Abstract
Autoimmunity may have its origins of early repertoire selection in developmental B cells. Such a primary repertoire is probably shaped by selecting B cells that can efficiently perform productive signaling, stimulated by self-antigens in the bone marrow, such as DNA. In support of that idea, we previously found a V segment from VH10 family that can form antibodies that bind to DNA independent of CDR3 usage. In this paper we designed four antibody fragments in a novel single-chain pre-BCR (scpre-BCR) format containing germinal V gene segments from families known to bind DNA (VH10) or not (VH4) connected to a murine surrogate light chain (SLC), lacking the highly charged unique region (UR), by a hydrophilic peptide linker. We also tested the influence of CDR2 on DNA reactivity by shuffling the CDR2 loop. The scpre-BCRs were expressed in bacteria. VH10 bearing scpre-BCR could bind DNA, while scpre-BCR carrying the VH4 segment did not. The CDR2 loop shuffling hampered VH10 reactivity while displaying a gain-of-function in the nonbinding VH4 germline. We modeled the binding sites demonstrating the conservation of a positivity charged pocket in the VH10 CDR2 as the possible cross-reactive structural element. We presented evidence of DNA reactivity hardwired in a V gene, suggesting a structural mechanism for innate autoreactivity. Therefore, while autoreactivity to DNA can lead to autoimmunity, efficiently signaling for B cell development is likely a trade-off mechanism leading to the selection of potentially autoreactive repertoires.
Highlights
Self-antigens are known for their participation in the development and progress of autoimmune diseases such as systemic lupus erythematosus (SLE) [1], rheumatoid arthritis [2] and type 1 diabetes [3] among others
The recombinant single-chain pre-B cell receptor (pre-B-cell receptor (BCR)) contained a heavy chain composed of a germline VH10, a synthetic CDR3, and a germline FW4
Sequence alignment of the VH10 chain used in scpre-BCR with the two anti-DNA antibodies 1CBV [24], 4Z8F [25] shows that the V chain sequences are very similar and share the adjacent arginine residue at position 50 and 52 (Figure 6).a potential saltbridge could be formed with DNA’s phosphate groups, allowing the VH10 germline chain the ability to bind DNA independently of CDR3 or the VL domain
Summary
Self-antigens are known for their participation in the development and progress of autoimmune diseases such as systemic lupus erythematosus (SLE) [1], rheumatoid arthritis [2] and type 1 diabetes [3] among others. The emergence of the rearranged light chain (VL) substituting the SLC drives the appearance of the membrane-associated B-cell receptor (BCR) During this developmental stage, the signaling of the newly formed immature B-cell relies on antigenic stimulus for survival and selection [18]. The signaling of the newly formed immature B-cell relies on antigenic stimulus for survival and selection [18] At this point, BCR auto and polyreactivity help to delineate the primary repertoire [19], and polyreactive V gene have been shown to be associated with immunological protection [20]. Germline V genes encoded DNA reactivity could help explain the observed part of the B-cell primary repertoire autoreactivity It is unknown if the germline-encoded VH10 gene segment is sufficient to encompass a DNA binding site as an innate (germline) property. We investigated structural features in the VH10 germline sequence and located its DNA binding capacity to CDR2
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