IgG1
TNP-BSA
TNP-BSA/C
FA
CD154 +
TNP-BSA/C
FA
α
TNP-LPS/CFA
CD154 +
TNP-LPS/CFA
α
TNP-BSA
TNP-BSA/C
FA
CD154 +
TNP-BSA/C
FA
α
TNP-LPS/CFA
CD154 +
TNP-LPS/CFA
α
0.0
0.5
1.0
1.5
2.0
2.5
d11 d25
anti-
TNP
1/10
0 (O
D45
0)
IgG2c
TNP-BSA
TNP-BSA/C
FA
CD154 +
TNP-BSA/C
FA
α
TNP-LPS/CFA
CD154 +
TNP-LPS/CFA
α
TNP-BSA
TNP-BSA/C
FA
CD154 +
TNP-BSA/C
FA
α
TNP-LPS/CFA
CD154 +
TNP-LPS/CFA
α
0.0
0.5
1.0
1.5
2.0
d11 d25
anti-
TNP
1/10
0 (O
D45
0)
(d)0
serum
TNP-BSATNP-BSA/CFATNP-BSA/CFATNP-LPS/CFATNP-LPS/CFA
-1 11 15
boost:
TNP-BSA/IFA
serum
25
TNP-LPS/IFA
PBSPBS
αCD154PBS
αCD154
d: -1, 2, 5, 8, 11, 14, 17, 20, 23
C D
IgG2b
TNP-BSA
TNP-BSA/C
FA
CD154 +
TNP-BSA/C
FA
α
TNP-LPS/CFA
CD154 +
TNP-LPS/CFA
α
TNP-BSA
TNP-BSA/C
FA
CD154 +
TNP-BSA/C
FA
α
TNP-LPS/CFA
CD154 +
TNP-LPS/CFA
α
0.0
0.5
1.0
1.5
2.0
2.5
d11 d25
anti-
TNP
1/10
0 (O
D45
0)
0
serum
TNP-LPSor
TNP-BSA/CFA
14 (d)
WT or Tcrb-/-mice
A B IgG1
TNP-LPS
TNP-BSA/C
FA
TNP-LPS
TNP-BSA/C
FA
0.0
0.1
0.2
0.3
0.4
WT Tcrb-/-an
ti-TN
P 1/
100
(OD
450)
IgG2c
TNP-LPS
TNP-BSA/C
FA
TNP-LPS
TNP-BSA/C
FA
0.0
0.2
0.4
0.6
WT Tcrb-/-
anti-
TNP
1/10
0 (O
D45
0)
IgG2b
TNP-LPS
TNP-BSA/C
FA
TNP-LPS
TNP-BSA/C
FA
0.0
0.1
0.2
0.3
0.4
0.5
WT Tcrb-/-
anti-
TNP
1/10
0 (O
D45
0)
Figure S1. The development of TD, but not TI, IgGs is dependent on TCRab+ T cells and CD154. (A) Graphical representation of the experimental approach followed in B. WT or Tcrb-/- mice were injected with 50 µg of TNP-LPS alone or TNP-BSA in CFA on d 0. (B) Anti-TNP IgG1, IgG2c and IgG2b serum Ab levels on d 14 were determined via ELISA. The symbols represent data from individual animals. Horizontal lines represent means + SEM. One representative of 2 independent experiments is shown. (C) Schematic plan of the experimental approach followed in D. WT mice were injected with 50 µg of TNP-BSA alone or in CFA on d 0 and boosted with 50 µg of TNP-BSA in IFA on d 15 or injected with 50 µg of TNP-LPS in CFA on d 0 and boosted with 50 µg of TNP-LPS in IFA on d 15. The mice were additionally treated with PBS or aCD154 Abs every third day starting on d -1. (D) Anti-TNP IgG1, IgG2c and IgG2b serum Ab levels on d 11 and 25 were determined via ELISA. One representative of 2 independent experiments is shown.
IgG1
3.000
0
1.000
0
0.333
3
0.111
1
0.037
0
0.012
3
0.004
1
0.001
40.0
0.2
0.4
0.6
anti-TNP IgG1
anti-TNP IgG1anti-TNP IgG2canti-TNP IgG2banti-TNP IgM
µg/ml
anti-
TNP
(OD
450)
IgG2c
3.000
0
1.000
0
0.333
3
0.111
1
0.037
0
0.012
3
0.004
1
0.001
40.0
0.2
0.4
0.6
0.8
1.0
anti-TNP IgG2c
anti-TNP IgG1anti-TNP IgG2canti-TNP IgG2banti-TNP IgM
µg/ml
anti-
TNP
(OD
450)
IgG2b
3.000
0
1.000
0
0.333
3
0.111
1
0.037
0
0.012
3
0.004
1
0.001
40.0
0.2
0.4
0.6
0.8
1.0
anti-TNP IgG2b
µg/ml
anti-
TNP
(OD
450)
IgM
3.000
0
1.000
0
0.333
3
0.111
1
0.037
0
0.012
3
0.004
1
0.001
40.0
0.2
0.4
0.6
0.8
1.0anti-TNP IgM
µg/ml
anti-
TNP
(OD
450)
A
B
(d)-12 0
footpadswelling
PBSTNP-LPSTNP-Ficoll
TNP-LPS/CFATNP-LPS/alum
NP-FicollTNP-BSA
orTNP-BSA/CFA
DTH:OVA
in the rightfootpad
TNP-OVAin alum
14
WT mice serum
-1 13
C
IgG1
PBS
NP-Ficoll
0.0
0.1
0.2
IgG2c
PBS
NP-Ficoll
0.0
0.2
0.4
0.6
IgG2b
PBS
NP-Ficoll
0.0
0.2
0.4
0.6
0.8
IgM
PBS
NP-Ficoll
0.0
0.2
0.4
0.6
0.8
anti-
NP
1/10
0 (O
D45
0)
IgG3
PBS
NP-Ficoll
0.0
0.2
0.4
IgA
PBS
NP-Ficoll
0.0
0.2
0.4
0.6
0.8
IgG
PBS
NP-Ficoll
0.00.10.20.30.40.50.6
D
Figure S2. TI antigen-specific B cell activation reduces a subsequent antigen-induced DTH reaction. (A) Graphical representation of the experimental approach followed in B-D and described in Figure 1. WT mice were injected i.p. with either PBS or 50 µg of TNP-Ficoll, TNP-LPS or TNP-LPS in CFA or alum or NP-Ficoll or TNP-BSA alone or TNP-BSA in CFA on d -12. DTH was induced through i.p. injection of TNP-OVA in alum on d 0 and OVA in the right footpad on d 14. (B)The TNP-reactivity of cloned anti-TNP monoclonal IgG1, IgG2c, IgG2b and IgM standard Abs with identical VDJ and VJ regions is shown, in addition to the specificity validation of secondary HRP-coupled anti-IgG1, anti-IgG2c, anti-IgG2b and anti-IgM Abs used in the anti-TNP ELISA experiments to calculate the anti-TNP Ab concentrations in Figure 1. Each ELISA was repeated at least 2 times. (C) Anti-TNP IgA Ab levels on d -1 were determined via ELISA. The symbols represent data from individual animals. Horizontal lines represent means + SEM. (D) Anti-NP serum isotype and IgG subclass Ab levels on day -1 induced with TI NP-Ficoll were determined via ELISA. One representative of 3 independent experiments is shown.
anti-TNP IgA, d -1
PBS
TNP-LPS
TNP-Ficoll
TNP-LPS/CFA
TNP-LPS/alum
NP-Ficoll
TNP-BSA
TNP-BSA/C
FA0.0
0.1
0.2
0.3
0.4
0.5
anti-
TNP
1/10
0 (O
D45
0)
A B
(d)-12 0
footpadswelling
PBSTNP-LPS
TNP-LPS/CFAor
TNP-BSA/CFA
DTH:OVA
in the rightfootpad
TNP-OVAin alum
14
Fcgr2b-/- mice
Figure S3. TI antigen-specific B cell activation reduces a subsequent antigen-induced DTH reaction independent of FcgRIIB. (A) Graphical representation of the experimental approach followed in B. Fcgr2b-/- mice were injected i.p. with either PBS (n=6) or 50 µg of TNP-LPS (n=5), TNP-LPS in CFA (n=6), or TNP-BSA in CFA (n=5) on d -12. DTH was induced through i.p. injection of TNP-OVA in alum on d 0 and OVA in the right footpad on d 14. (B) The differences in footpad thickness between the right and left footpad of the indicated Fcgr2b-/- mice were determined at 4 days after local DTH induction. A box-and-wisker diagram with median and sample minimum and maximum is shown. One representative of 3 independent experiments is shown.
DTH, d 18
PBS
TNP-LPS
TNP-LPS/CFA
TNP-BSA/C
FA-0.1
0.0
0.1
0.2
0.3
0.4
0.5 ** *
foot
pad
thic
knes
s(d
elta
mm
)
mass (m/z) abbreviation
Man GlcNAc 1172 G0 (human and mouse)GlcNAc Man
Man
Man GlcNAc 1417 G0GlcNAc Man
GlcNAc Man
Man GlcNAc 1662 G0GlcNAc Man
GlcNAc Man
Man GlcNAc 1621 G1GlcNAc Man
GlcNAc Man
Man GlcNAc 1866 G2GlcNAc Man
GlcNAc Man
Man GlcNAc 1825 G2GlcNAc Man
GlcNAc Man
Man GlcNAc 2070 G2GlcNAc Man
GlcNAc Man
Man GlcNAc 1376 G1GlcNAc Man
Man
Gal
Gal
Gal
Gal
Gal
Gal
Gal
Human and mouse IgG Fc glycan structures (EndoS)
B
A
Man GlcNAc 1737 G1 S1 (human)GlcNAc Man
Man
Neu5Ac Gal
Man GlcNAc 1767 G1 S1 (mouse)GlcNAc Man
Man
Neu5Gc Gal
Man GlcNAc 2012 G1 S1GlcNAc Man
GlcNAc Man
Neu5Gc Gal
Man GlcNAc 1982 G1 S1GlcNAc Man
GlcNAc Man
Neu5Ac Gal
, : GlcNAc
: mannose (Man) : galactose (Gal; G)
: sialic acid (Neu5Ac or Neu5Gc; S)
: fucose
GlcNAc
GlcNAc
GlcNAc
EndoS
Man GlcNAc 2257 G1 S1GlcNAc Man
GlcNAc Man
Man GlcNAc 2216 G2 S1GlcNAc Man
GlcNAc Man
Man GlcNAc 2461 G2 S1GlcNAc Man
GlcNAc Man
Neu5Gc Gal
Neu5Gc
Gal
Gal
Neu5Gc
Gal
Gal
Man GlcNAc 2607 G2 S2GlcNAc Man
GlcNAc Man
Man GlcNAc 2852 G2 S2GlcNAc Man
GlcNAc Man
Neu5Gc Gal
Neu5Gc Gal
Neu5Gc Gal
Neu5Gc Gal
mass (m/z) abbreviationHuman and mouse IgG Fc glycan structures (EndoS)
Man GlcNAc 2227 G1 S1GlcNAc Man
GlcNAc Man
Neu5Ac Gal
Man GlcNAc 2186 G2 S1GlcNAc Man
GlcNAc Man
Neu5Ac
Gal
Gal
Man GlcNAc 2431 G2 S1GlcNAc Man
GlcNAc Man
Neu5Ac
Gal
Gal
Man GlcNAc 2547 G2 S2GlcNAc Man
GlcNAc Man
Neu5Ac Gal
Neu5Ac Gal
Man GlcNAc 2792 G2 S2GlcNAc Man
GlcNAc Man
Neu5Ac Gal
Neu5Ac Gal
GlcNAc
GlcNAc
GlcNAc
GlcNAc
GlcNAc
GlcNAc
C
D EIgG glycosylation,
Fcgr2b-/- mice
untreate
d
OVA/CFA
TNP-LPS
TNP-Ficoll
TNP-LPS/CFA
0102030405060708090
100
G0 G1 G2G1 S1 G2 S1 G2 S2
IgG αOVA αTNP IgG IgG
% o
f tot
al g
lyca
ns
IgG sialylation,Fcgr2b-/- mice
untreate
d
OVA/CFA
TNP-LPS
TNP-Ficoll
TNP-LPS/CFA
0
3
6
9
12
15
18G1 S1 G2 S1 G2 S2
IgG αOVA αTNP IgG IgG
IgG agalactosylation,Fcgr2b-/- mice
untreate
d
OVA/CFA
TNP-LPS
TNP-Ficoll
TNP-LPS/CFA
0
10
20
30
40
50
60G0
IgG αOVA αTNP IgG IgG
F
Figure S4. TI immunization induces sialylated IgG Abs. (A) The largest IgG Fc glycan coupled to Asn 297 with the cleavage site of EndoS is depicted. Sialic acid: human N-acetylneuraminic acid [Neu5Ac] or murine N-glycolylneuraminic acid [Neu5Gc] depending on the source of the IgG Ab: murine serum or supernatant from human HEK 293 cells. (B)Potential human and murine Fc glycan structures released with EndoS from Asn297. The numbers show the molecular masses (m/z) of the potential human and mouse Fc glycan structures (permethylated) determined through MALDI-TOF MS. (C) The Fc glycosylation (the same analyses as in Figure 3, B and C) of purified total IgGs from the pooled sera of three 10 wk-old untreated WT (independent experiments, n=11) or Tcrbd-/- (n=3) mice or purified OVA- or TNP-specific IgGs from the pooled sera of 6-10 10-wk-old WT mice i.p. injected 14 days before with 2 mg of pure OVA (n=3); or 100 µg of OVA in CFA (n=18) or alum (n=3) or TNP-OVA in CFA (n=3); or 50 µg of TNP-LPS (n=4), TNP-Ficoll (n=5) or TNP-LPS in CFA (n=3) or alum (n=3) was analyzed. . The bars indicate the frequency of glycan structures with 0, 1 or 2 galactose (G) and 0, 1 or 2 sialic acid (S) residues and represent means + SEM for all independent experiments for each group. (D) The Fc glycosylation, (E) sialylation and (F) agalactosylation (G0) of purified total IgGs from the pooled sera of three 10 wk-old untreated Fcgr2b-/- mice (n=3) or purified OVA- or TNP-reactive IgGs from the pooled sera of six 10-wk-old Fcgr2b-/- mice i.p. injected 14 days before with 100 µg of OVA in CFA (n=3) or 50 µg of TNP-Ficoll (n=3), TNP-LPS (n=3) or TNP-LPS in CFA (n=3) were analyzed. The symbols represent data from individual animals.
IgG Fc glycosylation, WT mice
untreate
d wt
untreate
d TCRbd-/-
2 mg O
VA
OVA/CFA
OVA/alum
TNP-OVA/C
FA
TNP-LPS
TNP-Ficoll
TNP-LPS/CFA
TNP-LPS/alum
0102030405060708090
100
IgG αOVA IgG αTNP IgG
G0 G1 G2 G1 S1 G2 S1 G2 S2
% o
f tot
al g
lyca
ns
IgG1, d 14
WT
Ifngr1-
/-
Il17ra
-/-
Ifngr1-
/- x Il1
7ra-/-
0.0
0.5
1.0
1.5
anti-
OVA
1/1
00 (O
D45
0)
IgG2c, d 14
WT
Ifngr1-
/-
Il17ra
-/-
Ifngr1-
/- x Il1
7ra-/-
0.0
0.5
1.0
1.5
2.0 *** ***
anti-
OVA
1/1
00 (O
D45
0)
IgG2b, d 14
WT
Ifngr1-
/-
Il17ra
-/-
Ifngr1-
/- x Il1
7ra-/-
0.0
0.5
1.0
1.5
2.0an
ti-O
VA 1
/100
(OD
450)
B
A IgG Fc glycosylation, WT mice
untreate
d wt
WT
Ifngr1-
/-
Il17ra
-/-
Ifngr1-
/- x Il1
7ra-/-
0102030405060708090
100
IgG αOVA IgG
G0 G1 G2G1 S1 G2 S1 G2 S2
OVA in CFA
% o
f tot
al g
lyca
ns
Figure S5. IFNgRI and IL-17RA are important for the development of inflammatory asialylated IgGs. (A) The Fc glycosylation (the same analyses as in Figure 3, D and E). of purified total IgGs from the pooled sera of three 10-wk-old untreated WT mice (independent experiments; n=11) or purified OVA-specific IgGs from the pooled sera of six 10-wk-old WT (n=18), Ifngr1-/- (n=5), Il17ra-/- (n=3) and Ifngr1-/- x Il17ra-/- (n=3) mice i.p. injected 14 days before with 100 µg of OVA in CFA was analyzed through EndoS-treatment and MALDI-TOF MS (Supplemental Figure 4, A and B). The bars indicate the frequency of glycan structures with 0, 1 or 2 galactose (G) and 0, 1 or 2 sialic acid (S) residues and represent the means + SEM for all independent experiments for each group. The data from untreated and treated WT mice were used from our recent studies (13) (B) Anti-OVA serum IgG1, IgG2c and IgG2b levels on d 14 of the indicated groups were determined via ELISA. The symbols represent data from individual animals. Horizontal lines represent means + SEM. One representative of 3 ELISA experiments is shown.
D
A
(d)-1 0
footpadswelling
PBSαTNP IgG1 (H5 <1% sial)αTNP IgG1 (H5 5% sial)αTNP IgG1 (H5 15% sial)αTNP IgG1 (H5 50% sial)
αThy1.1 IgG1 (OX7 3% sial)or
αThy1.1 IgG1 (OX7 64% sial)
DTH:OVA
in the rightfootpad
TNP-OVAin alum
14
WT or Fcgr2b-/- mice
17
serum
BαThy1.1 IgG1 (OX-7)
native
+sial
0
20
40
60
80
100 S1S2
sial
ylat
ion
(% o
f tot
al g
lyca
ns)
DTH, d 17
PBS
H5 <1%
sial
H5 5% si
al
H5 15%
sial
H5 50%
sial
OX-7 <1
% sial
OX-7 64
% sial
PBS
H5 <1%
sial
H5 5% si
al
H5 15%
sial
H5 50%
sial
OX-7 <1
% sial
OX-7 64
% sial
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6 ** ** 0.09 ** *
WT Fcgr2b-/-
foot
pad
thic
knes
s (d
elta
mm
)
E
αTNP IgG1 (H5)
native
+sial
0
20
40
60
80
100 S1S2
sial
ylat
ion
(% o
f tot
al g
lyca
ns)
C
anti-OVA IgG2b, d 13
PBS
H5 <1%
sial
H5 5% si
al
H5 15%
sial
H5 50%
sial
OX-7 <1
% sial
OX-7 64
% sial
PBS
H5 <1%
sial
H5 5% si
al
H5 15%
sial
H5 50%
sial
OX-7 <1
% sial
OX-7 64
% sial
0.00.10.20.30.40.50.60.70.80.9 ** *
WT Fcgr2b-/-
** ****
anti-
OVA
1/1
00 (O
D45
0)
GFH5 and OX-7 IgG1
0.001 0.01 0.1 1 100.0
0.2
0.4
0.6
0.8
H5 +sialH5 native
OX7 +sialOX-7 native
µg/ml
IgG
1 (O
D 4
50)
H5 versus OX-7 IgG1
0.001 0.01 0.1 1 100.0
0.2
0.4
0.6
H5 +sialH5 native
OX-7 +sialOX-7 native
µg/ml
anti-
TNP
(OD
450
)
Figure S6. Transfer of antigen-specific sialylated monoclonal IgGs reduces a subsequent antigen-induced DTH response. (A) Graphical representation of the experimental approach followed in B-G. WT and Fcgr2b-/- mice were simultaneously injected i.v. on d -1 with either PBS or 100 µg of native non-sialylated (<1% sial) or 5%, 15% or 50% sialylated (sial; mix of native and in vitro sialylated H5 (70% sialylation; B)) monoclonal anti-TNP murine IgG1 (hybridoma clone H5) or with 100 µg of native low-sialylated (3% sial) or 64% in vitro sialylated antigen-unspecific anti-Thy1.1 murine IgG1 (hybridoma clone OX-7; C) (each group, n=7-8). DTH responses were induced through the injection of TNP-OVA in alum on d 0 and OVA in the right footpad on d 14. (B and C) The frequency of Fc sialic acid (S) modifications (Supplemental Figure 4) on native or in vitro sialylated (B) anti-TNP murine IgG1 (clone H5) or (C) anti-Thy1.1 murine IgG1 (clone OX-7) was determined through EndoS-treatment and MALDI-TOF MS. The bars represent means + SEM. (D) Serum IgG2b anti-OVA Ab levels on d 13 were determined via ELISA. Symbols represent data from individual animals. Horizontal lines represent means + SEM. (E) The differences in footpad thickness between the right and left footpad were determined at 3 days after local DTH induction. A box-and-wisker diagram with median and sample minimum and maximum is shown. One representative out of two independent experiments is shown. (F and G) Altered IgG glycosylation of anti-TNP murine IgG1 does not influence antigen binding. (F) Murine IgG1 ELISA and (G) TNP reactivity of native non-sialylated anti-TNP IgG1 (H5 native) and anti-Thy1.1 IgG1 (OX-7 native) Abs and their in vitro sialylated variants (H5 +sial; OX-7 +sial). One representative of 2 independent ELISA experiments is shown in F and G.
DB
(d)0 12
analysis:anti-TNP
serum IgG
TNP-Ficoll+
PBSαTNP IgM (native)
orαTNP IgM (de-sial)
WT mice
C
Figure S7. IgM Ab sialylation regulates antigen-induced B cell activation. (A) Graphical representation of the experimental approach followed in B-D. WT mice were injected i.p. with ICs containing 100 µg of TNP-Ficoll and 50 µg of either native sialylated or sialidase-treated (de-sial) monoclonal anti-TNP IgM (each group, n=6). The positive control group received PBS and TNP-Ficoll (no IC). (B) Coomassie blue staining of a SDS PAGE gel with 10 µg of heat-denatured native monoclonal anti-TNP IgM Ab. The lanes were run on the same gel but were noncontiguous. (C) The frequency of sialic acid (S) containing glycan structures on the indicated IgM Abs were determined through PNGaseF-treatment and MALDI-TOF MS (Supplemental Figure 8). The bars represent the means. (D) Anti-TNP serum IgG levels on d 12 were determined via ELISA. One representative of 2 independent experiments is shown.
A
anti-TNP IgG
PBS
anti-T
NP IgM (n
ative
)
anti-T
NP IgM (d
e-sial
)
0.00.20.40.60.81.01.21.4
TNP-Ficoll +
** **
anti-
TNP
1/10
00 (O
D 4
50)
100M [kDa]
75
50
37
25
20
αTNP IgMsialylation
native
de-sial
05
10152025303540 S1
S2
% o
f tot
al g
lyca
ns
Man GlcNAc GlcNAc 1172 M3Man
Man
mass (m/z) abbreviationHuman IgG Fc glycan structures (PNGaseF)B
A
Man GlcNAc GlcNAc 1346 F M3Man
Man
Fuc
2 GlcNAc
3 GlcNAc
4 GlcNAc
High-mannose structures
1580 M5
Man GlcNAc GlcNAc 1754 F M5Man
Man
Fuc
1784 M6Man GlcNAc GlcNAc
ManMan
Man
Man
Man
high-mannosestructures
complexstructures
hybridstructures
Man GlcNAc GlcNAc 1376 M4Man
Man
Man
Man GlcNAc GlcNAc 1550 F M4Man
Man
Fuc
Man
Man GlcNAc GlcNAc
Man
Man
Man
Man
Man
Man
PNGaseF
mass (m/z) abbreviationHuman IgG Fc glycan structures (PNGaseF)
Man GlcNAc GlcNAc 1591 F M3 A1GlcNAc
Man
Man
Fuc
High-mannose structures
Man GlcNAc GlcNAc 2162 F M7
ManMan
Man
ManFuc
Man GlcNAc GlcNAc 2192 M8
ManMan
Man
Man
Man GlcNAc GlcNAc 2366 F M8
ManMan
Man
ManFuc
Man GlcNAc GlcNAc 2396 M9
ManMan
Man
ManMan
Man GlcNAc GlcNAc 2570 F M9
ManMan
Man
ManMan
Man
Fuc
Hybrid structures
Man GlcNAc GlcNAc 1417 M3 A1GlcNAc
Man
Man
Man GlcNAc GlcNAc 1621 M3 A1 G1GlcNAc
Man
ManGal
Man GlcNAc GlcNAc 1795 F M3 A1 G1GlcNAc
Man
Man
Fuc
Gal
1958 F M6Man GlcNAc GlcNAc
ManMan
Man
ManFuc
Man
Man GlcNAc GlcNAc 1988 M7
ManMan
Man
Man
Man
Man
Man
Man
Man
2 Man
Man
2 Man
Man
Man
ManMan
Man
mass (m/z) abbreviationHuman IgG Fc glycan structures (PNGaseF)
Hybrid structures
Man GlcNAc GlcNAc 1982 M3 A1 G1 S1GlcNAc
Man
ManNeu5Ac Gal
Man GlcNAc GlcNAc 2156 F M3 A1 G1 S1GlcNAc
Man
Man
Fuc
Neu5Ac Gal
Man GlcNAc GlcNAc 2186 M4 A1 G1 S1GlcNAc
Man
ManNeu5Ac Gal
Man
Man GlcNAc GlcNAc 2360 F M4 A1 G1 S1GlcNAc
Man
ManNeu5Ac Gal
ManFuc
Man GlcNAc GlcNAc 2390 M5 A1 G1 S1GlcNAc
Man
ManNeu5Ac Gal
ManMan
Man GlcNAc GlcNAc 2564 F M5 A1 G1 S1GlcNAc
Man
ManNeu5Ac Gal
ManMan
Fuc
Man GlcNAc GlcNAc 2203 F M5 A1 G1GlcNAc
Man
ManGal
FucManMan
Man GlcNAc GlcNAc 2029 M5 A1 G1GlcNAc
Man
ManGal
ManMan
Man GlcNAc GlcNAc 1825 M4 A1 G1GlcNAc
Man
ManGal
Man
Man GlcNAc GlcNAc 1999 F M4 A1 G1GlcNAc
Man
ManGal
ManFuc
mass (m/z) abbreviationHuman IgG Fc glycan structures (PNGaseF)
Complex structures
Man GlcNAc GlcNAc 1662 A2GlcNAc Man
GlcNAc Man
Man GlcNAc GlcNAc 1836 F A2GlcNAc Man
GlcNAc Man
Fuc
Man GlcNAc GlcNAc 1907 A2 BGlcNAc Man
GlcNAc Man
Man GlcNAc GlcNAc 1866 A2 G1GlcNAc Man
GlcNAc Man
Gal
Man GlcNAc GlcNAc 2040 F A2 G1GlcNAc Man
GlcNAc Man
Fuc
Gal
Man GlcNAc GlcNAc 2081 F A2 BGlcNAc Man
GlcNAc Man
Fuc
GlcNAc
GlcNAc
Man GlcNAc GlcNAc 2070 A2 G2GlcNAc Man
GlcNAc ManGal
Gal
Man GlcNAc GlcNAc 2111 A2 B G1GlcNAc Man
GlcNAc Man
Gal
Man GlcNAc GlcNAc 2244 F A2 G2GlcNAc Man
GlcNAc Man
Fuc
Gal
Gal
Man GlcNAc GlcNAc 2285 F A2 B G1GlcNAc Man
GlcNAc Man
Fuc
Gal
Man GlcNAc GlcNAc 2315 A2 B G2GlcNAc Man
GlcNAc ManGal
Gal
GlcNAc
GlcNAc
GlcNAc
mass (m/z) abbreviationHuman IgG Fc glycan structures (PNGaseF)
Complex structures
Man GlcNAc GlcNAc 2227 A2 G1 S1GlcNAc Man
ManGlcNAc
Neu5Ac Gal
Man GlcNAc GlcNAc 2401 F A2 G1 S1GlcNAc Man
GlcNAc Man
Fuc
Neu5Ac Gal
Man GlcNAc GlcNAc 2431 A2 G2 S1GlcNAc Man
GlcNAc Man
Neu5Ac
Gal
Gal
Man GlcNAc GlcNAc 2472 A2 B G1 S1GlcNAc Man
GlcNAc Man
Neu5Ac
Man GlcNAc GlcNAc 2605 F A2 G2 S1GlcNAc Man
GlcNAc Man
Fuc
Neu5Ac
Gal
Gal
Man GlcNAc GlcNAc 2646 F A2 B G1 S1GlcNAc Man
GlcNAc Man
Fuc
Neu5Ac Gal
GlcNAc
GlcNAc
Man GlcNAc GlcNAc 2792 A2 G2 S2GlcNAc Man
GlcNAc Man
Neu5Ac Gal
Neu5Ac Gal
Man GlcNAc GlcNAc 2967 F A2 G2 S2GlcNAc Man
GlcNAc Man
FucNeu5Ac Gal
Neu5Ac Gal
Man GlcNAc GlcNAc 2676 A2 B G2 S1GlcNAc Man
GlcNAc Man
Neu5Ac
Gal
Gal
GlcNAc
Man GlcNAc GlcNAc 2850 F A2 B G2 S1GlcNAc Man
GlcNAc Man
Fuc
Neu5Ac
Gal
Gal
GlcNAc
Man GlcNAc GlcNAc 2489 F A2 B G2GlcNAc Man
GlcNAc Man
Fuc
Gal
Gal
GlcNAc
Gal
mass (m/z) abbreviationHuman IgG Fc glycan structures (PNGaseF)
Man GlcNAc GlcNAc 3038 A2 B G2 S2GlcNAc Man
GlcNAc Man
Neu5Ac Gal
Neu5Ac Gal
Figure S8. Possible N-glycans linked to IgM. (A) IgM Abs contain 5 conserved glycosylation sites, and 2 of these sites bear oligo-mannose glycans (65). The largest N-glycans bearing high-mannose, hybrid or complex structures with 2, 3 or 4 terminal GlcNAcs, respectively, are depicted. Dark-blue: N-acetyl-glucosamine (GlcNAc; A); green: mannose (Man; M); light-blue: bisecting GlcNAc (B); red: fucose (Fuc; F); yellow: galactose (Gal; G); pink: sialic acid (Neu5Ac; S). The monoclonal IgM contains only human Neu5Ac and no murine Neu5Gc because it was produced in human HEK 293 cells. MALDI-TOF MS was performed from PNGaseF-released N-glycans. The cleavage site of PNGaseF is indicated with an arrow. (B) Potential IgM N-glycan structures released with PNGaseF. The numbers show the molecular masses (m/z) of the possible N-glycan structures (permethylated).
Complex structures
GlcNAc
Man GlcNAc GlcNAc 3212 F A2 B G2 S2GlcNAc Man
GlcNAc Man
FucNeu5Ac Gal
Neu5Ac Gal
GlcNAc
41
Tables
Supplementary Tables
Table S1: Primer.
anti-TNP variable VDJ heavy chain part,
forward primer:
5´ tctaccggtgtacattccgaggtgcagcttcaggagtca
anti-TNP variable VDJ heavy chain part,
reverse primer:
5´ gcagggctagctgcagagacagtgaccagagtccc
anti-TNP complete VJ kappa light chain,
forward primer:
5´ gtcaccggtgtacattcagacattgtgatgtcacagtct
anti-TNP complete VJ kappa light chain,
reverse primer:
5´ ttattcggaagctttcaacactcattcctgttgaag
C57BL/6 IgG1 forward primer (NheI): 5´ cctcgcgctagcacgacacccccatctgtctatccac
C57BL/6 IgG1 reverse primer (BsiWI): 5´ ttattcggcgtacgcgtcatttaccaggagagtgggag
C57BL/6 IgG2c forward primer (NheI): 5´ cctcgcgctagcacaacagccccatcggtctatccac
C57BL/6 IgG2c reverse primer (BsiWI): 5´ ttattcggcgtacgcgtcatttacccagagaccgggag
C57BL/6 IgG2b forward primer (NheI): 5´ cctcgcgctagcacaacacccccatcagtctatccac
C57BL/6 IgG2b reverse primer (BsiWI): 5´ ttattcggcgtacgcgtcatttacccggagaccgggag
C57BL/6 IgM forward primer (NheI): 5´ cctcgcgctagccagtccttcccaaatgtcttccccctcg
C57BL/6 IgM reverse primer (BsiWI): 5´ ttattcggcgtacgcgtcaatagcaggtgccgcctgtg
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