HIV Tat Domain Improves Cross-correction of Human Galactocerebrosidase in a Gene- and Flanking Sequence-dependent Manner

Abstract

Krabbe disease is a devastating neurodegenerative lysosomal storage disorder caused by a deficiency of β-galactocerebrosidase (GALC). Gene therapy is a promising therapeutic approach for Krabbe disease. As the human brain is large and it is difficult to achieve global gene transduction, the efficacy of cross-correction is a critical determinant of the outcome of gene therapy for this disease. We investigated whether HIV Tat protein transduction domain (PTD) can improve the cross-correction of GALC. Tat-PTD significantly increased (~6-fold) cross-correction of GALC through enhanced secretion and uptake in a cell-culture model system. The effects of Tat-PTD were gene and flanking amino acids dependent. Tat-fusion increased the secretion of α-galactosidase A (α-gal A), but this did not improve its cross-correction. Tat-fusion did not change either secretion or uptake of β-glucocerebrosidase (GC). Tat-PTD increased GALC protein synthesis, abolished reactivity of GC to the 8E4 antibody, and likely reduced mannose phosphorylation in all these lysosomal enzymes. This study demonstrated that Tat-PTD can be useful for increasing cross-correction efficiency of lysosomal enzymes. However, Tat-PTD is not a mere adhesive motif but possesses a variety of biological functions. Therefore, the potential beneficial effect of Tat-PTD should be assessed individually on each lysosomal enzyme.Molecular Therapy-Nucleic Acids (2013) 2, e130; doi:10.1038/mtna.2013.57; published online 22 October 2013.

Figure 1

Schematic representations of the constructs. G4, 4-glycine spacer; HA, human influenza hemagglutinin epitope tag; His, hexa-histidine tag; HMM, an artificial secretion signal; TAT, HIV Tat protein transduction domain; α1AT and Fib, secretion signals from α1-antitrypsin and fibronectin respectively.

Figure 2

HIV Tat-fusion improves cross-correction of GALC. (a) GALC activities in lysates of Krabbe patient's fibroblasts (Pt. Fibro) and twitcher mouse Schwann cells (TwS1) incubated with conditioned media of 293T cells that were transfected with empty vector (mock), GALC-HA and GALC-TatHA (n = 3). (b) GALC in conditioned media of transfected 293T cells assessed by enzyme assay (upper, n = 3–4) and western blot analysis (lower). (c) GALC in lysates of transfected 293T cells assessed by enzyme assay (upper, n = 4–5) and western blot analysis (lower). (d) GALC activities in the lysates of Krabbe patient's fibroblasts (Pt. Fibro) and twitcher Schwann cells (TwS1) incubated with GALC-HA- or GALC-TatHA–containing conditioned media that have the same GALC activity (n = 3). Data are presented as mean ± SEM. *P < 0.05, ***P < 0.001 determined by Mann–Whitney test. GALC, β-galactocerebrosidase; HA, human influenza hemagglutinin epitope tag.

Figure 3

Tat-PTD increases protein expression of GALC. (a) Western blot analysis of GALC protein levels in the lysates and conditioned media of twitcher mouse fibroblasts (Tw2) stably expressing GALC-HA or GALC-TatHA. (b) mRNA levels of human GALC in transfected 293T cells and the retrovirus-infected Tw2 cells assessed by quantitative RT-PCR (n = 3). (c) Half-life of GALC protein in Tw2 cells expressing GALC-HA or GALC-TatHA assessed by cycloheximide chase assay. The representative western blot was shown (upper). Half-life was calculated using protein levels quantified by densitometry (lower, n = 3). (d) Western blot analysis of GALC protein in 293T cells that were treated with brefeldin A for 2 days after transfection. (e) Western blot analysis of GALC in 293T cells that were treated with tunicamycin for 2 days after transfection. Arrow and arrowhead indicate GALC with and without N-linked glycosylation. The molecular weight standards were shown on the left. Data are presented as mean ± SEM. *P < 0.05, determined by (b) Mann–Whitney test and (c) t-test. GALC, β-galactocerebrosidase; HA, human influenza hemagglutinin epitope tag; PTD, protein transduction domain.

Figure 4

Effect of Tat-PTD on improving cross-correction is gene- and flanking residues dependent. (a–e) Effects of Tat domain in α-gal A. (a) α-gal A activities in the lysates of transfected 293T cells (n = 4–5). (b) α-gal A activities in the conditioned media of transfected 293T cells (n = 4–5). (c) Western blot analysis of α-gal A protein in the lysates and media of transfected 293T cells. (d) Western blot analysis of α-gal A in 293T cells treated with brefeldin A after transfection. (e) Intracellular α-gal A activities of Fabry patient's fibroblasts that were fed with conditioned media from 293T cells transfected with various constructs (n = 5–9). (f–j) Effects of Tat domain in GC. (f) GC activities in the lysates of transfected 293T cells (n = 3). (g) GC activities in the conditioned media of transfected 293T cells (n = 3). (h) Intracellular GC activities of Gaucher patient's fibroblasts that were fed with conditioned media from 293T cells (n = 3). (i) GC protein in the cell lysates and conditioned media of transfected 293T cells analyzed by western blot with 8E4 or rabbit polyclonal antibody to GC. (j) Western blot analysis of GC in the lysates of transfected 293T cells after deglycosylation with neuraminidase, O-glycosidase and PNGase F. (k–m) Comparison of GALC-TatHA and GALC-HATat. (k) GALC activities in the lysates of transfected 293T cells (n = 4–5). (l) GALC activities in the conditioned media of transfected 293T cells (n = 3–4). (m) Intracellular GALC activities of Krabbe patient's fibroblasts loaded with media from transfected 293T (n = 3). Data are presented as mean ± SEM. *P < 0.05, ***P < 0.001 determined by (a,b,e,k–m) Mann–Whitney test and (f–h) t-test. GALC, β-galactocerebrosidase; GC, β-glucocerebrosidase; HA, human influenza hemagglutinin epitope tag; PTD, protein transduction domain; α-gal A, α-galactosidase A.

Figure 5

Heterogeneous secretion signals decrease secretion and expression of GALC. (a) GALC activities in the conditioned media of 293T cells transfected with constructs with various secretion signals (n = 3). (b) GALC activities in the lysates of transfected 293T cells (n = 3). (c) Western blot analysis of GALC protein in the conditioned media and cell lysate of 293T cells. Data are presented as mean ± SEM. *P < 0.05, determined by one-way analysis of variance followed by Bonferroni's multiple comparison test. GALC, β-galactocerebrosidase.