Acid sphingomyelinase activity is regulated by membrane lipids and facilitates cholesterol transfer by NPC2

Abstract

During endocytosis, membrane components move to intraluminal vesicles of the endolysosomal compartment for digestion. At the late endosomes, cholesterol is sorted out mainly by two sterol-binding proteins, Niemann-Pick protein type C (NPC)1 and NPC2. To study the NPC2-mediated intervesicular cholesterol transfer, we developed a liposomal assay system. (Abdul-Hammed, M., B. Breiden, M. A. Adebayo, J. O. Babalola, G. Schwarzmann, and K. Sandhoff. 2010. Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion. J. Lipid Res. 51: 1747–1760.) Anionic lipids stimulate cholesterol transfer between liposomes while SM inhibits it, even in the presence of anionic bis(monoacylglycero)phosphate (BMP). Preincubation of vesicles containing SM with acid sphingomyelinase (ASM) (SM phosphodiesterase, EC 3.1.4.12) results in hydrolysis of SM to ceramide (Cer), which enhances cholesterol transfer. Besides SM, ASM also cleaves liposomal phosphatidylcholine. Anionic phospholipids derived from the plasma membrane (phosphatidylglycerol and phosphatidic acid) stimulate SM and phosphatidylcholine hydrolysis by ASM more effectively than BMP, which is generated during endocytosis. ASM-mediated hydrolysis of liposomal SM was also stimulated by incorporation of diacylglycerol (DAG), Cer, and free fatty acids into the liposomal membranes. Conversely, phosphatidylcholine hydrolysis was inhibited by incorporation of cholesterol, Cer, DAG, monoacylglycerol, and fatty acids. Our data suggest that SM degradation by ASM is required for physiological secretion of cholesterol from the late endosomal compartment, and is a key regulator of endolysosomal lipid digestion.

Evaluation of plasma cholestane-3β,5α,6β-triol and 7-ketocholesterol in inherited disorders related to cholesterol metabolism

“…Therefore, a defi ciency of this enzyme would result in the lysosmal accumulation of esterifi ed cholesterol and in a relative depletion of free cholesterol, which may likely cause the activation of endogenous cholesterol biosynthesis by the upregulation of HMG-CoA reductase, resulting in the characteristic hypercholesterolemia observed in these patients and contributing to the formation of oxysterols ( 20,21 ). free cholesterol from the lysosomal compartment ( 18 ). The effects of SM excess on cellular cholesterol transport have been demonstrated in NP-B fi broblasts ( 19 ).…”

Membrane lipids regulate ganglioside GM2 catabolism and GM2 activator protein activity

“…In all intervesicular transfer experiments, we used 4 mol% quencher (rhodamine-PE) in the donor liposomes, twice as high as the level of the fl uorophore (2 mol%). Under these conditions, the highest level of dequench of NBD-fl uorescence reached by complete fusion of donor and acceptor vesicles was much lower than and secreted from these luminal vesicles mainly by two sterol binding proteins, NPC2 and NPC1 ( 11,31,47,69,70 ). Those intraendolysosomal vesicles have been identifi ed as platforms for lysosomal glycosphingolipid, sphingolipid, and membrane degradation (71)(72)(73).…”

“…In the case of the GM2AP, the C-terminal-tag places positive charges at the opposite side of the positively charged domain of the protein ( 43 ). This may infl uence lipid binding properties and the ability of GM2AP to escape from negatively charged membranes ( 11,16 ).…”

“…They all facilitate the interaction of the membrane-bound substrate with the water-soluble enzyme at the water-membrane interface and differ from sphingolipid transfer proteins of the cytosol ( 6 ). The anionic lysosomal phospholipid, BMP, is also known to dramatically enhance (glyco)sphingolipid hydrolysis by lysosomal hydrolases and sphingolipid activator proteins ( 7-9 ).Even at low pH values (pH <5.0), BMP conveys negative surface charge to luminal lysosomal vesicles ( 10,11 ). This favors an electrostatic binding of the positively charged activator proteins and the polycationic hydrolases, and enhances degradation of (glyco)sphingolipids substantially ( 7,9,10,(12)(13)(14)(15)(16).…”

“…Even at low pH values (pH <5.0), BMP conveys negative surface charge to luminal lysosomal vesicles ( 10,11 ). This favors an electrostatic binding of the positively charged activator proteins and the polycationic hydrolases, and enhances degradation of (glyco)sphingolipids substantially ( 7,9,10,(12)(13)(14)(15)(16).…”