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Molecular Profiling of Gaucher Disease by Fourier Transform Infrared Spectroscopy Hacettepe University Faculty of Medicine, Department of Medical Biology, ANKARA, TURKEY Serap DÖKMECİ (EMRE)

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Molecular Profiling of Gaucher Disease by Fourier Transform Infrared Spectroscopy Hacettepe University Faculty of Medicine, Department of Medical Biology , ANKARA, TURKEY . Serap DÖKMECİ (EMRE). 1. Gaucher Disease (GD). - PowerPoint PPT Presentation

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Page 1: Serap DÖKMECİ (EMRE)

Molecular Profiling of Gaucher Disease by Fourier Transform

Infrared Spectroscopy

Hacettepe University Faculty of Medicine, Department of Medical Biology, ANKARA, TURKEY

Serap DÖKMECİ (EMRE)

Page 2: Serap DÖKMECİ (EMRE)

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

Page 3: Serap DÖKMECİ (EMRE)

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

Page 4: Serap DÖKMECİ (EMRE)

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.

Page 5: Serap DÖKMECİ (EMRE)

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.

Page 6: Serap DÖKMECİ (EMRE)

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.

Page 7: Serap DÖKMECİ (EMRE)

700

2

WAVENUMBER(cm-1)

ABSORBANCE

1545

1

3

4

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 information4. Band width: Membrane fluidity

Page 8: Serap DÖKMECİ (EMRE)

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.

Page 9: Serap DÖKMECİ (EMRE)

4. Material and Methods

Page 10: Serap DÖKMECİ (EMRE)

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 + + - -

7 1 N370/RecAHI

+ +

- -(splenectomized) (osteopenia)

8 1 L296V/L290V

+

+ - -(splenectomized)

Page 11: Serap DÖKMECİ (EMRE)

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.

Page 12: Serap DÖKMECİ (EMRE)

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).

Page 13: Serap DÖKMECİ (EMRE)

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.

Page 14: Serap DÖKMECİ (EMRE)

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.

Page 15: Serap DÖKMECİ (EMRE)

5. RESULTS and DISCUSSION

5. 1. FTIR Fingerprinting5. 2. Comparison of the levels of biomolecules5. 3. Determination and comparison of protein secondary structures

Page 16: Serap DÖKMECİ (EMRE)

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.

Page 17: Serap DÖKMECİ (EMRE)

Abs

orba

nce

(Arb

itary

Uni

ts, A

. U.)

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

Fingerprint region

Page 18: Serap DÖKMECİ (EMRE)

1

9

876

5

4

32

Lipid Dominated Region

Peak Number Wavenumber (cm-1) Definition Organic compound1 3294 Amide A, mainly N–H stretching of proteins with contribution

from intermolecular H bondings and O–H stretching mode of polysaccharides

Mainly proteins

2 3060 Amide B, N–H stretching Proteins3 3011 Olefinic =CH stretching Unsaturated lipids4 2957 CH3 asymmetric stretching 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

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

ControlGD

Page 19: Serap DÖKMECİ (EMRE)

10

11

191817

1615

1413

12

20

Peak Number Wavenumber (cm-1) Definition Organic compound10 1732 Saturated ester C=O stretching Cholesterol esters, phospholipids, ester

functional groups in lipids11 1653 Amide I; 80% C=O stretching, 10% N–H bending, 10% C–

N stretchingProteins

12 1544 Amide II; 60% N–H bending, 40% C–N stretching Proteins13 1455 CH2 bending Lipids14 1396 COO– symmetric stretching Fatty acids15 1241 PO2

- asymmetric stretching (fully hydrogen-bonded) Nucleic acids, phophorylated proteins and phospholipids

16 1147 C–O stretching Carbonhydrates/glycogen, nucleic acids17 1094 PO2

- ionized symmetric stretching of phosphodiester groups, C–O stretcing

Nucleic acids, phospholipids, glycogen, oligosaccharides and glycolipids

18 1041 C–O stretching, coupled with C–O bending of the C–OH groups of carbonhydrates

Oligosaccharides, polysaccharides

19 1021 C–O stretching, coupled with C–O bending of the C–OH groups of carbonhydrates

Oligosaccharides, polysaccharides

20 976-875 C–N+ –C stretching Nucleic acids, ribose-phosphate main chain vibrations of RNA, phosphate monoesters

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

Control

GD

Fingerprint Region

Page 20: Serap DÖKMECİ (EMRE)

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.

Page 21: Serap DÖKMECİ (EMRE)

5. 2. Comparison of the levels of biomolecules

Calculated as a sum of mean band area values of the bands originated from lipids.

Page 22: Serap DÖKMECİ (EMRE)

5. 2. Comparison of the levels of biomolecules

Calculated as a sum of mean band area values of Amid I and II bands.

Page 23: Serap DÖKMECİ (EMRE)

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.

INDIVIDUAL VARIATIONINDIVIDUAL VARIATIONLipid Dominated Region Fingerprint region

Nor

mal

izatio

n ba

nd

Nor

mal

izatio

n ba

nd

1

1023

4

5

67 8

9

1314

15

16

17

18 19

20

11

12

Page 24: Serap DÖKMECİ (EMRE)

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.

Page 25: Serap DÖKMECİ (EMRE)

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

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.

Individual variationIndividual variation

Page 26: Serap DÖKMECİ (EMRE)

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.

Page 27: Serap DÖKMECİ (EMRE)

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.

Page 28: Serap DÖKMECİ (EMRE)

Naşit İGCİ1, Parisa SHARAFI2, Duygu ÖZEL DEMİRALP1, Aysel YÜCE3, Özerk DEMİRALP4

1 Ankara University Biotechnology Institute Proteomics Department2 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