Life science
Medical care, Drug development

Flippase and scramblase
WPI Immunology Frontier Research Center
Specially Appointed Professor Shigekazu Nagata
https://researchmap.jp/Nagata0306

Life science

Abstract
Phospholipids are asymmetrically distributed between the innerand outer leaflets of plasma membranes with exclusive phosphatidylserine (PtdSer) localization in the inner leaflet. This asymmetrical distribution is maintained by ATP-dependent flippases, which
specifically translocate PtdSer from outer to inner leaflets. Whencells undergo apoptosis, the asymmetrical distribution of phospholipids is disrupted by a scramblase, leading to PtdSer-exposure.
The PtdSer, thus exposed to the cell surface, works as an “eat me”
signal for apoptotic cells. The PtdSer-exposure is also observedin other processes, such as activated platelets. We found that apatient with neurological deterioration carries a point mutation in aflippase, causing the flipping of phosphatidylcholine (PtdCho). We
also determined the tertiary structure of the scramblase responsible for the apoptotic PtdSer exposure.

▍ Background & Results
Two P4-type ATPases (ATP11A and 11C) are flippases at plasma membranes. A group at Tohoku University detected a de novo
point mutation of ATP11A in a patient with neurological deterioration. This mutation caused an amino acid substitution (Q84E) in thefirst transmembrane segment of ATP11A, and the mutant ATP11A
flipped PtdSer and PtdCho. Aberrant PtdCho flipping decreased
PtdCho in the outer leaflet of plasma membranes and increased
sphingomyelin (SM). This change altered cell characteristics,
including cholesterol homeostasis and sensitivity to sphingomyelinase.
Two families of membrane proteins (TMEM16 and XKR) support
the scrambling of phospholipids at plasma membranes. XKR8forms a complex with Basigin and is cleaved by a caspase at theC-terminus to function as a scramblase. By applying cryo-EM analysis, we found that the XKR8-Basigin complex adopts a cuboidlike structure. A PtdCho molecule was present in a hydrophobic
cleft on the surface. Six charged residues inside the molecule wereessential for scrambling phospholipids in inward and outward directions, providing a path for the translocation of phospholipids.

Figure 1. A point mutation (Q84E) in human ATP11A flippase that causes the intracellular transport of PtdCho(PC) (1), up-regulation of sphingomyelin synthase(SGMS1)(2), and the synthesis of SM (3).

▍ Significance of the research and Future perspective
In this study, we have elucidated the etiology of a human patientwith neurological deterioration. Our results will lead to how thepatient should be treated. Our result also showed that the flippaseshould strictly follow substrate specificity and mislocalization ofphospholipids causes severe human disease. Missense and nonsense mutations have been identified in the various P4-ATPase ofhuman patients. Our results will give a hint to determine the etiology of these diseases.
Phospholipids have amphiphilic characteristics and are not easy
to move between the lipid bilayer. The structure of the Xkr8-Basigin
would provide insights into the molecular mechanisms underlying
phospholipid scrambling. Since PtdSer-exposure occurs in various
physiological and pathological settings, the design of the scramblase will help develop drugs that modulate this process.

Figure 2. The tertiary structure of human XKR8-Basigin scramblase complex (A),and the transmembrane regions of XKR8 (B). The XKR8 on the upper periphery
carries a hydrophobic cleft with a PtdCho(PC) molecule.

P a t e n t Japanese Patent No. 6525212
Segawa, Katsumori; Kikuchi, Atsuo; Noji,Tomoyasu et al. A sublethal ATP11A mutation associated with neurological deterioration causes aberrant
phosphatidylcholine flipping in plasma membranes. J. Clin. Invest. 2021, 131, e148005, doi: 10.1172/jci148005
Tr e a t i s e
Sakuragi, Takaharu; Kanai, Ryuta; Tsutsumi, Akihisa et al. The tertiary structure of the human Xkr8-Basigin complex that scrambles phospholipids at
plasma membranes. Nat. Struct. Mol. Biol. 2021, 28, 825-834, doi: 10.1038/s41594-021-00665-8
URL http://biochemi.ifrec.osaka-u.ac.jp
K e y w o r d flippase, scramblase, phospholipid, phosphatidylserine