Metabolic Lysosomal Enzyme Probes
1
Naleway ,
Introduction
Defects in lysosomal enzyme activity have been associated with a variety of diseases including
Parkinson’s, Tay-Sachs, Sandhoff, Krabbe and Gaucher syndromes. New lysosomal staining
probes useful for labeling lysosomes in a live-cell format and capable of monitoring lysosomal
metabolic activity have been developed. These new targeted substrates are based upon
fluorescent probes that have a low pKa value for optimum fluorescence at the lower physiological
pH values found in lysosomes, contain specific enzymatically-cleavable functions for specific
enzyme activities as well as targeting groups to direct their accumulation to the lysosomes using a
live-cell staining format.
Application to the staining of cells derived from blood samples of patients with Metachromatic
Leukodystrophy, Krabbe, Gaucher I, II and III, Tay-Sachs and Sandhoff Diseases as well as
healthy human fibroblast and leukocyte control cells are presented. In addition the ability to
monitor the effect of secondary therapeutic agents on enzyme activity is presented. This work was
supported by NIH Grant 5R44NS073225-04
O
O
+ 6-isomer
1o and 2o
1o alone
2o alone
N
Figure 6. Live
fibroblast cells were
stained with 5 uM
M1268 at 37oC
overnight. Cells were
washed 3x with
PBS, then OptiKlear™ Imaging
Buffer containing 1
ug/ml Hoechst 33342
was added for
imaging. Cells were
imaged on Zeiss
Axio Observer A1
fluorescence
microscope using
40X air objective.
O
+ 6-isomer
N
H
a) M1268
O
b) M1359
c) M1903
Figure 1. Structures of substrates for a) Acid Esterase, b) Aryl-Sulfatase and c) β-Glucosidase.
Krabbe
cells
Measurement of Metabolic Activity
Effect of Chloroquine
Treatment on M1268
Metabolism
1600
1400
Fluorescence
1200
1000
800
600
400
200
0
0
100
Non-diseased cells
Figure 3. Live Gaucher
III fibroblasts stained for
60 min with 5 uM M1268
then fixed, permeabilized
and blocked overnight at
4oC. Primary rabbit-antiLAMP1 pAb was applied
for 1 hour at 22oC. After
washing, an Alexa Fluor
555-goat-anti-rabbit Ab
conjugate was applied
for 30 minutes at 22oC.
Cells were stained with
Hoechst 33342 prior to
imaging on Zeiss Axio
Observer A1
fluorescence microscope
with 40X air objective.
Non-diseased
cells
Cl
N
N
H
No dye
OSO3Na
Cl
O
O
Drug Discovery
Figure 4. Live
AG06173
fibroblasts were
stained for 2
hours at 37oC
with 2 uM
M1268 and 75
nM
LysoTracker®
Red. Cells were
washed 3x with
PBS and
imaged in serum
free media on
Zeiss Axio
Observer A1
fluorescence
microscope with
40X air
objective.
M1268
LysoTracker®
Red
Merge
Diseased cells
200
300
Chloroquine (uM)
400
500
Figure 2. Live AGO6173 fibroblast
cells were treated with varying
amounts of chloroquine in complete
media, and incubated overnight at
37oC, 5% CO2. Cells were then
stained with 10uM M1268 for 60 min
at 37oC, 5% CO2, then washed and
media replaced with Opti-Klear™
Imaging Buffer containing 1ug/ml
Hoechst 33342. Fluorescence was
then measured using a Tecan Infinite
M200 Pro plate reader. Hoechst
33342 fluorescence was used to
normalize for cell number and the
data plotted. Error bars are one
standard deviation of the mean.
Methods and Materials
• Primary Human Fibroblasts from Gaucher II(F0381999) and Krabbe (F0461990) patients were
obtained from Istituto Giannina Gaslini (Genova, Italy). Cells were cultured in RPMI 1640
Medium supplemented with 10% FCS and 1X Antibiotic-Antimycotic Solution (Toku-e,
Bellingham, WA).
• Primary Human Fibroblasts from a healthy donor (AG06173), Metachromatic Leukodystrophy
(GM00243), and Mucopolysaccharidosis Type VI (GM00519) patients were obtained from the
Coriell Institute for Medical Research (Camden, NJ). Cells were cultured in Minimum Essential
Medium Eagle supplemented with 10% FCS and 1X Antibiotic-Antimycotic Solution.
• Immortalized B-lymphocytes from Gaucher I (GM10870), Gaucher II (GM08752), Gaucher III
(GM01769), Metachromatic Leukodystrophy (GM01017), and Mucopolysaccharidosis Type VI
(GM01022) patients were obtained from the Coriell Institute for Medical Research. Cells were
cultured in RPMI 1640 Medium supplemented with 15% FCS and 1X Antibiotic-Antimycotic
Solution.
• Immortalized B-lymphocytes from Glycine encephalopathy (73022, used as normal control cells)
and 3 Fabry (100121, 100810, 100975) patients were provided by Dr Longo. Cells were cultured
in RPMI 1640 Medium supplemented with 15% FCS and 1X Antibiotic-Antimycotic Solution.
• Cell lines were incubated at 37ºC, 5% CO2 humidification.
• Cell labeling solutions were prepared by diluting substrate stock solutions in the appropriate
medium for the cell line (see above). Hoechst 33342 was added to a final concentration of 1
ug/ml for use as a nuclear stain.
• Labeled cells were washed 3x with Phosphate Buffered Saline and media replaced with 1X OptiKlear™ Live Cell Imaging Buffer (M1919) prior to imaging. Cells were imaged using a Zeiss
Axio Observer A1 equipped with a Q Imaging QI Click digital camera.
• For flow cytometry, cells were stained as above without nuclear staining, then resuspended in 1X
MarkerGene™ Flow Holding and Sorting Buffer for Non-Adherent Cells (M1926) and then run on
a BD Accuri C6 Flow Cytometer with unstained cell controls.
Mucopolysaccharidosis
VI
Cells
Fibroblasts
A
Figure 5.
Live
diseased
(Pompe)
and nondiseased
cells were
stained with
M1268 and
BODIPYCeramide.
Cells were
imaged by
Confocal
microscopy.
M1268
BODIPYCeramide
B
60000
30000
Undiseased
25000
Mucopolysaccharidosis
VI
15000
Krabbe
10000
Gaucher II
5000
50uM
Undiseased
50000
Fabry
40000
Gaucher I
30000
Gaucher II
20000
Gaucher III
10000
0
Cell Type
0
-10000
Cell Type
Mucopolysaccharidosis
VI
Metachromatic
Leukodystrophy
Figure 7. Live fibroblast (A)
and B-lymphocytes (B) cells
were stained with 5uM M1268
at 37oC overnight. Cells were
washed 3x with PBS and
Opti-Klear™ Imaging Buffer
was added for imaging. Cells
were imaged on Zeiss Axio
Observer A1 fluorescence
microscope using 40X air
objective. The image intensity
was then measured using
CellProfiler (Broad Institute,
MIT) and the average
intensity plotted.
Non-diseased
Cells
Non-diseased
Figure 8. Live
fibroblast cells
were stained
with 200uM
M1359 at
37oC
overnight.
Cells were
washed in
PBS and
imaged on
Zeiss Axio
Observer A1
fluorescence
microscope
using 40X air
objective
Mucopolysaccharidosis
VI
Metachromatic
Leukodystrophy
Non-diseased
Mucopolysaccharidosis VI
Metachromatic
Leukodystrophy
Figure 9. Live Blymphocyte cells
were stained with
100uM M1359 at
37oC for 2 hours.
Cells were
washed, then
resuspended in
MarkerGene™
Flow Buffer.
Fluorescence of
10000 events
was then
measured using
the FL 1 channel
of the BD Accuri
C6 flow
cytometer.
Non-diseased
Gaucher I
B
Non-Diseased vs Gaucher II
1
2
3
4
Figure 13. Live AG06173 cells
were stained with 10 uM
M1268 for 60 min, followed by
4% paraformaldehyde fixation,
then staining with 1 ug/ml
Hoechst 33342 and 50 ng/ml
TAMRA-SE for 60 min. Cells
were imaged on Zeiss Axio
Observer A1 fluorescence
microscope using 40X
objective (A). Images can then
be processed in CellProfiler to
identify cells (B1), nuclei (B2),
cytoplasm (B3) and lysosomes
(B4). The lysosome intensity
and number can then be
measured on a per cell basis.
Non-diseased
Gaucher Type I
Gaucher Type II
Gaucher Type III
Discussion and Conclusions
Gaucher II
Gaucher III
Figure 10. Live B-lymphocyte
cells were stained with 50uM
M1903 at 37oC for 2 hours. Cells
were washed, then resuspended
in MarkerGene™ Flow Buffer.
Fluorescence of 10000 events
was then measured using the FL
1 channel of the BD Accuri C6
flow cytometer.
Mutation Screening by High Resolution Melt Analysis
Non-diseased vs Gaucher I
25uM
A
Disease-Specific Substrates
Metachromatic
Leukodystrophy
Cells
Figure 12. Live Gaucher
Type III B-lymphocytes
were incubated for 72 hours
with 0-200 uM M131941 at
37oC. The cells were then
stained for 1.25 hours with
100 uM M1903 at 37oC.
Cells were washed and
resuspended in
MarkerGene™ Flow Buffer
then analyzed on a BD
Accuri C6 Flow Cytometer.
After gating for
autofluorescence 0 uM drug
(black line) was plotted
against drug (red line).
100uM
Merge
B-Lymphocytes
35000
20000
200uM
Suitability for High Content Screening
Average Total Image Intensity
(fluorescence)
NaO3SO
Cl
O
5uM M1268
Average Total Image Intensity (fluorescence)
Cl
1
Batchelor .
Differences in Lysosomal Burden of Diseased Cells
Structures
OAc
2
Pasquali ,
Localization of Esterase Substrate to Lysosomes
Lysosomes are acidic cytoplasmic organelles that are present in nucleated mammalian cells and
are involved in a variety of cellular processes including repair of the plasma membrane, defense
against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell
signaling.
O
2
Longo ,
John Joseph
Fiona Karen
Nicola
Marzia
Robert Hardy
1 Research and Development, Marker Gene Technologies, Inc., Eugene, OR, 2 Medical
Genetics/Pediatrics, University of Utah School of Medicine, Salt Lake City, UT
576.1
AcO
1
Harlan ,
Non-Diseased vs Gaucher III
Figure 11. Genomic DNA was
extracted from diseased and
non-diseased B-lymphocytes.
Primers were designed to
flank region containing known
mutation 1226A>G/N370S (F:
GGATCGAGGGATGCAGTAC
AG, R:
GGTTCAGGGCAAGGTTCC
A). High Resolution Melt was
performed using Applied
Biosystems StepOne qPCR
Instrument. Data will be
verified by sequencing.
• The present substrates have been confirmed as localizing to the lysosome when compared
with known lysosomal stains.
•When cell metabolism is inhibited with chloroquine, staining with an esterase substrate is
reduced, demonstrating that the substrates can be used to measure cell metabolism.
•When applied to diseased cells, the intensity of M1268 staining is increased, reflecting the
increase of lysosomal burden found in diseased cells.
•Substrates specific to the enzyme deficiency in Gaucher or Metachromatic Leukodystrophy
exhibit reduced staining compared to that in non-diseased cells.
•Disease-causing mutations can be screened for quickly and easily using the molecular
biology technique of high resolution melt(HRM).
•With the development of a CellProfiler pipeline capable of identifying individual cell organelles
including lysosomes, a high content screening assay was developed to measure enzyme
activity with or without drug treatment and to screen compound libraries for new drug targets.
•Treatment of Gaucher III cells with the novel compound M131941 exhibited an increase in
lysosomal glucosidase enzyme activity over untreated cells.
In conclusion, the data presented here demonstrates that our new targeted substrates are
suitable for monitoring the effect of secondary therapeutic agents on lysosomal enzyme
activity.
References
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