Upload
others
View
5
Download
0
Embed Size (px)
Citation preview
Sphingomyelin
O CH3
HN CH3
O
OHP
ON
OOCH3
H3C
H3C
Konrad SandhoffLIMES, Membrane Biology and Lipid Biochemistry UnitKekulé-InstitutGerhard-Domagk-Str. 1, 53121, BonnUniversität Bonn
Membrane lipids regulate glycosphingolipid catabolism, its
enzymes and lipid binding proteinsPhiladelphia, Aug. 2015
Inside
Outside
3 Na+
2 K+
ATPase
Electro-
chemical
potential,
Gradients
CeramideGanglioside GM1OH
HOOH
O
OH
O
OH
OHHO
O
O
AcHN
O
OH
OOH
O
O
OHOH
O
OH
O
HOOC
HO
OH
HO
CH3
HN CH3
O
OH
AcHN
-Gangliosides, SM & Chol. stabilize
neuronal membranes.
-However, Chol. & SM inhibit lysos.
catabolism of ganglioside GM2.-
Plasma membrane
SAPCD1-
loading
HydrolysisGlycocalix
CD1b
NPC-1
ASMNPC-2
Late endosome
Early endosome
Golgi
ER
LysosomeTemporal fusionand discharge
Sorting
Sorting
Vesiculartransport? LLBPs
Principles of lysosomal sphingolipid catabolism
and membrane digestion
pH Chol BMP
7.4
6.0
5.0
4.5
Gallala et al., J. Neurochem. 2011
Luminal vesicles (LV) are
platforms for SL, GSL-
and membrane-
degradation
1. Late endosomes:
Maturration of IMs by
removal of Chol (NPC1,
NPC2, NPC disease)
2. Lysosomes:
Degradation by enzymes
and LLBPs (GM2AP, Sap
A,B,C,D) :
Inherited defects cause
fatal diseases, lipid and
membrane storage,
loss of permeability
barrier in the skin,
and affect CD1 loading
3. KO mice are available
LV
LV
At late endosomes: Membrane lipids & ASM regulate
cholesterol transfer by NPC-2
Abdul-Hammed et al., J. Lipid Res. 2010
DONOR ACCEPTOR
+ endolysosomal
lipid
binding protein
Separation of
donor (pellet) and
acceptor (supernatant)
Analysis of
acceptor in
supernatant
Fluorescence
Radiolabeled [14C] lipid NBD-PE Biotin-PE host lipids
DONOR ACCEPTOR
+ endolysosomal
lipid
binding protein
Separation of
donor (pellet) and
acceptor (supernatant)
Analysis of
acceptor in
supernatant
Fluorescence
Radiolabeled [14C] lipid NBD-PE Biotin-PE host lipids
0
20
40
60
0 10 20
lipid concentration [mol%]
Ch
ole
ste
rol tr
an
sfe
rre
d
(% o
f to
tal ra
dio
activity in
su
pe
rna
tan
t/ 1
0 m
in/
0.0
92
nm
ol N
PC
2/ 2
00
µl) Cer +
BMP
SM
B
20 mol% BMP
Cer
0
10
20
30
40
50
0 30 60 90 120
Pre-incubation time of vesicles with ASM
[min]
Ch
ole
ste
rol tr
an
sfe
r
[% o
f to
tal ra
dio
activity in
sup
ern
ata
nt/ 0
.5 m
M N
PC
2/
10
min
]
8.0
8.5
9.0
9.5
10.0
SM
co
nce
ntra
tion
[mo
l%]
0
10
20
30
40
50
0 30 60 90 120
Pre-incubation time of vesicles with ASM
[min]
Ch
ole
ste
rol tr
an
sfe
r
[% o
f to
tal ra
dio
activity in
sup
ern
ata
nt/ 0
.5 m
M N
PC
2/
10
min
]
8.0
8.5
9.0
9.5
10.0
SM
co
nce
ntra
tion
[mo
l%]
0
10
20
30
40
4.2 5.0 6.0 7.4pH
Lip
id t
ran
sfe
rre
d
(% o
f to
tal ra
dio
activity in
su
pe
rna
tan
t/ 1
0 m
in/
0.0
92
nm
ol N
PC
2/ 2
00
µl)
Chol Sulf
PE PCGM3 GalCer
Cer
A
0
10
20
30
40
4.2 5.0 6.0 7.4pH
Lip
id t
ran
sfe
rre
d
(% o
f to
tal ra
dio
activity in
su
pe
rna
tan
t/ 1
0 m
in/
0.0
92
nm
ol N
PC
2/ 2
00
µl)
Chol Sulf
PE PCGM3 GalCer
Cer
AC
Oninla,Breiden, Sandhoff, 2014
Chol.-storage in NPC disease triggers GM2 & GSL accumulation
ASM is regulated by membrane lipids
0
5
10
15
20
0 100 200 300 400 500 600
ASM [ng]
PA
hyd
roly
sed
[%/2
h /3
7°
C]
PA
PG
BMP
w/o
PA
C
0,0
0,2
0,4
0,6
0,8
1,0
0 100 200 300 400 500 600
ASM [ng]
PE
hyd
roly
sed
[pm
ol/ 2
h/ 3
7°
C]
PAPE
D
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)
0
50
100
150
200
250
0 100 200 300 400 500 600
hyd
roly
ze
d l
ipid
[p
mo
l/ h
/ p
H 5
.0/ 3
7°C
]
ASM [ng/50 µl]
SM
PA
PG
BMP
w/o
A
0
50
100
150
200
250
0 100 200 300 400 500 600
hyd
roly
ze
d l
ipid
[p
mol/ h
/ pH
5.0
/ 37
°C]
ASM [ng/50 µl]
PC
PA
PG
BMP
w/o
B
Oninla, Breiden et al. (2014) JLR
Electrostatic binding of ASM to liposomes
Oninla et al., JLR 2014
�
0 50 100 150 200 250 300 350
0
1000
2000
3000
4000
5000
6000
7000
Time [s]
Resp
on
ce U
nit
s [
RU
]
EPC
neutral
MVL5
DOTMA
control (w/o liposomes)
baseline: w/o ASM
PG
PA
BMP
DHPPIPS
Association phase (ASM) Dissociation phase (buffer)
10 mol% Cholesterol
10 mol% SM
20 mol% anionic or cationic lipid
60 mol% DOPC
Zeta potential [mV ]
of liposomes
PG - 24.65
PA - 24.48
BMP - 26.53
PS - 23.58
PI - 21.50
DHP - 22.45
Neutral
(PC)
DOTMA
0.11
22.21
EPC 23.44
Infantile Amaurotic Idiocy:
cherry red spot
Tay-Sachs Disease:
Neuronal MCBs, Lysosomal storage
TSD is a recessive and lethal ganglioside storage disease
Wright et al. (2003) J Mol Biol. 331, 951-64
Wendeler et al. (2004) Eur. J. Biochem. 271:614-27
C(n=5,1)-TPD-GM2
(CH2)n
O F3C
NNO
OH
HNHO
HOHO
O
O OH
O OH
O
OHHOO
OH
O
HN
OH
HOOC
O
OH
HONH
O
OH
O
h*
Mechanism of GM2AP-Liftase
IM, luminal
vesicle
Michaelis-
Menten
Complex
Surface,
Plasmon
Resonance,
Biacore
BMP (PA&PI) stimulate
hydrolysis of liposomal GM2
by Hex A and GM2AP
BMP mobilizes membrane
lipids by GM2AP
BMP stimulates inter-
vesicular lipid transfer
by GM2AP
Anheuser et al.(2015),
JLR
Cholesterol
inhibits hydrolysis of
liposomal GM2 by
Hex A & GM2AP
Cholesterol
Inhibits solubilization
of M-lipids by GM2AP
Cholesterol inhibits
Intervesicular transfer
M-lipids by GM2AP
Biacore
Anhheuser et al.,
(2015) JLR
NPC: Lysosomal
cholesterol
accumulation
inhibits lysosomal
GM2 catabolism,e.g.
In NPC disease
Q: Is solubilization of
M-lipids by SAPs
physiologically
relevant?
H N
O H
O
O
O
O H
O H
O
S
O
O
O
H
R
RLyso = H2
O
RC18
=
O
RC2
=
O
RC6
=
0
100
200
300
400
500
600
Lyso C2 C6 C18
Acyl chain length (R)
+ Sap-B
- Sap-B
Deg
rad
atio
n r
ate
(pm
ol/
h x
mU
en
zym
e)
Vogel et al. (1991) EJB 200, 591-597
Lysosomes:
Lipid phase problem, sulfatide cleavage
( Also LUV-bound GlcCers by beta-Glcase, GM2 by Hex A, GM1 by beta-Galase )
Glycosylation of Sap- B is essential for its
physiological functions
Sap-B
Glycosylated Unglycosylated
Causing MLD(glycosylation site variants)
▬1,2 + 1,2
Rescue of Sap-B deficient cells + 3,4 ▬
4
Stimulation of sulfatide
degradation
(micellar/ liposomal assays)+ 4 ++ 4
Lipid transfer (sulfatide etc.) + 5 (+) 6
SPR (liposomes adsorbed on chip):
Lipid binding
Lipid mobilization
+ 4
++ 4
+ 4
▬4
In vitro:
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.
Misbaudeen Abdul-Hammed
Bernadette Breiden
Susi Anheuser
Günter Schwarzmann
Coworkers
vi
vmax
Lysosome
Substrate influx: vi
Degradation rate: vmax
[S]eq/Km = 1/[(vmax/vi)-1]
A
B
Critical threshold activity .....
Ste
ady
sta
tesubstr
ate
con
ce
ntr
atio
n[S
] eq
/K
m
Hypothetical solubilityof substrate
0.5
1.0
1 5 10 2015
5
15
10
Turn
ove
rra
teo
fsu
bstr
ate
v/v
i
Enzyme activity Vmax/vi
0
0
10
20
30
40
50
200 400 600 800 1000 1200
Tu
rno
ve
r ,%
of
incor p
or a
ted
GM
2
Hexosaminidase activity, pmol cleaved GM2 / h x mg x AU
Normal
Heterozygotes
Adult
Juvenile
Infantile
GM2-Activator deficiency
Threshold Theory
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 Biochem200(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
(GM1-Gangliosidosis)
GM1 galactosidase-b-
Sialidase
Arylsulfatase A
b-
b-HexosaminidaseA
HexosaminidaseB
b-Galactosyl-ceramidase
Sphingomyelinase
(Niemann-PickType A, B)
GalCer galactosidase
GM1 galactosidase
-b-
-b-
Ceramidase
(GM2-Gangliosidoses,AB, B, 0 Variant)
(Sialidosis)
a-Galacto-sidase A
(Fabry)
(Sandhoff)
(Fabry)
(Krabbe)
(Metachromaticleukodystrophy)
(Gaucher)
(Farber)
GM1
GM2
GM3
LacCer
GlcCer
Cer
Sphingosine
Sulfatide
DiGalCer
Globotriaosylceramide
Globoside
GalCer
Glucosylceramidase
a- GalactosidaseA
b-Hexosaminidase
(GM1-Gangliosidosis)
GM1 galactosidase-b-GM1 galactosidase-b-
Sialidase
Arylsulfatase A
b-
b-HexosaminidaseA
HexosaminidaseB
b-Galactosyl-ceramidase
Sphingomyelinase
(Niemann-PickType A, B)
GalCer galactosidase
GM1 galactosidase
-b-
-b-
Ceramidase
(GM2-Gangliosidoses,
(Sialidosis)
a-Galacto-sidase A
(Fabry)
(Sandhoff)
(Fabry)
(Krabbe)
(Metachromaticleukodystrophy)
(Gaucher)
(Farber)
GM1
GM2
GM3
LacCer
GlcCer
SphingomyelinCer
Sphingosine
Sulfatide
DiGalCer
Globotriaosylceramide
Globoside
GalCer
Glucosylceramidase
a- GalactosidaseA
b-Hexosaminidase
Sap-C, Sap-D
GM2AP, Sap-
B
Sap-B
Sap-B, Sap-C
Sap-C,
Sap-B
Sap-B
Sap-B
Sap-C
GM2-AP
Lysosomal & extracellular GlcCer degradation: Protein & lipid modifiers
Sandhoff, K. and Kolter, T. (1995) Naturw., 82, 403-413
Amaurotic idiocy: 5 genetic
diseases:
GM1- and 4 forms GM2-
gangliosidoses (Var AB, B, B1, 0)
Hex A deficiency: various clinical
courses (inf. juv. adult chron.),
Threshold theory
Coworker:
Bernadette Breiden
Misbaudeen Abdul-Hammed
Günter Schwarzmann
Susi Anheuser
Konrad Sandhoff, LIMES,
University of Bonn
Skin biopsy of a pSap-
deficient patient
(W. Roggendorf)
Feeding of pSAP reverses membrane storage
Burkhardt et al. (1997)
Bis(monoacylglycero)phosphate (BMP, S-configuration, sn1)
Wilkening, Linke, Sandhoff (1998) J. Biol. Chem. 273, 30271-8
• Electrostatic interaction: Cationic
proteins bind to anionic vesicles
• SAPs stimulate enzymatic
hydrolysis of liposome-bound
sphingolipids.
• BMP and other anionic PLs
stimulate degradation of:
- Cer, GlcCer, SM, GM1, GM2:
3-15 fold in the presence of SAPs.
(Sensitivity to CADs)
Lysosome
-
-
-
-
-
-
-
-
-
-
-
--
HO
Sap-CSap-D
acid
Ceramidase
Gluco-
cerebrosidase
CeramideGlucosylceramide
BMPBMP
Intralysosomal vesicles
GlycocalixLysosomal membrane
active siteactive site
Activator protein
Lysosomal enzyme
P
O
O
O
OOHH
OC
O
ROHH
ORC
O
P
O
O
O
O
OHHO
C
O
ROHH
ORC
O
P
O
O
O
O
OHHO
C
O
ROHH
ORC
O
-
+
-
+
+
+++
++
+
+
+
+
+
+
-
-
-
-
+
Sphingolipid degradation at intralysosomal membrane surfaces:
Stimulation by SAPs and anionic lipids
Sandhoff ,Proc. Jpn. Acad. Ser. B, Vol. 88,
554-82 (2012)
Lysosomal anionic lipids enhance the degradation of GM1 by β-galactosidase, but only in the presence of SAPs
Wilkening G et al. (2000) J. Biol. Chem. 275, 35814-35819
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.
Lysosomal lipids in LUVs stimulate the enzymatic GlcCer hydrolysis in the presence and also in the absence of 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
+ Sap-C
- Sap-C
-- 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
Conclusions----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
-- 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
N
H
O
O
OO
OOH
NNNNNNNN
R HH
R
RHRH
R
R O
O
O
O
O
O
O
H H H H
OO
HN
O
OHOH
OO
HN
O
OOH
OHO
O O
OH
HH
O
OO HN
O
O
OHOHHO
HO
OH
Permeability barrier of the skin
Golgi
Stra
tum
co
rne
um
Skin surface
AcylCer
Lipid
matrix
?
?Trans-
esterification
AcylGlcCer
Lip
id-b
ou
nd
en
ve
lop
e
Corneocyte
AcylGlcCer
CETrans-
esterification
b-GlcCerase
Sap-C
Glc
Glc
Acid Ceramidase
Sap-D, -C b-GlcCerase
Sap-C
w-O
H-C
er
w-O
H-G
lcC
er
w-O
H-f
att
y a
cid
PM
AcylGlcCer (EOS)
Stra
tum
gra
nu
los
um
CE
Lamellar body
Breiden & Sandhoff, 2013
R12-LOXeLOX3
HOO
Cong. icht.
Collod. babies
Collod. babies
template
Ultrastructure of pSAPknockout epidermis
Ruthenium staining reveals that thepSAP-deficient interstices (B, betweenarrows) lack the regular, compactpattern of lamellar membranesobserved in normal, pSAP-replete SC(C, between open arrows)..
Doering, T. et al. J Biol Chem, 274, 11038-11045
Biosynthesis of AcylGlcCer
AcylGlcCer
Glc
Golgi
ER
L-Serine
Palmitoyl-CoA
OO
O
NHO
O
HO
HO
OO
OH
HH
OO
NHO
OH
HO
AcylGlcCer
AcylCer
OH
NHO
OH
HO
Sphinganine
Cytosol
w-OH C16 FA-CoA
w-OH FA (C28)-CoA
Lipid droplet
DAG TAG Linoleic acid
PM
CerS 3
w-Hydroxylation
FA-elongation
complex
(ELOVL 1,3,6,7)
ELOVL 4
DAGT2Lipase
CGI 58
GlcCer-
synthase
(Ugcg)
Lamellar
body
w-OH-CerSPT
ABCA12
Breiden & Sandhoff, 2013
w-OH FA (>C28)-CoA
Alternative pathway
1. UDP-Glucose
2. Linoleoyl CoA
w-OH FA (<C24)-CoAELOVL 1, 4 Harlequin icht.
Dorfman-Chanarin S.
Cong. ichthyosis
Schemes of CerS3d/d phenotypic alterations with proposed cascades of events.
Jennemann R et al…..Sandhoff R, (2012) Hum. Mol. Genet.; 21:586-608
© The Author 2011. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
• CerS3 ko mice die shortly after birthfrom TEWL
• Loss of all SLs with acyl residues longer than 24 C-atoms, lack of protein-bound ceramides
• Lack of continuous extracellularlipid lamellae, water loss, hyperkeratosis
• High skin susceptibility to Candida infection
• SG –SC interface: absense of LBexocytotic figures and lamellar lipid-free domains
• Impaired processing of epidermalproteins: (pro) filaggrin, involucrin, loricrin.
High skin susceptibility to Candida infection
Jennemann R et al. .Sandhoff R (2012) Hum. Mol. Genet. 21:586-608
© The Author 2011. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
(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.
Principles of molecular and cellular pathology
- 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 oflysosomes may contribute to clinical heterogeneiety of diseases
- ULC-Glc-Ceramides & ULC-Ceramides are key components of the water & immunebarrier of mammalian skin. Defects in their metabolism can cause ichtiosys and areoften 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.
0
0,1
0,2
0,3
0,4
0,5
PI PI +30 mmol/l
desipramine
PG PG +30 mmol/l
desipramine
GM2AP-His6
GM2AP
Tu
rno
ver
of
GM
2[p
mol/h/p
mol G
M2A
P]
Hydrolysis of liposomal GM2 by HexA
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.
0
500
1000
1500
2000
2500
3000
3500
0 200 400 600 800 1000time [s]
baseline
GM2AP desipramine30 mmol/l
buffer
RU
Desipramine strongly reduces membrane binding of GM2AP
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.
Surface plasmon resonance (SPR) study
Cell type specific biosynthesis of glycosphingolipids
van Echten et al. (1989)
J. Neurochem. 52, 207-214
GM1
OH
OHO
NH
HOOH
OH
O
O
HO
O
OH
O OH
HOO
HO
O
COOHO
OH
O
NH
O
OH
OHO
OH
OH
NH
O
HO
O
Combinatorial Ganglioside Biosynthesis
GM1b
GA1
GA2
LacCer
GlcCer
GD1a
GM1a
GM2
GM3
GT1b
GD1b
GD2
GD3 GT3
GT2
GT1c
GQ1c
GP1c GP1c aGQ1b GQ1b aGT1a Gt1GD1c GD1a
GalT I
SAT I SAT II SAT IIIGalNAcT
GalT II
SAT IV
SAT V, SAT x
NeuAc a 2,8-
NeuAc
NeuAc a2,6-
GalNAc
0-series a-series b-series c-series
Cer
GlcTA
B
C D
E
aPM
ER
Golgi
TGN
Kolter, Proia, Sandhoff (2002) J. Biol. Chem. 277, 25859-62
lum
ina
l sid
e
O
O
OH
OH
O
OH OH
OH
O
O
-OOC
OHHO
HO
OHAcHN
O (CH2)12CH3
OH
NH
O
(CH2)16CH3
HO
GM3
A: lethal at embryonic day 6,
apoptosis in ectoderm (*)
D: normal life spanE: infertile (testis), myelin
D+E: "GM3 only", sudden
death, audiogenic
seizures
C: early death, formation of
0-series glycolipids,
infantile epilepsy
C+E: early death,
LacCer-3-sulfate
(*) neuron-, hepatocyte-,
keratinocyte-specific ko-
mice are born
Linke T et al. J. Biol. Chem. 2001;276:5760-5768
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.
Sap-D, PI and increased membrane curvature enhance
ceramide hydrolysis by aCerase
Wilkening, et al. (1998)
1. Induction of lipidoses in rats Lüllmann et al. (1978)
2. Detachment of acid sphingomyelinase from anionic
liposomes Kölzer et al. (2004)
3. Proteolytic degradation of acid sphingomyelinase and
other lysosomal enzymes by desipramine-treated
cultured fibroblasts. Hurwitz et al. (1994)
Cytoplasmic Inclusion body; x 78400
Chromaffin cell of a rat treated with
1-chloro-amitriptyline (120 mg/kg 10 wk)
N
NH
CH3
N
CH3
CH3
Cl
Chloroquine(Antimalaria)
N
NCH3
H
Desipramine(Antidepressant)
Propranolol(b-Adrenoreceptor-antagonist)
Many drugs are cationic amphiphilic lipids
(CADs) :
CADs are partially neutral at pH 7, penetrate mem-
branes, and are protonated & trapped in lysosomes.
Lysosome
-
-
-
-
-
-
-
-
-
-
-
--
HO
SAP-CSAP-D
acid
Ceramidase
Gluco-
cerebrosidase
CeramideGlucosylceramide
BMPBMP
intralysosomal Vesicles
Glycocalixlysosomal Membrane
active siteactive site
activator protein
lysosomal enzyme
-
++
+
+++
++
+
+
+
+
+
+
-
-
-
-
+
LysosomeLysosome
-
-
-
-
-
-
-
-
-
-
-
--
HO
SAP-CSAP-D
acid
Ceramidase
Gluco-
cerebrosidase
CeramideGlucosylceramide
BMPBMP
intralysosomal Vesicles
Glycocalixlysosomal Membrane
-
-
-
-
-
-
--
HO
SAP-CSAP-D
acid
Ceramidase
Gluco-
cerebrosidase
CeramideGlucosylceramide
BMPBMP
intralysosomal Vesicles
Glycocalixlysosomal Membrane
active siteactive site
activator protein
lysosomal enzyme
--
++++
++
++++++
++++
++
++
++
++
++
++
--
--
--
--
++
O N CH3
CH3
OH H
CADs: Cationic amphiphilic drugs (lipophilic)
induce a phospholipidosis: the molecular view
BMP and PI stimulate the degradation of membrane-bound ceramide in the absence of SAPs
BMP
Linke T et al. J. Biol. Chem. 2001;276:5760-5768
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.
Rate increases with curvature of liposomes
Sap-D stimulates up to 3fold
PI
Skin biopsy of a pSap-
deficient patient
(W. Roggendorf)
Feeding of pSAP reverses membrane storage
Burkhardt et al. (1997)
0.0
0.5
1.0
1.5
2.0
Ce
r [mo
l%]
1000
1500
2000
2500
3000
0 20 40 60 80 100
Pre-incubation time of vesicles with
ASM [min]
Ch
ole
ste
roltr
an
sfe
r
[nm
ol/h/m
gN
PC
2atpH
5.0
]
8.0
8.5
9.0
9.5
10.0
SM
[mo
l%]
with ASM control: w/o ASM
Degradation of SM by ASM stimulates
intervesicular cholesterol transfer by NPC2
Sandhoff, K. and Kolter, T. (1995) Naturwissenschaften, 82, 403-413
Lysosomal Sphingolipid Degradation
GM 1 galactosidase-b-
SialidaseSAP -B
Arylsulfatase ASAP -B
SAP -B
b-
b-Hexosaminidase A
Hexosaminidase B
b-Galactosyl -ceramidaseSAP -C
Sphingomyelinase
GalCer galactosidase
GM1 galactosidase
-b-
-b-
Ceramidase
Sialidosis
a-Galacto -sidase A
SAP -B
GM1
GM2
GM3
LacCer
GlcCer
Cer
Sphingosine
Sulfatide
DiGalCer
Globotriaosylceramide
Globoside
GalCer
SAP -C, SAP -D
SAP -C
Glucosylceramidase
a- Galactosidase A
SAP -B, SAP -C
GM2 -AP, SAP -B
b-Hexosaminidase
GM2 -AP
GM 1 galactosidase-b-GM 1 galactosidase-b-
SialidaseSAP -B
Arylsulfatase ASAP -B
SAP -B
b-
b-Hexosaminidase A
Hexosaminidase B
b-Galactosyl -ceramidaseSAP -C
Sphingomyelinase
GalCer galactosidase
GM1 galactosidase
-b-
-b-
Ceramidase
a-Galacto -sidase A
SAP -B
GM1
GM2
GM3
LacCer
GlcCer
SphingomyelinCer
Sphingosine
Sulfatide
DiGalCer
Globotriaosylceramide
Globoside
GalCer
SAP -C, SAP -D
SAP -C
Glucosylceramidase
a- Galactosidase A
SAP -B, SAP -C
GM2 -AP, SAP -B
b-Hexosaminidase A
GM2 -AP
GM1 Gangliosidosis
GM2 Gangliosidosis
AB, B, O Variant
Gaucher
Farber
Niemann-Pick
Type A, B
Krabbe
Metachromatic
leukodystrophy
Fabry
Fabry
Sandhoff
, SAP-A
Coworkers:
Bernadette Breiden
Vincent Oninla
Günter Schwarzmann
Susi Anheuser
Previous Coworkers:
Misbaudeen Abdul-Hammed
Natascha Remmel
Silvia Locatelli-Hoops
Wiebke Möbius
Michaela Wendeler
Melanie Kölzer
Thomas Döring
rgl Sap-B (5 µM)
-2000
-1000
0
1000
2000
0 100 200 300 400
Time (s)
Resp
on
se U
nit
s (
RU
)
pH 4.2
pH 7.0
pH 5.5
Sap-B: variant forms
-2000
0
2000
4000
6000
0 100 200 300 400
Time (s)
Re
sp
on
se
Un
its
(R
U)
r Sap-B,
deglycosylated
r Sap-B, Asn215His
r Sap-B, Asn215Gln
rgl Sap-B
Patient:
rgl Sap-B / BMP 20 mol%
-5000
-3000
-1000
1000
3000
0 100 200 300 400
Time (s)
Re
sp
on
se
Un
its
(R
U)
0
40
20
Chol
(mol %)
5
3825
BMP 14 mol%
g-Sap-B mobilizes lipids from BMP rich and
cholesterol poor membranes at acidic pH values
Surf. Plasmon resonance, Remmel, N. et al. (2006)
rgl Sap-B / BMP 0 mol%
-5000
-3000
-1000
1000
3000
0 100 200 300 400
Time (s)
Re
sp
on
se
Un
its
(R
U)
0
40
20
Chol
(mol %)
5
3825
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=200 nm
*
OHHO
OHO
O
OH
ONH
OOH
HOO
O
O
HN
OH
OO
O
OHO
OH
OH
HOOH
COO-
HOOH
HO
O
HO
H
H
O
NH
O
NHS
HN
NHO
*
OHHO
OHO
O
OH
ONH
OOH
HOO
O
O
HN
OH
OO
O
OHO
OH
OH
HOOH
COO-
HOOH
HO
O
HO
H
H
O
NH
O
NHS
HN
NHO
* Position of radiocarbon
• 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.
Perspectives for endocytosis and sphingolipid digestion
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.
Lysosomal lipid binding proteins
(SAPs and NPC2)
IMs are adjusted for degradation, BMP is generated and
cholesterol leaves inner vesicles of late endosomes
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
SM
Sphingosine
pH
Glycocalix
NPC-2
NPC-1
Ring Finger
Lateendosome
intraendosomalvesicle
ASM
?
?
sphingomyelin ceramide cholesterol
GlcCer
Ceramide
+ Fatty acid
5.5
pH 4.8
NTD
SM
(IM)
•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.
Pathologic mechanisms caused by lipid storage in late endosomes and lysosomes
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
rgl Sap-B (5 µM)
-2000
-1000
0
1000
2000
0 100 200 300 400
Time (s)
Resp
on
se U
nit
s (
RU
)
pH 4.2
pH 7.0
pH 5.5
Sap-B: variant forms
-2000
0
2000
4000
6000
0 100 200 300 400
Time (s)
Re
sp
on
se
Un
its
(R
U)
r Sap-B,
deglycosylated
r Sap-B, Asn215His
r Sap-B, Asn215Gln
rgl Sap-B
rgl Sap-B / BMP 0 mol%
-5000
-3000
-1000
1000
3000
0 100 200 300 400
Time (s)
Re
sp
on
se
Un
its
(R
U)
0
40
20
Chol
(mol %)
Patient:
53825
rgl Sap-B / BMP 20 mol%
-5000
-3000
-1000
1000
3000
0 100 200 300 400
Time (s)
Re
sp
on
se
Un
its
(R
U)
0
40
20
Chol
(mol %)
5
3825
BMP 14 mol%
gSap-B mobilizes lipids from BMP rich and
cholesterol poor membranes at acidic pH values
Plasmon resonance, Remmel, N. et al. (2006)
Lysosomal lipids in LUVs stimulate the enzymatic hydrolysis of ganglioside GM2 in the presence of GM2AP.
Werth N et al. J. Biol. Chem. 2001;276:12685-12690
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.
BMP and PI stimulate the degradation of membrane-bound ceramide in the absence of SAPs
BMP
Linke T et al. J. Biol. Chem. 2001;276:5760-5768
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.
Rate increases with curvature of liposomes
Sap-D stimulates up to 3fold
PI
BASM
SM PC
Cer
Ch
ol
Sti
mu
lati
on
/ in
hib
itio
n b
y
DAG
Ce
rD
AG
FA
MA
G
0 10 20 30 40mol%
0 10 20 30 40mol%
Anionic lipids (20 mol%): PA, PG BMP
A
Choltransfer NPC2
SM
Cer DAG
ASM
PC
host lipid (PC)SM
Cer DAG
Cholesterol
anionic lipids
stimulation inhibition
Membrane lipids regulates ASM activity
and cholesterol transfer by NPC2
(GM1-Gangliosidosis)
GM1 galactosidase-b-
Sialidase
Arylsulfatase A
b-
b-Hexosaminidase A
Hexosaminidase B
b-Galactosyl-ceramidase
Sphingomyelinase
(Niemann-PickType A, B)
GalCer galactosidase
GM1 galactosidase
-b-
-b-
Ceramidase
(GM2-Gangliosidoses,AB, B, 0 Variant)
(Sialidosis)
a-Galacto-sidase A
(Fabry)
(Sandhoff)
(Fabry)
(Krabbe)
(Metachromaticleukodystrophy)
(Gaucher)
(Farber)
GM1
GM2
GM3
LacCer
GlcCer
Cer
Sphingosine
Sulfatide
DiGalCer
Globotriaosylceramide
Globoside
GalCer
Glucosylceramidase
a- Galactosidase A
b-Hexosaminidase
(GM1-Gangliosidosis)
GM1 galactosidase-b-GM1 galactosidase-b-
Sialidase
Arylsulfatase A
b-
b-Hexosaminidase A
Hexosaminidase B
b-Galactosyl-ceramidase
Sphingomyelinase
(Niemann-PickType A, B)
GalCer galactosidase
GM1 galactosidase
-b-
-b-
Ceramidase
(GM2-Gangliosidoses,
(Sialidosis)
a-Galacto-sidase A
(Fabry)
(Sandhoff)
(Fabry)
(Krabbe)
(Metachromaticleukodystrophy)
(Gaucher)
(Farber)
GM1
GM2
GM3
LacCer
GlcCer
SphingomyelinCer
Sphingosine
Sulfatide
DiGalCer
Globotriaosylceramide
Globoside
GalCer
Glucosylceramidase
a- Galactosidase A
b-Hexosaminidase
Sap-C, Sap-D
GM2AP, Sap-B
Sap-B
Sap-B, Sap-C
Sap-C,
Sap-B
Sap-B
Sap-B
Sap-C
GM2-AP
Lysosomal sphingolipid degradation
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.
Gal- (1-4)-Glc-Cerb
GalCer- -galactosidaseb
Sap-B
Sandhoff
Ceramide
Acid ceramidaseSap-D
Farber
FabrySialidosis
Glucosylceramid- -glucosidasebSap-C
Sphingomyelinase
Fabry
Krabbe
Gaucher
Niemann-Pick A and B
Lactosylceramide
Gal- (1-1)-Cerb
Galactosylceramide
Gal- (1-3)-Gal-Cera
Digalactosylceramide
GM1- -galactosidasebSap-B and Sap-C
Sap-B
SialidaseSap-B
a-Galactosidase A
Gal- (1-4)-Gal-Glc-Cera
Globotriaosylceramide
GalNAc- (1-3)-Gal-Gal-Glc-Cerb
Globoside
b-Hexos-aminidase A,B
Galactosylceramide- -galactosidasebSap-A
Tay-Sachs, SandhoffAB variant
GM1-Gangliosidosis
GalNAc- (1-4)-Gal-Glc-Cerb
GM2
NeuAc
GM3
b-Hexosaminidase AGM2-Activator
GM1- -galactosidasebGM2-Activator, Sap-B GM1-Gangliosidosis
GM1
-Hexosaminidase A, Bb
GM2-Activator
GM1- -galactosidaseb
Sandhoff
Glc- (1-1)-Cerb
Gal- (1-3)-GalNAc-Gal-Glc-Cerb
GA1
GalNAc- (1-4)-Gal-Glc-Cerb
GA2
Glucosylceramide
Metachromaticleukodystrophy
SulfatideO S-3-Gal-Cer3
Sphingomyelin
Sphingosine
Arylsulfatase ASap-B
a-Galactosidase A
NeuAc
Gal- (1-3)-GalNAc-Gal-Glc-Cerb
aNeuAc- (2-3)-Gal- (1-4)-Glc-Cerb
Lysosomal sphingolipid degradation
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.
• 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.
Perspectives:
Membrane lipids regulate activity of endolysosomal proteins
Human prosaposin
protein domain
precursor coding region
noncoding region n insertion of 0, 6, 9 bp of Exon 8, 8‘
glycosylation sites
cysteine residues
n
A B
ATG AAATAG
0 0,5 1,0 1,5 2,0 2,5 kb
prosaposin
deficiency
PS P S
P
S
phosphorylation of CHO
sulfatation of CHO
protein domain
precursor coding regionprecursor coding region
noncoding regionnoncoding regionnoncoding region n insertion of 0, 6, 9 bp of 8, 8‘n insertion of 0, 6, 9 bp of 8, 8‘
glycosylation sites
cysteine residues
n
A B
ATG AAATAG
0 0,5 1,0 1,5 2,0 2,5 kb
MLD Gaucher
PPSS PP SS
PP
Krabbe
disease causing mutations
Sandhoff et al (2001) in: The Metabolic
and Molecular Bases of Inherited Disease
Lipid Storage and Threshold Theory
Patient
(Infantile)
Residual catabolic activity
(juvenile)
]1max
[
1][
-
Vi
VKm
S eq
Normal
Lysosome
Vi
[S]eq
Conzelmann and Sandhoff (1983/84), Dev. Neurosci., 6, 58-71
Wilkening, et al. (1998)
1. Induction of lipidoses in rats Lüllmann et al. (1978)
2. Detachment of acid sphingomyelinase from anionic
liposomes Kölzer et al. (2004)
3. Proteolytic degradation of acid sphingomyelinase and
other lysosomal enzymes by desipramine-treated
cultured fibroblasts. Hurwitz et al. (1994)
Cytoplasmic Inclusion body; x 78400
Chromaffin cell of a rat treated with
1-chloro-amitriptyline (120 mg/kg 10 wk)
N
NH
CH3
N
CH3
CH3
Cl
Chloroquine(Antimalaria)
N
NCH3
H
Desipramine(Antidepressant)
Propranolol(b-Adrenoreceptor-antagonist)
Cationic Amphiphilic Drugs (CADs):
• Many drugs are CADs
• CADs are neutral at pH 7 and penetrate membranes
• They are protonated and trapped in lysosomes
Lysosome
-
-
-
-
-
-
-
-
-
-
-
--
HO
SAP-CSAP-D
acid
Ceramidase
Gluco-
cerebrosidase
CeramideGlucosylceramide
BMPBMP
intralysosomal Vesicles
Glycocalixlysosomal Membrane
active siteactive site
activator protein
lysosomal enzyme
-
++
+
+++
++
+
+
+
+
+
+
-
-
-
-
+
LysosomeLysosome
-
-
-
-
-
-
-
-
-
-
-
--
HO
SAP-CSAP-D
acid
Ceramidase
Gluco-
cerebrosidase
CeramideGlucosylceramide
BMPBMP
intralysosomal Vesicles
Glycocalixlysosomal Membrane
-
-
-
-
-
-
--
HO
SAP-CSAP-D
acid
Ceramidase
Gluco-
cerebrosidase
CeramideGlucosylceramide
BMPBMP
intralysosomal Vesicles
Glycocalixlysosomal Membrane
active siteactive site
activator protein
lysosomal enzyme
--
++++
++
++++++
++++
++
++
++
++
++
++
--
--
--
--
++
O N CH3
CH3
OH H
CADs: Cationic amphiphilic drugs(lipids!)
induce a phospholipidosis: the molecular view
Michaelis Menten
Complex
A
Intraendolysosomal Membrane
Mechanism of GM2AP-liftase
Wright et al. (2003), J. Mol. Biol. 331, 951-64
Wendeler et al. (2004),Eur. J.Biochem. 271, 614-27 BWerth et al. (2001) J. Biol. Chem. 276, 12685
BMP (mol %)
5
10
20
25
PA,PI,DP,Sulf
(Chol 35 inhibts)
C(n=5,1)-TPD-GM2
(CH2)nO F3C
NNO
OH
HNHO
HOHO
O
O OH
O OH
O
OHHOO
OH
O
HN
OH
HOOC
O
OH
HONH
O
OH
O
-8000
-4000
0
4000
8000
0 90 180 270 360
Time (s)
Resp
on
se U
nit
s (
RU
)
(R
U)
2.5µM Cyt.-c
50mM NaCitrate
2.5µM gSap-A
20mM Chaps
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.
20 mM Chaps
2.5 µM gSap-A
50 mM NaCitrate
2.5 µM Cyt-C
Locatelli Hoops, S. et al. JBC (2006)
Lipid solubilization: Sap-A releases lipids from
liposomes bound to a sensor chip
Start--
MAG -
ASM
lyso-PC
lyso-PE
lyso-PA
lyso-PG
lyso-PI
lyso-PC-Plasm
NBD-PE
PC-Plasm
Biotin-PE
Palm-PE
lyso-PE-Plasm
DHP
+ _ _ _ _ _
+ + + +
lysoglycerophospholipids
- MAG
- DAG
SMlyso-SM
lyso-CPE
CPE
ASM
- Cer
- So
+ _ _ _ _
+ + +
sphingophospholipids
PC PA PG PI PS CLBMP(R/R)
BMP(S/S)
BMP(R/S)
PE
+ _ _ _ _ _ _ _ _ _ _
+ + + + + + + + +
glycerophospholipids
- 1-alkenylglycerol- DAG
- 1-alkenyl-2-acylglycerol
+ + + _ ___ __ _
+ + + +
plasmalogenSynthetic
phospholipids
A
C D E
B
Breiden, Oninla, Sandhoff, 2012
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)
ASM is a nonspecific phosoholipase C
BMP and GM2AP stimulate the hydrolysis of LUV-bound ganglioside GM2.
Werth N et al. J. Biol. Chem. 2001;276:12685-12690
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).
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
PA and SAP-C stimulate hydrolysis of LUV-bound glucosylceramide.
Anionic lipids in LUVs stimulate the enzymatic GlcCer hydrolysis in the presence and absence of SAP-C.
Wilkening G et al. J. Biol. Chem. 1998;273:30271-30278
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. C, GlcCer carrying LUVs with or without PA were doped with increasing proportions of dolichol (0–10 mol %) and assayed for enzymatic GlcCer hydrolysis as follows: without SAP-C (○), with 2.5 μm SAP-C (•), with 10 mol % PA (▵), 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 (○).
+ Sap-C
- Sap-C
- Sap-C
+ Sap-C
Gal- (1-4)-Glc-Cerb
GalCer- -galactosidaseb
Sap-B
Sandhoff
Ceramide
Acid ceramidaseSap-D
Farber
FabrySialidosis
Glucosylceramid- -glucosidasebSap-C
Sphingomyelinase
Fabry
Krabbe
Gaucher
Niemann-Pick A and B
Lactosylceramide
Gal- (1-1)-Cerb
Galactosylceramide
Gal- (1-3)-Gal-Cera
Digalactosylceramide
GM1- -galactosidasebSap-B and Sap-C
Sap-B
SialidaseSap-B
a-Galactosidase A
Gal- (1-4)-Gal-Glc-Cera
Globotriaosylceramide
GalNAc- (1-3)-Gal-Gal-Glc-Cerb
Globoside
b-Hexos-aminidase A,B
Galactosylceramide- -galactosidasebSap-A
Tay-Sachs, SandhoffAB variant
GM1-Gangliosidosis
GalNAc- (1-4)-Gal-Glc-Cerb
GM2
NeuAc
GM3
b-Hexosaminidase AGM2-Activator
GM1- -galactosidasebGM2-Activator, Sap-B GM1-Gangliosidosis
GM1
-Hexosaminidase A, Bb
GM2-Activator
GM1- -galactosidaseb
Sandhoff
Glc- (1-1)-Cerb
Gal- (1-3)-GalNAc-Gal-Glc-Cerb
GA1
GalNAc- (1-4)-Gal-Glc-Cerb
GA2
Glucosylceramide
Metachromaticleukodystrophy
SulfatideO S-3-Gal-Cer3
Sphingomyelin
Sphingosine
Arylsulfatase ASap-B
a-Galactosidase A
NeuAc
Gal- (1-3)-GalNAc-Gal-Glc-Cerb
aNeuAc- (2-3)-Gal- (1-4)-Glc-Cerb
Lysosomal sphingolipid degradation