Membrane Lipids Regulate Glycosphingolipid Catabolism, Its Enzymes and Lipid Binding Proteins Philadelphia, Aug

Membrane lipids regulate glycosphingolipid catabolism, its enzymes and lipid binding proteins Philadelphia, Aug. 2015

HO OH OH O OH O HO O OH O AcHN OH O O O OH HN CH3 HOOC O HN CH3 H C O O CH O O 3 N P 3 OH HO O CH3 O H3C OH OH CH3 O O OH OH HO OH AcHN OH Ganglioside GM1 Ceramide Sphingomyelin

2 K+ Outside 

ATPase

Inside 3 Na+ Electro- Konrad Sandhoff -Gangliosides, SM & Chol. stabilize chemical LIMES, Membrane Biology and Lipid Biochemistry Unit neuronal membranes. potential, Kekulé-Institut Gerhard-Domagk-Str. 1, 53121, Bonn -However, Chol. & SM inhibit lysos. Gradients Universität Bonn catabolism of ganglioside GM2.- Principles of lysosomal sphingolipid catabolism and membrane digestion

pH Chol BMP Luminal vesicles (LV) are platforms for SL, GSL- 7.4 and membrane- degradation

1. Late endosomes: Maturration of IMs by Sorting removal of Chol (NPC1, 6.0 NPC2, NPC disease) Early endosome 2. Lysosomes: Degradation by enzymes NPC-2 Late ASM and LLBPs (GM2AP, Sap endosome Sorting ER 5.0 LV A,B,C,D) : Inherited defects cause Golgi NPC-1 fatal diseases, lipid and membrane storage, Temporal fusion Lysosome and discharge loss of permeability Vesicular LLBPs barrier in the skin, transport? SAP CD1- and affect CD1 loading loading Plasma CD1b 4.5 LV membrane 3. KO mice are available Hydrolysis Glycocalix

Gallala et al., J. Neurochem. 2011 At late endosomes: Membrane lipids & ASM regulate cholesterol transfer by NPC-2

Separation of + endolysosomal donor (pellet) and lipid acceptor (supernatant) binding protein

Fluorescence

DONOR ACCEPTOR Analysis of acceptor in supernatant Radiolabeled [14C] lipid NBD-PE Biotin-PE host lipids

40 60 50 10.0

SM concentration [mol%] Chol Sulf Cer + PE PC 30 20 mol% BMP 40 GM3 GalCer 40 9.5 Cer BMP 30 20 9.0 Cer

20 10 min] 20 Lipid transferred transferredLipid

Lipid transferred transferredLipid 8.5

supernatant/ 10 min/min/1010 supernatant/supernatant/

10 supernatant/10min/

Cholesterol transferCholesterol 10

(% of total radioactivity ininradioactivityradioactivity ofof totaltotal(%(%

Cholesterol transferred Cholesterol radioactivitytotal of (% in SM

0.092 nmol NPC2/ 200 µl) µl) 200200nmolnmol NPC2/ NPC2/ 0.0920.092

0.092nmol NPC2/200 µl)

[% of total radioactivity in of [%

0 supernatant/mM NPC2/ 0.5 0 8.0 0 0 10 20 0 30 60 90 120 4.2 5.0 6.0 7.4 B lipid concentration [mol%] A pH Pre-incubation time of vesicles with ASM C [min]

Abdul-Hammed et al., J. Lipid Res. 2010 Oninla,Breiden, Sandhoff, 2014 Chol.-storage in NPC disease triggers GM2 & GSL accumulation ASM is regulated by membrane lipids

SM PC 250 250 PA PA

200 200 C]

C] PG PG

° ° BMP BMP 150 150 w/o w/o

100 100

50 50

hydrolyzed lipid hydrolyzed

[pmol/ h/ pH 5.0/ 37 5.0/ pH h/ [pmol/ [pmol/ h/ pH 5.0/ 37 5.0/pH h/ [pmol/ 0 0 0 100 200 300 400 500 600 0 100 200 300 400 500 600 A ASM [ng/50 µl] B ASM [ng/50 µl]

20 1,0 PA PA PE PA PG 0,8

15 BMP C]

° C]

° w/o 0,6 10

hydrolysed 0,4

hydrolysed

[%/2h /37 [%/2h

[pmol/ 2h/ 37 2h/ [pmol/ PE PE PA PA 5 0,2

0 0,0 0 100 200 300 400 500 600 0 100 200 300 400 500 600 C ASM [ng] D ASM [ng] Liposomes: 10 mol% Cholesterol; 10 mol% SM ; 0 or 20 mol% anionic lipids ; 80 or 60 mol% DOPC ASM: recombinat human ASM expressed in insect Sf21 cells (Lansmann et al., 2000) Oninla, Breiden et al. (2014) JLR Electrostatic binding of ASM to liposomes

7000 Association phase (ASM) Dissociation phase (buffer) Zeta potential [mV ]

6000 of liposomes ]

U PG PG - 24.65

R 5000

[

t PA PA - 24.48 i

n 4000 U

BMP BMP - 26.53 e c PS

n 3000 PI PS - 23.58 o

p DHP s PI - 21.50 e 2000 R control (w/o liposomes) DHP - 22.45 neutral 1000 DOTMA Neutral 0.11 EPC (PC) MVL5 0 baseline: w/o ASM DOTMA 22.21 0 50 100 150 200 250 300 350 Time [s] EPC 23.44

10 mol% Cholesterol 10 mol% SM 20 mol% anionic or cationic lipid 60 mol% DOPC Oninla et al., JLR 2014 TSD is a recessive and lethal ganglioside storage disease

Infantile Amaurotic Idiocy: cherry red spot

Tay-Sachs Disease:

Neuronal MCBs, Lysosomal storage Mechanism of GM2AP-Liftase

OH OH O OH HO O OH OH NH O HO O O O O HN O OH (CH2)n HOOC OH O F3C O N N HO C(n=5,1)-TPD-GM2 HO OH h* HN HO O

Michaelis- Menten Complex

IM, luminal vesicle Wright et al. (2003) J Mol Biol. 331, 951-64 Wendeler et al. (2004) Eur. J. Biochem. 271:614-27 BMP (PA&PI) stimulate hydrolysis of liposomal GM2 by Hex A and GM2AP

BMP mobilizes membrane Surface, lipids by GM2AP Plasmon Resonance, Biacore

BMP stimulates intervesicular lipid transfer by GM2AP

Anheuser et al.(2015), JLR Cholesterol NPC: Lysosomal inhibits hydrolysis of cholesterol liposomal GM2 by accumulation Hex A & GM2AP inhibits lysosomal GM2 catabolism,e.g. In NPC disease

Q: Is solubilization of M-lipids by SAPs Cholesterol physiologically Inhibits solubilization of M-lipids by GM2AP relevant?

Biacore

Cholesterol inhibits Intervesicular transfer M-lipids by GM2AP

Anhheuser et al., (2015) JLR

597 - 591 , 200 EJB (1991) Vogel et al. al. et

bound GlcCers by beta by GlcCers bound - LUV Also ( Galase ) Galase - beta by GM1 A, Hex by GM2 Glcase, -

H O O H O

O S

O O

R N H O H O

H O

2 = = Lyso R = C R H = C R = C R

O O O

) R ( length chain Acyl

Lyso C6 C18 C2

B - Sap -

100 mU x enzyme (pmol/h

Degradation rate

200

300 B - Sap +

400

500 )

600

Lipid phase problem, sulfatide cleavage sulfatide problem, phase Lipid Lysosomes: Lysosomes: Glycosylation of Sap- B is essential for its physiological functions

Sap-B Glycosylated Unglycosylated

Causing MLD ▬ 1,2 (glycosylation site variants) 1,2 +

+ 3,4 Rescue of Sap-B deficient cells ▬ 4

In vitro: Stimulation of sulfatide degradation + 4 ++ 4 (micellar/ liposomal assays)

Lipid transfer (sulfatide etc.) + 5 (+) 6

SPR (liposomes adsorbed on chip):

4 4 Lipid binding + + ++ 4 Lipid mobilization ▬ 4 Lipid modifiers may contribute to clinical heterogeneity of lysosomal diseases

- BMP & other anionic PLs enhance a) cholesterol transfer by NPC2 b) the hydrolysis of SM by ASM, of GM1 by ß-Galase, and of GM2 by Hex A c) the mobilization of membrane lipids by SAPs d) transfer of 2-NBD-GM1 by GM2AP e) the activities of GM2AP, Sap A & B etc.

- Cholesterol levels inhibit: Hydrolysis of GM2 by Hex A (in NPC2), PC by ASM, mobilization of lipids by Sap A,B & GM2AP, transfer of 2-NBD-GM1 by GM2AP

- SM inhibits cholesterol transfer by NPC2 (in NPA&B), hydrolysis of GM2 by Hex A , and mobilization of membrane lipids by GM2AP

- Turnover of GM2 by Hex A, mobilization of membrane lipids and transfer of 2- NBD-GM1 by GM2AP are dependent on a narrow pH range of 4.2 +/- 0.4 (Hex A range: pH 3 – 7)

- The His6-tag changes capabilities of GM2AP : It stimulates of hydrolysis of liposomal GM2 by Hex A, but inhibits lipid mobilization completely. It triggers vesicle fusion. Coworkers

Misbaudeen Abdul-Hammed Bernadette Breiden Susi Anheuser Günter Schwarzmann

Threshold Theory m

e ] 15

[

A t ..... Critical threshold activity v

a v

r i

n 10 1.0

c s Lysosome

t o v

a max

r e

e [S] /K = 1/[(v /v )-1] s 5 Hypothetical solubility 0.5 eq m max i v

e of substrate

T Substrate influx: v y i

a Degradation rate: vmax

S 1 5 10 15 20 Enzyme activity Vmax/vi

2 f

M 40 o

Normal G %

d 30

e r

Heterozygotes t

a v

Adult r 20

p r

Juvenile r u

o 10

c T

Infantile n i GM2-Activator deficiency 0 0 200 400 600 800 1000 1200 Hexosaminidase activity, pmol cleaved GM2 / h x mg x AU 1. Wrobe D, Henseler M et al. (2000) A non-glycosylated and functionally deficient mutant (N215H) of the sphingolipid activator protein B (SAP-B) in a novel case of metachromatic leukodystrophy (MLD)." J Inherit Metab Dis 23(1): 63-76 2. Regis S., M. Filocamo, et al. (1999). "An Asn > Lys substitution in saposin B involving a conserved amino acidic residue and leading to the loss of the single N-glycosylation site in a patient with metachromatic leukodystrophy and normal arylsulphatase A activity." Eur J Hum Genet 7(2): 125-30 3. Matzner , U., B. Breiden, et al. (2009). "Saposin B-dependent reconstitution of arylsulfatase A activity in vitro and in cell culture models of metachromatic leukodystrophy." J Biol Chem 284(14): 9372-81 4. Remmel , N., S. Locatelli-Hoops, et al. (2007). "Saposin B mobilizes lipids from cholesterol-poor and bis(monoacylglycero)phosphate-rich membranes at acidic pH. Unglycosylated patient variant saposin B lacks lipid-extraction capacity." Febs J 274(13): 3405-20 5. Vogel A., G. Schwarzmann, et al. (1991). "Glycosphingolipid specificity of the human sulfatide activator protein." Eur J Biochem 200(2): 591-7 6. Abdul-Hammed, M., B. Breiden, et al. "Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion." J Lipid Res 51(7): 1747-60 Lysosomal & extracellular GlcCer degradation: Protein & lipid modifiers

Konrad Sandhoff, LIMES, GM1 University of Bonn GM1 - b - galactosidase GM2AP, Sap- (GM1-Gangliosidosis) B Globoside b-HexosaminidaseA (Sandhoff) b-HexosaminidaseB

GM2 Amaurotic idiocy: 5 genetic

b-Hexosaminidase diseases: (GM2-Gangliosidoses, GM2-AP Globotriaosylceramide AB, B, 0 Variant) GM1- and 4 forms GM2- Sap-B a- GalactosidaseA (Fabry) gangliosidoses (Var AB, B, B1, 0) GM3

(Sialidosis) Sialidase Hex A deficiency: various clinical Sap-B DiGalCer courses (inf. juv. adult chron.), (Fabry) a-Galacto- Sulfatide sidase A Threshold theory LacCer Sap-B GalCer-b-galactosidase Arylsulfatase A Sap-B GM1-b-galactosidase Sap-B, Sap-C (Metachromatic leukodystrophy) GalCer Coworker: GlcCer (Krabbe) b-Galactosyl- ceramidase Bernadette Breiden Glucosylceramidase (Gaucher) Sap-C Sap-C, Misbaudeen Abdul-Hammed

Sphingomyelinase Günter Schwarzmann (Niemann-Pick Susi Anheuser Cer Type A, B) Sphingomyelin (Farber) Ceramidase Sap-C, Sap-D Sandhoff, K. and Kolter, T. (1995) Naturw., 82, 403-413 Sphingosine Skin biopsy of a pSap- deficient patient (W. Roggendorf)

Feeding of pSAP reverses membrane storage Burkhardt et al. (1997) Sphingolipid degradation at intralysosomal membrane surfaces: Stimulation by SAPs and anionic lipids

Lysosome Activator protein Intralysosomal vesicles Lysosomal enzyme • Electrostatic interaction: Cationic proteins bind to anionic vesicles Glycocalix Lysosomal membrane • SAPs stimulate enzymatic hydrolysis of liposome-bound + + + sphingolipids. + + + Sap-D Sap-C • BMP and other anionic PLs

acid - Gluco-

Ceramidase - - - cerebrosidase + - + - + stimulate degradation of: active site HO + active site - - Cer, GlcCer, SM, GM1, GM2:

+ - - + +

+ - + 3-15 fold in the presence of SAPs. Ceramide - - Glucosylceramide - - (Sensitivity to CADs) -

- BMP - - BMP - Sandhoff ,Proc. Jpn. Acad. Ser. B, Vol. 88, O H OH H OH 554-82 (2012) O P O O R R O C C O O O Bis(monoacylglycero)phosphate (BMP, S-configuration, sn1)

Wilkening, Linke, Sandhoff (1998) J. Biol. Chem. 273, 30271-8 Lysosomal anionic lipids enhance the degradation of GM1 by β- galactosidase, but only in the presence of SAPs

Lysosomal anionic lipids (10 mol %) were incorporated in LUVs, composed of 10 mol % GM1, 20 mol % cholesterol, and 60 mol % PC. Assays in the absence of an activator protein and in the presence of 5 μm GM2-AP or 5 μm SAP-B were carried out.

Wilkening G et al. (2000) J. Biol. Chem. 275, 35814-35819 Lysosomal lipids in LUVs stimulate the enzymatic GlcCer hydrolysis in the presence and also in the absence of Sap-C.

+ Sap-C

- Sap-C

A: Assays were conducted with GlcCer as substrate in the absence (○) and presence of Sap-C (2.5 μm) (•), using LUVs with various proportions of synthetic BMP (0–40 mol %).

B: Assays were carried out with varying concentrations of PI in LUVs, with (•) and without (○) the addition of 2.5 μm Sap-C, keeping the total lipid concentration in the assays constant.

Wilkening G et al. J. Biol. Chem. 1998;273:30271-30278 Conclusions ------Several inherited diseases can generate the same clinical phenotype ( amaurotic idiocy caused by GM1&GM2 gangliosidoses ( TSD, SD, Var AB & B1 ) -- Mutations in the same gene (e.g.Hex A ) can cause different clinical courses (inf., juv., adult & chronic courses due to different levels of residual catabolic activities )

-- Catabolic activity of an enzyme ( e.g. Hex A ) is strongly modified by the microenvironment in which the enzymic reaction occurs, e.g. at the surface of liposomes ( or in vivo at the surface of luminal lysosomal vesicles ) ( pH-value, ionic strength, LTPs or SAPs, lipid composition of the GM2 carrying membrane: anionic M-lipids stimulate ( up to 15 fold ), cholesterol inhibits strongly -- No direct correllation between gene mutations and the clinical course of a disease, the microenvironment of the enzymic reaction modifies the catabolic rate. Ceramide metabolism: Key to skin barrier function

Epidermis is important against desiccation and infections

Barrier : Extracellular lipid layers :ULC-Cers ,ULC-FA, Cholesterol Permeability barrier of the skin

O H R O H R O H R O H R O H Skin surface N N N N N N N N N CE O H R O H O H R O H O O O O O O Lipid

Corneocyte matrix

Stratum Stratum corneum Cer

- ? AcylCer

GlcCer -

fatty fatty acid ?

- OH

- Trans-

OH bound bound envelope

w esterification -

- b-GlcCerase

w Sap-C Collod. babies Lipid HO O Glc template PM Stratum granulosum R12-LOX AcylGlcCer CE H H Trans- eLOX3 OH N N esterification Cong. icht. O O O OH OH OH O HO O OH Acid Ceramidase Glc Sap-D, -C b-GlcCerase HO AcylGlcCer Sap-C OH Collod. babies Lamellar body

O Golgi OH O O HN HO O O HO AcylGlcCer (EOS) OH OH Breiden & Sandhoff, 2013 Ultrastructure of pSAP knockout epidermis

Ruthenium staining reveals that the pSAP-deficient interstices (B, between arrows) lack the regular, compact pattern of lamellar membranes observed in normal, pSAP-replete SC (C, between open arrows)..

Doering, T. et al. J Biol Chem, 274, 11038-11045 Biosynthesis of AcylGlcCer

ER Alternative pathway PM 1. UDP-Glucose 2. Linoleoyl CoA OH O L-Serine w-OH-Cer AcylCer H SPT OH HO OH O Sphinganine OH O NH HO O NH NH CerS 3 HO O HO O Palmitoyl-CoA Cong. ichthyosis w-Hydroxylation w-OH FA (>C28)-CoA

w-OH C16 FA-CoA ELOVL 4 FA-elongation Glc complex w-OH FA (C28)-CoA AcylGlcCer (ELOVL 1,3,6,7) GlcCer- ABCA12 Lamellar ELOVL 1, 4 synthase Harlequin icht. body w-OH FA (

Lipase DAGT2 CGI 58 DAG TAG Linoleic acid Dorfman-Chanarin S. AcylGlcCer Lipid droplet Breiden & Sandhoff, 2013 Golgi Cytosol Schemes of CerS3d/d phenotypic alterations with proposed cascades of events.

• CerS3 ko mice die shortly after birth from TEWL

• Loss of all SLs with acyl residues longer than 24 C-atoms, lack of protein-bound ceramides

• Lack of continuous extracellular lipid lamellae, water loss, hyperkera tosis

• High skin susceptibility to Candida infection

• SG –SC interface: absense of LB exocytotic figures and lamellar lipid-free domains

• Impaired processing of epidermal proteins: (pro) filaggrin, involucrin, loricrin.

Jennemann R et al…..Sandhoff R, (2012) Hum. Mol. Genet.; 21:586-608

(A–C′) Cultured skin samples of CerS3d/d mice reveal after inoculation with C. albicans increased microbial adhesion and growth after 6 h (A′), microbial invasion of all epidermal layers after 24 h (B′) and microbial colony formation within SS in parallel to migration of pseudohyphae into the dermis after 66 h (C′, the residual basement membrane, red arrowheads). Note the abundance of immune cells in mutant dermis after 66 h. PAS-hemalaun.

Jennemann R et al. .Sandhoff R (2012) Hum. Mol. Genet. 21:586-608

- Cell type specificity of GSL- & SL- biosynthesis

- Promiscuity (and redundancy) of hydrolases and SAPs

- SAPs are multifunctional proteins (lipid binding & mobilization, intermembrane lipid transfer and vesicle fusion)

- Membrane lipids are strong regulators of lipid degradation at vesicular surfaces

-Unknown transient levels of cholesterol & anionic lipids in luminal vesicles of lysosomes may contribute to clinical heterogeneiety of diseases

- ULC-Glc-Ceramides & ULC-Ceramides are key components of the water & immune barrier of mammalian skin. Defects in their metabolism can cause ichtiosys and are often fatal

Liposomal activity assay

- Uncharged liposomes contained: 5 mol% cholesterol, [14C]-GM2 and PC as a host lipid - negatively charged liposomes contained additional 20 mol% BMP, PI or PG, positively charged liposomes contained 20 mol% MVL5 or EPC

Adding negative charged lipids in the liposomal membranes resulted in a strong enhanced hydrolysis of of GM2 by HexA in presence of GM2AP. Using positively charged liposomes, the hydrolysis rate of GM2 was as low as that observed with unchaged vesicles. Hydrolysis of liposomal GM2 by HexA

0,5

0,4

0,3

GM2AP-His 0,2 6 GM2AP

Turnover GM2of [pmol/h/pmol GM2AP] 0,1

0 PI PI + PG PG + 30 mmol/l 30 mmol/l desipramine desipramine

Liposomal activity assay

- The negatively charged liposomes contained 20 mol% PI or PG, 5 mol% cholesterol, [14C]-GM2 and PC as a host lipid. Adding desipramine to the assay preparation (30 mmol/l final concentration), the hydrolysis of GM2 by HexA in presence of GM2AP was strongly reduced.

- The cationic anphiphilic drug desipramine is desturbing the interaction of GM2AP with the vesicle membrane, most likely by its positive netcharge. Desipramine strongly reduces membrane binding of GM2AP

3500 3000 2500 2000

RU 1500 1000 500

0 baseline 0 200 400 600 800 1000 time [s] GM2AP desipramine buffer 30 mmol/l

Surface plasmon resonance (SPR) study Negatively charged liposomes containing 5 mol% cholesterol, 5 mol% BMP and 10 mol% GM2 were immobilized on a Pioneer HPA-chip. Running buffer was 20 mM sodium citrate buffer (pH 4.2). Response signals measured after binding of membrane lipids were defined as zero (baseline: orange). GM2AP (0.2 mM) in running buffer was injected into the flow cells at a rate of 20 µl/min. Afterwards, desipramine (30 mmol/l, dissolved in water) was injected for 3 min (flow rate 20 µl/min), followed by buffer alone.

The cationic anphiphilic drug desipramine is desturbing the interaction of GM2AP with the vesicle membrane, most likely by its positive netcharge. Cell type specific biosynthesis of glycosphingolipids

OH HO OH

O HO

O OH HO OH O OH O OH NH N O COOH H O HO O O

OH HO O

O HO

HO O OH

NH O OH

GM1

van Echten et al. (1989) J. Neurochem. 52, 207-214 Combinatorial Ganglioside Biosynthesis ER OH Cer OH OH OH O - HO O (CH ) CH A GlcT OOC O O 2 12 3 O NH HO OH OH (CH2)16CH3 GlcCer O HO O Golgi AcHN OH GM3 B GalT I OH D C A: lethal at embryonic day 6, LacCer GM3 GD3 GT3 apoptosis in ectoderm (*) SAT I SAT II SAT III E GalNAcT C: early death, formation of 0-series glycolipids, GA2 GM2 GD2 GT2 infantile epilepsy

luminal side luminal D: normal life span GalT II E: infertile (testis), myelin C+E: early death, GA1 GM1a GD1b GT1c LacCer-3-sulfate TGN SAT IV D+E: "GM3 only", sudden death, audiogenic GM1b GD1a GT1b GQ1c seizures SAT V, SAT x (*) neuron-, hepatocyte-, GQ1b a GP1c a PM GD1c GD1a GT1a Gt1a GQ1b GP1c keratinocyte-specific ko- NeuAc a 2,8- NeuAc a2,6- NeuAc GalNAc mice are born 0-series a-series b-series c-series

Kolter, Proia, Sandhoff (2002) J. Biol. Chem. 277, 25859-62 Sap-D, PI and increased membrane curvature enhance ceramide hydrolysis by aCerase

LUVs and SUVs with varying mean diameter either contained none or 25 mol % PI. In addition, incubation mixtures contained either none or 2.5 μm SAP-D.

Linke T et al. J. Biol. Chem. 2001;276:5760-5768 CADs: Cationic amphiphilic drugs (lipophilic) induce a phospholipidosis: the molecular view

Many drugs are cationic amphiphilic lipids (CADs) : CADs are partially neutral at pH 7, penetrate membranes, and are protonated & trapped in lysosomes.

Cytoplasmic Inclusion body; x 78400 CH3 CH3 Chromaffin cell of a rat treated with CH3 1-chloro-amitriptyline (120 mg/kg 10 wk) H CH N N 3 O N CH3 Lysosome OH H N activator protein CH3 intralysosomal Vesicles N lysosomal enzyme N Cl H

Glycocalix lysosomal Membrane Chloroquine Propranolol Desipramine (Antimalaria) (b-Adrenoreceptor-antagonist) (Antidepressant) + + + + + Lüllmann et al. (1978)

+ SAP-D SAP-C 1. Induction of lipidoses in rats

acid - Gluco- -

- - - cerebrosidase -

+ Ceramidase - + - + 2. Detachment of acid sphingomyelinase from anionic active site HO + active site

- liposomes Kölzer et al. (2004) -

+ - - + +

+ - + 3. Proteolytic degradation of acid sphingomyelinase and

- Ceramide - - Glucosylceramide other lysosomal enzymes by desipramine-treated - -

- - cultured fibroblasts. Hurwitz et al. (1994) - BMP - BMP Wilkening, et al. (1998) BMP and PI stimulate the degradation of membrane-bound ceramide in the absence of SAPs

BMP

Rate increases with curvature of liposomes

Sap-D stimulates PI up to 3fold

Acid ceramidase activity was measured in the presence of increasing concentrations of BMP (▪) and PI (●) in ceramide-bearing LUVs in the absence of SAPs. The data presented are the means of three determinations. All individual values were in the range of ±5 up to ±10% of the mean.

Linke T et al. J. Biol. Chem. 2001;276:5760-5768 Skin biopsy of a pSap- deficient patient (W. Roggendorf)

Feeding of pSAP reverses membrane storage Burkhardt et al. (1997) Degradation of SM by ASM stimulates intervesicular cholesterol transfer by NPC2

with ASM control: w/o ASM

]

0 . 3000 10.0 2.0

Cer [mol%] Cer

S a 2500 9.5 1.5

t 2

[

N 2000 9.0 1.0

s g

l m

]

h 1500 8.5 0.5

n [ 1000 8.0 0.0 0 20 40 60 80 100 Pre-incubation time of vesicles with ASM [min] Lysosomal Sphingolipid Degradation

GM1 GM 1 - b - galactosidase GM1 Gangliosidosis GM2 -AP, SAP -B Globoside b-Hexosaminidase A Sandhoff b-Hexosaminidase B Coworkers: GM2 Bernadette Breiden b-Hexosaminidase A Vincent Oninla GM2 Gangliosidosis AB, B, O Variant GM2 -AP Globotriaosylceramide Günter Schwarzmann SAP -B a- Galactosidase A Fabry Susi Anheuser GM3

Sialidase Previous Coworkers: Sialidosis DiGalCer SAP -B Misbaudeen Abdul-Hammed a-Galacto - Natascha Remmel Sulfatide Fabry sidase A SAP -B LacCer Silvia Locatelli-Hoops GalCer -b- galactosidase Arylsulfatase A GM1 -b-galactosidase SAP -B Wiebke Möbius SAP -B, SAP -C Metachromatic leukodystrophy GalCer Michaela Wendeler

GlcCer b-Galactosyl - Melanie Kölzer Krabbe ceramidase Glucosylceramidase Gaucher SAP -C, SAP-A Thomas Döring SAP -C

Sphingomyelinase Niemann-Pick Cer Type A, B Sphingomyelin Ceramidase Farber SAP -C, SAP -D

Sandhoff, K. and Kolter, T. (1995) Naturwissenschaften, 82, 403-413 Sphingosine g-Sap-B mobilizes lipids from BMP rich and cholesterol poor membranes at acidic pH values

rgl Sap-B (5 µM) Sap-B: variant forms 2000 6000

1000 4000

pH 7.0 r Patient:Sap-B, Asn215His 0 2000 r Sap-B, Asn215Gln pH 5.5 -1000 r Sap-B, 0 deglycosylated

Response Units (RU) pH 4.2 Response Units (RU) Response rgl Sap-B -2000 -2000 0 100 200 300 400 0 100 200 300 400 Time (s) Time (s) rgl Sap-B / BMP 0 mol% rgl Sap-B / BMP 2014 mol% mol% Chol 3000 3000 Chol (mol %) (mol %)

1000 1000 5 0 3840 25 -1000 20 -1000 3840 2520

-3000 -3000

Response Units (RU) Response Units (RU) 5 0 -5000 -5000 0 100 200 300 400 0 100 200 300 400 Time (s) Time (s) Surf. Plasmon resonance, Remmel, N. et al. (2006) Localization of Biotin-GM1 on Cryosections: Human fibroblasts were incubated with 10µM Biotin-GM1 for 72 h at 37°C. B: double labeling with anti-caveolin-1 (arrowheads) and anti-biotin (large gold) D: double labeling with anti-acid phosphatase (arrowheads) and anti- biotin (large gold) E: double labeling with anti-LIMP (arrowheads) and anti-biotin (large gold) Möbius, Herzog, Sandhoff, Schwarzmann (1999). J. Histochem. Cytochem. 7 bar=200O nm NH H HO HN O OH H S NH OH NH COO- OH O O O HO OH HN O O * O O O HO OH OH O O OH OH HO OH NH O O * Position of radiocarbon HO O OH HO Perspectives for endocytosis and sphingolipid digestion

• Cholesterol transfer by NPC2 between vesicles (and cholesterol egress from late endosomes) is stimulated by BMP and other anionic lipids, and inhibited by SM down to 10%. • ASM is stimulated by anionic lipids (PG, PA, BMP) up to 14 fold, but not inhibited by cholesterol. • Results suggest a sequential pathway of lipid degradation at intraendosomal vesicles during endocytosis: - At late endosomes PM derived anionic PLs (PG, PA) stimulate ASM to degrade SM, thereby releasing the block of NPC2 mediated cholesterol egress. ASM also degrades PG, PA, and other PLs, triggering loss of bilayer structure and barrier function of luminal membranes. - Generation of BMP and reduction of cholesterol levels in intralysosomal vesicles activates SAPs (and hydrolases) needed for effective degradation GLS and ceramides. - Cationic lipids are toxic and inhibit catabolic steps (GalSo (Krabbe), GlcSo (Gaucher), So, Sa). CADs trigger proteolysis of ASM & other hydrolases and induce a phospholipidoses. Lysosomal lipid binding proteins (SAPs and NPC2)

LLBPs are small glycoproteins, generated from big precursors by proteolysis in endolysosomes. Their activity is regulated at acidic pH by membrane lipids:

Some (NPC2, Saps A, B, C) bind and lift membrane lipids from surfaces of liposomes. Some form soluble stochiometric lipid protein complexes (GM2 – GM2AP; sulfatides - (Sap-B)2 ) (and Michaelis complexes with their catabolic hydrolases).SapA forms lipoprotein discs (2 SapA, 8-10 PL, Prive).

Inherited deficiencies of LLBPs (NPC-2, GM2AP, Sap A, B, C, D) cause fatal diseases (prosaposin (-/-) : SL and membrane storage, defective skin barrier).

LLBPs (GM2AP, Saps A,B,C…) are regulated by lipids and

• transfer lipids from donor to acceptor vesicles,

• are fusogenic (GM2AP, Sap-D, Sap-C…..), and

• disintegrate BMP rich and cholesterol poor vesicles. IMs are adjusted for degradation, BMP is generated and cholesterol leaves inner vesicles of late endosomes

Glycocalix Late endosome SM GlcCer NPC-2 SM ASM pH 5.5 intraendosomal vesicle (IM) Ceramide

? pH 4.8 NTD Sphingosine + Fatty acid

? NPC-1 sphingomyelin ceramide cholesterol Ring Finger

1. Membrane lipids regulate cholesterol transfer by NPC-2 (BMP… , Cer , SM ) 2. Digestion of SM(15%) by ASM enhances cholesterol transfer by NPC-2 (3fold) 3. ASM is a phospholipase C, cleaves PA, PG, PC, lyso-PC, SM and 17 other PLs 4. Anionic membrane lipids stimulate ASM: PA and PG 7-14fold; BMP 3fold 5. Diacylglycerol – a hydrolysis product of PLs by ASM- stimulates ASM activity 6. High cholesterol content inhibits PC, hydrolysis by ASM, but less SM cleavage Pathologic mechanisms caused by lipid storage in late endosomes and lysosomes

•Expansion of lysosomal compartment at the expense of cytosol and other organelles

•Impaired digestion of macromolecules: Inhibition of catabolic enzymes and proteins (SM inhibits NPC-2 and lipid sorting, cholesterol inhibits Sap-A, -B etc., CADs inhibit catabolic steps and trigger proteolysis of ASM etc.)

•Starvation of neurons by impaired digestion and traffic jams in lysosomes (cellular stomachs reduce their nutrient supply: Fe++, B12, FAs, AAs, CHO, sphingoid bases, salvage pathways).

•Further mechanisms: - Accumulation of toxic cationic lipids; galactosylsphingosine (GalSo) (psychosine hypothesis, Krabbe), GlcSo, So, Sa. -Release of cytokines,activation of microglia, influx of macrophages. - CADs (Desipramine) impair lysosomal digestion, trigger proteolysis of ASM and other catabolic hydrolases. Lysosomal lipids in LUVs stimulate the enzymatic GlcCer hydrolysis in the presence and also in the absence of Sap-C.

+Sap-C

-Sap-C

A, assays were conducted with GlcCer as substrate in the absence (○) and presence of Sap-C (2.5 μm) (•), using LUVs with various proportions of synthetic BMP (0–40 mol %). B, assays were carried out with varying concentrations of PI in LUVs, with (•) and without (○) the addition of 2.5 μm Sap-C, keeping the total lipid concentration in the assays constant.

Wilkening G et al. J. Biol. Chem. 1998;273:30271-30278 gSap-B mobilizes lipids from BMP rich and cholesterol poor membranes at acidic pH values

rgl Sap-B (5 µM) Sap-B: variant forms 2000 6000

1000 4000 Patient: pH 7.0 r Sap-B, Asn215His 0 2000 r Sap-B, Asn215Gln pH 5.5 -1000 r Sap-B, 0 deglycosylated

1000 1000 5 0 3840 25 -1000 20 -1000 3840 2520

-3000 -3000

Response Units (RU) Response Units (RU) 5 0 -5000 -5000 0 100 200 300 400 0 100 200 300 400 Time (s) Plasmon resonance, Remmel, N. et al. (2006) Time (s)

Lysosomal lipids in LUVs stimulate the enzymatic hydrolysis of ganglioside GM2 in the presence of GM2AP.

All assays were conducted with ganglioside GM2-carrying LUVs as substrates in the absence (□) and presence (■) of GM2AP (0.5 μm) using LUVs with various proportions of synthetic BMP (0– 25 mol %) (A) or PI (0–30 mol %) (B).

PA, sulfatides and BMP plus Chol stimulate to a

similar extent.

Werth N et al. J. Biol. Chem. 2001;276:12685-12690 BMP and PI stimulate the degradation of membrane-bound ceramide in the absence of SAPs

BMP

Rate increases with curvature of liposomes

Sap-D stimulates PI up to 3fold

Linke T et al. J. Biol. Chem. 2001;276:5760-5768 Membrane lipids regulates ASM activity and cholesterol transfer by NPC2

A B SM PC ASM

Cer DAG

C b

SM PC

i e

C b

Chol i

transfer NPC2 ASM h

A l

Cer DAG F

A M

0 10 20 30 40 0 10 20 30 40 mol% mol% Cholesterol SM host lipid (PC) Anionic lipids (20 mol%): PA, PG BMP anionic lipids Cer DAG stimulation inhibition Lysosomal sphingolipid degradation

GM1 GM1 - b - galactosidase GM2AP, Sap-B (GM1-Gangliosidosis) Globoside b-Hexosaminidase A (Sandhoff) b-Hexosaminidase B

GM2

b-Hexosaminidase (GM2-Gangliosidoses, GM2-AP Globotriaosylceramide AB, B, 0 Variant) Sap-B a- Galactosidase A (Fabry) GM3

(Sialidosis) Sialidase Sap-B DiGalCer

(Fabry) a-Galacto- Sulfatide sidase A LacCer Sap-B GalCer -b-galactosidase Arylsulfatase A Sap-B GM1-b-galactosidase Sap-B, Sap-C (Metachromatic leukodystrophy) GalCer

GlcCer (Krabbe) b-Galactosyl- ceramidase Glucosylceramidase (Gaucher) Sap-C Sap-C,

Sphingomyelinase

(Niemann-Pick Cer Type A, B) Sphingomyelin (Farber) Ceramidase Sap-C, Sap-D

Sphingosine Sandhoff, K. and Kolter, T. (1995) Naturwissenschaften, 82, 403-413 Summary

 NPC-2 is a cholesterol-binding and -transfer protein needed for secretion of cholesterol from intraluminal vesicles (IMs) of late endosomes (SM inhibits, PA, PG & BMP stimulate). NPC-2 deficiency causes NPC, ASM deficiency NPA & NPB disease  LLBPs (GM2AP, saposins A, B, C, D) are needed for sphingolipid- and membrane-degradation at intraluminal lysosomal membranes (lipid phase problem). They bind lipids, transfer lipids, some fuse vesicles, and disintegrate lipid vesicles at low pH.  rgSap-A and rgSap-B solubilize liposomal lipids at low pH, low cholesterol and high BMP levels. Variant saposins of patients are inactive.  Blocks in SL-catabolism cause fatal neurodegenerative lipid storage diseases. Prosaposin and ßGlcase(-/-):skin barier broken; Collodion babies; O-acyl-VLC-GlcCers up.  Cationic lipids (GalSo in Krabbe, GlcSo in Gaucher, So&Sa)are cytotoxic. Desipramine (CAD, a lipid !) interferes with lipid catabolism and triggers proteolytic degradation of ASM and other hydrolases. Lysosomal sphingolipid degradation

NeuAc Gal- b(1-3)-GalNAc-Gal-Glc-Cer Gal-b (1-3)-GalNAc-Gal-Glc-Cer GM1 GA1

GM1-Gangliosidosis GM1-b -galactosidase GM2-Activator, Sap-B GM1-Gangliosidosis GM1-b -galactosidase

NeuAc GalNAc- b (1-4)-Gal-Glc-Cer GalNAc-b(1-3)-Gal-Gal-Glc-Cer GalNAc-b (1-4)-Gal-Glc-Cer GA2 Globoside GM2

b-Hexos- Tay-Sachs, Sandhoff b-Hexosaminidase A, B b-Hexosaminidase A Sandhoff Sandhoff aminidase A,B AB variant GM2-Activator GM2-Activator

NeuAc-a (2-3)-Gal-b (1-4)-Glc-Cer Sialidosis Gal- b (1-4)-Glc-Cer Fabry Gal-a(1-4)-Gal-Glc-Cer GM3 Sialidase Lactosylceramide a-Galactosidase A Globotriaosylceramide Sap-B Sap-B GalCer-b-galactosidase GM1- b -galactosidase Sap-B and Sap-C Gal- a(1-3)-Gal-Cer Glc-b(1-1)-Cer Digalactosylceramide Glucosylceramide

Fabry a-Galactosidase A Glucosylceramid-b-glucosidase Sap-B Gaucher Sap-C b Galactosylceramide- -galactosidase Gal-b(1-1)-Cer Sphingomyelinase Sap-A Sphingomyelin Galactosylceramide Niemann-Pick A and B Ceramide Krabbe

Farber Acid ceramidase Sap-D Metachromatic Arylsulfatase A leukodystrophy Sap-B

Sphingosine O3 S-3-Gal-Cer Sulfatide Lipid substrate (*GM2) : Its turnover in patient`s cells correlates with:

- Patient`s celluar Hex A & GM2AP (GM2 cleaving) activity and with the clinical course of the disease.

- But the clinical course hardly correlates with patient`s enzyme activity on soluble, artificial, synthetic substrates & the gene mutations.

The latter concepts ignore the influence of : - (Patient`s) enzyme specificity against lipids, - Activity and specificity of essential, promiscuous and multifunctional SAPs, - Extensive regulation of lipid turnover by membrane lipids at luminal vesicular surfaces. Perspectives: Membrane lipids regulate activity of endolysosomal proteins

• NPC-2 transfer of cholesterol between vesicles: - Is stimulated by BMP, PA, PG and inhibited by SM down to 10%. - PG, PA and BMP stimulate ASM up to 14 fold and release the block by SM. • Results suggest a sequential pathway of lipid degradation at luminal IMs during endocytosis: - At late endosomes PM derived anionic PLs (PG, PA) stimulate ASM to degrade SM, thereby releasing the block of cholesterol egress by NPC-2 inhibition. ASM also degrades PG, PA, and other PLs, triggering loss of bilayer structure and barrier function of luminal membranes. - Generation of BMP and reduction of cholesterol levels in IMs activates SAPs (and hydrolases) needed for effective degradation GLS and ceramides. - Cationic lipids are toxic and inhibit catabolic steps (GalSo (Krabbe), GlcSo, So, Sa). CADs trigger proteolysis of ASM & other hydrolases and induce a phospholipidoses. Human prosaposin

0 0,5 1,0 1,5 2,0 2,5 kb

S P S P

A B

ATG n TAG AAA

prosaposin Krabbe MLD Gaucher deficiency

cysteine residues protein domain

precursor coding region glycosylation sites

noncoding region n insertion of 0, 6, 9 bp of Exon 8, 8‘

P phosphorylation of CHO disease causing mutations S sulfatation of CHO

Sandhoff et al (2001) in: The Metabolic and Molecular Bases of Inherited Disease Lipid Storage and Threshold Theory

Lysosome

[S]eq

[S] 1 eq  V max Km [ -1] Vi Residual catabolic activity Normal Patient (juvenile) (Infantile)

Conzelmann and Sandhoff (1983/84), Dev. Neurosci., 6, 58-71 CADs: Cationic amphiphilic drugs(lipids!) induce a phospholipidosis: the molecular view

Cationic Amphiphilic Drugs (CADs): • Many drugs are CADs • CADs are neutral at pH 7 and penetrate membranes • They are protonated and trapped in lysosomes

Glycocalix lysosomal Membrane Chloroquine Propranolol Desipramine (Antimalaria) (b-Adrenoreceptor-antagonist) (Antidepressant) + + + + + Lüllmann et al. (1978)

+ SAP-D SAP-C 1. Induction of lipidoses in rats

acid - Gluco- -

- - - cerebrosidase -

+ Ceramidase - + - + 2. Detachment of acid sphingomyelinase from anionic active site HO + active site

- liposomes Kölzer et al. (2004) -

+ - - + +

+ - + 3. Proteolytic degradation of acid sphingomyelinase and

- Ceramide - - Glucosylceramide other lysosomal enzymes by desipramine-treated - -

- - cultured fibroblasts. Hurwitz et al. (1994) - BMP - BMP Wilkening, et al. (1998) Mechanism of GM2AP-liftase

OH OH O OH HO O OH OH NH O HO O O O O HN O OH HOOC OH (CH2)n A O F3C O N N HO HO OH C(n=5,1)-TPD-GM2 HN HO O

Michaelis Menten Complex

BMP (mol %)

Intraendolysosomal Membrane 5

PA,PI,DP,Sulf Wright et al. (2003), J. Mol. Biol. 331, 951-64 Wendeler et al. (2004),Eur. J.Biochem. 271, 614-27 B (Chol 35 inhibts) Werth et al. (2001) J. Biol. Chem. 276, 12685 Lipid solubilization: Sap-A releases lipids from liposomes bound to a sensor chip

8000

4000 2.5 µM Cyt-C

2.5µM Cyt.-c 0 50 mM50mM NaCitrate NaCitrate 0 90 180 270 360 2.5µM gSap-A 2.5 µM20mM gSap- AChaps

Response Units (RU) (RU) Units Response (RU) -4000

Start-- 20 mM Chaps -8000 Time (s)

Plasmon resonance studies: Liposomes were reversibly bound to the hydrophobic surface of the sensor chip (dextran matrix derivatized with alkyl chains) and can be totally removed by detergent (Chaps). Proteins that are not membrane active (cytochrome-C) bind to liposomes. Their curves reach a plateau value and do not fall below the base line. Locatelli Hoops, S. et al. JBC (2006) ASM is a nonspecific phosoholipase C

A sphingophospholipids B glycerophospholipids

- DAG - Cer - MAG

- So

______ASM + + + + + + + + + + + + + + lyso- lyso- BMP BMP BMP SM CPE PC PE PA PG PI PS CL SM CPE (R/R) (S/S) (R/S)

Synthetic C lysoglycerophospholipids D plasmalogen E phospholipids - 1-alkenyl-2-acylglycerol - DAG MAG - - 1-alkenylglycerol

______ASM + + + + + + + + + + + + lyso- lyso- lyso- PC- lyso-PE- Palm- NBD- lyso- lyso- lyso-PC- Biotin- DHP PC PE PA PG PI Plasm Plasm Plasm PE PE PE

Micellar assay: 15 µg lipid/10 µg ASM/ 24 h at 37°C pH 4.5 ASM: recombinat HUMAN ASM expressed in insect Sf21 cells (Lansmann et al., 2000) Breiden, Oninla, Sandhoff, 2012 BMP and GM2AP stimulate the hydrolysis of LUV-bound ganglioside GM2.

BMP and GM2AP stimulate the hydrolysis of LUV-bound ganglioside GM2. The degradation of ganglioside GM2 inserted into LUVs doped with 0 mol % BMP (○), 5 mol % BMP (■), 10 mol % BMP (●), 20 mol % BMP (▴), or 25 mol % BMP (▪) was measured in the presence of increasing concentrations of GM2AP (0–2.2 μm).

Werth N et al. J. Biol. Chem. 2001;276:12685-12690 PA and SAP-C stimulate hydrolysis of LUV-bound glucosylceramide.

The degradation was measured with LUVs doped with 0 mol % PA (×), 5 mol % PA (•), 10 mol % PA (▴), or 20 mol % PA (▪) and increasing concentrations of SAP-C. All assay mixtures were prepared as described under “Experimental Procedures” and contained increasing proportions of PA in LUVs.

Wilkening et al, 2000, JBC 273, 30271-8 Anionic lipids in LUVs stimulate the enzymatic GlcCer hydrolysis in the presence and absence of SAP-C.

A, assays were conducted with + Sap-C GlcCer as substrate in the absence (○) and presence of SAP-C (2.5 μm) (•) using LUVs with various proportions of synthetic BMP (0–40 - Sap-C mol %). B, assays were carried out with varying concentrations of PI in LUVs, with (•) and without (○) the addition of 2.5 μm SAP-C, keeping the total lipid concentration in the assays constant. C, GlcCer carrying LUVs with or without PA were doped with increasing proportions of dolichol (0–10 mol + Sap-C %) and assayed for enzymatic GlcCer hydrolysis as follows: without SAP-C (○), with 2.5 μm SAP-C (•), with 10 mol % PA (▵), - Sap-C and with 10 mol % PA and 2.5 μm SAP-C (▴).D, assays were performed with varying concentrations of dolichol phosphate (DP) (0–10 mol %) with 2.5 μm SAP-C (•) or no SAP-C (○).

Wilkening G et al. J. Biol. Chem. 1998;273:30271-30278 Lysosomal sphingolipid degradation

NeuAc Gal- b(1-3)-GalNAc-Gal-Glc-Cer Gal-b (1-3)-GalNAc-Gal-Glc-Cer GM1 GA1

GM1-Gangliosidosis GM1-b -galactosidase GM2-Activator, Sap-B GM1-Gangliosidosis GM1-b -galactosidase

NeuAc GalNAc- b (1-4)-Gal-Glc-Cer GalNAc-b(1-3)-Gal-Gal-Glc-Cer GalNAc-b (1-4)-Gal-Glc-Cer GA2 Globoside GM2

b-Hexos- Tay-Sachs, Sandhoff b-Hexosaminidase A, B b-Hexosaminidase A Sandhoff Sandhoff aminidase A,B AB variant GM2-Activator GM2-Activator

Farber Acid ceramidase Sap-D Metachromatic Arylsulfatase A leukodystrophy Sap-B

Sphingosine O3 S-3-Gal-Cer Sulfatide