Structure-function analysis of a lupus anti-DNA autoantibody: central role of the heavy chain complementarity-determining region 3 Arg in binding of double- and single-stranded DNA

Abstract

To determine the contribution of the somatic point mutations and that of the complementarity-determining region (CDR)3 Arg to DNA binding, we engineered the germline V(H) and V(kappa) gene revertant and site-mutagenized the CDR3 Arg residues of the mutated and "antigen-selected" mAb 412.67. This anti-DNA autoantibody was derived from B-1 cells of a lupus patient and bore two H-CDR3 Arg, Arg105 and Arg107, encoded by N segment additions, and one kappa-CDR3 Arg, Arg97, resulting from a point mutation (Kasaian et al. 1994. J. Immunol. 152: 3137-3151; Kasaian et al. 1995. Ann. N.Y Acad. Sci. 764: 410-423). The germ-line revertant bound double-stranded (ds) DNA and single-stranded (ss) DNA as effectively as its wild-type counterpart (relative avidity: 6.4x10(-7) and 9.9x10(-9) vs. 6.7x10(-7) and 9.1 x10(-9) g/microl), raising the possibility that an antigen other than DNA was responsible for the selection of the mAb 412.67 V(H) and V(kappa) point mutations. H-CDR3 Arg105 and Arg107 were both required for dsDNA binding, but either Arg105 or Arg107 was sufficient for ssDNA binding. The central role of Arg105 and Arg107 in DNA binding reflected their solvent-exposed orientation at the apex of the H-CDR3 main loop. Consistent with its inward orientation afar from the antigen-binding surface, the kappa-CDR3 Arg97 played no role in either dsDNA or ssDNA binding.

Fig. 1

Nucleotide sequences of the wild-type, variously substituted, and germ-line (top row) mAb 412.67 V_HDJ_H and V_κJ_κ gene segments. 412.67.gl.5a is the autologous germ-line gene sequence. Dashes indicate identities. Solid lines on top of each cluster depict CDR. Lowercase letters denote vector sequences. The sequences or reverse complementary sequences encompassed by the H-Ov1, H-Ov2, H-FR3, 67cFR3, H-FR4, 67-CDR3 A, and 67-CDR3 B or κ-Ov1, κ-Ov2, κIII-FR3 A, κIII-FR3 B, and κ-FR4 mutagenized primers are underlined.

Fig. 2

Deduced amino acid sequences of the wild-type, variously substituted, and germ-line (top row) mAb 412.67 V_HDJ_H and V_κJ_κ gene segments. 412.67.gl.5a is the autologous germ-line sequence. Dashes indicate identities. Solid lines on top of each cluster depict CDR.

Fig. 3

Generation of the germ-line revertant and mutagenized mAb 412.67 V_HDJ_H and V_κJ_κ DNA, their insertion into the pcDNAIG and the pSXRDIG vectors and expression in F3B6 cells.

Fig. 4

Binding of the original IgA1κ mAb 412.67 (A), recombinant “wild-type” V_H67-D(R105, R107)J_H67/V_κ67-(R97) J_κ67 (B), “germ-line” V_H67gl.5a-D(R105, R107) J_H67/V_κKv325(R97)J_κ67 (C), “wild-type CDR3 Gly-substituted” V_H67-D(G105, G107)J_H67/V_κ67-(W97)J_κ67 (D), “Trp-CDR3” revertant V_H67-D (R105, R107) J_H67/V_κ67(W97)J_κ67 (E), “wild-type CDR3 Gly-substituted” V_H67-D(G105, G107) J_H67/V_κ67(R97)J_κ67 (F), “single CDR3 Gly-substituted” V_H67gl.5a-D(G105, R107)J_H67/V_κ67(R97)J_κ67 (G), “single CDR3 Gly-substituted” V_H67gl.5a-D(R105, G107)J_H67/V_κ67(R97)J_κ67 (H) IgG1κ to ssDNA (■) and dsDNA (□). Bars schematize the composition of original wild-type IgA1κ, the wild-type IgG1κ, and the various recombinant and mutagenized molecules (black rectangles depict the wild-type sequences derived from the original mAb 412.67; white rectangles depict the gene sequence derived from autologous germ-line gene segments or the H-CDR3 Gly-substituted sequences). Antigen-binding activity is expressed as absorbance at 492 nm.

Fig. 5

Ribbon drawing of mAb 412.67 Fv showing the κ chain on the left and the H chain on the right. All Arg and Lys in the H-CDR and κ-CDR are indicated.

Fig. 6

Stereo images of α-carbon trace drawings of the modeled mAb 412.67 Fv with B form DNA positioned above the binding site, drawn to scale. The molecular surface covering the CDR residues is depicted as dots (A). The H-CDR3 Arg 105 (R105) and 107 (R107) are depicted as full circles; the κ-CDR3 Arg97 (R97) and the H-FR3 Arg98 (R98) are depicted as open circles (B). The H-CDR3 Arg105 and Arg107 are substituted with Gly residues (C).