Novel Green Fluorescent Polyamines to Analyze ATP13A2 and ATP13A3 Activity in the Mammalian Polyamine Transport System

Abstract

Cells acquire polyamines putrescine (PUT), spermidine (SPD) and spermine (SPM) via the complementary actions of polyamine uptake and synthesis pathways. The endosomal P_5B-type ATPases ATP13A2 and ATP13A3 emerge as major determinants of mammalian polyamine uptake. Our biochemical evidence shows that fluorescently labeled polyamines are genuine substrates of ATP13A2. They can be used to measure polyamine uptake in ATP13A2- and ATP13A3-dependent cell models resembling radiolabeled polyamine uptake. We further report that ATP13A3 enables faster and stronger cellular polyamine uptake than does ATP13A2. We also compared the uptake of new green fluorescent PUT, SPD and SPM analogs using different coupling strategies (amide, triazole or isothiocyanate) and fluorophores (symmetrical BODIPY, BODIPY-FL and FITC). ATP13A2 promotes the uptake of various SPD and SPM analogs, whereas ATP13A3 mainly stimulates the uptake of PUT and SPD conjugates. However, the polyamine linker and coupling position on the fluorophore impacts the transport capacity, whereas replacing the fluorophore affects polyamine selectivity. The highest uptake in ATP13A2 or ATP13A3 cells is observed with BODIPY-FL-amide conjugated to SPD, whereas BODIPY-PUT analogs are specifically taken up via ATP13A3. We found that P_5B-type ATPase isoforms transport fluorescently labeled polyamine analogs with a distinct structure-activity relationship (SAR), suggesting that isoform-specific polyamine probes can be designed.

Keywords: P5B-type ATPases; fluorescently labeled polyamines; mammalian polyamine transport systems; radiolabeled polyamines.

Figure 1

Purification and ATPase activity of hATP13A2. (A). Coomassie staining showing the purified human ATP13A2 protein present in the different elution fractions (E1 to E4) from total membrane fraction (TMF) and onto the beads (B). (B). Immunoblot analysis of ATP13A2 purified protein from the fractions depicted in (A,C). ATPase activity of purified ATP13A2 (1.25 µg) measured with increasing concentrations of unlabeled spermine (SPM), clickable spermine (azide SPM), free BODIPY (alkyne BODIPY) and BODIPY-SPM, which structures are presented on the right (N = 2 to 7, technical duplicates, two-way ANOVA with Tukey’s multiple comparisons test; ns: not significant; ****: p < 0.001). The Y axis depicts the slope in optical density at 340 nm reflecting NADH consumption and further ATP consumption, normalized to the highest dose of SPM. V_max and K_m shown in the table were determined using the Michaelis–Menten fitting in GraphPad Prism software. N.D.— not determined.

Figure 2

Time- and dose-dependency of BODIPY-SPM uptake in ATP13A2-expressing SH-SY5Y cell models. (A,B). SH-SY5Y cells were incubated with 5 µM BODIPY-SPM for the indicated times ((A), time dependency) or with increasing concentrations of BODIPY-SPM for 2 h ((B), dose dependency). The mean fluorescence intensity (MFI) of 10,000 events was recorded using a BD Canto II HTS flow cytometer. Graphs represent the MFI recorded in ATP13A2 WT OE (A2 WT-OE) and ATP13A2 D508N-OE (A2 D508N-OE) cells, normalized to the 0 h incubation time in A2 D508N-OE cells (in A) or to the lowest concentration of BODIPY-SPM (i.e., 100 µM) in A2 D508N-OE cells (in B; ns: not significant, **: p < 0.01; ***: p < 0.001; ****: p < 0.0001). (C). Graph depicts the fold change in BODIPY-SPM uptake (5 µM; 2 h) between A2 WT-OE and A2 D508N-OE cells (N = 10, technical duplicates, unpaired t-test). (D). Comparison of the uptake of 5µM BODIPY-conjugated (BDP-SPM) versus 5 µM radiolabeled spermine (¹⁴C-SPM) in SH-SY5Y A2 WT-OE and D508N-OE cells for 30 min (N = 3, technical duplicates, one-way ANOVA with Tukey’s multiple comparisons test).

Figure 3

Time- and dose-dependency of BODIPY-PUT and BODIPY-SPD uptake in ATP13A3-expressing HMEC-1 cell models. (A,C). Time dependent BODIPY-polyamine uptake in HMEC-1 cells incubated with 5 µM BODIPY-PUT (A) or BODIPY-SPD (C). The mean fluorescence intensity (MFI) of 10,000 events was recorded using a BD Canto II HTS flow cytometer. Graphs represent the fold change between the MFI recorded in A3 WT-OE versus A3 D498N-OE cells, normalized to the 0 h incubation time in A3 D498N-OE cells. (N = 3 to 9, with technical duplicates, two-way ANOVA with Sidak’s multiple comparisons test). (B,D). Dose–response of BODIPY-PUT (B) or BODIPY-SPD (D) uptake in HMEC-1 cells at 30 min. The MFI of 10,000 events was recorded using a BD Canto II HTS flow cytometer. Graphs represent the fold change difference between the MFI recorded in A3 WT-OE and A3 D498N-OE cells, normalized to the highest concentration of BODIPY-polyamine (i.e., 100 µM) in A3 D498N-OE cells (N = 3 to 9, with technical duplicates, two-way ANOVA with Sidak’s multiple comparison test; ns: not significant, *: p < 0.1; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001). (E,F). Comparison of the uptake of 1 and 5 µM BODIPY-conjugated (BDP-PUT and BDP-SPD) versus radiolabeled putrescine and spermidine (¹⁴C-PUT and ¹⁴C-SPD) in HMEC-1 A3 WT-OE and D498N-OE cells for 30 min (N = 3 to 9, technical duplicates, two-way ANOVA with Tukey’s multiple comparisons test).

Figure 4

Simplified scheme representing the chemical synthesis of the different green fluorescent polyamine conjugates used in the study. A detailed description of the synthesis is given in Supplementary Information.

Figure 5

Comparison of the cellular uptake of BODIPY-conjugated versus BODIPY-FL-A, BODIPY-FL-T and FITC-conjugated polyamines in ATP13A2 and ATP13A3 cell models. (A–D). SH-SY5Y, ATP13A2, WT-OE and D508N-OE cells were incubated for 2 h with 5 µM of each fluorescently labeled polyamine. (E–H). HMEC-1, ATP13A3, WT-OE and D498N-OE cells were incubated for 30 min with 5 µM of each fluorescently labeled polyamine. The mean fluorescence intensity (MFI) was recorded using a BD Canto II flow cytometer based on the settings used for BODIPY-polyamines and normalized as indicated on the Y axis. Experiments were performed four independent times (N = 4) in ATP13A2 cell model and three independent times (N = 3) in ATP13A3 cell model, with technical duplicates. Statistical analysis was conducted using GraphPad Prism and a two-way ANOVA test with Sidak’s multiple comparison test.