) as well as of triply and
quadruply charged ions (IgG1: TKPREEQYNSTYR, IgG2/3: TKPREEQFNSTFR
and IgG4/(3): TKPREEQFNSTYR (or TKPREEQYNSTFR)), Table S2. For
additional validation of glycopeptide identities, retention times of glycopeptides
from the IgG standard (Sigma Aldrich, St Louis, MO, Table S3) as well as MS/MS
and deglycosylated peptides obtained by glycosidase treatments of ACPA and FT
were used . The close sequence homology of EEQFNSTYR (IgG4) and
EEQYNSTFR (IgG3) around Asn297 results in identical monoisotopic masses and
overlapping retention times of the corresponding glycopeptides (Figure S1).
Therefore, these glycopeptides cannot per se be differentiated by LC-MS/MS.
However, IgG3: EEQFNSTFR is more frequently found in individuals of European
descent [17, 18]. Via protein A column separation of IgG3 and IgG4 with
subsequent LC-MS/MS analysis (Figure S1), this information was confirmed.
Thus, EEQFNSTFR glycopeptides are referred to as IgG2/3 and EEQFNSTYR (or
EEQYNSTFR) glycopeptides are referred to as IgG4/(3).
Quantification of glycoforms was performed in a label-free manner using Quanti
[28, 31]. Glycopeptide ion abundances were integrated over respective
chromatographic monoisotopic ion peaks (,10 ppm from the theoretical values) at the charged
states described above and within a 1.5 min interval around the expected retention
times. Results were validated by manual qualitative and quantitative investigation of
the.raw files. Glycoform abundances were normalized to total content (100%) of
Fcglycosylated IgG1 peptides, total content (100%) of Fc-glycosylated IgG2/3 peptides and
total content (100%) of Fc-glycosylated IgG4/(3) peptides, respectively.
Univariate statistical analysis was performed using paired two-tailed Students
ttest on matched FT versus ACPA data using mean values of the triplicate
measurements for each individual (Table S3). Both serum and plasma enriched FT
and ACPA fractions obtained from the same individual (subject 9, Table S1) were
used to validate that the IgG profile was not affected by the matrix origin. The
validation confirmed that the correlation between the measured glycan and
protein levels in the serum and plasma generally was very good (details are given
in Table S1). The mean of the combined plasma and serum measurements for
subject 9 was used when comparing ACPA and FT statistically. As described
above, the study included SF samples from three individuals that were tested at
two occasions (one year between sampling dates). As shown in Figure S2 the
samples show intra-individual similarities but are distinct in terms of FT and
ACPA specificity. To avoid skewed statistical results, the mean values for the
samples were used. Principal component analysis (PCA) and orthogonal
projections to latent structures discriminate analysis (OPLS-DA) were
performed using SIMCA 13.0 (Umetrics, Umea, Sweden) following log
transformation, mean centering and UV scaling. Model performance was reported
as cumulative correlation coefficients for the model (R2X[cum]) and predictive
performance based on seven-fold cross validation calculations (Q2[cum]).
When comparing the relative abundances (%) of the heavy chains from IgG14,
(IGHG1, IGHG2, IGHG3 and IGHG4), the most prominent IgG type was IgG1 in
all samples, (FT: 76%10%, ACPA: 78%11%). The abundance of IgG4 was
significantly higher (p50.004) in ACPA (6%8%) compared to FT (2%1%).
In contrast, the abundance of IgG2 was significantly lower (p50.0000001) in the
ACPA fractions (7%3%) compared to the FT fractions (17%8%). An
overview of the IgG type distributions and intra-individual distribution change in
FT and ACPA samples of SF or S/P is shown in Figure 2.
We also observed differences in the abundance of heavy and light chain variants
between the ACPA and FT fractions. Significantly elevated levels of a peptide
previously found in the variable region of l-chains LV603 and LV601 were found
in the ACPA fractions (p50.0001, Figure 3). The sequence was identified as
DFMLTQPHSVSESPGK via MS/MS with a score of 72 in SF (subject 15) and a
score of 66 in S/P (subject 1), Figure S3. Additionally, significantly lower levels
(p,0.04) of two l-chains (LV301 and LV302), three k-chains (KV119, KV106
and KV204), and one heavy chain (HV304), were observed in the ACPA fractions,
Table S4. For identified peptide sequences, see Table S5.
The IgG heavy and light chains contributed to approximately 99.9% of all
detected proteins in the FT fractions and 99.0% of all proteins in the ACPA
fractions. Additionally, traces of IgM, IgA, complement 1 q (C1q) and CD5
antigen like protein (CD5L), could be detected (Table S5). All four of these
proteins were observed with significantly higher abundances in the ACPA
fractions compared to the FT fractions (Table S4).
Figure 2. Composition (%) of IgG heavy chain isotypes. (A) FT isotype composition. (B) ACPA isotype composition. (C) Log10 fold change of the
intraindividual ACPA/FT-ratio. Values >0 (dashed line) indicate an increase in ACPA. Given p-values (comparing FT and ACPA) were obtained with paired t-test.
NS: Not Significant.
Significant intra-individual variations in Fc-glycans of ACPA-IgG1 compared to FT-IgG1
A total of 19 glycans substituting Asn 297 of IgG1 were searched after in the
LCMS/MS data using Quanti (Table S2). With the exception of FA2BG2S2, all
glycoforms were detected in quantifiable amounts (Table 1). Four out of five of the
identified bisected glycans (FA2B, FA2BG1, FA2BG2 and FA2BG2S1) were observed
at significantly lower levels (p,0.01) in the ACPA (Table 1). Similarly, the sum of
all bisected glycans (n55) were significantly decreased (p50.0001) in the
ACPAIgG1 as shown in Table 1, Figure 4 and Figure S4. Notable are also the differences
observed when comparing FT-IgG1 and ACPA-IgG1 distributions of the
monoantennary forms (i.e. FA1, FA1G1 and FA1G1S1). All three of these glycans were
significantly elevated (p,0.04) in ACPA-IgG1, Table 1, Figure 4 and Figure S4. As
previously reported, we could confirm a significant decrease in afucosylated
ACPAIgG1 glycans (n54, p50.001), and a significant increase (p50.002) in the main FA2
glycan (Table 1, Figure 4 and S4, Figure 5 and S5, respectively). With the exception
of FA2G2S2 (p50.01, S/P only, Table 1), ACPA-IgG1 were not found to be
significantly less sialylated compared to the FT-IgG1.
Significant intra-individual variations in Fc-glycans of ACPA-IgG4/(3)
compared to FT-IgG4/(3)
Nine glycopeptides N-linked to either Asn297 of IgG4 or (IgG3 with peptide
sequence EEQYNSTFR) were detected in quantifiable amounts, Table 1. In
contrast to ACPA IgG1 which showed significantly elevated FA2 levels compared
to FT, FA2 abundances in ACPA-IgG4/(3) were significantly lower (p50.002,
Table 1, Figure 5, Figure S5). However, note that this difference possibly
generates more similar FA2 levels in ACPA-IgG1 and ACPA-IgG4/(3). Thus, in S/P,
the FA2 distributions in ACPA for IgG1 and IgG4/(3) were 35%9% vs 38%9%
respectively, compared to the FT levels at 30%8% vs 44%8%, respectively. A
similar trend is observed for the SF FA2 distribution in FT and ACPA (Table 1).
In addition to the change in distribution of the FA2 glycan, FA2G2S1 as well as the
combined abundance of glycoforms FA2G1S1 and FA2G2S1, were significantly
elevated in the ACPA-IgG4/(3) fractions (p50.001 and p50.001), Table 1,
Figure 5 and Figure S5.
In order to confirm that IgG3, with peptide sequence EEQYNSTFR, is a minor
component of the IgG4/(3) glycopeptide pool, ACPA and FT samples were further
Table 1. IgG Fc glycans in serum or plasma (S/P) and synovial fluid (SF) samples of ACPA versus FT.
IgG Type Glycana SFc
Relative distributions (%), and their respective standard deviations are indicated. In addition to the individual glycan species the sum of the mono-antennary
(SA1), sum of sialylated (SS), sum of bisected (SB) and sum of afucosylated (SaF) forms are shown. P-values comparing FT and ACPA were obtained using
paired t-test; significant p-values (p,5.0E-2) are bolded.
Abbreviations: aGlycan acronyms are provided in Figure 1A, bSerum or Plasma, cSynovial Fluid, dFlow Through, eAnti-Citrullinated Peptide Antibody,
fAverage; gStandard Deviation.
purified using a Protein A column, digested and reanalyzed by LC-MS/MS.
Compared to IgG2 (20%12%) and IgG4 (3%2%), IgG3 is the main isotype
that is measured in the protein A column FT, 72%12%. This, combined with
the distribution of glycopeptides EEQYNSTFR (5%9%) and EEQFNSTFR
(95%9%) in the protein A column FT, indicates that the majority of IgG3
contains the EEQFNSTFR sequence (Figure S1). The results, i.e. a decrease of FA2
and an increase in the sialylated forms in ACPA vs FT were consistent following
protein A column analysis of IgG4 in the protein A column elutes.
Significant intra-individual variations in Fc-glycans of ACPA-IgG2/3
compared to FT-IgG2/3
With the exception of FA2B, which was significantly decreased (p50.01) in the
ACPA-IgG2/3, no other significant differences were observed.
Differences between the S/P and SF glycan-profiles
We could confirm that similarly to what has previously been reported , the
general trend in the SF IgG1 glycans indicated lower levels of sialylation and
galactosylation and a higher abundance of FA2 (Table 1, Figure S6). Notably, the
same trend was also observed in both the IgG2/3 and in the IgG4/(3) glycan profile.
No attempt was done to test for statistically significant differences in the SF
profiles compared to the S/P profiles due to the low number of SF samples in the
study (n54). All individual data is provided in Table S3.
Differences in the ACPA and FT glycan-profiles according to
With the exception of FA2G1S1 from ACPA-IgG1 (S/P) that was significantly
more abundant (p50.03) in patients with short disease duration, no significant
differences were observed in either ACPA or FT when comparing patients with
long and short disease duration.
Multivariate statistical modeling to identify overall trends in the
Multivariate statistical modeling integrating Fc-glycan (n539), IgG heavy and
light chain (n525) as well as IgM, IgA, C1Q and CD5L data (Table S3) was
performed in order to find overall trends in the data set. The PCA model
constructed from the three first components (R250.46, Q250.16), showed a
distinct separation between ACPA and FT samples in component t ,
(Figure 6A). Components t  and t  were mainly affected by inter-individual
differences such as age and matrix type (SF or S/P). No distinct effects caused by
disease- or symptom duration were observed.
An OPLS-DA model (R250.93, Q250.85, CV ANOVA p-value52.3E-12) was
constructed in order to differentiate FT and ACPA samples according to the
abundances of glycans and proteins. The high Q2-value (accounting for
predictability) as well as the low model p-value, indicate that the model is robust.
The separation of the predictive variance (ACPA or FT specific) from the
orthogonal variance (unspecific noise caused by other inter-individual
differences), greatly improves the interpretability of the OPLS-DA model compared to
a PCA model. In the OPLS-DA scores plot (Figure 6B), the FT (2) and ACPA (+)
samples are distinctly separated on the predictive X-axis (t ) while the
nonACPA/FT specific sample differences are indicated via the separation along the
Yaxis (to ). The glycopeptides and proteins that distinguish the FT and ACPA
samples with 95% confidence are shown in the loading column plot, Figure 6C.
Proteins/Fc-glycans with positive pq  values correlate positively with ACPA,
while those with negative pq  values correlate negatively with ACPA (i.e. they
are positively FT-correlating). The OPLS-DA loading plot confirms and gives an
overview of the core results obtained via the univariate statistical analyses (Table 1
and Table S4). Namely, that the main significantly elevated glycans in IgG1 (FA2,
and the mono-antennary forms), as well as in IgG4/(3) (sialylated glycoforms),
have a strong correlation with the ACPA samples. Similarly, afucosylated forms
and bisected forms from IgG1 and FA2 of IgG4/(3) strongly anti-correlate with the
ACPA samples. Notably, in addition to the IgG4 isotype and the variable chain
sequence LV603/LV601, IgG1 and IgG3 also positively correlate with the ACPA
samples with 95% confidence.
The present study uses a single analytical method (LC-MS/MS) to simultaneously
investigate features on the level of peptide abundances and Fc-glyosylation of
IgG14. Our data show distinct features of ACPA-IgG eluted from CCP2 affinity
columns as compared to non-CCP2 reactive FT-IgGs from the same patients. The
differences concern IgG subclasses, peptide-chain sequence repertoire as well as
We have previously reported that the relative ACPA (anti-CCP IgG)
distribution in the overall IgG pool of ACPA positive patients are approximately
1.5% in plasma and 2.2% in SF  and correspond to a median ACPA
concentration of 1 mM in plasma and 0.4 mM in SF, respectively. Since IgG has a
high occupancy of Fc-glycans in vivo [28, 32], the concentration of
Fcglycosylated IgG would be in a range proportional to these numbers. However, in
terms of the distribution of the different glycoforms, it is important to take into
account that (and as shown in Figure 1A), each IgG molecule can be substituted
by two glycans and that differences in the combination of substituted glycotypes
(FA2+FA2G2, FA2+FA2, e.g.) introduces an additional and potentially important
level of complexity.
In line with previous findings [8, 9], we could demonstrate that IgG4 is
significantly elevated (p50.004) in ACPA fractions as compared to FT fractions
(Figure 2), and we provide further novel intra-individual data showing that
ACPA-IgG4 Fc-glycans are significantly more sialylated (p50.001) and contain
less amounts of FA2 (p50.002) compared to FT-IgG4 (Table 1, Figure 5). The
differences in biological properties of IgG4 compared to IgG1 and IgG3 suggest
that the effects of IgG4 ACPA may be different from the effects of ACPAs of the
other subclasses. For example, IgG4 antibodies are poor inducers of complement
and Fc-receptors [11, 12, 33], but have anti-inflammatory properties, including
Fab-arm exchange , i.e. the ability to swap one heavy and light chain pair
with another molecule, resulting in bi-specific antibodies. IgG4 antibodies also
have the ability to target the Fc-region of other IgGs via its Fc-, rather than its
Fab-region, thereby contributing to the clearance of IgG and IgG-bound material
. It could be hypothesized that the composition of Fc-glycans from
ACPAIgG4 may influence ACPA-IgG4-Fc/ACPA-IgGx-Fc interactions, and that the
distinct shift towards sialylated glycan species in the ACPA-IgG4 glycopeptide
profile indicates a preference towards sialylated IgG4 glycans in such interactions.
However, it is important to point out that similarly to ACPA, IgG rheumatoid
factor (RF) has been reported to contain elevated levels of IgG4 , and that the
RF-mimicking activity of IgG4 is a confounding factor when measuring
IgG4RF . Likewise, the IgG4 in the ACPA fractions may not all necessarily be
ACPAs, as antibodies with other specificities could have been co-purified due to
their Fc-Fc-binding capacity .
The other main observation in the IgG4 glycan profile was the significantly
lower abundance of FA2 in the ACPA eluate, compared to FT. Thus, an inverse
trend compared to the IgG1 glycans is observed. However, this trend results in a
more similar FA2 distribution in ACPA-IgG4 and ACPA-IgG1 (Table 1). At
present we cannot be certain that this finding represents a specific ACPA glycan
feature with specific FA2 functionality (independent of the IgG subclass), but it is
an interesting possibility. Even though the distributions of FA2 are indeed similar
among the ACPA isotypes, the overall profiles/substitution patterns
(galactosylation, sialylation and bisected species), show major variations and are not shifting
to a general homologous ACPA profile.
In addition to the significantly elevated FA2 levels in ACPA IgG1, two
significant differences in glycan profiles were observed compared to FT IgG1: I) an
under-representation of all bisected glycoforms, and II) a higher degree of all
mono-antennary glycoforms (Figure 4, Table 1). Additionally, an
under-representation of all afucosylated glycoforms was confirmed . Noteworthy, in
in vitro studies, both bisected- and core afucosylated Fc-glycans have been shown
to increase the affinity to FccII and III receptors, potentially resulting in more
potent ADCC [14, 21, 22]. In accordance with our data, elevated levels of
fucosylated N-glycans have previously been found in sera from RA patients, both
for all serum proteins as well as specifically for IgG [24, 27]. Furthermore, a recent
study demonstrated that ACPA-IgG1 Fc core-fucosylation was elevated compared
to total IgG1 prior to RA disease onset, and then further elevated when the disease
was initiated .
In contrast to the bisected and afucosylated glycans, little is known about the
mono-antennary (A1) Fc glycans, and their effect on IgG functionality. Most
likely this is due to the low amounts of these forms in IgG . Furthermore,
when analyzed by mass spectrometry, these glycans can be generated inside the
instrument via in-source fragmentation. This could possibly explain the
significantly increased amounts of FA1 in the ACPA fractions (since FA2 is also
significantly increased), but does not explain the significantly higher abundance of
FA1G1 (p50.04) and FA1G1S1 (p50.03) in ACPA-IgG1 compared to FT-IgG1.
Further research is needed in order to investigate the potency and biological effect
of these potentially important monoantennary glycans.
Due to the limited number (n54) of SF samples, no statistical comparisons
were made between the SF- and S/P-samples in terms of Fc-glycan or IgG-isotype
distribution. Individual values are provided in Table S3. Generally the shift from
higher to lower (or lower to higher) distribution comparing FT and ACPA is the
same independent of whether the ACPA was enriched from S/P or SF. However,
from the acquired data we can conclude that high distributions of FA2 and
monoantennary forms, as well as low distributions of afucosylated and bisected forms,
are most prominent in IgG1-SF and/or ACPA-IgG1-SF (Figure S6). Furthermore,
it is likely that SF-IgG4 has a higher distribution of FA2 and a lower distribution of
FA2G2S1 compared to S/P-IgG4 (Figure S6). Notable is also that the elevated
levels of IgG4-ACPA is prominent in ACPA-S/P and that the IgG2 isotype
distribution generally is low in both FT-SF and ACPA-SF (Figure S6). The
relatively higher proportion of ACPA in the inflamed joints combined with the
observation that as much as 30% of the total memory B-cell pool in RA SF is
ACPA-specific, suggests a local autoantibody production in these compartments
and an ongoing auto-immune response [30, 40, 41]. Hence, it may not be
surprising that the glycoforms that are characteristic for ACPA-IgG1 (high: FA2
and monoantennary forms, as well as low: afucosylated and bisected forms) are
particularly enhanced in the SF samples. On the contrary, the increased IgG4 levels
in the ACPA eluted fractions which were particularly pronounced in S/P might
indicate that IgG4 is having a different, peripheral and/or secondary function.
In addition to the variation in ACPA isotype and Fc-glycosylation pattern, a
majority of the studied patients had significantly elevated levels (p50.0001) of one
variable region l-chain peptide in the ACPA (Figure 3). The old paradigm in
immunological sciences states that antigen specificity is determined by a
completely random process which will result in unique antigen binding peptide
sequence regions in antibodies (of similar target) in different individuals. A
number of studies, several of which were MS-based, have in recent years
challenged this dogma . Even though MS methods are not as sensitive as
genomic studies, MS-based methodologies may still give a truer picture of the
expression levels of the abundant IgG-peptide repertoire. It is possible that the
increased abundance of this l-peptide is yet another example indicating sequence
homology between antibodies of particular specificity. It is important to point out
that the identified peptide (DFMLTQPHSVSESPGK, Figure S3) is not part of any
of the three complementary determining regions (CDRs). The sequence homology
is from the first variable framework region of LV603 and LV601; the full sequences
can be found at http://www.uniprot.org/ (UniProt IDs: P06317 and P01721).
Noteworthy is that the l-chains and DFMLTQPHSVSESPGK previously were
found and identified in immunoglobulin l light chain type amyloid fibril proteins
The sensitivity of our method further allowed us to detect and quantify in the
ACPA and FT fractions traces of the following proteins (ACPA:FT): C1q
(,0.1%:0.03%), IgM (,0.9%:0.1%), IgA (,0.1%:0.03%) and CD5L
(,0.1%:0.02%), with numbers in parentheses referring to the relative abundance
(total IgG5100%). It is noteworthy that all four of these proteins were detected
following both IgG and ACPA purifications, with significantly higher abundances
in the ACPA fractions compared to FT (p,0.002, Table S4). The presence of these
proteins can be explained by the likely formation of immune complexes. C1q as
the initiator of the classical complement pathway , IgM and IgA as RF ,
and CD5L as associated with IgM , or potentially as a citrullinated ACPA
target . It should be pointed out that serum albumin was also detectable in the
samples, but never exceeded 0.1% in abundance.
Compared to other studies investigating Fc-glycosylation patterns in ACPA,
our methodology has the advantage of measuring IgG-isotype and peptide
sequence distribution as well as IgGx-Fc glycan profiles simultaneously. As
demonstrated herein, we succeeded in one single analysis in both confirming the
results of multiple previous studies as well as obtaining novel data on the
ACPAIgG4-Fc glycosylation profile and new information on ACPA-IgG1 glycans. From
the generated data, it is evident that IgG Fc-glycosylation patterns are highly
complex but with particular distinct features. Hence, each glycan profile
represents a specific pattern that likely affects different functions depending on the
IgG-type and/or IgG-specificity. Increased knowledge of ACPA glycosylation
profiles in different IgG isotypes will thus likely be important for future in vitro
and in vivo functional studies. This knowledge should also be put in relation to
the B-cell status (activation, differentiation, cytokine and T-cell effects,
localization, etc.). Combined, such knowledge can improve our understanding of
disease mechanisms and pathways in ACPA-positive RA. Prospectively, the IgG
Fc-glycosylation profile could potentially be used for clinical diagnostics and
Figure S1. Extracted ion chromatograms. (A) Extracted ion chromatograms of
FA2, FA2G1 and FA2G2 glycopeptides, from ACPA and FT IgG1, IgG2/3 and IgG4/
(3) from subject 1. The solid lines indicate integrated ions from glycopeptides with
one misscleavage. (B) Extracted ion chromatograms of merged FA2, FA2G1 and
FA2G2 glycopeptide ions of IgG2 (EEQFNSTFR), IgG3 (EEQFNSTFR or
EEQYNSTFR) and IgG4, (EEQFNSTYR) from the ACPA and the FT extracted S/P
pool following protein A column separation of IgG3 (found in the protein A FT
fraction). It was concluded that the majority of IgG3 has the EEQFNSTFR
sequence since EEQFNSTFR and not EEQYNSTFR was the main glycopeptide
found in the protein A FT.
Figure S2. Multivariate analysis scores plots of the SF samples based on both
the glycan and protein data. Subjects are labeled according to Table S1. (A) PCA
model constructed from two components (R250.34, Q250.03). Samples cluster
according to individual. (B) OPLS-DA model constructed from two components
(R250.97, Q250.70). Samples separate distinctly along the x-axis according to FT
and ACPA specificity.
Figure S3. MS/MS peptide spectra. Spectra were obtained from precursors m/z
880.4204 (SF-ACPA) and m/z 880.4191 (S/P-ACPA) corresponding to [M+2]2+ of
DFMLTQPHSVSEPGK. Assigned b- and y-ions are indicated in the figure.
Figure S4. IgG1-Fc-glycan distribution and intra-individual differences in
bisected (n55), afucosylated (n54) and mono-antennary (n53) forms. Shown
p-values were obtained using paired Students T-test. S/P samples (gray), SF
Figure S5. Fc-glycan distribution and intra-individual differences of FA2 in
IgG1 and IgG4/(3), as well as FA2G2S1 in IgG4/(3). Shown p-values were obtained
using paired Students T-test. S/P samples (gray), SF samples (red).
Figure S6. Mean and standard error of mean (SEM) of the different sample
types. Data include control IgG standard (Sigma Aldrich) run in duplicates in 0.3
to 2 pmol/5 uL injections (n510) as well as FT-S/P, ACPA-S/P, FT-SF and
ACPA-SF samples, respectively. (A) FA2 distribution in IgG1. (B) Sum of
monoantennary form in IgG1. (C) Sum of afucosylated forms in IgG1. (D) Sum of
bisected forms in IgG1. (E) FA2 distribution in IgG4. (F) FA2G2S1 distribution in
IgG4. (G) IgG4 isotype distribution. (H) IgG2 isotype distribution.
Table S1. Clinical data of participating subjects. Disease duration times and
symptoms duration times were based on criterias given by Raza et al 
according to Initial fulfillment of RA criteria based on rheumatologists
assessment and First musculoskeletal symptoms relevant, (in the opinion of the
assessing rheumatologist), to the current complaint, respectively. Subject 1517
were sampled at two occasions, approximately 1 year between sampling dates. In
order to investigate potential differences between the blood matrixes, both plasma
and serum extracted ACPA and FT were obtained from subject 9. The correlation
between the measured glycan and protein levels in the serum and plasma FT was
very good (R250.99). Two outliers in the ACPA samples (HV301 and HV308)
affected the overall correlation, (R250.66 compared to R250.94, if the outliers
Table S2. Glycopeptide ions searched for. In total 19 glycan structures
substituting 6 different peptides (determined by monoisotopic mass and retention
times) were searched for via two charge states.
Table S3. Individual ACPA and FT Fc-glycan distributions (%) and protein
abundances. Protein abundances are given as log10[ion intensity/average ion
intensity]. Values from IgG control standard (Sigma Aldrich) are also included. D:
detected, -: not detected.
Table S4. List of quantified IgG chains/peptides and other proteins found in
the proteomics analysis of the FT and ACPA samples. Protein levels,
(normalized to the average abundance  and log-transformed), and their
respective standard deviations are shown. P-values comparing FT and ACPA were
obtained with paired t-test (p,5.0E-2 is significant, bolded). Peptide sequences
are given in Table S5. LV603, CD5L, KV106, LAC3, HV320, HV308 were not
found in subject 17 and 18. LV102 was not found in any of the SF samples (subject
Table S5. Identified peptide sequences.
We thank our patients and colleagues at the Rheumatology clinic at Karolinska
University Hospital Solna in Stockholm, Sweden, for contributing to the care of
the patients and to the capture of patient derived biological materials. Yngve
Sommarin, Euro Diagnostica AB, is kindly acknowledged for the generous
donation of CCP2 affinity columns. Fredrik Wermeling is kindly acknowledged
for his input and comments.
Conceived and designed the experiments: KL LK SLL. Performed the experiments:
SLL CFC EO. Analyzed the data: SLL CFC AJY AHH. Contributed reagents/
materials/analysis tools: RAZ LK VM AIC P-JJ. Wrote the paper: SLL KL LK RAZ
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