Point Mutations of Single Amino Acids Abolish Ability of α3 NC1 Domain to Elicit Experimental Autoimmune Glomerulonephritis in Rats

Thomas Hellmark,Lanlin Chen,Sophie Ohlsson,Jörgen Wieslander,Warren Kline Bolton

doi:10.1074/jbc.m211951200

Thomas Hellmark, Lanlin Chen + Show 3 more

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https://doi.org/10.1074/jbc.m211951200

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Abstract

We previously showed concordance between Goodpasture syndrome antibody binding and production of experimental glomerulonephritis using human chimeric proteins. We now examine a more limited amino-terminal region of alpha3(IV) non-collagenous domain (NC1) and the impact of single amino acid (AA) mutations of this region on glomerulonephritis induction. Rats were immunized with collagenase-solubilized glomerular basement membrane (csGBM), D3, an alpha1(IV)NC1 chimeric protein with 69 AA of alpha3(IV)NC1 (binds Goodpasture sera), D4, the D3 construct shortened by 4 AA (non-binding), P9, P10, single AA mutants (non-binding), and S2, alpha1(IV)NC1 with 9 AA of alpha3(IV)NC1 (binding). All rats immunized with csGBM and S2 and 50% of D3 rats developed glomerulonephritis. csGBM rats had intense GBM-bound IgG deposits, but S2 and D3 rats had minimal deposits. None of the D4, P9, or P10 rats developed glomerulonephritis. Lymphocytes from nephritic rats proliferated with csGBM, S2, and D3, but not with D4, P9, or P10. Discrete segments of alpha3(IV)NC1 within the alpha1(IV)NC1 backbone can induce glomerulonephritis. Single AA mutations within that epitope render the antigen unresponsive to Goodpasture sera and incapable of inducing glomerulonephritis. These studies support the concordance of glomerulonephritis inductivity and Goodpasture serum binding. Further, they define a critical limited AA sequence within alpha3(IV)NC1 of nine or fewer AA, which confers nephritogenicity to the nonnephritogenic alpha1(IV)NC1 without in vivo antibody binding. This region may be a T-cell epitope responsible for induction of glomerulonephritis in this model in rats and Goodpasture syndrome in man.

Highlights

We previously showed concordance between Goodpasture syndrome antibody binding and production of experimental glomerulonephritis using human chimeric proteins
Rats were immunized with collagenase-solubilized glomerular basement membrane, D3, an ␣1(IV)NC1 chimeric protein with 69 amino acid (AA) of ␣3(IV)NC1, D4, the D3 construct shortened by 4 AA, P9, P10, single AA mutants, and S2, ␣1(IV)NC1 with 9 AA of ␣3(IV)NC1
Induction of Glomerulonephritis by collagenase-solubilized glomerular basement membrane (csGBM) and Goodpasture Serum Binding Chimeric Protein Constructs—Animals immunized with csGBM and S2 construct all developed hematuria, and half of the animals immunized with D3 developed hematuria

Summary

Introduction

We previously showed concordance between Goodpasture syndrome antibody binding and production of experimental glomerulonephritis using human chimeric proteins. Single AA mutations within that epitope render the antigen unresponsive to Goodpasture sera and incapable of inducing glomerulonephritis These studies support the concordance of glomerulonephritis inductivity and Goodpasture serum binding. They define a critical limited AA sequence within ␣3(IV)NC1 of nine or fewer AA, which confers nephritogenicity to the nonnephritogenic ␣1(IV)NC1 without in vivo antibody binding This region may be a T-cell epitope responsible for induction of glomerulonephritis in this model in rats and Goodpasture syndrome in man. Point mutations of various AA in this region of ␣3(IV)NC1 are capable of abolishing Goodpasture syndrome antibody binding [18] Whether these same epitopes are responsible for the induction of disease cannot be studied directly in man. The close approximation of these epitopes within a narrow region in the absence of in vivo bound antibody suggests that this area may contain an epitope responsible for T-cell-induced glomerulonephritis

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