Neuronal sphingolipidoses: Membrane lipids and sphingolipid activator proteins regulate lysosomal sphingolipid catabolism

Accepted Manuscript Membrane lipids and sphingolipid activator proteins regulate lysosomal sphingolipid catabolism Prof. Konrad Sandhoff PII:

S0300-9084(16)30079-7

DOI:

10.1016/j.biochi.2016.05.004

Reference:

BIOCHI 4990

To appear in:

Biochimie

Received Date: 2 February 2016 Accepted Date: 3 May 2016

Please cite this article as: K. Sandhoff, Membrane lipids and sphingolipid activator proteins regulate lysosomal sphingolipid catabolism, Biochimie (2016), doi: 10.1016/j.biochi.2016.05.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Neuronal Sphingolipidoses Membrane lipids and sphingolipid activator proteins regulate lysosomal

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sphingolipid catabolism 1

Abstract

Glycosphingolipids and sphingolipids of cellular plasma membranes (PMs) reach

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luminal intra-lysosomal vesicles (LVs) for degradation mainly by pathways of

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endocytosis. After a sorting and maturation process (e. g. degradation of sphingomyelin (SM) and secretion of cholesterol), sphingolipids of the LVs are digested by soluble enzymes with the help of activator (lipid binding and transfer) proteins. Inherited defects of lipid-cleaving enzymes and lipid binding and transfer

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proteins cause manifold and fatal, often neurodegenerative diseases.

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A short version of the presentation given at the 11th GERLI Lipidomic Meeting, Oct. 25-28, 2015, in Bischoffsheim, France 1

ACCEPTED MANUSCRIPT The review summarizes recent findings on the regulation of sphingolipid catabolism and cholesterol secretion from the endosomal compartment by lipid modifiers, an essential stimulation by anionic membrane lipids and an inhibition of crucial steps by cholesterol and SM. Reconstitution experiments in the presence of all proteins

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needed, hydrolase and activator proteins, reveal an up to 10-fold increase of

ganglioside catabolism just by the incorporation of anionic lipids into the ganglioside carrying membranes, whereas an additional incorporation of cholesterol inhibits GM2

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catabolism substantially. It is suggested that lipid and other low molecular modifiers affect the genotype-phenotype relationship observed in patients with lysosomal

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diseases. 2

Abbreviations: ASM, acid sphingomyelinase; BMP, bis(monoacylglycero)-phosphate; CAD,

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1. Introduction

cationic amphiphilic drug; DOPC, dioleoyl-L-α-phosphatidylcholine; ESCRT, endosomal sorting complexes required for transport; GM2AP, GM2 activator protein; g-rGM2AP, glycosylated recombinant human GM2AP; GSL, glycosphingolipid; HexA, hexosaminidase A; LTP, lipid transfer protein; LV, luminal intralysosomal vesicle; NBD, 7-nitrobenz-2-oxa-1,3diazol-4-yl; NPC, Niemann-Pick disease type C; NPC1, Niemann-Pick disease type C 1 protein; NPC2, Niemann-Pick disease type C 2 protein; PA, phosphatidic acid; PC, phosphatidylcholine; PG, phosphatidylglycerol; PM, plasma membrane; SAP, sphingolipid activator protein; SM, sphingomyelin 2

ACCEPTED MANUSCRIPT Most sphingolipid storage diseases are caused by inherited defects in lysosomal sphingolipid catabolism, often due to defective hydrolases [1]. Their catabolic activities are modified by the micro-environment of the enzyme reaction, known modifiers are pH-value, temperature and ionic strength. In case of sphingolipid

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degradation, lipid transfer proteins (LTPs) are also of utmost importance. Due to the lipid phase problem, water soluble lysosomal hydrolases can hardly attack their

membrane bound lipid substrates effectively. They need the help of lipid binding and

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transfer proteins that distort membrane structures, bind membrane lipids, especially sphingolipids, and present them to the water-soluble hydrolases [2]. Inherited defects

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of these lysosomal sphingolipid activator proteins (SAPs) also cause fatal lysosomal storage diseases [3]. Mostly unknown, however, is the strong impact of membrane lipids of the substrate carrying membrane on sphingolipid catabolism, which I will

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discuss in this mini review.

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2. Lipid and protein composition of eukaryotic membranes is organelle specific [4].

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ACCEPTED MANUSCRIPT Neuronal plasma membranes (PMs) are enriched and stabilized by gangliosides, sphingomyelin and cholesterol. A high cholesterol content of about 40 mol % of all membrane lipids is essential for the function of many membrane proteins, e.g. for the Na+- K+ ATPase to generate electrochemical gradients and to keep the sodium ion

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permeability down, close to zero [5]. A decrease in the cholesterol content of the PM would attenuate the generation of action potentials. On the other hand, cholesterol appears to be inhibitory for lysosomal degradation of sphingolipids [6] and, therefore,

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must be removed along the endosomal pathway. During endocytosis, luminal

vesicles (LVs) are generated with the help of ESCRT proteins [7]. The endosomal

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perimeter membrane forms invaginations which can pinch off to generate LVs (Fig. 1). We identified these intra-endosomal LVs as platforms for lipid and membrane degradation [8-10]. At the level of late endosomes, LVs mature by removal and efflux of cholesterol, mediated mainly by two sterol-binding and transfer proteins, the small

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water soluble glycoprotein, NPC2, and the perimeter membrane-spanning NPC1 [11]. A deficiency of either of these proteins causes a modest lysosomal accumulation of cholesterol in fatal Niemann-Pick disease type C (NPC) patients, though the overall

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cholesterol content of the brain tissue appears to be almost unchanged [12]. Furthermore, bis(monoacylglycero)-phosphate (BMP), an anionic lysophospholipid, is

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generated during endocytosis exclusively in the LVs of late endosomes and lysosomes, stimulating several degradative steps in sphingolipid catabolism [13].

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ACCEPTED MANUSCRIPT NPC2 can pick up cholesterol directly from vesicle surfaces and transfer it to other vesicles or hand it over to NPC1 for efflux from the late endosomes [14]. Unexpectedly, the cholesterol transfer activity of NPC2 is effectively regulated by the lipid composition of the donor and acceptor vesicles as studied in vitro. Whereas

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ceramide (Cer) and anionic membrane lipids like BMP stimulate the intervesicular cholesterol transfer by NPC2 at low pH values, the additional incorporation of sphingomyelin (SM) strongly inhibits the transfer of cholesterol [6]. Indeed,

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degradation of vesicular SM to Cer by pre-incubation of the vesicles with acid

sphingomyelinase (ASM) increases cholesterol transfer considerably by NPC2 [6].

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This observation can explain the substantial secondary cholesterol accumulation in the lysosomal compartment of patients with Niemann-Pick disease type A and B [15], who suffer from an inherited loss of ASM activity that induces a severe

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endolysosomal SM storage.

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3. Anionic lipids stimulate sphingolipid catabolism

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ACCEPTED MANUSCRIPT Also the hydrolytic activity of ASM against membrane bound SM and a few other lipids such as phosphatidylcholine (PC) and phosphatidylglycerol (PG), is strongly regulated by the lipid composition of the vesicular membranes. Again anionic membrane lipids, like PG, phosphatidic acid (PA), and phosphatidylinositol (PI), are

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strong stimulators in the acidic environment prevailing in the endolysosomal

compartment [16]. The binding of the enzyme ASM to vesicular surfaces is enhanced at pH5 by incorporation of anionic lipids, but is inhibited by incorporation of cationic

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amphiphilic drugs (CADs) [17, 18]. This suggests that there is an electrostatic

interaction between negatively charged vesicular surfaces containing anionic lipids

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and polycationic proteins, as demonstrated by analysis of their zeta potentials at pH 5.0 [6].

CADs, such as the antidepressant desipramine, can be taken up by cultured human fibroblasts as neutral, unprotonated amphiphiles. In the acidic lysosomal

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compartment, they are protonated and trapped. As components of the LVs they compensate their negative surface charge, releasing ASM and other catabolic

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proteins from their vesicular surfaces. Released proteins are then proteolytically

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degraded inducing a phospholipidosis [17, 18].

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ACCEPTED MANUSCRIPT We now know all the major proteins essential for lysosomal degradation of ganglioside GM2, the main storage compound in Tay-Sachs disease. Reconstitution experiments employing GM2-carrying liposomal vesicles as mimics of LVs of the endolysosomal system, purified hexosaminidase A (HexA) and GM2 activator protein

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(GM2AP) as essential LTP, show that in addition to the known proteins, anionic

membrane lipids are needed in the vesicular GM2-carrying membranes to achieve physiologically relevant turnover rates for GM2 [19] (Fig.2 A).

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Anionic lipids like BMP, PG and PA are also required for GM2AP to solubilize and

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mobilize lipids from membranes (Fig. 2B) and to stimulate the inter-vesicular lipid transfer (Fig. 2 C), identifying GM2AP as a multifunctional glycoprotein.

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4. High cholesterol levels inhibit glycosphingolipid (GSL) catabolism

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ACCEPTED MANUSCRIPT NPC is characterized by a lysosomal accumulation of both, cholesterol and GSLs like glucosylceramide, lactosylceramide, gangliosides GM3 and GM2 [11, 15]. It had been suggested that GSL and cholesterol accumulation are both triggered by NPC1 deficiency in late endosomes [20]. The identification of the defective proteins NPC1 in

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NPC type 1 and NPC2 in NPC type 2 as cholesterol binding and transfer proteins [14, 21, 22] suggested a primary defect in cholesterol efflux from late endosomes and lysosomes. Therefore, our recent finding that high cholesterol concentrations, as they

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occur in cellular plasma membranes and as storage material in the lysosomes of NPC patients, inhibit the BMP-enhanced hydrolysis of GM2 significantly (Fig.3 A), is

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important to explain the increased lysosomal levels of GM2 in NPC as a secondary accumulation. The strong cholesterol-induced inhibition of SAPs like Sap A [23], Sap B [24] and GM2AP [19] may well contribute to the additional secondary storage of minor glycosphingolipids such as glucosylceramide, lactosylceramide and

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ganglioside GM3 as observed in NPC patients [11]. The secondary accumulation of GSLs seems to be important for the molecular pathogenesis of NPC, as miglustat, an

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patients [25, 26]

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inhibitor of sphingolipid biosynthesis indeed improves the clinical course of the

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ACCEPTED MANUSCRIPT Cholesterol also inhibits other functions of GM2AP, such as the solubilization of membrane lipids and their inter-vesicular transfer (Fig. 3 B and C), identifying the small glycoprotein GM2AP as an efficient lysosomal LTP which, however, is not fusogenic [27]. Some lysosomal SAPs mediate an intervesicular lipid transfer and

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others can trigger fusion of vesicles at low pH values [9, 28, 29]. Since the vesicle markers we used so far, NBD-PE and Biotin-PE, are not resistant against extraction and transfer by GM2AP, we had to synthesize novel vesicle markers, stable against

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transfer by SAPs in order to study lipid transfer and vesicle fusion separately. We prepared novel bipolar lipids, which span the lipid bilayer and carry hydrophilic head

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groups at each end. They are stably anchored in the vesicular membranes, even in the presence of SAPs. [27]. To assay the transfer of fluorescent NBD labelled lipids from donor to acceptor vesicles, the donor may contain membrane-spanning marker lipids with fluorescence quencher residues as headgroups suitable for real time

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FRET assays. A pair of membrane-spanning lipids in the same liposomal membrane, one containing a fluorescent group, the other a quenching head group, allows the

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analysis of vesicle fusion with a surplus of unlabeled liposomes [27].

5. SAPs and anionic lipids are strong stimulators of lysosomal sphingolipid degradation

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ACCEPTED MANUSCRIPT All steps in lysosomal sphingolipid digestion we studied so far are strongly enhanced by both, lysosomal LTPs, the SAPs, and anionic lipids [1, 2]. To demonstrate the extent of regulation, Fig. 4 presents quantitative data on the stimulation of the enzymatic degradation of membrane-bound ganglioside GM1 by lysosomal β-

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galactosidase in the presence and absence of lysosomal LTPs (GM2AP and Sap-B) and anionic lipids.

Membrane-bound complex glycosphingolipids like GM1 (Fig. 4) and GM2 (Fig. 2 A)

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are hardly hydrolyzed by their respective hydrolases in the absence of anionic lipids,

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and even less in the absence of both, SAPs and anionic lipids, whereas the smaller glucosylceramide is already cleaved by ß-glucosidase in the presence of anionic lipids and absence of SAPs [30]. Carbohydrate free sphingolipids, like SM [16] and Cer [31] are, however, slowly cleaved by their respective hydrolases in the absence of SAPs and anionic lipids, but they reach physiological rates of degradation only in

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the presence of SAPs and anionic lipids. The absence of all four saposins A, B, C and D caused by the inherited absence of their precursor protein prosaposin, triggers

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a fatal perinatal disease, mainly induced by the loss of the water permeability barrier in the skin [1, 10, 32]. The absence of all four saposins causes a massive

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accumulation of ceramides, glucosylceramides, ganglioside GM3 and many other lipids [10, 33, 34], despite the presence of anionic lipids and active hydrolases in the tissue of the patients.

6. Lipid modifiers affect the catabolic activity of lysosomal hydrolases on native lipid substrates. 10

ACCEPTED MANUSCRIPT The lipid composition of eukaryotic organelles and vesicular membranes is particle specific and apparently adjusted to their individual functions. Disturbed lipid composition of organelle membranes may contribute to clinical heterogeneity of lysosomal diseases. Data obtained so far indicate that the catabolic activity of a

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lysosomal enzyme as exemplified above for hexosaminidase A involved in

ganglioside GM2 hydrolysis, is strongly modified by the microenvironment in which the enzymic reaction occurs, e. g in free solution acting on a water-soluble synthetic

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substrate like 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide or on a membrane

ganglioside GM2 by HexA are:

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bound glycolipid. Important modifiers for the catabolism of a lipid substrate like

the presence or absence of a lipid binding and transfer protein like GM2AP,

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the surface charge and lipid composition of the GM2 carrying membranes,

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the nature of stimulating anionic lipids in the lipid substrate (GM2) carrying membrane [6],

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the presence of cholesterol and other inhibiting lipids in the GM2 carrying

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membrane, -

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the local pH value (whereas hexosaminidase A cleaves soluble synthetic

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substrates in a wide pH range of 3 - 7, cleavage of membrane bound GM2 in the presence of GM2AP occurs only in a narrow pH range of 4.0 – 4.6) [19, 35, 36],

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the ionic strength and ion composition in the microenvironment of the catabolic reaction.

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ACCEPTED MANUSCRIPT The strong and cumulative influence of these modifiers on the rate of enzyme catalyzed lipid cleavage, e.g. of GM2 hydrolysis, may well affect the correlation

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between genotype and phenotype obtained for patients with lysosomal diseases.

Acknowledgements: This work was supported by the German Research Foundation (DFG) (SFB 645, TRR83), and the Fonds der Chemischen Industrie (FCI). I thank

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Bryan Winchester for English language editing.

Legends

Fig. 1. Proposed topology of endocytosis and lysosomal degradation. Patches of the plasma membrane are internalized by way of coated pits or caveolae,

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carrying glycosphingolipids (GSLs, red) and receptors such as EGFR (epidermal growth factor receptor, blue) to form transport vesicles which fuse with early endosomes and mature to late endosomes. Endosomal perimeter membranes form

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invaginations, controlled by ESCRT proteins [7], which bud off, generating intraendosomal luminal vesicles (LVs). In the late endosomes, lipid sorting occurs at the

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LVs. At a low pH around pH 5, acid sphingomyelinase (ASM) degrades sphingomyelin of the LVs to ceramide, whereas the perimeter membrane is protected against the action of ASM by a “glycocalyx” facing the lumen of the endosomes and lysosomes. The decrease in sphingomyelin is coupled with an increase in ceramide, facilitating the binding of cholesterol (Chol) to NPC2 and its transport to other membranes or NPC1 of the perimeter membrane of the late endosome for further export [14]. LVs of the late endosomes may reach the lysosomes by temporal fusion 12

ACCEPTED MANUSCRIPT and discharge, by maturation of late endosomes to lysosomes or possibly also by vesicular transport. A vesicular transport between late endosomes and lysosomes has been suggested but not been proven. After reaching the lysosomes, LVs and their GSLs are degraded by hydrolases with the assistance of SAPs (sphingolipid

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activator proteins). Their degradation products like monosaccharides, fatty acids, sphingoid bases etc., are exported to the cytosol of the cell or loaded on to CD1b immunoreceptors. In the course of endocytosis, the pH of the lysosol and the

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cholesterol content of the LV drop, whereas the bis(monoacylglycero)phosphate

(BMP) content of the LV membranes increases. Gradients are shown [modified after

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[2]].

Fig. 2 BMP enhances hydrolysis of liposomal GM2 by hexosaminidase A and GM2AP, solubilization of membrane lipids and transfer of 2-NBD-GM1 by

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GM2AP.

A: Turnover of GM2 was investigated in an in vitro liposomal activity assay using negatively charged liposomes with 5–35 mol% bis(monoacylglycero)phosphate

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(BMP, colored columns) and uncharged liposomes (0 mol% BMP (black column), all containing 5 mol% cholesterol and dioleoyl-L-α-phophatidylcholine (DOPC) as host

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lipid. Increase of BMP stimulated the hydrolysis of [14 C]GM2 by hexosaminidase A using glycosylated recombinant human GM2AP (g-rGM2AP) as activator protein. Mean error was determined to be less than 10%. B: Surface Plasmon Resonance (SPR) binding studies were carried out by binding a monolayer of liposomes to the gold surface of a chip, thereby raising RU (relative units as measure for bound material) from ‘-2000’ to the dashed zero (0) line. Bound to the chip were uncharged liposomes containing no BMP (black line) and negatively charged liposomes 13

ACCEPTED MANUSCRIPT containing 5–35 mol% BMP (colored lines), each of them containing 5 mol% cholesterol and DOPC as host lipid. Using liposomes with 0–5 mol% BMP, the injected g-rGM2AP flowing over the surface adsorbed to the lipid layer (black and purple lines) but did not solubilize lipids; using liposomes with 10 mol% BMP (green

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line) or 35 mol% BMP (red line) the RU values dropped below the dashed zero (0) line, indicating mobilization of small amounts of lipids. Substantial lipid solubilization could be detected only with liposomes containing 20 (blue line) and 10 mol% BMP,

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especially when GM2AP-free buffer was injected (after arrow). C: Intervesicular

transfer of fluorescent 2-NBD-GM1 by recombinant glycosylated GM2AP (g-rGM2AP)

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was measured by a Förster Resonance Energy Transfer (FRET) assay using negatively charged liposomes with varying BMP content (colored lines) and uncharged liposomes (black lines). Without BMP in the liposomes, no increase of fluorescence could be measured (black line) indicating no transfer of 2-NBD-GM1.

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Increase of BMP content up to 35 mol% (red line) led to a successive enhancement of 2-NBD-GM1 transfer by g-rGM2AP. Fluorescence obtained in a control by total

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fusion of donor and acceptor liposomes is given by the dashed line (total fusion).

In the FRET assay for intervesicular transfer of NBD-GM1, donor and acceptor

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vesicles were prepared separately . Negatively charged liposomes (containing 5 mol% cholesterol and 20 mol% BMP) with 2 mol% fluorescent 2-NBD-GM1 and 4 mol% rhodamine-PE as quencher in 20 mM sodium citrate (pH 4.2) were used as donor vesicles. The acceptor vesicles were made of the same lipids without 2-NBDGM1 and rhodamine PE. The final lipid concentrations in the assay were 4 and 24 nmol for the donor and acceptor vesicles, respectively (donor-acceptor vesicle ratio is 1:6). The experiment was started by the addition of 0.083 pmol (1.5 µg) g-rGM2AP. 14

ACCEPTED MANUSCRIPT Increase in NBD-fluorescence in the acceptor vesicles was measured continuously. Figure and legend were modified after [19]

Fig. 3. High cholesterol levels inhibit hydrolysis of liposomal GM2 by

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hexosaminidase A, solubilization of membrane lipids and transfer of 2-NBDGM1 by GM2AP.

A: A high cholesterol content of 40 mol% strongly inhibits the hydrolysis of [14

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C]GM2 by hexosaminidase A in the presence of glycosylated recombinant human GM2AP (g-rGM2AP, colored columns) using negatively charged liposomes,

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containing 20mol % BMP, 5 mol% cholesterol and DOPC as host lipid. However, a cholesterol content below 20 mol% had negligible influence on the turnover of [14 C]GM2 by g-rGM2AP (green column). Mean error was determined to be less than 10%. B: Using negatively charged liposomes with high cholesterol levels of 35 and

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40 mol%, the injected g-rGM2AP adsorbed to the liposomal layer but almost no material was released. Release occurred only when negatively charged liposomes with low cholesterol levels ≤ 20 mol% were used. Lines below dashed zero line (0)

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indicate lipid solubilization. g-rGM2AP adsorbed to the lipid layer and lipids were solubilized, especially when GM2AP-free buffer was injected (green and black lines,

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after arrow). C: High cholesterol levels reduced the rate of intervesicular transfer of 2-NBD-GM1 by g-rGM2AP in the FRET assay (green and blue lines) compared with liposomes with 5 mol% cholesterol (black line) using negatively charged liposomes. This study was conducted as described in Fig. 2. Figure and legend were modified after [19]

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Fig. 4 Lysosomal anionic lipids and SAPs enhance the degradation of ganglioside GM1 by β-galactosidase up to 100-fold. Using phosphatidylcholine (PC) containing neutral liposomes in the absence of

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anionic lipids and SAPs, β-galactosidase hardly hydrolyzes any detectable amounts of ganglioside GM1 (column far left). However, incorporation of anionic lipids (BMP, phosphatidylinositol (PI), dolicholphosphate (DP), phosphatidylserine (PS)) into the

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GM1-carrying liposomes and the addition of a SAP, either GM2AP or Sap-B,

stimulated the GM1 hydrolysis up to 100-fold. As indicated, lysosomal anionic lipids

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(10 mol %) were incorporated in lipid unilamellar vesicles, composed of 10 mol % ganglioside GM1, 20 mol % cholesterol, and 60 mol % PC. Assays were carried out in the absence of an activator protein and in the presence of 5 micromolar of GM2AP isolated from human spleen (GM2-AP) or 5 micromolar of saposin B isolated from

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human spleen (Sap-B). Figure and legend were modified after [37].

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[37] G. Wilkening, T. Linke, G. Uhlhorn-Dierks, K. Sandhoff, Degradation of membranebound ganglioside GM1. Stimulation by bis(monoacylglycero)phosphate and the activator proteins SAP-B and GM2-AP, J Biol Chem, 275 (2000) 35814-35819.

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ACCEPTED MANUSCRIPT 1. Anionic lipids are essential stimulators to reach physiological rates of lysosomal sphingolipid degradation. 2. High levels of cholesterol and sphingomyelin in the luminal vesicles of endosomes and lysosomes inhibit crucial steps of lysosomal sphingolipid catabolism. 3. Accumulation of cholesterol in Niemann-Pick type C patients triggers a secondary

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storage of glycosphingolipids.