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Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag
Published on: December 14, 2017
Biochemical analysis of catalytic and disease-associated residues in mouse Golgi-resident
Ryota Urushihara1, Yasuhisa Maruno1, Saki Maruoka1
1Laboratory of Biophysical Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
Abstract:
Golgi-resident 3'-phosphoadenosine-5'-phosphate (PAP) phosphatase BPNT2/gPAPP plays an essential role in maintaining sulfation homeostasis, but its biochemical characterization has been limited by the difficulty of in vitro analyses. Here, we established a stable expression and activity assay system using a Trigger factor-fused BPNT2 construct. Using this system, we performed AlphaFold 3-guided mutational analyses. The predicted model and mutational analyses showed that several acidic residues are required for efficient PAP phosphatase activity, likely through their contribution to Mg²⁺ coordination. In addition, T177 was suggested to help maintain an active-site geometry that could accommodate a catalytic water molecule. Conservation analysis and lithium inhibition supported the idea that BPNT2 shares a conserved Mg²⁺-dependent catalytic framework with related PAP phosphatases. Disease-associated variants D175N and T181P showed substantially reduced activity, possibly due to impaired Mg2+ coordination and local structural distortion. These findings provide a biochemical framework for understanding BPNT2 function in Golgi sulfation regulation.
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