Limits...
The critical role of arginine residues in the binding of human monoclonal antibodies to cardiolipin.

Giles I, Lambrianides N, Latchman D, Chen P, Chukwuocha R, Isenberg D, Rahman A - Arthritis Res. Ther. (2004)

Bottom Line: Of four arginine residues in IS4VH CDR3 substituted to serines, two residues at positions 100 and 100 g had a major influence on the strength of CL binding while the two residues at positions 96 and 97 had no effect.In CDR exchange studies, VL containing B3VL CDR1 were associated with elevated CL binding, which was reduced significantly by substitution of a CDR1 arginine residue at position 27a with serine.In contrast, arginine residues in VL CDR2 or VL CDR3 did not enhance CL binding, and in one case may have contributed to inhibition of this binding.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Rheumatology, Department of Medicine, University College London, UK. i.giles@ich.ucl.ac.uk

ABSTRACT
Previously we reported that the variable heavy chain region (VH) of a human beta2 glycoprotein I-dependent monoclonal antiphospholipid antibody (IS4) was dominant in conferring the ability to bind cardiolipin (CL). In contrast, the identity of the paired variable light chain region (VL) determined the strength of CL binding. In the present study, we examine the importance of specific arginine residues in IS4VH and paired VL in CL binding. The distribution of arginine residues in complementarity determining regions (CDRs) of VH and VL sequences was altered by site-directed mutagenesis or by CDR exchange. Ten different 2a2 germline gene-derived VL sequences were expressed with IS4VH and the VH of an anti-dsDNA antibody, B3. Six variants of IS4VH, containing different patterns of arginine residues in CDR3, were paired with B3VL and IS4VL. The ability of the 32 expressed heavy chain/light chain combinations to bind CL was determined by ELISA. Of four arginine residues in IS4VH CDR3 substituted to serines, two residues at positions 100 and 100 g had a major influence on the strength of CL binding while the two residues at positions 96 and 97 had no effect. In CDR exchange studies, VL containing B3VL CDR1 were associated with elevated CL binding, which was reduced significantly by substitution of a CDR1 arginine residue at position 27a with serine. In contrast, arginine residues in VL CDR2 or VL CDR3 did not enhance CL binding, and in one case may have contributed to inhibition of this binding. Subsets of arginine residues at specific locations in the CDRs of heavy chains and light chains of pathogenic antiphospholipid antibodies are important in determining their ability to bind CL.

Show MeSH
Effect of complementarity determining region exchange in the light chains. Cardiolipin binding of IgG in COS-7 cell supernatants containing wild-type heavy chains expressed with wild-type or hybrid light chain constructs. (a) Light chains expressed with IS4 variable heavy chainregion (VH). (b) Light chains expressed with B3VH. Presented as concentration of IgG in the supernatant versus optical density (OD) at 405 nm in the anti-cardiolipin ELISA.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC1064879&req=5

Figure 2: Effect of complementarity determining region exchange in the light chains. Cardiolipin binding of IgG in COS-7 cell supernatants containing wild-type heavy chains expressed with wild-type or hybrid light chain constructs. (a) Light chains expressed with IS4 variable heavy chainregion (VH). (b) Light chains expressed with B3VH. Presented as concentration of IgG in the supernatant versus optical density (OD) at 405 nm in the anti-cardiolipin ELISA.

Mentions: For each heavy chain/light chain combination that bound CL, the linear portion of the binding curve for absorbance against antibody concentration was determined empirically, by dilution of antibody over a wide range of concentrations. Similar patterns of binding were obtained for each combination from repeated expression experiments, hence representative results from a single experiment only are shown in Figs 2,3,4.


The critical role of arginine residues in the binding of human monoclonal antibodies to cardiolipin.

Giles I, Lambrianides N, Latchman D, Chen P, Chukwuocha R, Isenberg D, Rahman A - Arthritis Res. Ther. (2004)

Effect of complementarity determining region exchange in the light chains. Cardiolipin binding of IgG in COS-7 cell supernatants containing wild-type heavy chains expressed with wild-type or hybrid light chain constructs. (a) Light chains expressed with IS4 variable heavy chainregion (VH). (b) Light chains expressed with B3VH. Presented as concentration of IgG in the supernatant versus optical density (OD) at 405 nm in the anti-cardiolipin ELISA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1064879&req=5

Figure 2: Effect of complementarity determining region exchange in the light chains. Cardiolipin binding of IgG in COS-7 cell supernatants containing wild-type heavy chains expressed with wild-type or hybrid light chain constructs. (a) Light chains expressed with IS4 variable heavy chainregion (VH). (b) Light chains expressed with B3VH. Presented as concentration of IgG in the supernatant versus optical density (OD) at 405 nm in the anti-cardiolipin ELISA.
Mentions: For each heavy chain/light chain combination that bound CL, the linear portion of the binding curve for absorbance against antibody concentration was determined empirically, by dilution of antibody over a wide range of concentrations. Similar patterns of binding were obtained for each combination from repeated expression experiments, hence representative results from a single experiment only are shown in Figs 2,3,4.

Bottom Line: Of four arginine residues in IS4VH CDR3 substituted to serines, two residues at positions 100 and 100 g had a major influence on the strength of CL binding while the two residues at positions 96 and 97 had no effect.In CDR exchange studies, VL containing B3VL CDR1 were associated with elevated CL binding, which was reduced significantly by substitution of a CDR1 arginine residue at position 27a with serine.In contrast, arginine residues in VL CDR2 or VL CDR3 did not enhance CL binding, and in one case may have contributed to inhibition of this binding.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Rheumatology, Department of Medicine, University College London, UK. i.giles@ich.ucl.ac.uk

ABSTRACT
Previously we reported that the variable heavy chain region (VH) of a human beta2 glycoprotein I-dependent monoclonal antiphospholipid antibody (IS4) was dominant in conferring the ability to bind cardiolipin (CL). In contrast, the identity of the paired variable light chain region (VL) determined the strength of CL binding. In the present study, we examine the importance of specific arginine residues in IS4VH and paired VL in CL binding. The distribution of arginine residues in complementarity determining regions (CDRs) of VH and VL sequences was altered by site-directed mutagenesis or by CDR exchange. Ten different 2a2 germline gene-derived VL sequences were expressed with IS4VH and the VH of an anti-dsDNA antibody, B3. Six variants of IS4VH, containing different patterns of arginine residues in CDR3, were paired with B3VL and IS4VL. The ability of the 32 expressed heavy chain/light chain combinations to bind CL was determined by ELISA. Of four arginine residues in IS4VH CDR3 substituted to serines, two residues at positions 100 and 100 g had a major influence on the strength of CL binding while the two residues at positions 96 and 97 had no effect. In CDR exchange studies, VL containing B3VL CDR1 were associated with elevated CL binding, which was reduced significantly by substitution of a CDR1 arginine residue at position 27a with serine. In contrast, arginine residues in VL CDR2 or VL CDR3 did not enhance CL binding, and in one case may have contributed to inhibition of this binding. Subsets of arginine residues at specific locations in the CDRs of heavy chains and light chains of pathogenic antiphospholipid antibodies are important in determining their ability to bind CL.

Show MeSH