Comparison of five peptide vectors for improved brain delivery of the lysosomal enzyme arylsulfatase A - PubMed (original) (raw)
Comparative Study
Comparison of five peptide vectors for improved brain delivery of the lysosomal enzyme arylsulfatase A
Annika Böckenhoff et al. J Neurosci. 2014.
Abstract
Enzyme replacement therapy (ERT) is a treatment option for lysosomal storage disorders (LSDs) caused by deficiencies of soluble lysosomal enzymes. ERT depends on receptor-mediated transport of intravenously injected recombinant enzyme to lysosomes of patient cells. The blood-brain barrier (BBB) prevents efficient transfer of therapeutic polypeptides from the blood to the brain parenchyma and thus hinders effective treatment of LSDs with CNS involvement. We compared the potential of five brain-targeting peptides to promote brain delivery of the lysosomal enzyme arylsulfatase A (ASA). Fusion proteins between ASA and the protein transduction domain of the human immunodeficiency virus TAT protein (Tat), an Angiopep peptide (Ang-2), and the receptor-binding domains of human apolipoprotein B (ApoB) and ApoE (two versions, ApoE-I and ApoE-II) were generated. All ASA fusion proteins were enzymatically active and targeted to lysosomes when added to cultured cells. In contrast to wild-type ASA, which is taken up by mannose-6-phosphate receptors, all chimeric proteins were additionally endocytosed via mannose-6-phosphate-independent routes. For ASA-Ang-2, ASA-ApoE-I, and ASA-ApoE-II, uptake was partially due to the low-density lipoprotein receptor-related protein 1. Transendothelial transfer in a BBB cell culture model was elevated for ASA-ApoB, ASA-ApoE-I, and ASA-ApoE-II. Brain delivery was, however, increased only for ASA-ApoE-II. ApoE-II was also superior to wild-type ASA in reducing lysosomal storage in the CNS of ASA-knock-out mice treated by ERT. Therefore, the ApoE-derived peptide appears useful to treat metachromatic leukodystrophy and possibly other neurological disorders more efficiently.
Keywords: arylsulfatase A; blood–brain barrier; enzyme replacement therapy; lysosomal storage disease; metachromatic leukodystrophy; peptide vectors.
Figures
Figure 1.
Biochemical characterization of ASA fusion proteins. A, Schemes of ASA fusion proteins. Amino acid sequences of the peptide vectors are given in the one-letter code. Domains are not drawn to scale. B, Western blot analysis. Fifty nanograms of purified ASA and 2.5–10 μl of conditioned medium from CHO-S cells stably expressing ASA fusion proteins were loaded per lane and reacted with a polyclonal anti-human ASA antibody. C, Deglycosylation of ASA fusion proteins with EndoH. Digested and undigested protein was separated by SDS-PAGE and stained with PageBlue Protein Staining solution (Thermo Scientific). D, M6P contents and specific activities as indicated.
Figure 2.
Cellular uptake of ASA fusion proteins. A, Uptake by CHO-K1, MEF, and bEND.3 cells as indicated. Cells were either coincubated with 7.5 m
m
M6P to block MPR300-mediated endocytosis (lower values) or, as a control, with 7.5 m
m
G6P (upper values). Uptake is expressed as nanograms of intracellular ASA per microgram of input ASA and micrograms of extractable protein. Numbers represent mean ± SD of n = 3 wells per condition. S-signs denote statistically significant differences between M6P and G6P feedings (Student's t test, p < 0.05). Asterisks and plus signs indicate statistical differences between an ASA fusion protein and the ASA control in the presence of G6P and M6P, respectively. B, Uptake by MEF-mpr−/− cells (deficient for MPR300). Bars represent mean ± SD of n = 4–6 wells per condition. Asterisks indicate statistically significant differences from wild-type ASA (Student's t test, p < 0.05). C, LRP1-dependent endocytosis. CHO-K1 wild-type cells (closed bars) and CHO-lrp−/− cells (deficient for LRP1; open bars) were incubated with 7.5 m
m
M6P to block M6P-dependent endocytosis. Bars represent mean ± SD of n = 3 wells per condition. Asterisks indicate a statistically significant difference between LRP1-deficient and wild-type CHO-K1 cells (Student's t test, p < 0.05).
Figure 3.
Immunofluorescence staining of MEF-asa−/− (ASA-deficient) cells after feeding of ASA fusion proteins. Cells were fed with wild-type ASA (A–C), ASA-Tat (D–F), ASA-ApoB (G–I), ASA-ApoE-I (J–L), ASA-ApoE-II (M–O), and ASA-Ang-2 (P–R), respectively. Recombinant enzyme and LAMP-2 were stained in red (middle) and green (right), respectively. Merge panels (left) also show nuclei counterstained with DAPI (blue). Scale bars, 10 μm.
Figure 4.
Basolateral transfer of ASA fusion proteins in an in vitro BBB model. A, Scheme of the experimental setup. B, Basolateral concentrations of ASA (closed bars) and ASA fusion proteins (open bars) were measured 24 h after the addition of 0.1 mg/ml purified protein to the apical medium. Bars represent mean ± SD of n = 3–5 wells per condition. Asterisks indicate statistically significant differences between ASA and ASA fusion proteins (Student's t test, p < 0.05).
Figure 5.
Tissue distribution of ASA fusion proteins. ASA-knock-out mice were injected with ASA-Tat (A), ASA-ApoB (B), ASA-ApoE-I (C), ASA-ApoE-II (D), and ASA-Ang-2 (E), respectively (open bars). In F, APOE-knock-out mice were injected with ASA-ApoE-II. For each fusion protein, a separate set of control mice was treated with ASA (closed bars). Bars represent mean ± SD of n = 4–5 mice. Asterisks indicate statistically significant differences between wild-type ASA and ASA fusion proteins (Student's t test, p < 0.05).
Figure 6.
Reduction of sulfatide storage by ERT. Sulfatide and cholesterol levels were quantified in kidney (A) and total brain (B) of ASA-knock-out mice (ASA−/−) treated with vehicle (mock), wild-type ASA (wt ASA), and ASA-ApoE-II, respectively. Untreated wild-type mice (ASA+/+) were used as controls. Sulfatide levels were normalized on cholesterol levels and are expressed as the fold increase above mean levels of wild-type tissues. Bars represent mean ± SD of n = 3 mice per group. ASA-ApoE-II reduced sulfatide storage more efficiently than wild-type ASA (Student's t test, _p-_values indicated).
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