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, 20 (18)

Inhibition of Mitochondrial Complex I Impairs Release of α-Galactosidase by Jurkat Cells

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Inhibition of Mitochondrial Complex I Impairs Release of α-Galactosidase by Jurkat Cells

Jonathan R A Lambert et al. Int J Mol Sci.

Abstract

Fabry disease (FD) is caused by mutations in the GLA gene that encodes lysosomal α-galactosidase-A (α-gal-A). A number of pathogenic mechanisms have been proposed and these include loss of mitochondrial respiratory chain activity. For FD, gene therapy is beginning to be applied as a treatment. In view of the loss of mitochondrial function reported in FD, we have considered here the impact of loss of mitochondrial respiratory chain activity on the ability of a GLA lentiviral vector to increase cellular α-gal-A activity and participate in cross correction. Jurkat cells were used in this study and were exposed to increasing viral copies. Intracellular and extracellular enzyme activities were then determined; this in the presence or absence of the mitochondrial complex I inhibitor, rotenone. The ability of cells to take up released enzyme was also evaluated. Increasing transgene copies was associated with increasing intracellular α-gal-A activity but this was associated with an increase in Km. Release of enzyme and cellular uptake was also demonstrated. However, in the presence of rotenone, enzyme release was inhibited by 37%. Excessive enzyme generation may result in a protein with inferior kinetic properties and a background of compromised mitochondrial function may impair the cross correction process.

Keywords: Fabry; cross correction; mitochondria.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Increase in intracellular α-gal-A activity following transfection and with no effect on other lysosomal hydrolases or cell viability. Intracellular α-gal-A activity increased with GLA transgene dose. Linear regression R2 = 0.835, p < 0.0001 (A). The activity of three other lysosomal enzymes was measured and shows no correlation with virus dose (B). Cell viability showed no evidence of cell toxicity following transduction. (C). Activity of α-gal-A and β-galactosidase in Jurkats transduced with lentivirus containing GFP did not correlate with virus dose (D).
Figure 1
Figure 1
Increase in intracellular α-gal-A activity following transfection and with no effect on other lysosomal hydrolases or cell viability. Intracellular α-gal-A activity increased with GLA transgene dose. Linear regression R2 = 0.835, p < 0.0001 (A). The activity of three other lysosomal enzymes was measured and shows no correlation with virus dose (B). Cell viability showed no evidence of cell toxicity following transduction. (C). Activity of α-gal-A and β-galactosidase in Jurkats transduced with lentivirus containing GFP did not correlate with virus dose (D).
Figure 2
Figure 2
Release of α-gal-A by GLA Jurkats. A-gal-A activity was measured in the culture media supernatant collected after zero, two and three days incubation at 37 °C with GLA-Jurkats containing between zero and 0.5 vg/cells. The extracellular activity in supernatant was plotted against intracellular activity within the GLA-Jurkats. Secretion of α-gal-A activity into the surrounding media by GLA-Jurkats increased with both intracellular activity and duration of incubation. Linear regression found that at day 0, R2 = 0.607 (p < 0.001); day 2, R2 = 0.786 (p < 0.0001); and day 3, R2 = 0.796 (p < 0.0001).
Figure 3
Figure 3
Uptake of alpha-galactosidase A activity by wild-type naive Jurkats. (A). Bathing (donor) media, from over expressing α-gal-A Jurkats, were taken after two days in culture. Recipient cells (wild-type Jurkats) were incubated for two days in the donor media. Intracellular α-galA was plotted against the initial α-gal-A activity in the donor media. There was significant increase in α-gal-A activity in the recipient cells with increasing donor media activity. Linear regression R2 = 0.450 (p < 0.01) (B). Comparison of the reduction in α-galA activity in donor media with and without cells present. There was a significant difference between the slopes of the lines (p < 0.0001), consistent with take-up of enzyme from donor media to recipient cells.
Figure 4
Figure 4
The effect of rotenone on mitochondrial complex I activity in wild-type Jurkats. An amount of 100 nM rotenone significantly (*** p < 0.0001) decreased complex I activity (protein baseline) when compared with untreated controls (A). There was no significant (ns) effect on citrate synthase activity (B). The ratio of complex I activity to citrate synthase also showed a significant (*** p < 0.0001) reduction in the presence of rotenone (C).
Figure 5
Figure 5
Effect of mitochondrial complex I inhibition on (A) lentivirus-mediated transduction efficiency, (B) enzyme release (** p < 0.001) and (C) enzyme uptake.

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