Transcript presentation_ewggd_2..

Molecular Profiling of Gaucher Disease by
Fourier Transform
Infrared Spectroscopy
Serap DÖKMECİ (EMRE)
Hacettepe University Faculty of Medicine,
Department of Medical Biology, ANKARA, TURKEY
1. Gaucher Disease (GD)
• Gaucher disease is defined as an autosomal
recessive disorder resulting from deficiency of
the glucocerebrosidase (E.C.3.2.1.45), GBA,
also known as acid-β-glucosidase
Diagnosis
• Three recognized types:
– Type I (Noncerebral juvenile)
• Most common in Ashkenazi Jew lineage (1:450)
– Type II (Infantile cerebral)
• 1 in 100,000 live births
• Death usually occurs w/in 1 year
– Type III (Chronic neuropathic/Norbottnian)
• 1 in 50,000 live births
1. Gaucher Disease (GD)
• Glucocerebrosidase is a lysosomal hydrolase
responsible for the degradation of the natural
glycosphingolipid, glucosylceramide, into ceramide
and glucose.
• Deficiency of this enzyme results in the accumulation
of undegraded glucosylceramide, almost exclusively
in macrophages.
2. Fourier-Transform Infrared
Spectroscopy (FT-IR)
• With FT-IR, complete molecular diversity of the
samples can be studied comparatively with a
knowledge of origins of the peaks (such as
glycolipids, lipids, proteins etc.) as well as the
amount of the particular materials can be
determined.
• Also secondary structure ratios of proteins can be
determined by analyzing the amide bands. These
features provide invaluable information about
functional and structural changes in cells underlying
disease mechanisms.
2. Fourier-Transform Infrared
Spectroscopy (FT-IR)
• Infrared (IR) radiation is a noninvasive and
nondestructive type of radiation and when absorbed
by the tissues, fluids or cells, it causes vibration of
the covalent bonds of molecules within the sample.
• Fourier Transform Infrared (FT-IR) spectroscopy is a
widely used and preferred method of infrared
spectroscopy due to its speed and sensitivity.
A
B
S
O
R
B
A
N
C
E
2
4
1
3
WAVENUMBER(cm-1)
1545
1. Band position (Frequency/wavenumber): Definition of a
functional group
2. Signal intensity and band area: Concentration of a particular
molecule
3. Frequency shift: Structural information
4. Band width: Membrane fluidity
70
0
3. Aim of the Study
• The aim of this study is to achieve molecular
characterization of biomolecules in GD in
comparison with controls by using FTIR-ATR
spectroscopy and cluster analysis.
4. Material and Methods
Patient No
Gaucher
Type
Genotype
hepato/
splenomegaly
Bone involment
Neurologic
involvement
Cardiac
involvement
1
1
N370S/ -
+
+
-
-
+
2
3
D409H/D409H
+
+
(oculomotor
apraxia)
-
3
1
N370S/ -
+
+
-
-
4
1
N370S/ -
+
+
-
-
+
5
3
L444P/L444P
+
+
(oculomotor
apraxia)
-
6
3
L444P/L444P
+
+
-
-
+
+
(splenectomized)
(osteopenia)
-
-
-
-
7
1
N370/RecAHI
+
8
1
L296V/L290V
(splenectomized)
+
4. 1. Patients and Cell Culture
• Fibroblast were cultured from skin biopsy.
• Human skin fibroblast from unaffected
individuals and these patients were cultured
in BME supplemented with 10 % FCS in 75
cm2 culture flasks in a humidified atmosphere
containing 5 % CO2 at 37 0C.
4. 2. FTIR-ATR
• Cultured skin fibroblast cell samples were used for
FT-IR measurements.
• Infrared spectra were obtained by a Bruker Tensor 27
FT-IR (Bruker Optics, GmbH, Germany) equipped
with a liquid nitrogen cooled photovoltaic MCP
detector and universal attenuated total reflectance
(ATR) cell (Pike Technologies, Wisconsin, U.S.A).
4. 2. FTIR-ATR
• 2.5 µl of cell in PBS were spotted onto ZnSe
ATR crystal. Sample was dried on the crystal by
very low pressure nitrogen gas for 3 minutes.
• Spectra recorded in the mid-infrared region,
between 4500-850 cm-1 wavenumbers.
4. 2. FTIR-ATR
• Baselined absorbance spectra were used for
exact integration calculations.
• Relative intensity values of second derivative
peaks in the Amid I region (1700-1600 cm-1)
which were obtained by automated peak
picking were used to compare protein
secondary structures.
• Spectral analyses were carried out by OPUS
software and t-test was performed using SPSS
software.
5. RESULTS and DISCUSSION
5. 1. FTIR Fingerprinting
5. 2. Comparison of the levels of biomolecules
5. 3. Determination and comparison of protein
secondary structures
5. 1. FT-IR Fingerprinting
• The spectra were complex with many characteristic
bands.
• Two main regions:
– Lipid dominated region (between3500–2840 cm-1)
– Fingerprint region (1800-850 cm-1)
• 20 major absorption bands were assigned to various
biomolecules such as proteins, lipids, cholesterol
esters, nucleic acids and carbonhydrates in the
present study.
Absorbance (Arbitary Units, A. U.)
Fingerprint region
Lipid dominated region
W A V E N U M B E R (cm-1)
Representative FT-IR absorbance spectrum between 4500–850 cm-1
obtained by averaging all the spectra used and average absorbance
spectra of GD and control groups.
Lipid Dominated Region
5
GD
1
Control
4
6
2
7 8
3
9
Average FT-IR absorbance spectrum of GD and controls between 3600-2750 cm-1 . The
spectrum was normalized with respect to the Amid A band between 3500-3100 cm-1
Peak Number
1
Wavenumber (cm-1)
3294
Definition
Amide A, mainly N–H stretching of proteins with contribution
from intermolecular H bondings and O–H stretching mode of
polysaccharides
Organic compound
Mainly proteins
2
3
4
3060
3011
2957
Amide B, N–H stretching
Olefinic =CH stretching
CH3 asymmetric stretching
Proteins
Unsaturated lipids
Mainly lipids, low signal from proteins
5
2929
CH2 asymmetric streching
Mainly lipids, low signal from proteins
6
2907
CH2 asymmetric streching
Minly lipids, low signal from proteins
7
2871
CH3 symmetric streching
Mainly proteins, low signal from lipids
8
2858
CH2 symmetric streching
Mainly lipids, low signal from proteins
9
2843
C–H stretching
Mainly lipids
11
Fingerprint Region
12
Average FT-IR absorbance spectrum of GD and controls between
1800-850 cm-1 cm-1 . The spectrum was normalized with respect to
the Amid I band between 1700-1600 cm-1
16
15
13
14
Control
17
18 19
10
20
GD
Peak Number
10
Wavenumber (cm-1)
1732
Definition
Saturated ester C=O stretching
11
1653
12
13
14
15
1544
1455
1396
1241
Amide I; 80% C=O stretching, 10% N–H bending, 10% C–N
stretching
Amide II; 60% N–H bending, 40% C–N stretching
CH2 bending
COO– symmetric stretching
PO2- asymmetric stretching (fully hydrogen-bonded)
16
17
1147
1094
18
1041
19
1021
20
976-875
C–O stretching
PO2- ionized symmetric stretching of phosphodiester groups,
C–O stretcing
C–O stretching, coupled with C–O bending of the C–OH
groups of carbonhydrates
C–O stretching, coupled with C–O bending of the C–OH
groups of carbonhydrates
C–N+ –C stretching
Organic compound
Cholesterol esters, phospholipids, ester
functional groups in lipids
Proteins
Proteins
Lipids
Fatty acids
Nucleic acids, phophorylated proteins and
phospholipids
Carbonhydrates/glycogen, nucleic acids
Nucleic acids, phospholipids, glycogen,
oligosaccharides and glycolipids
Oligosaccharides, polysaccharides
Oligosaccharides, polysaccharides
Nucleic acids, ribose-phosphate main chain
vibrations of RNA, phosphate monoesters
5. 2. Comparison of the Levels of
Biomolecules
• We used integration values (band area) of each peak
using OPUS software for comparative purposes.
• As a result of the comparative evaluation, lipid and
protein levels are seen to increased in GD.
• Also we observed individual variation.
• Additionally, bandwidth of CH2 symmetric stretching
of lipids is slightly decreased in GD, indicating the
descrease in mebrane fluidity.
5. 2. Comparison of the levels of
biomolecules
Calculated as a sum of mean band area values of the bands originated from lipids.
5. 2. Comparison of the levels of
biomolecules
Calculated as a sum of mean band area values of Amid I and II bands.
INDIVIDUAL VARIATION
Lipid Dominated Region
11
5
Fingerprint region
12
4
16
1
15
78
2
3
9
10
Normalization band
Normalization band
6
13
14
17
18 19
Individual absorbance spectra of GD patients (G1-G8) and controls between (A)
3600–2750 cm-1 and (B) 1800-850 cm-1 wavenumbers. The spectral range
between 3600–2750 cm-1 and 1800-850 cm-1 were normalized with respect to
the Amid A band between 3500-3100 cm-1 and Amid I band between 1700-1600
cm-1, respectively after baseline correction.
20
5. 3. Determination and Comparison of
Protein Secondary Structures
• We determined protein secondary structures
using second derivative spectrum of Amide I
band.
• Band intensities were used for comparative
purposes.
• We observed an increase in the ratio of
antiparallel β-sheet and α-helix structures in
GD, while β-sheet was decreased.
• Also individual variation was observed.
Individual variation
Second derivative spectra of Amid I absorption band between 1700–1600 cm-1 wavenumbers which
demonstrates main protein secondary structures. Absorption maxima appear as minima and the
spectra were vector normalized. (A) Mean spectra of GD and control groups. (B) Individual spectra of all
the samples.
Peak Number
Wavenumber (cm-1)
Protein secondary structure
I
1697
β-turns/edge
II
1684
Antiparallel β-sheet, β-turns
III
1672
Turn, bend, β-turn
IV
1654
α-helix
V
1636
β-sheet
VI
1615
Amino acid side chain vibrations, intermolecular β-sheets
6. CONCLUSION
• FTIR stretoscopy is an valuable tecnique for
the investigation of biological structure due to
its sensitivity and ability to give valuable
information about the functional groups
which might have diagnostic value for
biological systems.
6. CONCLUSION
• We report the first FT-IR spectrum of GD patient
fibroblast cells in the mid-infrared region with their
spectral assignments. Also this is the first FT-IR
spectroscopic work aimed to determine molecular
alterations in GD.
• Lipid and proteins are important parametres for the
proper functioning of biological membranes, which, in
turn, influence celluler processes and disease states.
• Minor alterations of these lipids have a significant affect
on vesicular trafficing and intercellular signaling further
contributing to the complex pathology manifest in GD.
Naşit İGCİ1, Parisa SHARAFI2, Duygu ÖZEL DEMİRALP1, Aysel
YÜCE3, Özerk DEMİRALP4
1 Ankara University Biotechnology Institute Proteomics Department
2 Hacettepe University Faculty of Medicine, Department of Medical Biology
3 Hacettepe University İhsan Dogramacı Children Hospital, Pediatric Gastroenterology,
Hepatology and Nutrition Unit
4 Atatürk Training and Research Hospital, Department of Plastic and Reconstructive Surgery