EFFECTS OF THE ANTIBODY CONSTANT REGION ON ANTIGEN RECOGNITION It is well accepted that specificity...
1
EFFECTS OF THE ANTIBODY CONSTANT REGION ON ANTIGEN RECOGNITION It is well accepted that specificity and affinity of antigen-antibody interactions are driven by the variable regions (V) of immunoglobulins (Ig), while the constant regions of the heavy chains (CH) are responsible for effector functions and avidity properties. However, recent reports provided evidence that antibodies with identical V regions, but different CH exhibited important changes concerning the affinity and avidity against the antigen (Ag). These results suggest that the CH could have a role in Ag recognition, but at the present the structural evidences that explain how this phenomenon occur remains elusive. With the purpose to understand the mechanism of this change in the affinity constants of two Ig with identical V regions but different CH, we studied the serum of a patient with an immunocytic sarcoma containing monoclonal antibodies of the IgA1 and IgG1 isotypes. Both Igs, either the entire molecule or the Fab fragment, presented differences in the affinity constants against their Ag evaluated by Surface Plasmon Resonance (SPR), indicating that the CH1 constant domain could have a role in Ag recognition. Using these Ab, we were able to solve the crystallographic structures of the Fab fragments from the IgA1, IgG1 and IgG1 in complex with their Ag (tubulin autoantigen). Also we generated preliminary results by SPR that suggest a possible effect of the glycosylation in the hinge region of the IgA1 in Ag recognition. Additionally, this work reports at a high resolution the first crystallographic structure of a human IgA1 Fab fragment. Overall, our structural and kinetic studies provided evidence on the mechanism by which the constant regions could affect the antigen recognition site. Fab-Cys 220 -Pro-Val-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser-Cys 241 -Fc O-glycan O-glycan O-glycan O-glycan O-glycan Hinge region C. ramosum AK183 N. gonorrhoeae 1 N. meningitidis 1 N. gonorrhoeae 2 N. meningitidis 2 H. Influenzae S. Pneumoniae S. sangis IgA1 proteases Figure 1. Schematic representation of the hinge region of the human IgA1. The O- glycoylated sites and the sites of cleavage by IgA proteases are indicated. The highlighted proteases are the ones that were produced for the different studies. 0 100 200 300 400 0 50 100 150 200 250 300 R esponse (R U s) Tim e (s) 0 100 200 300 400 0 50 100 150 200 250 300 R esponse (R U s) Tim e (s) 0 2 4 6 8 10 12 14 16 0 50 100 150 200 250 300 R eq (R U s) C o n cen tratio n ( M) 0 5 10 15 20 25 30 0 50 100 150 200 250 300 R eq (R U s) C o n cen tratio n ( M) FabA ramosum FabA gono K D : 3.7 x 10 -6 M K D : 2.9 x 10 -6 M Steady state Steady state 0 100 200 300 400 500 -2 0 0 20 40 60 80 100 120 140 160 180 200 R esponse (R U s) Tim e (s) FabA ramosum FabA gonorrhoeae 0 100 200 300 400 500 0 20 40 60 80 100 120 140 160 R esponse (R U s) Tim e (s) K D : 1.64 x 10 -7 M FabG1 Kinetic annalisis Figure 2. A-B and D. Kinetic annalisis of the two FabA fragments and FabG1 fragments by SPR. Althogh the FabG1 has the same antigen binding site, it has a higher affinity than the FabA fragments. C. Superposition of one of the curves of both FabA showing that despite having similar affinties, the dissociation phase is different. A C B D Correa, Agustín ; Trajtenberg, Felipe 1 ; Obal, Gonzalo 2 ; Larrieux, Nicole 1 ; Dighiero, Guillermo; Pristch, Otto 2 ; Buschiazzo, Alejandro 1 ; Oppezzo, Pablo. G A F C B E D CLκ VL CH1α VH CDRH2 CDRH1 CDRH3 CDRL1 CDRL3 CDRL2 CH1α domain VH-VL region FabA structure D E A Figure 3. A, Structure of the human IgA1 Fab fragment (1.5 Å); the four domains of the FabA are indicated. B. Crystal obtained for the FabA. C. Disulphide bond between Cys195 and Cys219, that fix the carboxiterminal part of the CH1 domain (Pro221). D.VH-VL region, (CDR’s position is indicated). E. CH1α, with the internal disulphide bonds depicted. Cys 195 Cys 219 Pro 221 B C K207 P208 T122 L11 P122 L11 Y209 FabG1 FabA FabE FabM FabG1 FabA FabAm FabA FabAm Fab E FabM FabG 1 Y209 D207 K20 9 P212 K207 P122 A121 T12 4 A123 T122 CDRH2 CDRH3 CDRH1 CDRL2 CDRL1 CDRL3 CH1γ CLκ VL VH R101H H35H N33H R50H K52H L101L N96L K97L T99L Figure 4. A. Structure of the human IgG1 Fab fragment (2.4 Å); the four domains of the FabG1 are indicated. B. Structure of the human IgG1 Fab fragment (2.2 Å) in complex with part of the antigen (green map). The electronic density map of the residues that participate in the interaction are indicated in violet. FabG1 and FabG1-peptide structure A B Peptide sequence: TAEEEEDFGE - - - - - - R 67 CDRH2 CDRH3 V 104 Y 105 VH VL C D E A B Figure 5. A-C. Surface map of the variable region of the FabG1-peptide, FabG1 and FabA respectively. The residues V104 and Y105 are indicated in green. D. Charge map of the variable domain of the FabG1. E-F. Structure alignment of the CDRH2 and CDRH3 regions of the FabA (sky-blue) and FabG1 (red). F IgA1-IgG1 Variable domain comparison IgA1-IgG1 switch region comparison Figure 6. A. Structure alignment of the switch region between the FabA (sky-blue) and FabG1 (red). B-C. Surface map of the switch region (VH-CH1 interface) of the FabG1 and FabA respectively. D. Structure alignment of the switch region of different Fab fragments. FabAm, mouse FabA, all the others are from human. A B C D Recombinant Protein Unit, 1 Protein Crystallography Unit, Protein Biophisics Unit; Institut Pasteur de Montevideo, Uruguay. Conclusions: The structural analysis of the two Fab fragments, indicate that changes in the aminoacidic composition between the IgA1 and IgG1 in the hinge region could be responsible for the differences in affinity obtained by SPR. In the case of the FabA, the presence of an hidrofobic core stabilized by the interaction of L11, P122 and Y209, rigidify this region changing the relative orientation of the VH and CH1 domains. This change induce an asymmetric rearrangement in the configuration of the variable domains, altering in this way the antigen binding site.
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Transcript of EFFECTS OF THE ANTIBODY CONSTANT REGION ON ANTIGEN RECOGNITION It is well accepted that specificity...
EFFECTS OF THE ANTIBODY CONSTANT REGION ON ANTIGEN RECOGNITION
It is well accepted that specificity and affinity of antigen-antibody interactions are driven by the variable regions (V) of immunoglobulins (Ig), while the constant regions of the heavy chains (CH) are responsible for effector functions and avidity properties. However, recent reports provided evidence that antibodies with identical V regions, but different CH exhibited important changes concerning the affinity and avidity against the antigen (Ag). These results suggest that the CH could have a role in Ag recognition, but at the present the structural evidences that explain how this phenomenon occur remains elusive. With the purpose to understand the mechanism of this change in the affinity constants of two Ig with identical V regions but different CH, we studied the serum of a patient with an immunocytic sarcoma containing monoclonal antibodies of the IgA1 and IgG1 isotypes. Both Igs, either the entire molecule or the Fab fragment, presented differences in the affinity constants against their Ag evaluated by Surface Plasmon Resonance (SPR), indicating that the CH1 constant domain could have a role in Ag recognition. Using these Ab, we were able to solve the crystallographic structures of the Fab fragments from the IgA1, IgG1 and IgG1 in complex with their Ag (tubulin autoantigen). Also we generated preliminary results by SPR that suggest a possible effect of the glycosylation in the hinge region of the IgA1 in Ag recognition. Additionally, this work reports at a high resolution the first crystallographic structure of a human IgA1 Fab fragment. Overall, our structural and kinetic studies provided evidence on the mechanism by which the constant regions could affect the antigen recognition site.
Fab-Cys220-Pro-Val-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser-Pro-Ser-Cys241-Fc
O-glycan O-glycanO-glycan O-glycanO-glycan
Hinge region
C. ramosum AK183 N. gonorrhoeae 1N. meningitidis 1N. gonorrhoeae 2
N. meningitidis 2
H. InfluenzaeS. PneumoniaeS. sangis
IgA1proteases
Figure 1. Schematic representation of the hinge region of the human IgA1. The O-glycoylated sites and the sites of cleavage by IgA proteases are indicated. The highlighted proteases are the ones that were produced for the different studies.
0 100 200 300 400
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Us)
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0 100 200 300 400
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pons
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Us)
Time (s)
0 2 4 6 8 10 12 14 160
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s)
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0 5 10 15 20 25 300
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(RU
s)
Concentration (M)
FabA ramosum FabA gono
KD: 3.7 x 10-6 M KD: 2.9 x 10-6 M
Steady state Steady state
0 100 200 300 400 500-20
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FabA ramosumFabA gonorrhoeae
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pons
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Time (s)
KD: 1.64 x 10-7 M
FabG1
Kinetic annalisis
Figure 2. A-B and D. Kinetic annalisis of the two FabA fragments and FabG1 fragments by SPR. Althogh the FabG1 has the same antigen binding site, it has a higher affinity than the FabA fragments. C. Superposition of one of the curves of both FabA showing that despite having similar affinties, the dissociation phase is different.
A CB
D
Correa, Agustín; Trajtenberg, Felipe 1; Obal, Gonzalo 2; Larrieux, Nicole 1; Dighiero, Guillermo; Pristch, Otto 2; Buschiazzo, Alejandro 1; Oppezzo, Pablo.
G
A F C
B ED
CLκ VL
CH1αVH
CDRH2
CDRH1
CDRH3
CDRL1
CDRL3
CDRL2
CH1α domainVH-VL region
FabA structure
D E
A
Figure 3. A, Structure of the human IgA1 Fab fragment (1.5 Å); the four domains of the FabA are indicated. B. Crystal obtained for the FabA. C. Disulphide bond between Cys195 and Cys219, that fix the carboxiterminal part of the CH1 domain (Pro221). D.VH-VL region, (CDR’s position is indicated). E. CH1α, with the internal disulphide bonds depicted.
Cys195
Cys219
Pro221
B
C
K207P208
T122
L11
P122
L11
Y209FabG1
FabA
FabEFabMFabG1FabAFabAm
FabA FabAm FabE FabM FabG1
Y209 D207 K209 P212 K207
P122 A121 T124 A123 T122
CDRH2
CDRH3
CDRH1
CDRL2
CDRL1
CDRL3
CH1γ
CLκVL
VH
R101H
H35H
N33H
R50H
K52H
L101L
N96L
K97L
T99L
Figure 4. A. Structure of the human IgG1 Fab fragment (2.4 Å); the four domains of the FabG1 are indicated. B. Structure of the human IgG1 Fab fragment (2.2 Å) in complex with part of the antigen (green map). The electronic density map of the residues that participate in the interaction are indicated in violet.
FabG1 and FabG1-peptide structureA B
Peptide sequence:TAEEEEDFGE - - - - - -
R67
CDRH2
CDRH3
V104
Y105
VH
VL
C
D E
A B
Figure 5. A-C. Surface map of the variable region of the FabG1-peptide, FabG1 and FabA respectively. The residues V104 and Y105 are indicated in green. D. Charge map of the variable domain of the FabG1. E-F. Structure alignment of the CDRH2 and CDRH3 regions of the FabA (sky-blue) and FabG1 (red).
F
IgA1-IgG1 Variable domain comparison
IgA1-IgG1 switch region comparison
Figure 6. A. Structure alignment of the switch region between the FabA (sky-blue) and FabG1 (red). B-C. Surface map of the switch region (VH-CH1 interface) of the FabG1 and FabA respectively. D. Structure alignment of the switch region of different Fab fragments. FabAm, mouse FabA, all the others are from human.
A B
C
D
Recombinant Protein Unit, 1 Protein Crystallography Unit, Protein Biophisics Unit; Institut Pasteur de Montevideo, Uruguay.
Conclusions:The structural analysis of the two Fab fragments, indicate that changes in the aminoacidic composition between the IgA1 and IgG1 in the hinge region could be responsible for the differences in affinity obtained by SPR. In the case of the FabA, the presence of an hidrofobic core stabilized by the interaction of L11, P122 and Y209, rigidify this region changing the relative orientation of the VH and CH1 domains. This change induce an asymmetric rearrangement in the configuration of the variable domains, altering in this way the antigen binding site.
