A subset of monoclonal anti-DNA autoantibodies enters a variety of living cells. Here, we aimed to identify the endocytic receptors recognized by an internalizing anti-nucleic acid autoantibody, the 3D8 single-chain variable fragment (scFv). We found that cell surface binding and internalization of 3D8 scFv were inhibited markedly in soluble heparan sulfate (HS)/chondroitin sulfate (CS)-deficient or -removed cells and in the presence of soluble HS and CS. 3D8 scFv colocalized intracellularly with either HS proteoglycans (HSPGs) or CSPGs in HeLa cells. 3D8 scFv was co-endocytosed and co-precipitated with representative individual HSPG and CSPG molecules: syndecan-2 (a transmembrane HSPG), glypican-3 (a glycosylphosphatidylinositol (GPI)-anchored HSPG); CD44 (a transmembrane CSPG); and brevican (a GPI-anchored CSPG). Collected data indicate that 3D8 scFv binds to the negatively charged sugar chains of both HSPGs and CSPGs and is then internalized along with these molecules, irrespective of how these proteoglycans are associated with the cell membrane. This is the first study to show that anti-DNA antibodies enter cells via both HSPGs and CSPGs simultaneously. The data may aid understanding of endocytic receptors that bind anti-DNA autoantibodies. The study also provides insight into potential cell membrane targets for macromolecular delivery.

3D8 single-chain Fv (scFv) is a catalytic nucleic acid antibody with anti-viral activity against a broad spectrum of viruses. Here we investigated the functional stability of 3D8 scFv to provide a basis for engineering a 3D8 scFv derivative and for developing stable formulations with improved stability and potential use as an anti-viral agent. The stability of 3D8 scFv was assessed by measuring its DNA-hydrolyzing activity under different biochemical and physical conditions using a fluorescence resonance energy transfer (FRET)-based method. In addition, the anti-influenza (H9N2) effect of 3D8 scFv was evaluated in A549 cells. 3D8 scFv was stable at 50°C for 6h at pH 7.2, for 3 days at pH 4-10 at 37°C and 30 days at pH 4-8 at 37°C. The stability was not affected by a reducing condition, freeze-thawing for up to 30 cycles, or lyophilization. Evaluation of the anti-virus effect showed that cells treated with 32-128 units of 3D8 scFv showed a 50% decrease in influenza replication compared to untreated cells. Based on its enzymatic stability in various biochemical and physical environments, 3D8 scFv holds good potential for development as an anti-viral therapeutic.

No abstract

Intrabodies, antibodies expressed within cells, offer an interesting way to target intracellular molecules, making them potentially useful for biotechnology and medicine. However, it remains controversial whether full-size IgG intrabodies expressed in the reducing environment of the cytosol of mammalian cells are workable and structurally sound. Herein, we settle this issue with a systematic investigation of the structure and functionality of four chimeric IgG1s with distinct variable (V) domains but identical constant (C) domains. Full-size IgGs expressed in the cytosol of HEK293 cells were either assemblycompetent or -incompetent, depending on the intrinsic properties of the V regions. Structural integrity of the C region is required for H:L association and the formation of a functional antigen-binding site. Partial intrachain disulfide bond formation occurs in both H and L chains of cytosolic IgG intrabodies, whereas interchain disulfide bond formation was absent and dispensable for functional assembly. IgG1s expressed in the cytosol and via the ER were shown to assemble differently. Our findings provide insight into the features and possible utilization of full-size IgGs as cytosolic antibodies in biotechnological and medical applications.

Background: A subset of anti-DNA antibodies have DNA-hydrolysis and/or cell-penetration activity.Results: Deactivation of different complementarity-determining regions (CDRs) in a multifunctional anti-nucleic acid antibody alters the properties of the antibody.Conclusion: CDR1 of the light chain plays a crucial role in maintaining the properties of 3D8 scFv.Significance: Single complete CDR deactivation allows exploration of the molecular features of multifunctional anti-DNA antibodies.