γ-secretase directly sheds the survival receptor BCMA from plasma cells Submitted by Sarah Anne Laurent st 1 of June 2015 Dissertation der Graduate School of Systemic Neurosciences der Ludwig-Maximilians-Universität München, 2015 First reviewer and supervisor: Prof. Dr. Edgar Meinl Second reviewer: Prof. Dr. Nikolaus Plesnila Third reviewer: Prof. Dr. Hermann Eibel Date of oral examination: 8th October 2015 CONTENTS Abstract ................................................................................................................................................... 1 Abbreviations .......................................................................................................................................... 3 1. Introduction ......................................................................................................................................... 5 1.1 Multiple sclerosis ..................................................................................................................... 5 1.2 Humoral immunity in MS ........................................................................................................ 6 1.3 Systemic lupus erythematosus ................................................................................................ 8 1.4 The BAFF/APRIL-system .......................................................................................................... 8 1.4.1 Structure, expression and signaling of BAFF, APRIL and their receptors ........................ 8 1.4.2 Physiological relevance of the BAFF/APRIL pathway .................................................... 11 1.4.2.1 B cell homeostasis and tolerance .............................................................................. 11 1.4.2.2 Role of BCMA in plasma cell survival and antigen presentation ............................... 11 1.4.3 The BAFF/APRIL-system in autoimmunity ..................................................................... 13 1.4.3.1 In mice ....................................................................................................................... 13 1.4.3.2 In human .................................................................................................................... 13 1.5 Soluble receptors ................................................................................................................... 14 1.5.1 Mechanisms of generations .......................................................................................... 14 1.5.1.1 Shedding .................................................................................................................... 14 1.5.1.2 Alternative splicing .................................................................................................... 15 1.5.1.3 Exosomes ................................................................................................................... 15 1.5.2 Functional relevance ..................................................................................................... 15 1.6 The γ-secretase complex ....................................................................................................... 16 1.6.1 Subunits and assembly .................................................................................................. 17 1.6.2 The role of γ-secretase in biology and disease .............................................................. 18 2 Objectives ...................................................................................................................................... 21 3 Materials and Methods ................................................................................................................. 23 3.1 Materials ................................................................................................................................ 23 3.1.1 Clinical samples ............................................................................................................. 23 3.1.2 Antibodies ...................................................................................................................... 26 3.1.3 Cytokines ....................................................................................................................... 27 3.1.4 Protease inhibitors ........................................................................................................ 28 3.1.5 ELISA .............................................................................................................................. 28 3.1.6 Reagents and buffers ..................................................................................................... 29 3.1.7 Gels ................................................................................................................................ 30 3.2 Cell culture ............................................................................................................................. 31 3.2.1 General cell culture method .......................................................................................... 31 3.2.2 Cell lines ......................................................................................................................... 31 3.2.3 Isolation of human peripheral blood mononuclear cells by density gradient centrifugation ................................................................................................................................ 32 3.2.4 Human B cell isolation ................................................................................................... 32 3.2.5 Human B cell stimulation and differentiation ............................................................... 33 3.2.5.1 CD40L/IL-21 stimulation ............................................................................................ 33 3.2.5.2 Resiquimod/IL-2 stimulation ..................................................................................... 33 3.2.6 Mouse B cell isolation and survival assay ...................................................................... 33 3.3 General protein analytics ...................................................................................................... 34 3.3.1 Cell lysates ..................................................................................................................... 34 3.3.2 Immunoprecipitation..................................................................................................... 34 3.3.2.1 Samples ..................................................................................................................... 34 3.3.2.2 Magnetic beads preparation, antibody coupling and crosslinking ........................... 35 3.3.2.3 Immunoprecipitation................................................................................................. 35 3.3.2.4 Elution ........................................................................................................................ 36 3.3.3 Gel electrophoresis, Coomassie staining and Western blot ......................................... 36 3.3.3.1 Western blot detection of soluble BCMA upon immunoprecipitation ..................... 36 3.3.3.2 Western blot detection of presenilin 1 and nicastrin................................................ 37 3.3.4 Mass spectrometric analysis and sample preparation .................................................. 37 3.3.4.1 In solution digestion by trypsin followed by mass spectrometry ............................. 37 3.3.4.2 Silver staining, in gel digestion by trypsin and chymotrypsin followed by mass spectrometry ............................................................................................................................. 38 3.4 Molecular cloning .................................................................................................................. 38 3.4.1 General molecular cloning methods ............................................................................. 38 3.4.1.1 Transformation of bacterial cells ............................................................................... 38 3.4.1.2 Plasmid purification from bacterial cells ................................................................... 39 3.4.1.3 Agarose gel electrophoresis ...................................................................................... 39 3.4.1.4 DNA extraction from agarose gel .............................................................................. 39 3.4.1.5 Cloning primers ......................................................................................................... 39 3.4.1.6 Plasmids ..................................................................................................................... 40 3.4.1.7 Polymerase chain reaction ........................................................................................ 40 3.4.1.8 DNA digestion with restriction endonuclease ........................................................... 41 3.4.1.9 DNA ligation ............................................................................................................... 41 3.4.1.10 DNA quantification ................................................................................................ 41 3.4.1.11 Transfection protocol ............................................................................................ 42 3.4.2 Soluble BCMA generation.............................................................................................. 42 3.4.3 NF-κB