Novel Green Fluorescent Polyamines to Analyze ATP13A2 and ATP13A3 Activity in the Mammalian Polyamine Transport System.
Marine Houdou, Nathalie Jacobs, Jonathan Coene, Mujahid Azfar, Roeland Vanhoutte, Chris Van den Haute, Jan Eggermont, Veronique Daniëls, Steven H L Verhelst, Peter Vangheluwe
Author Information
Marine Houdou: Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium. ORCID
Nathalie Jacobs: Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium.
Jonathan Coene: Laboratory of Chemical Biology, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium. ORCID
Mujahid Azfar: Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium.
Roeland Vanhoutte: Laboratory of Chemical Biology, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium.
Chris Van den Haute: Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA. ORCID
Jan Eggermont: Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium. ORCID
Veronique Daniëls: Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA. ORCID
Steven H L Verhelst: Laboratory of Chemical Biology, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium. ORCID
Peter Vangheluwe: Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium. ORCID
Cells acquire polyamines putrescine (PUT), spermidine (SPD) and spermine (SPM) via the complementary actions of polyamine uptake and synthesis pathways. The endosomal P-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-type ATPase isoforms transport fluorescently labeled polyamine analogs with a distinct structure-activity relationship (SAR), suggesting that isoform-specific polyamine probes can be designed.
