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

Abstract

Intrabodies can be powerful reagents to effect modulation of aberrant intracellular proteins that underlie a range of diseases. However, their cytoplasmic solubility can be limiting. We previously reported that overall charge and hydrophilicity can be combined to provide initial estimates of intracellular solubility, and that charge engineering via fusion can alter solubility properties experimentally. Additional studies showed that fusion of a proteasome-targeting PEST motif to the anti-huntingtin intrabody scFv-C4 can degrade mutant huntingtin proteins by directing them to the proteasome, while also increasing the negative charge. We now validate the generality of this approach with intrabodies against α-synuclein (α-syn), an important target in Parkinson disease. In this study, fusion of the PEST sequence to a set of four diverse, poorly soluble anti-α-syn intrabodies (D5E, 10H, D10 scFv, VH14 nanobody) significantly increased steady-state soluble intrabody protein levels in all cases, despite fusion with the PEST proteasomal-targeting signal. Furthermore, adding this PEST motif to the least soluble construct, VH14, significantly enhanced degradation of the target protein, α-syn~GFP. The intrabody-PEST fusion approach thus has dual advantages of potentially solubilizing intrabodies and enhancing their functionality in parallel. Empirical testing of intrabody-PEST fusions is recommended for enhancement of intrabody solubility from diverse sources.

Keywords: Parkinson disease; intrabodies; intrabody-PEST fusions; proteasome; α-synuclein.

Figure 1. Intracytoplasmic soluble expression of intrabody-PEST and intrabody-SCRPEST constructs is increased significantly with respect to intrabody alone. (A) D5E scFv, designated E; (B) 10H scFv, designated H; (C) D10 scFv, designated D; (D) VH14, designated V. ST14A cells were transiently transfected with Intrabody-hemagluttinin (HA), Intrabody-HA-PEST or Intrabody-HA-SCRPEST constructs. 48 h post-transfection, cells were harvested, cell lysates prepared, soluble protein separated and transferred on western blots. Proteins were identified using anti-HA monoclonal antibodies, with actin as a loading control. Proteins were quantified densitometrically and normalized to actin. Negative control lanes, empty vector only (pcDNA3.1-), were always blank. At least 3 independent experiments were performed and representative gels are illustrated. One-way ANOVA with Minitab statistical software was used to perform statistical significance. (*p < 0.05, **p < 0.01, compared with intrabody-HA)

Figure 2. Intracytoplasmic soluble expression of intrabody-PEST and intrabody-scrambled PEST constructs is increased significantly with respect to intrabody alone when treated with epoxomicin. (A) D5E scFv, designated E; (B) 10H scFv, designated H; (C) D10 scFv, designated D; (D) VH14, designated V. For all intrabodies, the level of soluble protein is significantly higher in epoxomicin treated cells compared with that of vehicle (DMSO) treated cells in Figure 1; Ratio of Figure 2 (+inhibitor) to Figure 1 (no inhibitor) values are shown at the bottom of each graph in Figure 2. Multiple fold increase is not apparent by just looking at the y axis scales of the Figure 1 and 2 because multiple exposures were taken for each treatment. Actin was used as a control to evaluate protein levels across various exposures. ST14A cells were transiently transfected with intrabody, intrabody-PEST or intrabody-scrambled-PEST constructs. Twelve hours prior to harvest cells were treated with 9 μM epoxomicin and 48 h after the transfection cells were harvested, cell lysates prepared, soluble protein separated and run on western blots. Proteins were identified using anti-HA, with actin as a loading control. Proteins were quantified densitometrically and normalized to actin. Inhibitor and non-inhibitor lanes actin bands were used separately from different exposures to calculate ratios, using only non-saturated exposures. At least 3 independent experiments were performed and representative gels are illustrated. One-way ANOVA with Minitab statistical software was used to perform statistical significance. (*p < 0.05, **p < 0.01, compared with intrabody-HA)

Figure 3. α-syn-GFP is degraded significantly in presence of VH14-PEST when compared with control (empty vector: pcDNA3.1- only treatment), VH14 or VH14-scrambled-PEST treated ST14A cells. ST14A cells were transiently co-transfected with α-syn-GFP and control or VH14 or VH14-PEST or VH14-scrambled-PEST constructs. 48 h. post-transfection, cells were harvested, cell lysates prepared, soluble protein separated and run on western blots. Proteins were identified using GFP antibody, and actin was used as a loading control. Proteins were quantified densitometrically and normalized to actin. At least 6 independent experiments were performed. One-way ANOVA with Minitab statistical software was used to perform statistical significance. (*p < 0.05, **p < 0.01, compared with intrabody-HA)