in Gaucher disease fibroblasts

tanktherapistΒιοτεχνολογία

23 Οκτ 2013 (πριν από 3 χρόνια και 11 μήνες)

61 εμφανίσεις

Sandro Sonnino


Department
of Medical

Biotechnology and Translational Medicine,

Centre of Excellence on Neurodegenerative Diseases,

University
of Milan
,
Italy


Plasma membrane associated glycohydrolases

in
Gaucher

disease fibroblasts

Sphingolid

metabolic

pathways


(?)

MUB
-
substrate

solubilized

in appropriate
cell

culture medium

Free MUB

1
hrs

at 37
°
C

Cell

medium

Fluorimeter

MUB
-
β
-
D
-
galactopyranoside

b
-
galac瑯sidase

MUB
-

β
-
D
-
glucopyranoside

+ AMP
-
DNM

b
-
glucosidase

GBA1

MUB
-

β
-
D
-
glucopyranoside

+ CBE

b
-
glucosidase

GBA2

High

Throughput

Assay

for

the

detection

in

living

cells

of

hydrolase

activities

associated

to

the

external

site

of

the

p
lasma

m
embrane

by

the

use

of

artificial

substrates

solubilized

into

the

cell

culture

medium
.

(
Aureli

M
.

et

al
.

(
2011
)

J
.

Neurochem
)


MUB
-
N
-
acetyl
-

β
-
D
-
Glucosaminide
-
6
-
sulfate

b
-
Hexosaminidase


6
-
hexadecanoylamino
-
4
-
MUB
-
phosphorylcholine

卍ase


PM associated hydrolytic activities were found in all
cell lines tested

The

fluorescence

detected

in

the

cell

medium

was

not

due

to

a

release

of

the

enzymes

in

the

medium

during

the

assay

and

this

means

that

the

measured

activity

was

really

due

to

cell

surface

enzymes

No

fluorescence

was

found

associated

to

cell

homogenates
:

the

artificial

substrates

do

not

enter

into

the

cells
.

P group< 0.0035

Experimental

design

18

lines

of

human

fibroblasts

derived

from

patients

affected

by

Gaucher

disease

(
6

for

each

of

the

three

GD

types)

displaying

several

distinct

genotypes
.


Cell Lines
:

3 lines of human fibroblasts derived from healthy donors (CTRL)

Experimental tools
:

Evaluation of the total cell associated
glycohydrolase

activities

Evaluation of the
glycohydrolase

activities associated to the cell
surface in living cells

b
-
Glucosidase

residual

activity

in GD
fibroblasts

nmoles
/(
10
6

cells

*h
)

* p<0,0023 vs CTRL

°

p<0,03 vs GD1

# p<0,003 vs GD2

nmoles
/(
10
6

cells

*h
)

* p<0,0001 vs CTRL


A possible
genic

cross
-
talk between GBA1 and GBA2.

GBA2 total
cell

activity

°

p<0,003 vs GD1

nmoles
/(
10
6

cells

*h
)

GBA2/GAPDH

GBA2
mRNA

expression

level


What about the other PM glycohydrolases?

* p<0,004 vs CTRL

°

p<0,0035 vs GD1

nmoles
/(
10
6

cells

*h
)

Total
cell

activities

b
-
g慬慣ao獩d慳a

b
-
h數e獡浩nid慳a



慣瑩癩瑩敳

nmoles
/(
10
6

cells

*h
)

β
-
gal
activity is up
-
regulated in GD2

β
-
hex

activity

is

up
-

regulated

depending

on

the

clinical

phenotype

considered
.

No

significant

changes

are

found

for

both

enzymes

in

the

total

cell

associated

activity

@
p<0,0035 vs
GD2

We

found

significant

differences

in

the

plasma

membrane

enzymes

activity

among

the

three

clinical

types

of

the

disease

that

could

provide

a

cell

“fingerprint”

specific

for

each

of

the

clinical

types

thus

representing

a

potential

new

tool

useful

for

a

better

clinical

typing

of

this

pathology
.


We

found

significant

differences

of

the

plasma

membrane

enzyme

activities

among

the

three

clinical

types

of

the

disease,

that

could

provide

a

cell

“fingerprint”

for

each

of

the

clinical

types

thus

representing

a

potential

new

tool

useful

for

a

better

clinical

typing

of

this

pathology


In the PM of fibroblasts derived from patients affected by
Gaucher

disease
type 2, the form of the disease characterized by a severe impairment of
the central nervous system, we found the major increase of two enzymes:
β
-
galactosidase

and GBA2.

Is the increased activity of the PM

β
-
galactosidase

and GBA2 correlated with the
neuronal impairment?

GBA2

b
-
g慬慣ao獩d慳a

Changes of PM
glycohydrolase

activities in neuronal
differentiation of rat granule cells in culture

*p< 0.003

2

8

17

Days

in
culture (DIC)

Initial stage of
neuronal
differentiation

Morphologically and
biochemically fully
differentiated neurons

Late stage of neuronal
development
(aging
)

Analyzing

the

PM

glychohydrolases

activity

during

in

vitro

neuronal

differentiation,

we

found

that

the

PM

associated

activities

of

GBA
2

and

β
-
galactosidase

strongly

increase

in

senescent

neurons

with

respect

to

the

fully

differentiated

neurons
.

Neu3

β
-
Gal

GBA1
and
GBA2

PM ceramide
production

Apoptosis

GlcCer

LacCer

+

+

+

PM
sphingolipid

enzyme activities and the
neuronal impairment of
Gaucher

disease

As

already

demonstrated

for

other

cells,

also

in

neurons

the

increased

activity

of

the

PM

associated

glycohydrolases

leads

to

the

ectopic

production

of

ceramide

that

determines

the

onset

of

apoptotic

phenotype
.


PM
associated
activities in
living
fibroblasts at
different
pHs

4.5

5

5.5

6

6.5

7

7.5

8

8.5

200

0

400

600

1200

800

1000

GBA2

pmoles
/mg
cell

prot*

h

4.5

5

5.5

6

6.5

7

7.5

8

8.5

1000

0

2000

3000

4000

GBA1

pH

pH

4.5

5

5.5

6

6.5

7

7.5

8

8.5

0

400

800

1200

1600

2000

b
-
Hexosaminidase

4.5

5

5.5

6

6.5

7

7.5

8

8.5

0

1000

2000

3000

4000

b
-
Galactosidase

The

PM

associated

β
-
hexosaminidase
,

β
-
galactosidase
,

and

β
-
glucosidase

GBA
1

and

GBA
2
,

detected

at

the

cell

surface

of

living

cell,

are

characterized

by

a

strictly

dependence

on

the

pH

showing

the

best

working

condition

at

acidic

pH
.

pmoles
/mg
cell

prot*

h

Na
+
, H
+

antiporter

Na
+

PKC

+

PIP
2

IP
3

DAG

+

extracellular

signals

Model of the possible modulation of the SL metabolism at the cell
surface trough the control of the extracellular pH

b
-
gal

H
+

LacCer

+


GBA2

GlcCer

Cer

intracellular

signals

H
+


phospholipase

C

Na
+

Ca
++

Na
+
, H
+

antiporter

Na
+

Model of the possible modulation of the SL metabolism at the cell
surface through the control of the extracellular pH

b
-
gal

H
+

LacCer


GBA2

GlcCer

H
+


Na
+

It is reasonable to hypothesize
that, the activity of the proton
transporters might be responsible
for the creation of a transient local
acidic microenvironment close to
the cell surface, enough to cause
the activation of PM
glycohydrolases and thus
triggering a local change in the
cell surface
glycosphingolipid

composition

Cer

Na
+
, H
+

antiporter

Na
+

b
-
gal

H
+

LacCer


GBA2

H
+


Na
+

-

-

5
-
(
N
-
Ethyl
-
N
-
isopropyl
)
-

amiloride

(EIPA)

The

use

of

inhibitors

of

the

proton

transporters

could

block

or

reduce

the

activity

of

the

PM

glycohydrolases

Proton transporter activators/inhibitors

ΔpH
* in
fibroblasts

ΔpH
* in
glioma cells

ΔpH
* in
neuroblastoma

cells

acetazolamide

-
0.35
±
0.01

-
0.39
±
0.01

-
0.29
±
0.04

EGTA

-
0.28
±
0.03

-
0.38
±
0.07

-
0.31
±
0.02

EIPA

+0.38
±
0.07

+0.34
±
0.02

+0.28
±
0.09

esomeprazole

+0.31
±
0.05

+0.29
±
0.09

+0.36
±
0.03

EGTA
:

cell

surface

acidification

by

r
emoval

of

extracellular

Ca
++

induces

a

marked

spike

in

O
2

consumption

associated

to

the

reduction

of

mitochondrial

and

cytosolic

Ca
++
,

membrane

depolarization

and

influx

of

extracellular

Na
+

with

release

of

protons

Acetazolamide
:

cell

surface

acidification

by

inhibition

of

the

carboxy
-
anhydrase


EIPA
:

cell

surface

alkalinization

by

inhibition

of

the

NHEs


Esomeprazole
:

cell

surface

alkalinization

by

inhibition

of

the

K
+

proton

pump


Changes in the plasma membrane pH measured in living cells after
drug treatments

*The

reported

ΔpH

respect

to

the

physiological

pH

7
.
4

represent

the

mean

±

S
.
D
.

obtained

by

3

independent

experiments

each

consisting

of

a

mi nimum

of

6
-
9

wells

stained

with

pH

sensitive

fluorescent

probe

DHPE

in

presence

of

different

drugs

able

to

modulate

the

acti vity

of

the

PM

proton

exchanger
.

The

fluoresce

was

detected

by

the

microplate

fluorescence

reader


0,1
m
M

1
m
M

pmoles
/(10
6
cells*h)

*

*

GBA1

GBA2

0,1
m
M

1
m
M

0

2000

4000

6000

0

200

400

600

0

100

200

300

400

β
-
galactosidase

0,1
m
M

1
m
M

*

*

+

*

*

0,1
m
M

1
m
M

β
-
Hexosaminidase


0

150

300

450

*

GBA1

1mM

2mM

*

GBA2

1mM

2mM

*

β
-
Hexosaminidase

1mM

2mM

*

1mM

2mM

β
-
galactosidase

0

150

300

450

600

750

900

pmoles
/(10
6
cells*h)

EIPA:
inhibitors

of

the
NHEs

proton

pump
,
alkalinization

of

the
cell

surface

EGTA
:

Removal

of

extracellular

Ca
++

induces

a

marked

spike

in

O
2

consumption

associated

to

reduction

of

mitochondrial

and

cytosolic

Ca
++
,

membrane

depolarization

and

influx

of

extracellular

Na
+

with

release

of

protons

Cell surface proton pump modulation. A new opportunity for
changes of PM glycosidase activities.

Dr Massimo Aureli


Prof
. Alessandro Prinetti

Prof. Vanna
Chigorno

Dr. Nicoletta Loberto

Dr. Rosaria
Bassi

Dr. Maura Samarani

Dr. Valentina Murdica

Dr
. Elena Chiricozzi



Thanks

to
:

Dr. Mirella Filocamo

Dr. Stefano Regis

Prof.
Johannes M.
Aerts

Dr.
Rolf

G.
Boot

Department of Medical
Chemistry, Biochemistry
and Biotechnology

The Medical School

University

of

Milan

"Diagnosi
Pre
-
Postnatale

Malattie Metaboliche"
Laboratory
,

G. Gaslini
Institute

Department of Medical
Biochemistry, Academic
Medical
Center
, University
of Amsterdam

Telethon Genetic
Biobank

Network “Cell Line
and DNA
Biobank

from Patients affected by
Genetic Disease” (G.
Gaslini

Institute).

y=y0*e
(K*X)

y0= 0.01484

K= 0.4982

X= (Ln y
-

Ln
0.01484)/0.4982

Fluor pH X/ fluor pH7.4

R
2
=
0,99

CTRL
: pH
7,33

±
0.05

EIPA
: pH 7,81
±
0.07

EGTA
: pH 7,05
±
0.03

Calibration

curve

Fluorimetric determination of the pH at the cell surface after the use of
NHEs modulators

EIPA:
inhibitors

of

the
NHEs

proton

pump
,
alkalinization

of

the
cell

surface

EGTA
:

Removal

of

extracellular

Ca
++

induces

a

marked

spike

in

O
2

consumption

associated

to

reduction

of

mitochondrial

and

cytosolic

Ca
++
,

membrane

depolarization

and

influx

of

extracellular

Na
+

with

release

of

protons

Work in
progress

Apply the method to
amniocytes

or chorionic villous for the
prenatal diagnosis and prognosis of the
Gaucher

Disease

Preliminary results on human chorionic villous

Cell surface associated
activities on human chorionic
villous cells derived from
normal and subject affected by
Gaucher

disease type 2.

GBA1

GBA2

β
-
Hex


β
-
Gal

*
p<0,05
vs CTRL

*

*

*

*

The cell surface enzymatic
pattern is similar to that found
in fibroblasts

pmoles
/(
10
6

cells

*h
)