# 65938-RG-1 Point-by-Point response 22.05 · Man GlcNAc 2852 G2 S2 GlcNAc Man GlcNAc Man Neu5Gc...

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


GlcNAc Man


GlcNAc Man


GlcNAc Man


GlcNAc Man


GlcNAc 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


GlcNAc Man

Neu5Ac Gal

, : GlcNAc

: mannose (Man) : galactose (Gal; G)

: sialic acid (Neu5Ac or Neu5Gc; S)

: fucose

GlcNAc

GlcNAc

GlcNAc

EndoS


GlcNAc Man


GlcNAc Man


GlcNAc Man

Neu5Gc Gal

Neu5Gc

Gal

Gal

Neu5Gc

Gal

Gal


GlcNAc Man


GlcNAc Man

Neu5Gc Gal

Neu5Gc Gal

Neu5Gc Gal

Neu5Gc Gal

mass (m/z) abbreviationHuman and mouse IgG Fc glycan structures (EndoS)


GlcNAc Man

Neu5Ac Gal


GlcNAc Man

Neu5Ac

Gal

Gal


GlcNAc Man

Neu5Ac

Gal

Gal


GlcNAc Man

Neu5Ac Gal

Neu5Ac Gal


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 agalactosylation,Fcgr2b-/- mice

untreate

d

OVA/CFA

TNP-LPS

TNP-Ficoll

TNP-LPS/CFA

0

10

20

30

40

50

60G0


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

Fuc

1784 M6Man GlcNAc GlcNAc

ManMan

Man

Man

Man

high-mannosestructures

complexstructures

hybridstructures

Man GlcNAc GlcNAc 1376 M4Man

Man

Man


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


ManMan

Man

ManFuc


ManMan

Man

ManMan


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


ManMan

Man

Man

Man

Man

Man

Man

Man

2 Man

Man

2 Man

Man

Man

ManMan

Man


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

ManNeu5Ac Gal

Man


Man

ManNeu5Ac Gal

ManFuc


Man

ManNeu5Ac Gal

ManMan


Man

ManNeu5Ac Gal

ManMan

Fuc


Man

ManGal

FucManMan


Man

ManGal

ManMan


Man

ManGal

Man


Man

ManGal

ManFuc


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


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


GlcNAc Man

Neu5Ac

Gal

Gal

Man GlcNAc GlcNAc 2472 A2 B G1 S1GlcNAc Man

GlcNAc Man

Neu5Ac


GlcNAc Man

Fuc

Neu5Ac

Gal

Gal

Man GlcNAc GlcNAc 2646 F A2 B G1 S1GlcNAc Man

GlcNAc Man

Fuc

Neu5Ac Gal

GlcNAc

GlcNAc


GlcNAc Man

Neu5Ac Gal

Neu5Ac Gal


GlcNAc Man

FucNeu5Ac Gal

Neu5Ac Gal


GlcNAc Man

Neu5Ac

Gal

Gal

GlcNAc


GlcNAc Man

Fuc

Neu5Ac

Gal

Gal

GlcNAc

Man GlcNAc GlcNAc 2489 F A2 B G2GlcNAc Man

GlcNAc Man

Fuc

Gal

Gal

GlcNAc

Gal



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


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

# 65938-RG-1 Point-by-Point response 22.05 · Man GlcNAc 2852 G2 S2 GlcNAc Man GlcNAc Man Neu5Gc...

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Transcript of # 65938-RG-1 Point-by-Point response 22.05 · Man GlcNAc 2852 G2 S2 GlcNAc Man GlcNAc Man Neu5Gc...