Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody - PubMed (original) (raw)

Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody

David W Colby et al. Proc Natl Acad Sci U S A. 2004.

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Abstract

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion in the number of polyglutamine-encoding CAG repeats in the gene that encodes the huntingtin (htt) protein. A property of the mutant protein that is intimately involved in the development of the disease is the propensity of the glutamine-expanded protein to misfold and generate an N-terminal proteolytic htt fragment that is toxic and prone to aggregation. Intracellular antibodies (intrabodies) against htt have been shown to reduce htt aggregation by binding to the toxic fragment and inactivating it or preventing its misfolding. Intrabodies may therefore be a useful gene-therapy approach to treatment of the disease. However, high levels of intrabody expression have been required to obtain even limited reductions in aggregation. We have engineered a single-domain intracellular antibody against htt for robust aggregation inhibition at low expression levels by increasing its affinity in the absence of a disulfide bond. Furthermore, the engineered intrabody variable light-chain (V(L))12.3, rescued toxicity in a neuronal model of HD. We also found that V(L)12.3 inhibited aggregation and toxicity in a Saccharomyces cerevisiae model of HD. V(L)12.3 is significantly more potent than earlier anti-htt intrabodies and is a potential candidate for gene therapy treatment for HD. To our knowledge, this is the first attempt to improve affinity in the absence of a disulfide bond to improve intrabody function. The demonstrated importance of disulfide bond-independent binding for intrabody potency suggests a generally applicable approach to the development of effective intrabodies against other intracellular targets.

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Figures

Fig. 1.

Fig. 1.

A single-domain intrabody against htt was engineered for high affinity in the absence of a disulfide bond. (A) Histograms of yeast cell-surface expression levels for VL and VL,C22V,C89A, indicating comparable levels of expression with and without the disulfide bond. mfu, mean fluorescence units. (B) Antigen-binding curves for YSD VL mutants measured by flow cytometry. Values normalized to maximal intensity were measured, except for VL,C22V,C89A, which was normalized to maximal intensity measured for VL.VL (♦) has a _K_d of ≈30 nM, whereas VL with cysteine mutations (VL,C22V,C89A; •) has significantly lower binding affinity (>10 μM). Repeated rounds of random mutagenesis of VL,C22V,C89A, followed by sorting for improved binding resulted in the mutant VL12.3, which has a _K_d of ≈3 nM. (C) Effect of VL and VL,C22V,C89A on htt aggregation in ST14A cells transiently transfected with indicated intrabody or vector control and httex1Q97-GFP at a 2:1 plasmid ratio. Both intrabodies are equally capable of partially blocking aggregation when overexpressed at high levels. ***, P < 0.001. (D) Homology model showing mutations obtained during engineering; the model contains residues present before mutagenesis. Cysteine residues are yellow. Mutations observed after mutagenesis and sorting are F37I (red), Y51D (pink), K67R (green), and A75T (orange).

Fig. 2.

Fig. 2.

Engineered VL12.3 robustly blocks htt aggregation in several different cell lines. (A) ST14A cells were transiently cotransfected with httex1Q97-GFP and either an intrabody [C4 (3) or VL12.3] or an empty control vector, and cells with visible aggregates were counted 1, 2, and 3 days posttransfection (5:1 intrabody:htt plasmid ratio, n = 3). VL12.3 (•) persistently eliminated htt aggregation over 3 days. (B) Dose–response of VL12.3 was measured at 2 days by using various intrabody:htt plasmid ratios (n = 3). (C) Fluorescence microscopy images of typical cells. (D) Flow cytometry histograms showing expression level per cell of httex1Q97-GFP in transfected cells in the presence of intrabody compared with empty vector (mean fluorescence intensity 82 vs. 76 mfu, respectively; transfection efficiencies were comparable in both samples, at 13% and 11%, respectively). (E) Comparison of intrabody activity for the intrabodies mentioned above and a non-htt binding intrabody (scFv ML3.9) and wild-type VL at a 1:1 intrabody:htt plasmid ratio (***, P < 0.001) in SH-SY5Y human neuroblastoma cells. (F) Partial dose–response for the same intrabodies in HEK293 cells. (G) Western blot of Triton X-100-soluble and -insoluble fractions of cells lysed 24 h after cotransfection at a 2:1 intrabody:htt ratio. (H) Anti-His6 Western blot of intrabody expression levels in transiently transfected HEK293 cells.

Fig. 3.

Fig. 3.

Engineered intrabody VL12.3 inhibits metabolic dysfunction in neuronal model of HD. ST14A cells were transfected with a plasmid encoding GFP, httex1Q25-GFP, httex1Q97-GFP, or httex1Q97-GFP with VL12.3 in a 2:1 ratio. (A) Live GFP-positive cells were collected by FACS in a 96-well plate, 35,000 cells per well, at 48 h posttransfection; typical dot plot is shown for a GFP sample. Other samples showed a similar pattern, and the sorting gate (boxed area) was the same in all instances. (B). Cells were incubated with MTT reagent for 3 h, solubilized, and the A570 was measured; mean values from three separate experiments containing all four samples are shown. Statistics directly over error bars are for comparison with GFP. ns, not significant; *, P < 0.05; **, P < 0.01. Statistics over brackets are comparisons between the two samples indicated. Four additional pairwise comparisons may be made between httex1Q97-GFP and httex1Q97-GFP plus VL12.3; the pooled results indicate a 56 ± 25% increase in A570; P < 0.001. Expression of httex1Q97-GFP significantly reduced the ability of cells to reduce MTT, but this effect was reversed by the coexpression of VL12.3.

Fig. 4.

Fig. 4.

VL12.3 suppresses aggregation and rescues toxicity in an S. cerevisiae model of HD. (A) Filter retardation assay showing httex1Q72-CFP aggregates (dark) from lysates of cells expressing httex1Q25-CFP or httex1Q72-CFP with either VL12.3 or an empty vector control. Dashed circles indicate where insoluble material would appear. Difference between 25Q with and without VL12.3 is insignificant and within the variance usually observed for the assay. (B) Spottings of yeast strains, indicating ability to grow on solid media. (C) Growth curves obtained by measuring the OD600 of yeast cultures. Yeast expressing VL12.3-YFP along with httex1Q72-CFP grow at rates comparable with those expressing htt with nonpathological polyglutamine repeat lengths, in contrast to those carrying an empty vector only.

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