The Unique Relationship between Niemann Pick Type C2 (NPC2) Protein and Lysobisphosphatidic Acid (LBPA)

Niemann Pick Type C (NPC) disease is a lysosomal storage disorder characterized by cholesterol accumulation in the late endosome/lysosome (LE/LY) caused by genetic defects in either NPC1 or NPC2 proteins. Wild type (WT) NPC2 is a 16 kDa soluble protein that binds cholesterol with 1:1 stoichiometry. We previously showed that NPC2 interacts directly with membranes to transport cholesterol; structure‐function analysis has shown that several regions of the NPC2 surface are involved in cholesterol transport, suggesting that NPC2 can not only interact with membranes in various orientations, but can also bind multiple membranes simultaneously. Indeed, we demonstrated that WT NPC2 promotes vesicle‐vesicle interactions. Kinetic analysis of cholesterol transport suggests that NPC2 functionally interacts with the unique lysosomal phospholipid lysobisphosphatidic acid (LBPA), also known as bis(monoacylglycero)phosphate (BMP), as LBPA increases WT NPC2 cholesterol transport rates dramatically. Here, we used point mutagenesis to examine NPC2 sterol transfer rates with LBPA‐containing membranes, focusing on the “hydrophobic knob” motif, predicted to be membrane‐interactive. In silico interrogation of NPC2‐membrane interactions was performed using molecular dynamics simulations. Further studies examined sterol trafficking in npc1−/− and npc2−/− patient fibroblasts with varying levels of LBPA. The results show that specific point mutations in the “hydrophobic knob” cause defective sterol transport to and from membranes containing LBPA, suggesting that this is the LBPA‐sensitive region of NPC2. Molecular dynamics simulations of NPC2 with an explicit model membrane showed that while both the apo and holo states of NPC2 interact directly with the model membrane, each has different mechanisms and orientations of binding. The observation of multiple stable NPC2‐membrane binding orientations strongly supports experimental evidence that NPC2 interacts with multiple membranes. Interestingly, Chevallier, et al. (2008) demonstrated that an increase in lysosomal LBPA content of npc1−/− fibroblasts increased cholesterol efflux from the lysosomes and relieved the phenotypical cholesterol accumulation in the npc1−/− patient cells. Based on our demonstration that LBPA interacts with NPC2 and dramatically accelerates its cholesterol transfer rate, we hypothesized that LBPA would not be able to ‘rescue’ the cholesterol accumulation in npc2−/− fibroblasts. We successfully increased cellular LBPA levels by supplementing cells with phosphatidylglycerol. In agreement with Chevallier et al., increased LBPA levels normalized the sterol content of npc1−/− fibroblasts. In contrast, however, increased LBPA levels did not result in cholesterol clearance from npc2−/− fibroblasts. Overall, these studies indicate that NPC2 and LBPA have a specific, functional, and likely direct interaction, which is essential to efficiently clear cholesterol from the LE/LY compartment. Support or Funding Information Funding was provided by grants from the Ara Parseghian Medical Research Foundation and the American Heart Association.