MECHANISMS OF CELLULAR PENETRATION AND NUCLEAR-LOCALIZATION OF AN ANTI-DOUBLE STRAND DNA AUTOANTIBODY

An anti-dsDNA Ab, mAb 3E10, was identified that bound membranes of fix ed human renal tubular cells, penetrated live murine renal tubular cel ls in vivo, and localized in the cell nucleus, mAb 3E10 binds both dsD NA and an extracellular matrix protein, HP8/HEVIN, expressed in high e ndothelial venules. Previous studies showed both shared and distinct b inding determinants of mAb 3E10 V-H for DNA and HP8/HEVlN. To independ ently assess the requirement of DNA and HP8/HEVIN in cellular penetrat ion, site-directed mutants of mAb 3E10 V-H and V kappa were studied fo r penetrating kidney cell lines, The results showed that residues requ ired for binding DNA, but not HP8/HEVIN, were necessary for Ab penetra tion, indicating that cellular penetration required the presence of DN A or binding of Ab to a membrane determinant precisely resembling DNA, Ab Fab penetrated cells, indicating that neither the Fe nor multivale nt Ab binding is necessary for cellular penetration, Ab synthesized in the cytoplasm as a result of deleting heavy and light chain signal pe ptides was not translocated to the nucleus; indicating a mechanism dis tinct from the usual protein nuclear localization signals and suggesti ng the need for a membrane-mediated pathway or for posttranslational m odification of the Ab.