Cellular interaction and cytotoxicity of the iowa mutation of apolipoprotein A-I (ApoA-IIowa) amyloid mediated by sulfate moieties of heparan sulfate

Abstract

The single amino acid mutation G26R in human apolipoprotein A-I (apoA-I) is associated with familial amyloid polyneuropathy III. ApoA-I carrying this mutation (apoA-IIowa) forms amyloid fibrils in vitro. Heparan sulfate (HS) is a glycosaminoglycan that is abundant at the cell surface and in the extracellular matrix. Although HS and its highly sulfated domains are involved in aggregation of amyloid-β and accumulate in cerebral amyloid plaques of patients with Alzheimer disease and mouse models of this disease, the role of HS in familial amyloid polyneuropathy III has never been addressed. Here, we used cell models to investigate the possible role of HS in the cytotoxicity of apoA-IIowa amyloid. Wild-type CHO cells, but not pgsD-677 cells, an HS-deficient CHO mutant, demonstrated uptake of apoA-IIowa amyloid after incubation with the amyloid. Addition of sulfated glycosaminoglycans to culture media prevented interaction with and cytotoxicity of apoA-IIowa amyloid to CHO cells. Elimination of cell surface HS or inhibition of HS sulfation with chemical reagents interfered with interaction of apoA-IIowa amyloid with CHO cells. We also found that cellular interaction and cytotoxicity of apoA-IIowa amyloid were significantly attenuated in CHO cells that stably expressed the human extracellular endoglucosamine 6-sulfatases HSulf-1 and HSulf-2. Our results thus suggest that cell surface HS mediates cytotoxicity of apoA-IIowa amyloid and that enzymatic remodeling of HS mitigates the cytotoxicity.

Keywords: HSulf; amyloid; amyloidosis; apolipoprotein; familial amyloid polyneuropathy; glycobiology; heparan sulfate; proteoglycan.

FIGURE 1.

Cellular interaction and cytotoxicity of apoA-I amyloid depend on HS. A, wild-type CHO (CHO-WT) and pgsD-677 cells were incubated with 1 μm apoA-I amyloid with or without the Iowa mutation, after which apoA-I amyloid levels in whole cell lysates were determined by using Western blotting and dot blotting. β-Actin was used as a loading control. The graph shows quantification of cellular apoA-I amyloid. Data are means ± S.E. of three independent experiments. Where no error bars appear, the experimental error was smaller than the symbol itself. **, p < 0.01 versus CHO cells. B, cellular apoA-I amyloid levels were determined by dot blotting after treating CHO cells with 1–10 μm apoA-I_Iowa amyloid. β-Actin was used as a loading control. C, CHO cells were plated and treated with apoA-I_Iowa fibrils for 12 h at 37 °C. The cells were further treated with 0.25% trypsin/EDTA for 10 min at 37 °C to remove the cell surface-associated fibrils, after which whole cell lysates were prepared. The apoA-I content in the whole cell lysate was analyzed by dot blotting. β-Actin was used as a loading control. D, cells were plated and treated with 1 μm apoA-I_Iowa fibrils for 12 h. The cells were then washed with PBS three times and cultured for an additional 12 h in fresh DMEM/F-12 in the presence or absence of chloroquine at 37 °C. The apoA-I content in the whole cell lysates was analyzed by dot blotting. β-Actin was used as a loading control. E, CHO and pgsD-677 cells were plated on 24-well plates and treated with 1 μm apoA-I amyloid, and cell viability, assessed with the MTT assay, was expressed as a percentage of untreated cells. Where no error bars appear, the experimental error was smaller than the symbol itself. Results represent means ± S.E. (n = 5). *, p < 0.05 versus CHO cells.

FIGURE 2.

Cellular interaction of apoA-I amyloid is dependent on HS in CHO cells. A and B, CHO and pgsD-677 cells were plated on poly-l-lysine-coated cover glasses and incubated with 1 μm apoA-I amyloid, after which cells were permeabilized and stained with an anti-apoA-I antibody (red) and an anti-EEA1 antibody (B, green). DAPI counterstaining is shown in blue. Images at low (A) and high (B) magnifications are shown. Scale bar, 50 μm (A) and 10 μm (B).

FIGURE 3.

Mitochondrial depolarization induced by apoA-I_Iowa amyloid is attenuated in HS-deficient CHO cells. A, CHO and pgsD-677 cells plated on poly-l-lysine-coated cover glasses were treated with 1 μm WT apoA-I amyloid or apoA-I_Iowa amyloid for 12 h. Mitochondrial membrane potential was measured by using the TMRE fluorescent dye. Representative TMRE fluorescence (red) and differential interference contrast (DIC) images are shown. Scale bar, 50 μm. B, quantification of the TMRE signals. Data are means ± S.E. of three independent experiments. ***, p < 0.0001 versus CHO cells.

FIGURE 4.

Lysosomal dysfunction induced by apoA-I_Iowa amyloid is attenuated in HS-deficient CHO cells. A, pH changes in lysosomes are measured by using Lysosensor in CHO and pgsD-677 cells after treatment with 1 μm WT apoA-I amyloid or apoA-I_Iowa amyloid for 12 h. Representative acidic signals of Lysosensor (red) and differential interference contrast (DIC) images are shown. Scale bar, 50 μm. B, quantification of the signals rising from acidic organelles. Data are means ± S.E. of three independent experiments. **, p < 0.001 versus CHO cells; ***, p < 0.0001 versus CHO cells.

FIGURE 5.

Exogenously added heparin and HS inhibit cellular interaction of apoA-I_Iowa amyloid and protect CHO cells from apoA-I_Iowa amyloid cytotoxicity. A, CHO cells were treated with 1 μm apoA-I_Iowa amyloid with or without heparin (5 μg/ml), and whole cell lysates were prepared, followed by Western and dot blotting to determine apoA-I_Iowa amyloid levels in the lysates. β-Actin was used as a loading control. The graph shows quantification of cellular apoA-I_Iowa amyloid. Data are means ± S.E. of three independent experiments. Where no error bars appear, the experimental error was smaller than the symbol itself. **, p < 0.001 versus heparin (−). B, apoA-I_Iowa amyloid interacted with heparin. ApoA-I_Iowa amyloid (0.1 mg/ml) was incubated with or without fluorescent heparin (12.5 μg/ml). Aliquots (2.5 μl) of the samples were blotted on a nitrocellulose filter. After visualization of the fluorescent signals, apoA-I_Iowa amyloid on the filter was probed with an anti-apoA-I antibody (lower panel). C, ELISA for the binding of the Iowa mutant apoA-I fibrils to immobilized heparin. Results represent means ± S.E. (n = 5). Where no error bars appear, the experimental error was smaller than the symbol itself. E and F, CHO cells were treated with 1 μm apoA-I_Iowa amyloid with or without HS (D) or HA (E) (5 μg/ml), and whole cell lysates were prepared, followed by dot blotting to determine apoA-I_Iowa amyloid levels in the lysates. β-Actin was used as a loading control. The graph shows quantification of cellular apoA-I_Iowa amyloid. Data are means ± S.E. of three independent experiments. **, p < 0.001 versus GAG (−). F, CHO cells were plated on 24-well plates and treated with 1 μm apoA-I_Iowa amyloid with or without heparin (left), HS (middle), or HA (right) (5 μg/ml), and cell viability, assessed with the MTT assay, was expressed as a percentage of untreated cells. Results represent means ± S.E. (n = 5). **, p < 0.001 versus GAG (−).

FIGURE 6.

Heparin attenuates cellular interaction of apoA-I_Iowa amyloid. A and B, CHO cells were plated on poly-l-lysine-coated cover glasses and incubated with 1 μm apoA-I_Iowa amyloid with or without heparin (5 μg/ml), after which cells were permeabilized and stained with antibodies, and bound antibodies were visualized. DAPI counterstaining is shown in blue. Images at low (A) and high (B) magnifications are shown. Scale bar, 50 μm (A) and 10 μm (B).

FIGURE 7.

β-Xyloside inhibits cellular interaction of apoA-I_Iowa amyloid. A, CHO cells were plated on poly-l-lysine-coated cover glasses, treated with 2.5 mm β-xyloside, incubated with 1 μm apoA-I_Iowa amyloid, permeabilized, and stained with anti-apoA-I antibody, after which bound antibodies were visualized. DAPI counterstaining is shown in blue. Scale bar, 50 μm. B, graph shows quantification of amyloid-positive cell numbers. Data are means ± S.E. of three independent experiments. ***, p < 0.0001 versus β-xyloside (−).

FIGURE 8.

Inhibition of cellular sulfation modifications suppresses cellular interaction of apoA-I_Iowa amyloid. A, effect of sodium chlorate on sulfation modifications of HS. CHO cells were treated with 100 mm sodium chlorate for 24 h, after which whole cell lysates were prepared. RB4CD12 signals were eliminated by treatment with sodium chlorate. β-Actin was used as a loading control. B, CHO cells were plated on poly-l-lysine-coated cover glasses, treated with 100 mm sodium chlorate, incubated with 1 μm apoA-I_Iowa amyloid, permeabilized, and stained with anti-apoA-I antibody, after which bound antibodies were visualized. DAPI counterstaining is shown in blue. Scale bar, 50 μm. C, graph shows quantification of amyloid-positive cell numbers. Data are means ± S.E. of three independent experiments. **, p < 0.001 versus sodium chlorate (−).

FIGURE 9.

Stable expression of HSulf-1 or HSulf-2 inhibits cellular interaction of apoA-I_Iowa amyloid and ameliorates cytotoxicity of this amyloid. A, nontransfectant CHO cells and transfectants stably expressing HSulf-1 or HSulf-2 were plated on poly-l-lysine-coated cover glasses, treated with 1 μm apoA-I_Iowa amyloid, permeabilized, and stained with anti-apoA-I antibody, after which bound antibodies were visualized. DAPI counterstaining is shown in blue. Scale bar, 50 μm. B, graph shows quantification of amyloid-positive cell numbers. Data are means ± S.E. of three independent experiments. **, p < 0.001 versus nontransfectants. C, CHO cells and their transfectants were plated on 24-well plates and treated with 5 μm apoA-I_Iowa amyloid, and cell viability, assessed with the MTT assay, was expressed as a percentage of untreated cells. Results represent means ± S.E. (n = 5). **, p < 0.001 versus nontransfectants.