Molecular Profiling of Gaucher Disease by Fourier Transform Infrared Spectroscopy

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

2 1. Gaucher Disease (GD) Gaucher disease is defined as an autosomal recessive disorder resulfng from deficiency of the glucocerebrosidase (E.C ), 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 (InfanFle cerebral) 1 in 100,000 live births Death usually occurs w/in 1 year Type III (Chronic neuropathic/norbo%nian) 1 in 50,000 live births

4 1. Gaucher Disease (GD) Glucocerebrosidase is a lysosomal hydrolase responsible for the degradafon of the natural glycosphingolipid, glucosylceramide, into ceramide and glucose. Deficiency of this enzyme results in the accumulafon 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 comparafvely with a knowledge of origins of the peaks (such as glycolipids, lipids, proteins etc.) as well as the amount of the parfcular materials can be determined. Also secondary structure rafos of proteins can be determined by analyzing the amide bands. These features provide invaluable informafon about funcfonal and structural changes in cells underlying disease mechanisms.

6 2. Fourier- Transform Infrared Spectroscopy (FT- IR) Infrared (IR) radiafon is a noninvasive and nondestrucfve type of radiafon and when absorbed by the Fssues, fluids or cells, it causes vibrafon 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 sensifvity.

7 A B S O R B A N C E WAVENUMBER(cm - 1 ) Band posikon (Frequency/wavenumber): DefiniKon of a funckonal group 2. Signal intensity and band area: ConcentraKon of a parkcular molecule 3. Frequency shir: Structural informakon 4. Band width: Membrane fluidity

8 3. Aim of the Study The aim of this study is to achieve molecular characterizafon of biomolecules in GD in comparison with controls by using FTIR- ATR spectroscopy and cluster analysis.

9 4. Material and Methods

10 PaKent No Gaucher Type Genotype hepato/ splenomegaly Bone involment Neurologic involvement Cardiac involvement 1 1 N370S/ D409H/D409H (oculomotor apraxia) N370S/ N370S/ L444P/L444P + + (oculomotor apraxia) L444P/L444P N370/RecAHI (splenectomized) (osteopenia) L296V/L290V + (splenectomized) + - -

11 4. 1. PaKents and Cell Culture Fibroblast were cultured from skin biopsy. Human skin fibroblast from unaffected individuals and these pafents were cultured in BME supplemented with 10 % FCS in 75 cm 2 culture flasks in a humidified atmosphere containing 5 % CO 2 at 37 0 C.

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 OpFcs, GmbH, Germany) equipped with a liquid nitrogen cooled photovoltaic MCP detector and universal a%enuated total reflectance (ATR) cell (Pike Technologies, Wisconsin, U.S.A).

13 4. 2. FTIR- ATR 2.5 µl of cell in PBS were spo%ed 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 cm - 1 wavenumbers.

14 4. 2. FTIR- ATR Baselined absorbance spectra were used for exact integrafon calculafons. RelaFve intensity values of second derivafve peaks in the Amid I region ( cm - 1 ) which were obtained by automated peak picking were used to compare protein secondary structures. Spectral analyses were carried out by OPUS sonware and t- test was performed using SPSS sonware.

15 5. RESULTS and DISCUSSION FTIR FingerprinKng Comparison of the levels of biomolecules DeterminaKon and comparison of protein secondary structures

16 5. 1. FT- IR FingerprinKng The spectra were complex with many characterisfc bands. Two main regions: Lipid dominated region (between cm - 1 ) Fingerprint region ( cm - 1 ) 20 major absorpfon 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 ) RepresentaFve FT- IR absorbance spectrum between cm - 1 obtained by averaging all the spectra used and average absorbance spectra of GD and control groups.

18 Lipid Dominated Region 1 GD 4 5 Control Average FT- IR absorbance spectrum of GD and controls between cm - 1. The spectrum was normalized with respect to the Amid A band between cm - 1 Peak Number Wavenumber (cm -1 ) Definition Organic compound Amide A, mainly N H stretching of proteins with contribution from intermolecular H bondings and O H stretching mode of polysaccharides Mainly proteins Amide B, N H stretching Proteins Olefinic =CH stretching Unsaturated lipids CH 3 asymmetric stretching Mainly lipids, low signal from proteins CH 2 asymmetric streching Mainly lipids, low signal from proteins CH 2 asymmetric streching Minly lipids, low signal from proteins CH 3 symmetric streching Mainly proteins, low signal from lipids CH 2 symmetric streching Mainly lipids, low signal from proteins C H stretching Mainly lipids

19 10 11 Fingerprint Region 12 Average FT- IR absorbance spectrum of GD and controls between cm - 1 cm - 1. The spectrum was normalized with respect to the Amid I band between cm - 1 Control GD Peak Number Wavenumber (cm -1 ) Definition Organic compound Saturated ester C=O stretching Cholesterol esters, phospholipids, ester functional groups in lipids Amide I; 80% C=O stretching, 10% N H bending, 10% C N Proteins stretching Amide II; 60% N H bending, 40% C N stretching Proteins CH 2 bending Lipids COO symmetric stretching Fatty acids PO - 2 asymmetric stretching (fully hydrogen-bonded) Nucleic acids, phophorylated proteins and phospholipids C O stretching Carbonhydrates/glycogen, nucleic acids PO - 2 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 Nucleic acids, phospholipids, glycogen, oligosaccharides and glycolipids Oligosaccharides, polysaccharides Oligosaccharides, polysaccharides C N + C stretching Nucleic acids, ribose-phosphate main chain vibrations of RNA, phosphate monoesters

20 5. 2. Comparison of the Levels of Biomolecules We used integrafon values (band area) of each peak using OPUS sonware for comparafve purposes. As a result of the comparafve evaluafon, lipid and protein levels are seen to increased in GD. Also we observed individual variafon. AddiFonally, bandwidth of CH 2 symmetric stretching of lipids is slightly decreased in GD, indicafng the descrease in mebrane fluidity.

21 5. 2. Comparison of the levels of biomolecules Calculated as a sum of mean band area values of the bands originated from lipids.

22 5. 2. Comparison of the levels of biomolecules Calculated as a sum of mean band area values of Amid I and II bands.

23 INDIVIDUAL VARIATION Lipid Dominated Region 5 11 Fingerprint region 1 NormalizaFon band NormalizaFon band Individual absorbance spectra of GD pafents (G1- G8) and controls between (A) cm - 1 and (B) cm - 1 wavenumbers. The spectral range between cm - 1 and cm - 1 were normalized with respect to the Amid A band between cm - 1 and Amid I band between cm - 1, respecfvely aner baseline correcfon.

24 5. 3. DeterminaKon and Comparison of Protein Secondary Structures We determined protein secondary structures using second derivafve spectrum of Amide I band. Band intensifes were used for comparafve purposes. We observed an increase in the rafo of anfparallel β- sheet and α- helix structures in GD, while β- sheet was decreased. Also individual variafon was observed.

25 Individual varia4on Second derivafve spectra of Amid I absorpfon band between cm - 1 wavenumbers which demonstrates main protein secondary structures. AbsorpFon 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

26 6. CONCLUSION FTIR stretoscopy is an valuable tecnique for the invesfgafon of biological structure due to its sensifvity and ability to give valuable informafon about the funcfonal groups which might have diagnosfc value for biological systems.

27 6. CONCLUSION We report the first FT- IR spectrum of GD pafent fibroblast cells in the mid- infrared region with their spectral assignments. Also this is the first FT- IR spectroscopic work aimed to determine molecular alterafons in GD. Lipid and proteins are important parametres for the proper funcfoning of biological membranes, which, in turn, influence celluler processes and disease states. Minor alterafons of these lipids have a significant affect on vesicular trafficing and intercellular signaling further contribufng to the complex pathology manifest in GD.

28 Naşit İGCİ 1, Parisa SHARAFI 2, Duygu ÖZEL DEMİRALP 1, Aysel YÜCE 3, Özerk DEMİRALP 4 1 Ankara University Biotechnology InsFtute Proteomics Department 2 Hace%epe University Faculty of Medicine, Department of Medical Biology 3 Hace%epe University İhsan Dogramacı Children Hospital, Pediatric Gastroenterology, Hepatology and NutriFon Unit 4 Atatürk Training and Research Hospital, Department of PlasFc and ReconstrucFve Surgery

Foundation Trust, London, UK Published online: 04 Feb 2008.

Foundation Trust, London, UK Published online: 04 Feb 2008. This article was downloaded by: [University of Illinois at Urbana-Champaign] On: 18 March 2015, At: 21:27 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954