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. 2016 Nov 23:6:37583.
doi: 10.1038/srep37583.

Identification of Small Molecule Compounds for Pharmacological Chaperone Therapy of Aspartylglucosaminuria

Antje Banning  1 , Christina Gülec  1 , Juha Rouvinen  2 , Steven J Gray  3 , Ritva Tikkanen  1
Affiliations

Identification of Small Molecule Compounds for Pharmacological Chaperone Therapy of Aspartylglucosaminuria

Antje Banning et al. Sci Rep. .

Abstract

Aspartylglucosaminuria (AGU) is a lysosomal storage disorder that is caused by genetic deficiency of the enzyme aspartylglucosaminidase (AGA) which is involved in glycoprotein degradation. AGU is a progressive disorder that results in severe mental retardation in early adulthood. No curative therapy is currently available for AGU. We have here characterized the consequences of a novel AGU mutation that results in Thr122Lys exchange in AGA, and compared this mutant form to one carrying the worldwide most common AGU mutation, AGU-Fin. We show that T122K mutated AGA is expressed in normal amounts and localized in lysosomes, but exhibits low AGA activity due to impaired processing of the precursor molecule into subunits. Coexpression of T122K with wildtype AGA results in processing of the precursor into subunits, implicating that the mutation causes a local misfolding that prevents the precursor from becoming processed. Similar data were obtained for the AGU-Fin mutant polypeptide. We have here also identified small chemical compounds that function as chemical or pharmacological chaperones for the mutant AGA. Treatment of patient fibroblasts with these compounds results in increased AGA activity and processing, implicating that these substances may be suitable for chaperone mediated therapy for AGU.

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Figures

Figure 1
Figure 1. Characterization of the novel T122K aspartylglucosaminuria mutation.
(A) Mutations that result in T122K and Arg161Gln plus Cys163Ser amino acid changes in AGU. Please note that Cys163Ser is the disease causing mutation, whereas Arg161Gln is a functionally neutral polymorphism. (B) AGA activity in control and AGU fibroblasts. N ≥ 7, shown as the mean of the data ± SD. Statistical analysis by One-Way Anova. (C) Processing of AGA in fibroblasts of AGU patients. (D) Localization of the mutated residues R116 and T122 in the structure of human AGA. The two αβ heterodimers are in cyan/blue and red/purple. (E) Processing of overexpressed, untagged AGA in HeLa cells. (F) AGA activity in cell lysates of transfected HeLa cells, N ≥ 10, shown as the mean of the data ± SD. Statistical analysis by One-Way Anova.
Figure 2
Figure 2. Lysosomal abnormalities in AGU fibroblasts.
Staining of control, T122K and AGU-Fin fibroblasts for (A) AGA (red) and LAMP3 (green); (B) Lysotracker-red and (C): LAMP-1. Scale bar 20 μm.
Figure 3
Figure 3. Mutated AGA proteins do not accumulate in the Golgi or ER.
Staining of control, T122K and AGU-Fin fibroblasts for AGA (red) and (A) calnexin (green) or (B) cation-independent Mannose-6-phosphate receptor (MPR, green). Scale bar 20 μm.
Figure 4
Figure 4. Coexpression of wildtype AGA results in enhanced activation of T122K and AGU-Fin mutated AGA proteins.
Strep-tagged wildtype, AGU-Fin, T122K and R116W AGA proteins were coexpressed or not with the untagged wildtype AGA protein. Upper part: Western blot with the anti-Strep-tag antibody (recognizes tagged constructs), lower part: anti-AGA antibody (recognizes both tagged and untagged proteins). Please note that the Strep tag was localized in the C-terminus of the β subunit, and untagged α subunit is thus not detectable with the strep-tag antibody. Enhanced processing of AGU-Fin and T122K can be observed upon co-expression of the wildtype protein, whereas R116W remains as a precursor. N = 6.
Figure 5
Figure 5. Chaperone treatment enhances AGA activity in AGU fibroblasts.
(A–C) Wildtype, T122K and AGU-Fin fibroblasts were treated for 48 h with 10 mM chaperone substances, and the AGA activity was measured. (A) Glycine, (B) betaine, (C) Asp. (D) Activity increase by the chemical chaperones is comparable to that obtained by treatment (48 h) with 50 ng/ml recombinant human AGA. (E) HEK 293 T cells stably transfected with Strep-tagged T122K AGA were treated as indicated and the processing of AGA was detected by Western blot using anti-AGA antibodies. GAPDH was used as a loading control. (F–H) Treatment of purified, recombinant AGA enzymes with the chaperone substances. (F): Wildtype AGA, (G) T122K AGA, (H): AGU-Fin AGA. (A–C) & (F–H) N ≥ 6, graphs show the mean of the data ± SD. Statistical analysis by Two-Way Anova.
Figure 6
Figure 6. Treatment with chaperone substances ameliorates lysosomal pathology in AGU fibroblasts.
(A) Lysotracker-red staining of wildtype, T122K and AGU-Fin fibroblasts treated with Gly or betaine. (B) Quantification of the Lysotracker intensity after chaperone treatment. At least 60 cells from 4 experiments were quantified. The data are shown as mean ± SD, statistical analysis by Two-Way Anova.
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