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. 2009;30(5):474-80.
doi: 10.1159/000242476. Epub 2009 Sep 28.

Modeling of human anti-GBM antibody-alpha3(IV)NC1 interactions predicts antigenic cross-linking through contact of both heavy chains with repeating epitopes on alpha3(IV)NC1

Kevin E C Meyers  1 , Mette Christensen, Michael P Madaio
Affiliations

Modeling of human anti-GBM antibody-alpha3(IV)NC1 interactions predicts antigenic cross-linking through contact of both heavy chains with repeating epitopes on alpha3(IV)NC1

Kevin E C Meyers et al. Am J Nephrol. 2009.

Abstract

Background/aims: Patients with anti-glomerular basement membrane diseases produce pathogenic autoantibodies (autoAb) that deposit in the kidney and initiate severe inflammation. Restricted antigenic specificity of the autoAb against 2 regions (with related sequences) within alpha3(IV)NC1, along with shared idiotypes (i.e. structural determinants), among pathogenic human autoAb suggested that common genetic elements encode the autoAb. The aim of this study was to determine whether the idiotypic relatedness of the autoAb was due to the fact that unique and similar genes were used to encode them, divergent genes were used to produce Ab with similar Ag-binding properties and conformation, or if other mechanisms were operative.

Methods: The encoding V gene sequences of pathogenic human anti-alpha3(IV)NC1 Ab, derived following immunization of XenoMice which produce human but not murine IgG, with alpha3(IV)NC1 were determined. Predicted conformations of autoAb-alpha3(IV)NC1 interactions were derived using the Ab sequences and molecularmodels of the alpha3(IV)NC1 structure.

Results: The pathogenic Ab were encoded by multiple, common V(H) and V(L) gene families indicating that they were not encoded by a unique subset of genes and that normal individuals have the capacity to produce them. However, modeling of the Ag-Ab interactions suggested that although the contact regions varied for individual Ab, the optimized energy constraints facilitate interaction of both Ab-binding regions with pathogenically relevant epitopes on alpha3(IV)NC1.

Conclusions: The results suggest that the repetitive nature and relatedness of the alpha3(IV)NC1 antigenic epitopes facilitate cross-linking of pathogenic Ab, in vivo, by allowing both IgG Fab to bind to the basement membrane. This most likely accounts for the high-affinity Ab binding we and others observed among human anti-alpha3(IV)NC1 Ab. Based on these observations, we postulate that this interaction provides for the stability of the Ab interaction, resulting in a high-affinity interaction that serves as an ideal scaffold for optimal FcR engagement and complement activation, thereby accelerating inflammation and contributing to the rapidly progressive nature of this disease.

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Figures

Fig. 1.
Fig. 1.
Anti-α3(IV)NC1 mAb bind to human α3(IV)NC1. Human mAb produced from XenoMouse II® after immunization with α3(IV)NC1 preparations bound to human α3(IV)NC1 by Western blot. Greater binding of m anti-α3(IV) to intact human α3(IV)NC1 with lesser binding to reduced human α3(IV)NC1, similar to that observed for human polyclonal serum-derived Ab. 4–6 μg Ag were used in these experiments.
Fig. 2.
Fig. 2.
XenoMouse II pathogenic mAb compete for binding with human-derived polyclonal anti-GBM Ab to α3(IV). Negative control mAb showed less than 5% inhibition at any time and was equivalent to ‘none’ as shown in the figure(+/– error bars = SD).
Fig. 3.
Fig. 3.
Model of human m anti-α3(IV)NC1 F1.1 binding to α3(IV)NC1. a In this model, the heavy chain CDR1 (shown in blue) and light chain CDR1 (shown in green) are shown interacting with the C2 epitope of α3(IV)NC1 (shown in red). b In this model, the heavy chain CDR1 (shown in blue) and light chain CDR1 (shown in green) are shown interacting with the C6 epi- tope C6 of α3(IV)NC1 (shown in red). Note the multiple contact Ab contact regions of the Ab with the repeating epitopes on α3(IV)NC1.
Fig. 3.
Fig. 3.
Model of human m anti-α3(IV)NC1 F1.1 binding to α3(IV)NC1. a In this model, the heavy chain CDR1 (shown in blue) and light chain CDR1 (shown in green) are shown interacting with the C2 epitope of α3(IV)NC1 (shown in red). b In this model, the heavy chain CDR1 (shown in blue) and light chain CDR1 (shown in green) are shown interacting with the C6 epi- tope C6 of α3(IV)NC1 (shown in red). Note the multiple contact Ab contact regions of the Ab with the repeating epitopes on α3(IV)NC1.
Fig. 4.
Fig. 4.
Model of human monoclonal anti-α3(IV)NC1 F2.1 binding to α3(IV)NC1. F2.1 is shown interacting at multiple sites with both the C2 and C6 epitopes α3(IV)NC1 (shown in red). Note the multiple contact Ab contact regions of the Ab with the repeating epitopes on α3(IV)NC1. The contact regions, however, are different from those of either F1.1. or F3.1.
Fig. 5.
Fig. 5.
Model of human m anti-α3(IV)NC1 F3.1 binding to α3(IV)NC1. F3.1 is shown interacting through heavy chain CDR2 (shown in green) and light chain CDR2 (shown in blue) sites with both the C2 (shown in red) and C6 (shown in brown) epitopes. Note the multiple contact Ab contact regions of the Ab with the repeating epitopes on α3(IV)NC1. The contact regions, however, are different from those of either F1.1. or F2.1.
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