A nucleic acid-hydrolyzing antibody penetrates into cells via caveolae-mediated endocytosis, localizes in the cytosol and exhibits cytotoxicity

Abstract

Abstract. Many natural anti-DNA antibodies (Abs) have the ability to translocate across the plasma membrane and localize in the nucleus of mammalian cells, frequently leading to cytotoxicity to cells. Herein, we report detailed intracellular trafficking routes and cytotoxicity in HeLa cells for a single chain variable fragment (scFv) Ab, 3D8, which is an anti-DNA Ab capable of hydrolyzing both DNA and RNA. The intracellular penetration of 3D8 scFv occurred by caveolae/lipid raft endocytosis. The time-course chasing experiments revealed that 3D8 scFv escaped directly from the caveosome into the cytosol and remained in the cytosol without further trafficking into endosomes, lysosomes, endoplasmic reticulum, Golgi, or nucleus. The cytosolically localized 3D8 scFv maintained its nuclease activity to hydrolyze cellular RNAs, mainly mRNAs, eventually triggering apoptotic cell death. Our results demonstrate that 3D8 scFv has a unique intracellular trafficking route of localizing in the cytosol, thereby exhibiting cytotoxicity due to its nuclease activity.

An RNA-hydrolyzing recombinant antibody exhibits an antiviral activity against classical swine fever virus

“…3D8 scFv is somewhat cytotoxic. Overexpression of 3D8 scFv in PK-15 cells by transient transfection with the 3D8 scFv gene has caused cell death, possibly resulting from inhibition of protein synthesis by intense damage to host RNAs [7]. Our trials of several times to establish stable clones that constitutively express 3D8 scFv have allowed us to acquire the clones which express 3D8 scFv protein only in subdetectable levels on Western blots, including C26 clone.…”

“…Confocal microscopy and flow cytometry were performed as previously described [7]. 3D8 scFv expressed in C26 cells was detected using rabbit anti-3D8 scFv antibody and secondary TRITClabeled anti-rabbit IgG antibody.…”

“…FRET-based RNA cleavage assay was performed as previously described [7]. Random ribo-oligonucleotides 21 bases long (500 nM), labeled with 6-carboxyfluorescein (FAM) at the 5 0 terminus and a black hole quencher (BHQ) at the 3 0 terminus, were synthesized (5 0 -FAM-CGA TGA GTG CCA TGG ATAT AC-BHQ-3 0 ), annealed to the non-labeled complementary ribo-oligonucleotide, and then delivered into cells in 96-black-well plates using Lipofectamine™ transfection reagent (Invitrogen).…”

“…2A). Under a microscopic observation of the cells at 72 hpi, brownish-stained CSFV foci were observed in control PK- Based on the report that the purified 3D8 scFv protein is able to penetrate cell membrane by itself [7], we also tested antiviral activity by exogenous treatment of 3D8 scFv protein to PK-15 cells.…”

“…Polyclonal antibodies, purified by affinity chromatography on DNA-cellulose, were able to hydrolyze both DNA and RNA [4]; conversely, polyclonal antibodies directed against RNA hydrolyzed both RNA and DNA [5]. Previously, we have introduced the 3D8 single-chain variable (3D8 scFv) antibody as a DNA-and RNA-hydrolyzing recombinant antibody [6,7]. The recent studies showing that canonical RNases can exert anti-viral effect prompted us to investigate the antiviral activity of the 3D8 scFv.…”