Proteolysis of the Receptor for Advanced Glycation End Products by Matrix Metalloproteinases Dissertation Zur Erlangung des Grades Doktor der Naturwissenschaften Am Fachbereich Biologie Der Johannes Gutenberg-Universität Mainz Ling Zhang geb. am 22-10-1972 in Wuhan, China Mainz 2006 i Table of Content Table of Contents Table of Contents ...................................................................................................................................ii 1. Introduction ....................................................................................................................................... 1 1.1 The A β peptide ................................................................................................................................. 1 1.1.1 A β peptide production and its pathologic role in Alzheimer’s disease (AD) ........................... 1 1.1.2 A β peptide clearance from the brain .......................................................................................... 2 1.2 RAGE (receptor for advanced glycation end products) .............................................................. 4 1.2.1 Structure ....................................................................................................................................... 4 1.2.2 Expression patterns ...................................................................................................................... 5 1.2.3 Extracellular ligands and their pathophysiolologic functions .................................................. 5 1.2.4 Intracellular signaling .................................................................................................................. 7 1.2.5 RAGE-Aβ interaction .................................................................................................................. 8 1.2.6 Isoforms of RAGE ........................................................................................................................ 9 1.2.6.1 Full-length RAGE ...................................................................................................................... 9 1.2.6.2 C-terminal truncated RAGE .................................................................................................... 10 1.2.6.3 Soluble RAGE (sRAGE) .......................................................................................................... 10 1.2.6.4 N-truncated RAGE ................................................................................................................... 12 1.3 Protein Ectodomain Shedding ..................................................................................................... 12 1.3.1 Ectodomain shedding ................................................................................................................. 12 1.3.2.1 A disintegrin and metalloproteinases (ADAMs) ...................................................................... 14 1.3.2.2. Matrix metalloproteinases (MMPs) ........................................................................................ 14 2. Aim of the study .............................................................................................................................. 19 3. Materials and Methods ................................................................................................................... 21 3.1 Materials ........................................................................................................................................ 21 3.1.1 Chemicals and Media ................................................................................................................. 21 3.1.2 Enzyme and Kit systems ............................................................................................................ 22 3.1.3 Laboratory instruments and accessories .................................................................................. 23 3.1.4 Solutions, buffers and media ..................................................................................................... 24 3.1.5 Antibodies ................................................................................................................................... 26 3.1.6 Bacterial strains and cell lines ................................................................................................... 28 3.1.7 Plasmids ...................................................................................................................................... 29 3.1.8 Oligonucleotides ......................................................................................................................... 32 3.1.9 Inhibitors and Activators .......................................................................................................... 33 3.2 Methods .......................................................................................................................................... 34 3.2.1 Molecular biology ....................................................................................................................... 34 3.2.1.1 Maintenance of bacterial strains ............................................................................................. 34 3.2.1.2 Preparation of competent bacteria ........................................................................................... 34 3.2.1.3 Transformation of E. coli ......................................................................................................... 34 3.2.1.4 Plasmid Minipreparation ......................................................................................................... 34 3.2.1.5 Plasmid Maxipreparation ........................................................................................................ 35 3.2.1.6 Enzymatic modification of DNA .............................................................................................. 36 3.2.1.7 DNA electrophoresis ................................................................................................................ 37 3.2.1.8 DNA purification ...................................................................................................................... 38 3.2.1.9 DNA Sequencing ...................................................................................................................... 38 3.2.1.10 Polymerase Chain Reaction (PCR) ....................................................................................... 38 3.2.2 Protein biochemical methods .................................................................................................... 40 3.2.2.1 Determination of protein concentration (Bradford assay) ..................................................... 40 3.2.2.2 TCA precipitation of proteins .................................................................................................. 40 3.2.2.3 Chloroform-Methanol precipitation of proteins ..................................................................... 41 3.2.2.4 SDS-polyacrylamide gel electrophoresis ................................................................................. 41 ii Table of Content 3.2.2.5 Western Blot ............................................................................................................................. 42 3.2.2.6 Coomassie staining of polyacrylamide gels ............................................................................. 43 3.2.2.7 Biotinylation of cell surface proteins ....................................................................................... 43 3.2.2.8 Gelatin zymography assay ........................................................................................................ 44 3.2.3 Cell biology methods .................................................................................................................. 44 3.2.3.1 Cell culture ............................................................................................................................... 44 3.2.3.2 Trypsinizing adhesive cells ....................................................................................................... 44 3.2.3.3 Subculture adhesive cells ......................................................................................................... 45 3.2.3.4 Freezing cells ............................................................................................................................ 45 3.2.3.5 Thawing cells ............................................................................................................................ 45 3.2.3.6 Transfect cells with lipofectamine 2000 .................................................................................. 45 3.2.3.7 Transfect cell with DEAE-Dextran reagent ............................................................................ 46 3.2.3.8 Selection of stable transfectants .............................................................................................. 46 3.2.4 Methods for analysis of ectodomain shedding ......................................................................... 46 3.2.4.1 General procedures .................................................................................................................. 46 3.2.4.2 Handling of secretion medium ................................................................................................ 48 3.2.4.3 Handling of cells .....................................................................................................................