title: Cell- and nuclear-penetrating anti-dsDNA autoantibodies have multiple arginines in CDR3 of VH and increase cellular level of pERK and Bcl-2 in mesangial cells.
abstract: Investigation of characteristics of cell- and nuclear-penetrating anti-double stranded (ds)DNA autoantibodies (autoAbs) is important to understand pathogenesis of lupus nephritis, but has not been clearly explored. The present study reports that three anti-dsDNA monoclonal autoAbs, which contain more than two arginine residues in their CDR3s of variable heavy domain (VH), penetrated into living murine mesangial cells and the cell nuclei. However, an anti-dsDNA monoclonal Ab (mAb) having only one arginine in the CDR3-VH did not penetrate cells. To assess the contribution of antigen-binding sites, especially the VH, in cell- and nuclear-penetration, we evaluated the characteristics of recombinant single chain Fv(scFv), VH, and variable light domain (VL) of a penetrating mAb. The scFv and VH domain, containing arginine in CDR3-VH maintained the ability to penetrate cells and the cell nuclei, whereas the VL domain, having no arginine in CDR3, did not penetrate cells. The penetratingm Abs, scFv, and VH activated ERK and increased cellular protein levels of Bcl-2, whereas the non-penetrating Ab and VL did not. The cell survival was decreased by the penetrating mAbs, scFv and VH, not by the non-penetrating mAb and VL. The data indicate that an antigen-binding site is required for cell-penetration and that positively-charged arginine residues in CDR3-VH contribute to the cell- and nuclear-penetrating ability of a subset of anti-dsDNA autoAbs. Furthermore,the nuclear-penetrating anti-dsDNA autoAbs could possibly function as a pathogenic factor for lupus nephritis by up-regulating ERK activation and Bcl-2 production in mesangial cells. The cell- and nuclear-penetrating VH domain may be exploited as a vehicle for the intra cellular delivery of various useful molecules.
authors.affiliation: Department of Biomedical Sciences, Graduate School of Medicine and Department of Microbiology, School of Medicine, Ajou University, Suwon 443-721, Republic of Korea.
authors.forename: Sae-Ran
authors.initials: SR
authors.lastname: Im
authors.affiliation: Department of Biomedical Sciences, Graduate School of Medicine and Department of Microbiology, School of Medicine, Ajou University, Suwon 443-721, Republic of Korea.
authors.forename: Sun-Woo
authors.initials: SW
authors.lastname: Im
authors.affiliation: Department of Biomedical Science, Graduate School of Biomedical Science & Engineering, Hanyang University, Seoul 133-791, Republic of Korea.
authors.forename: Hee-Yong
authors.initials: HY
authors.lastname: Chung
authors.affiliation: Department of Biomedical Sciences, Graduate School of Medicine and Department of Microbiology, School of Medicine, Ajou University, Suwon 443-721, Republic of Korea.
authors.forename: Pavithra
authors.initials: P
authors.lastname: Pravinsagar
authors.affiliation: Department of Biomedical Sciences, Graduate School of Medicine and Department of Microbiology, School of Medicine, Ajou University, Suwon 443-721, Republic of Korea. Electronic address: jangyj@ajou.ac.kr.
authors.forename: Young-Ju
authors.initials: YJ
authors.lastname: Jang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000974:Antibodies, Antinuclear / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D001120:Arginine / Q000378:metabolism*; D001323:Autoantibodies / Q000737:chemistry* / Q000276:immunology; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival; D022801:Complementarity Determining Regions / Q000737:chemistry* / Q000276:immunology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005434:Flow Cytometry; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000276:immunology; D050527:Mesangial Cells / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000276:immunology; D013329:Structure-Activity Relationship
chemical_list: D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D022801:Complementarity Determining Regions; D007135:Immunoglobulin Variable Region; D019253:Proto-Oncogene Proteins c-bcl-2; D057127:Single-Chain Antibodies; D001120:Arginine; D048049:Extracellular Signal-Regulated MAP Kinases
keywords: Anti-DNA autoantibody; Arginine; Bcl-2; CDR3-VH; ERK; Lupus nephritis; Nuclear-penetrating; Pathogenesis; Recombinant VH domain; Recombinant scFv