Four subtypes of disease-causing missense mutations underlie pathogenic protein interactions in neurodegenerative VPS13A disease
Xing Lin, Yuta Ryoden, Chigure Suzuki, Hiroyuki Ishikawa, Takaharu Sakuragi, Yasuo Uchiyama, Shigekazu Nagata
Xing Lin, Yuta Ryoden, Chigure Suzuki, Hiroyuki Ishikawa, Takaharu Sakuragi, Yasuo Uchiyama, Shigekazu Nagata
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Research Article Genetics Neuroscience

Four subtypes of disease-causing missense mutations underlie pathogenic protein interactions in neurodegenerative VPS13A disease

Abstract

VPS13A is an intracellular lipid transfer protein comprising more than 3,000 amino acids. Mutations in human VPS13A cause VPS13A disease, a neurodegenerative disorder that affects movement and cognition. VPS13A forms a complex with the membrane protein XK to mediate ATP-induced phospholipid scrambling in the plasma membrane. Here, we established a mouse cell system expressing full-length mouse VPS13A and examined its interaction with XK. Mutational analysis revealed that VPS13A binds to XK through a C-terminal β-strand that interacts with a β-hairpin in the central region of XK, an interaction essential for scramblase activity. The XK paralog XKR2, which contains a similar β-hairpin structure, also associates with VPS13A and supports phospholipid scrambling. We analyzed 10 mouse VPS13A variants corresponding to human patient mutations and classified them into 4 groups: (a) L67P, I90K, and W2453R, which showed reduced expression; (b) A1091P and M3080R, which were normally expressed but lacked scramblase activity; (c) S1446P, Q2689H, Y2713C, and R3084H, which modestly impaired expression or activity; and (d) I2763R, which altered cell size and disrupted ER independently of XK. These findings define the VPS13A–XK interaction interface, clarify the functional impact of disease-causing mutations, and reveal an unexpected gain-of-function mutation of a VPS13A variant.

Authors

Xing Lin, Yuta Ryoden, Chigure Suzuki, Hiroyuki Ishikawa, Takaharu Sakuragi, Yasuo Uchiyama, Shigekazu Nagata

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Figure 3

Interaction of mouse XKR2 with VPS13A and its ability to support ATP-induced PtdSer exposure.

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Interaction of mouse XKR2 with VPS13A and its ability to support ATP-ind...
(A) Structural comparison of XK and XKR2. AlphaFold 3–predicted structures of mouse XK (AlphafoldDB, Q9QXY7) and XKR2 (AlphafoldDB, Q5GH68), excluding their N- and C-terminal regions, are shown. The β-hairpin region is enclosed in a red box. (B–E) Functional analysis of XKR2. Xk–/–DKO-P2X7 cells were transfected with mEGFP-tagged mouse XK or XKR2. (B) Whole-cell lysates were separated by SDS-PAGE and immunoblotted with an anti-GFP Ab or stained with CBB. (C) GFP fluorescence was observed using a microscope. Scale bar: 5 �m. (D) Membrane fractions were separated by BN-PAGE and immunoblotted with anti-VPS13A or anti-GFP Abs or stained with colloidal gold protein stain. Red arrowhead indicates VPS13A-XK-mEGFP or XKR2-mEGFP complex; band marked with an asterisk is an unidentified protein; black arrowhead indicates uncomplexed XK or XKR2. (E) Cells were treated with ATP and analyzed by flow cytometry for PtdSer exposure. (F–J) Effect of XKR2 missense mutations on VPS13A binding and scramblase activity. Xk–/–DKO-P2X7 cells were transformed with WT, E151A, E153A, or E151A/E153A DM mouse XKR2-mEGFP. (F) Whole-cell lysates were separated by SDS-PAGE and immunoblotted with anti-GFP Ab. (G–I) Membrane fractions were separated by BN-PAGE (G and I) and SDS-PAGE (H) and immunoblotted with anti-GFP Ab (G) or anti-VPS13A Ab (H and I). Red arrowheads indicate the VPS13A-XKR2 complex; the black arrowhead indicates uncomplexed XKR2. The band marked by an asterisk is an unidentified protein. (J) Cells were stimulated with ATP, and PtdSer exposure was analyzed by flow cytometry using Cy5–annexin V. In (E and J), PtdSer exposure is expressed as MFI. Data are presented as the mean ± SD (bar) of 3 independent experiments. Statistical significance was determined using Šídák’s multiple comparison test. **P < 0.01, ****P < 0.0001.