(GM1-Gangliosidosis) GM1 galactosidase -b- Sialidase Arylsulfatase A b- b- HexosaminidaseA HexosaminidaseB b-Galactosyl- ceramidase Sphingomyelinase (Niemann-Pick Type A, B) GalCer galactosidase GM1 galactosidase -b- -b- Ceramidase (GM2-Gangliosidoses, AB, B, 0 Variant) (Sialidosis) a- Galacto- sidase A (Fabry) (Sandhoff) (Fabry) (Krabbe) (Metachromatic leukodystrophy) (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- HexosaminidaseA HexosaminidaseB b-Galactosyl- ceramidase Sphingomyelinase (Niemann-Pick Type A, B) GalCer galactosidase GM1 galactosidase -b- -b- Ceramidase (GM2-Gangliosidoses, (Sialidosis) a- Galacto- sidase A (Fabry) (Sandhoff) (Fabry) (Krabbe) (Metachromatic leukodystrophy) (Gaucher) (Farber) GM1 GM2 GM3 LacCer GlcCer Sphingomyelin Cer 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 & extracellular degradation of GlcCer: 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 Coworkers: Bernadette Breiden Misbaudeen Abdul-Hammed Günter Schwarzmann Susi Anheuser Konrad Sandhoff, LIMES, University of Bonn
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Lysosomal & extracellular degradation of GlcCer: Protein ......Lysosomal lipids stimulate the enzymatic hydrolysis of liposomal GlcCer in the presence and absence of Sap-C. A: Assays
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(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 degradation of GlcCer: Protein & lipid modifiers
Sandhoff, K. and Kolter, T. (1995) Naturw., 82, 403-413
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 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.
neutral a n i o i c v e s c l e s
Lysosomal lipids stimulate the enzymatic hydrolysis of liposomal GlcCer in the presence and 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
Glucosylceramide metabolism:
Key to skin barrier function
Lipid barrier in the epidermis
is important against desiccation and
infections
Barrier is formed by extracellular
lipid layers in the SC: 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
(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-Glucosylceramides & ULC-Ceramides are key components of the water & immunebarrier of mammalian skin. Defects in their metabolism can cause ichtiosys and areoften fatal
-- 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
-- No direct correllation between gene mutations and the clinical
course of a disease, the microenvironment of the enzymic
reaction with the GSL- substrate modifies the catabolic rate.
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.
Misbaudeen Abdul-Hammed
Bernadette Breiden
Susi Anheuser
Günter Schwarzmann
Coworkers
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
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
Lysosomal Glycosphingolipid Storage Diseases
Christian de Duve (1949)Primary lysosomes (diameter: 25 nm -1 µm) originate from trans-GolgiSecondary Lysosomes result from fusion with endocytic vesicles.Stomachs of the cell, hydrolytic enzymes act at low pH (5.0-4.2) to degrademacromolecules (Endocytosis, Autophagy, Heterophagy) feeding the cell:
Renate Lüllmann-Rauch in P. Saftig (Hrsg.): Lysosomes (2005)
Bar = 0.5 µm
Philadelphia, Aug. 2015, Konrad Sandhoff, LIMES, University Bonn
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
Membrane lipids regulate hydrolysis of liposomal GM2
by HexA in presence of GM2AP
Membrane lipids regulate hydrolysis of GM2 by HexA in presence of GM2AP. (BMP , Chol , SM )
The His6-tag changes the abilities of GM2AP, it enhances liposomal GM2 turnover, but blocks
- 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)
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
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
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
Summary
NPC-2 is a cholesterol-binding and -transfer protein needed for secretion of
cholesterol from intraluminal vesicles (IMs) of late endosomes (SM inhibits,