FATEMEH BOOTORABI (AHMAD) Production and Characterization of Carbonic Anhydrase VII Acetaldehyde-derived modifications and comparison to the other cytosolic isozymes ACADEMIC DISSERTATION To be presented, with the permission of the board of Institute of Biomedical Technology of the University of Tampere, for public discussion in the Jarmo Visakorpi Auditorium, of the Arvo Building, Lääkärinkatu 1, Tampere, on June 3th, 2011, at 12 o’clock. UNIVERSITY OF TAMPERE ACADEMIC DISSERTATION University of Tampere, Institute of Biomedical Technology and School of Medicine Tampere University Hospital Tampere Graduate Program in Biomedicine and Biotechnology (TGPBB) Finland Supervised by Reviewed by Professor Seppo Parkkila Docent Tiina Laitala-Leinonen University of Tampere University of Turku Finland Finland Docent Varpu Marjomäki University of Jyväskylä Finland Professor Juha Rouvinen University of Eastern Finland Finland Distribution Tel. +358 40 190 9800 Bookshop TAJU Fax +358 3 3551 7685 P.O. Box 617 [email protected] 33014 University of Tampere www.uta.fi/taju Finland http://granum.uta.fi Cover design by Mikko Reinikka Acta Universitatis Tamperensis 1617 Acta Electronica Universitatis Tamperensis 1078 ISBN 978-951-44-8459-9 (print) ISBN 978-951-44-8460-5 (pdf) ISSN-L 1455-1616 ISSN 1456-954X ISSN 1455-1616 http://acta.uta.fi Tampereen Yliopistopaino Oy – Juvenes Print Tampere 2011 ``In the name of God`` ` The most important thing I have learned from science is: falling nine times and getting up ten, even though the world may write me down a failure. To Adnan: for your support, encourage, and patience To mom: for helping me get to where I am today And to the loving memory of my father 3 CONTENTS CONTENTS....................................................................................................................................4 LIST OF ORIGINAL COMMUNICATIONS ............................................................................6 ABBREVIATIONS ........................................................................................................................7 ABSTRACT ....................................................................................................................................9 1. INTRODUCTION ....................................................................................................................10 2. REVIEW OF THE LITERATURE .......................................................................................11 2.1 Carbonic anhydrases ...........................................................................................................11 2.1.1 General aspects of carbonic anhydrase isozymes .....................................................11 2.1.2 Cytosolic carbonic anhydrase isozymes ...........................................................13 2.1.2.1 Carbonic anhydrase isozyme I ................................................................... 13 2.1.2.2 Carbonic anhydrase isozyme II ................................................................. 13 2.1.2.3 Carbonic anhydrase isozyme III................................................................ 15 2.1.2.4 Carbonic anhydrase isozyme VII .............................................................. 16 2.1.2.5 Carbonic anhydrase isozyme XIII ............................................................. 16 2.1.3 Other carbonic anhydrases ........................................................................................17 2.1.3.1 Membrane-associated and secretory isozymes (IV, VI, IX, XII, XIV, and XV) ............................................................................. 17 2.1.3.2 Mitochondrial isozymes (VA and VB) ...................................................... 21 2.2 Alcoholism and alcohol metabolism ...................................................................................22 2.3 Formation and role of acetaldehyde-protein adducts ..........................................................25 3. AIMS OF THE STUDY...........................................................................................................27 4. MATERIALS AND METHODS ............................................................................................28 4.1 Distribution of carbonic anhydrase VII (II, III) ..................................................................28 4.1.1 RT-PCR (II, III) ........................................................................................................28 4.1.2 Western blotting (II) .................................................................................................28 4.1.3 Immunohistochemistry (II, III) .................................................................................29 4.2 Production and purification of recombinant human cytosolic CAs (I, II, IV) ....................30 4.2.1 Construction and cloning of CA I, III, and VII on bacterial expression system (II, IV)…………………………………………………………………………...…30 4.2.2 Production and purification of human recombinant CA I, II, III, VII, and XIII proteins (I, II, IV)…………………………………………………………….31 4.3 Biochemical characterization of human recombinant CAs (I, II, IV).................................32 4.3.1 Structural modeling of CA VII (II) ...........................................................................32 4.3.2 Mass spectrometry (I, II, IV) ....................................................................................32 4.4 Modifications of human cytosolic CA I, II, III, VII, and XIII with acetaldehyde (I, IV) ..33 4.4.1 Labeling and acetaldehyde treatment of CA I, II, III, VII, and XIII (I, IV). ............33 4.4.2 Activity measurements of CA I, II, III, VII, and XIII (I, IV) ...................................33 4.5 Expression of carbonic anhydrase VII in gliomas (III) ......................................................34 4.5.1 Tumor materials and immunohistochemistry (III) ....................................................34 4.5.2 Statistical analyses (III).............................................................................................35 4.6 Carbonic anhydrase autoantibodies (IV) ............................................................................35 4 4.6.1 Serum samples (IV) ..................................................................................................35 4.6.2 Detection of autoantibodies against CA I, II, and VII enzymes (IV) .......................35 5. RESULTS .................................................................................................................................36 5.1 Sequence and RT-PCR analyses of CA VII short and long form (II) ................................36 5.2 Distribution of human CA VII (II)......................................................................................36 5.3 Production and purification of recombinant human cytosolic CA I, III, and VII (I, II, IV) ..............................................................................................................................37 5.4 Characterization of human CA VII (II) ..............................................................................37 5.4.1 Mass spectrometry ....................................................................................................37 5.5 Modifications of human cytosolic CAs I, II, III, VII, and XIII with acetaldehyde (I, IV) .38 5.5.1 Mass spectrometry (I, IV) .........................................................................................38 5.5.2 Activity measurement (I, IV) ....................................................................................39 5.6 Expression of CA VII in gliomas (III) ................................................................................39 5.6.1 Oligodendroglial and diffuse astrocytic tumors ........................................................39 5.7 Carbonic anhydrase autoantibodies ....................................................................................40 6. DISCUSSION ...........................................................................................................................41 6.1 Carbonic anhydrase VII long and short forms ....................................................................41 6.2 CA VII expression in malignant tissues ..............................................................................42 6.3 Acetaldehyde-derived modifications in cytosolic CAs.......................................................43 ACKNOWLEDGEMENTS ........................................................................................................46 REFERENCES .............................................................................................................................48 5 LIST OF ORIGINAL COMMUNICATIONS This thesis is based on the following papers, which are referred to in the text by the Roman numerals I-IV: I Bootorabi F, Jänis J, Valjakka J, Isoniemi S, Vainiotalo P, Vullo D, Supuran CT, Waheed A, Sly WS, Niemelä O, Parkkila S. (2008): Modification of carbonic anhydrase II with acetaldehyde, the first metabolite of ethanol, leads to decreased enzyme activity. BMC Biochem 27:9-32. II Bootorabi F, Jänis J, Smith E, Waheed A, Kukkurainen S, Hytönen V, Valjakka J, Supuran CT, Vullo D, Sly WS, Parkkila S. (2010): Analysis of a shortened form of human carbonic anhydrase VII expressed in vitro compared to the full-length enzyme. Biochimie 92(8):1072-80. III Bootorabi F, Haapasalo J, Smith E, Haapasalo H, Parkkila S. (2011): Carbonic anhydrase VII – a potential prognostic marker in gliomas.