For My Family Promotor: Prof. dr. Els J. M. Van Damme Laboratory of Biochemistry and Glycobiology Department of Molecular Biotechnology Faculty of Bioscience Engineering, Ghent University Dean: Prof. dr. ir. Guido Van Huylenbroeck Rector: Prof. dr. Anne De Paepe Ghent University Faculty of Bioscience Engineering Department of Molecular Biotechnology Chenjing Shang Biological activity of ribosome-inactivating proteins from Malus domestica (apple) and Sambucus nigra (elderberry) Thesis submitted in fulfillment of the requirements for the degree of Doctor (PhD) of Applied Biological Sciences: Cell and Gene Biotechnology Nederlandse vertaling van de titel: Biologische activiteiten van ribosoom-inactiverende proteïnen uit Malus domestica (appel) en Sambucus nigra (vlier). Cover illustration: EGFP fluorescence in protoplasts expressing type 2 RIP::EGFP fusion protein. Refer to this thesis: Shang C. (2015) Biological activity of ribosome-inactivating proteins from Malus domestica (apple) and Sambucus nigra (elderberry). PhD thesis, Faculty of Bioscience engineering, Ghent University, Ghent, Belgium. ISBN: 978-90-5989-810-3 The author and the promotor give the authorization to consult and to copy parts of this work for personal use only. Any other use is limited by the Laws of Copyright. Permission to reproduce any material contained in this work should be obtained from the author. The promotor, The author, Prof. dr. Els J. M. Van Damme Chenjing Shang Members of the examination committee: Prof. dr. ir. Nico Boon (chairman) Lab. Microbial Ecology and Technology, Dept. Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Belgium Prof. dr. Els J. M. Van Damme (promotor) Lab. Biochemistry and Glycobiology, Dept. Molecular Biotechnology Faculty of Bioscience Engineering, Ghent University, Belgium Prof. dr. ir. Monica Höfte (secretary) Lab. Phytopathology, Dept. Crop Protection Faculty of Bioscience Engineering, Ghent University, Belgium Prof. dr. Wim Van den Ende Lab. Molecular Plant Biology, Institute of Botany and Microbiology KU Leuven, Belgium Prof. dr. ir. Winnok H. De Vos Lab. Cell Biology and Histology, Dept. Veterinary Sciences University of Antwerp, Belgium Prof. dr. ir. Guy Smagghe Lab. Agrozoology, Dept. Crop Protection Faculty of Bioscience Engineering, Ghent University, Belgium Prof. dr. Danny Geelen Dept. Plant Production Faculty of Bioscience Engineering, Ghent University, Belgium Prof. dr. ir. Kris Audenaert Dept. Applied Biosciences Faculty of Bioscience Engineering, Ghent University, Belgium Prof. dr. ir. John Van Camp Dept. Food Safety and Food Quality Faculty of Bioscience Engineering, Ghent University, Belgium Table of contents Table of Contents List of abbreviations ................................................................................................................... i Scope ......................................................................................................................................... iii Chapter 1 General introduction ............................................................................................... 1 1.1 General introduction of ribosome-inactivating proteins ............................................ 2 1.1.1 Historical note ...................................................................................................... 2 1.1.2 Definition .............................................................................................................. 2 1.1.3 Classification of RIPs ............................................................................................. 2 1.1.4 Occurrence of RIPs ............................................................................................... 4 1.2 Biological activities of RIPs .......................................................................................... 5 1.2.1 The enzymatic activity .......................................................................................... 5 1.2.2 Role of RIPs in plant defense ................................................................................ 7 1.2.3 Animal studies .................................................................................................... 22 1.2.4 Effects of RIPs on mammalian cells .................................................................... 23 1.3 Distribution and evolution of RIPs ............................................................................ 30 1.3.1 Distribution ........................................................................................................ 30 1.3.2 Molecular evolution of RIP genes ...................................................................... 33 1.4 Applications of RIPs ................................................................................................... 35 1.4.1 RIPs in agriculture .............................................................................................. 35 1.4.2 RIPs in medicine ................................................................................................. 35 Chapter 2 Characterization of a type 1 RIP and a type 2 RIP from Malus domestica ............ 39 2.1 Abstract ..................................................................................................................... 40 2.2 Introduction .............................................................................................................. 40 2.3 Materials and methods ............................................................................................. 41 2.3.1 Cell culture ......................................................................................................... 41 2.3.2 Expression of type 1 RIP in Pichia pastoris ......................................................... 42 2.3.3 Transformation of Pichia pastoris, selection, and expression analysis .............. 42 2.3.4 Purification of recombinant type 1 RIP .............................................................. 44 2.3.5 Construction of the binary vector for expression of type 2 RIP in BY-2 cells .... 44 2.3.6 Stable transformation of BY-2 cells and expression analysis ............................. 45 2.3.7 Purification of recombinant type 2 RIP .............................................................. 45 2.3.8 N-terminal sequence analysis ............................................................................ 46 2.3.9 Agglutination assay ............................................................................................ 46 2.3.10 Carbohydrate inhibition test ............................................................................ 46 2.3.11 Glycan microarray screening assay .................................................................. 47 2.3.12 Protein deglycosylation.................................................................................... 47 2.3.13 Protein synthesis inhibition activity ................................................................. 47 2.3.14 Cytotoxicity assay ............................................................................................. 48 2.3.15 Preparation of crude extracts from apple tissue ............................................. 48 2.3.16 Analytical methods .......................................................................................... 49 2.3.17 Statistical analyses ........................................................................................... 49 2.4 Results ....................................................................................................................... 50 2.4.1 Identification and sequence analysis of RIP genes in the apple (Malus domestica) genome ............................................................................................ 50 2.4.2 Purification and characterization of recombinant type 1 RIP from P. pastoris . 54 2.4.3 Purification and characterization of recombinant type 2 RIP from BY-2 cells ... 55 2.4.4 Glycosylation analysis ........................................................................................ 57 2.4.5 Agglutination activity and carbohydrate binding properties of recombinant Table of contents type 2 RIP .......................................................................................................... 57 2.4.6 Apple RIPs inhibit protein synthesis in vitro ...................................................... 61 2.4.7 Cytotoxicity of apple RIPs to mammalian cells .................................................. 61 Chapter 3 Subcellular and tissue localization of ribosome-inactivating proteins from apple (Malus domestica) ................................................................................................................... 67 3.1 Abstract ..................................................................................................................... 68 3.2 Introduction .............................................................................................................. 68 3.3 Materials and methods ............................................................................................. 70 3.3.1 In silico analysis .................................................................................................. 70 3.3.2 Plant materials ................................................................................................... 70 3.3.3 EGFP fusion constructs ....................................................................................... 71 3.3.4 Transient expression of EGFP fusion proteins