Extended half‐life upon binding of destabilized intrabodies allows specific detection of antigen in mammalian cells

Abstract

Intracellular antibodies or intrabodies are antibody fragments used inside cells for interaction with targets. In case intrabodies interfere with antigen function, they can mediate cell killing following binding (recently reviewed in [1]). Whilst a variety of cell types including plant cells, fungal cells and even bacteria [2] have been described as hosts, mammalian cells are the most commonly used target cells [3], implying that intrabodies with functional ablation capabilities might be a useful antibody-based format for disease-specific reagents. Until now, the preferred intrabody is the recombinant single-chain Fv antibody fragment (scFv), expressed from a single cDNA and composed of an antibody variable heavy-chain (VH) sequence tethered to a variable light-chain (VL) sequence by a flexible linker. ScFv carries the specificity inherent in the antibody combining site, namely the three hypervariable complementary-determining regions (CDRs) of each variable (V) region that form the antigen-binding pocket. The ectopic expression of antibody fragments inside mammalian cells (intrabodies) is a challenging approach for probing and modulating target activities. We previously described the shuttling activity of intracellularly expressed Escherichia coli b-galactosidase conferred by the single-chain Fv (scFv) fragment 13R4 equipped with nuclear import ⁄ export signals. Here, by appending to scFvs the proteolytic PEST signal sequence (a protein region rich in proline, glutamic acid, serine and threonine) of mouse ornithine decarboxylase, we tested whether short-lived or destabilized intrabodies could affect the steady-state level of target by redirecting it to the proteasomes. In the absence of antigen, the half-life of the modified scFv 13R4, relative to untagged molecules, was considerably reduced in vivo. However, after coexpression with either cytoplasmic or nuclear antigen, the destabilized 13R4 fragments were readily maintained in the cell and strictly colocalized with b-galactosidase. Analysis of destabilized site-directed mutants, that were as soluble as 13R4 in the intracellular context, demonstrated that binding to antigen was essential for survival under these conditions. This unique property allowed specific detection of b-galactosidase, even when expressed at low level in stably transformed cells, and permitted isolation by flow cytometry from a transfected cell mixture of those living cells specifically labeled with bound intrabody. Altogether, we show that PESTtagged intrabodies of sufficient affinity and solubility are powerful tools for imaging the presence and likely the dynamics of protein antigens that are resistant to proteasomal degradation in animal cells.Abbreviations b-gal, b-galactosidase; CDR, complementary-determining region; (E)GFP, (enhanced) green fluorescent protein; mODC, mouse ornithine decarboxylase; ODC, ornithine decarboxylase; scFv, single-chain Fv antibody fragment; VH, variable heavy-chain; VL, variable light-chain; V, variable.

Fusion to a highly charged proteasomal retargeting sequence increases soluble cytoplasmic expression and efficacy of diverse anti-synuclein intrabodies

“…In fact, given the intrinsic insolubility of the constructs in this study, the overall soluble expression is greatly increased. These data are in contrast to the data published on anti-β-galactosidase 13R4 scFv-PEST intrabody, 26 where the mODC-PEST fusion rendered the intrabody-PEST protein itself unstable, and subsequent binding to its β-gal target stabilized the complex. We have now shown that 6 different intrabodies fused to the mODC-PEST degron do not result in a destabilization of the intrabody-PEST fusion protein, although two of them do enhance the turnover of their monomeric targets.…”

“…10,26 It is critical to consider the effect of the fusion on the steady-state level of intrabody protein because long half-life has been reported to be as or more In a yeast surface display library selection, the strongest binder to the critical hydrophobic interaction region (non-amyloid component; NAC) of α-syn was determined to be a single domain nanobody, VH14. 17 As an unprotected heavy chain only intrabody, VH14 is extremely insoluble, and is prone to intracellular aggregation.…”

Monitoring Interactions and Dynamics of Endogenous Beta-catenin With Intracellular Nanobodies in Living Cells*

“…Recent reports have described intrabodies modified by the addition of a degradation promoting domain. Upon ectopical expression of their antigens these intrabodies become stabilized (73). In our approach we observed an enrichment of the chromobody independently of the introduction of a destabilizing domain.…”

Suppression of cervical carcinoma cell growth by intracytoplasmic codelivery of anti-oncoprotein E6 antibody and small interfering RNA

“…The cells were washed with PBS and either fixed with 4% paraformaldehyde for microscopy or harvested by trypsinization for preparing total cell extracts as described previously (19). The transduced antibodies were visualized with Alexa Fluor 488 -labeled goat anti-mouse immunoglobulins (Molecular Probes), and p53 was detected with anti-p53 rabbit polyclonal serum FL-393 (Santa Cruz Biotechnology) followed by Alexa Fluor 568 -labeled goat anti-rabbit immunoglobulins (Molecular Probes).…”