Serap DÖKMECİ (EMRE)

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

2. 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

3. 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

4. 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.

5. 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.

6. 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.

7. ABSORBANCE 2 4 1 700 3 1545 WAVENUMBER(cm-1) • Bandposition (Frequency/wavenumber):Definition of a functionalgroup • Signalintensity and bandarea:Concentration of a particularmolecule • Frequencyshift:Structuralinformation • Bandwidth:Membranefluidity

8. 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.

9. 4. Material and Methods

11. 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 370C.

12. 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).

13. 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.

14. 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.

15. 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

16. 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.

17. Fingerprint region Absorbance (Arbitary Units, A. U.) 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.

18. 5 LipidDominatedRegion GD Control 1 4 6 7 8 3 2 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

19. 11 FingerprintRegion 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 12 Control 16 15 13 14 17 18 19 20 10 GD

20. 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.

21. 5. 2. Comparison of the levels of biomolecules Calculated as a sum of meanbandareavalues of thebandsoriginatedfromlipids.

22. 5. 2. Comparison of the levels of biomolecules Calculated as a sum of meanbandareavalues of Amid I and II bands.

23. INDIVIDUAL VARIATION LipidDominatedRegion 11 Fingerprintregion 5 12 4 16 1 6 15 7 8 17 13 14 18 19 Normalizationband Normalizationband 3 2 9 10 20 Individualabsorbancespectra of GD patients (G1-G8) andcontrolsbetween(A) 3600–2750 cm-1and(B) 1800-850 cm-1wavenumbers. Thespectralrangebetween 3600–2750 cm-1and 1800-850 cm-1werenormalizedwithrespecttotheAmid A bandbetween 3500-3100 cm-1andAmid I bandbetween 1700-1600 cm-1, respectivelyafterbaselinecorrection.

24. 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.

25. Individualvariation 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.

26. 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.

27. 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.

28. Naşit İGCİ1,Parisa SHARAFI2, Duygu ÖZEL DEMİRALP1, Aysel YÜCE3, Özerk DEMİRALP4 1 Ankara UniversityBiotechnologyInstituteProteomicsDepartment 2 Hacettepe UniversityFaculty of Medicine, Department of MedicalBiology 3 Hacettepe University İhsan DogramacıChildrenHospital, PediatricGastroenterology, Hepatology and NutritionUnit 4 Atatürk Training and ResearchHospital, Department of Plastic and ReconstructiveSurgery