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Autoreactivity of Human VH Domains from cDNA Libraries: Analysis with a Bacterial Expression System¹

Department of Biochemistry, Tufts University School of Medicine and Sackler School of Graduate Biomedical Sciences, Boston, MA

Previous studies showed that VH domains of several anti-DNA Abs can bind DNA in the absence of VL. In the current work, we tested the VH autoreactive potential more generally, examining VH domains that did not come from known autoantibodies. Using a bacterial expression system, we produced 11 fusion proteins, each containing a VH domain and a B domain of staphylococcal protein A. The VH domains were coded in cDNA libraries from circulating B cells of healthy young adult humans. Thus, binding properties of the Ig molecules from which they came were unknown. The B cells had not been stimulated in vitro. Seven cDNA clones combined the frequently expressed V_H3-23 gene segment with varied D_H and J_H segments. The other clones contained unmutated V_H3-7, V_H3-9, V_H3-53, and V_H4-39 segments. We compared these bacterial expression products with single-chain Fv, VH and VL domains of IgM mAb 18/2, a V_H3-23-encoded, DNA-binding autoantibody. Submicromolar concentrations of 5 of the 11 VH domains bound to ssDNA. Those and one more also bound to immobilized poly(dT), and two bound to circular plasmid dsDNA. Soluble poly(dT) was the most potent inhibitor in competitive ELISA. Seven of the VH domains also bound to immobilized nuclear ribonucleoprotein, four to histone and none to thyroglobulin. Two interacted with the matrix of a Sephacryl S-100 column. The polyreactive autoantigen-binding properties of these VH domains raise the question of whether these properties may play a role in the formation of the VH repertoire of circulating B cells.

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