Department of Chemistry and Biomolecular Sciences Effect of carbon source on the glycosylation pathway of Trichoderma reesei RUT-C30 Chris Ashwood Bachelor of Forensic Science 10th of October, 2014 A dissertation submitted in partial fulfilment of the Master of Research degree Table of Contents Declaration ....................................................................................................................................... iii Acknowledgements .......................................................................................................................... iv Abstract ............................................................................................................................................. v Abbreviations and symbol nomenclature ........................................................................................ vi Chapter 1 – Introduction ................................................................................................................... 1 1.1 The fungus Trichoderma reesei RUT-C30: a protein production machine. ............................. 1 1.1.1 History of the strain .......................................................................................................... 1 1.1.2 Protein glycosylation in Trichoderma reesei ..................................................................... 2 1.2 Metabolomics .......................................................................................................................... 6 1.2.1 Metabolomics in cellular physiology ................................................................................ 7 1.2.2 Targeted and untargeted metabolomics .......................................................................... 7 1.2.3 Nucleotide-sugars ............................................................................................................. 8 1.2.4 ATP, ADP and AMP nucleotides ...................................................................................... 11 1.3 Methods for metabolite analysis ........................................................................................... 12 1.3.1 Microbial cell culture ...................................................................................................... 13 1.3.2 Cell quenching and metabolite extraction ..................................................................... 14 1.3.3 Metabolite separation and analysis ............................................................................... 15 1.4 Evaluation of protein N- and O-glycosylation ........................................................................ 16 1.5 Research aims ........................................................................................................................ 17 Chapter 2 - Materials and Methods ................................................................................................ 18 2.1 Chemicals, proteins and other materials............................................................................... 18 2.2 Fungal strain .......................................................................................................................... 18 2.3 Biolog assay ........................................................................................................................... 18 2.4 Liquid cultivation in shake flasks ........................................................................................... 19 2.5 Monitoring of culture pH and mycelial growth ..................................................................... 19 2.6 CBHI activity ........................................................................................................................... 20 2.7 Protein concentration ............................................................................................................ 20 2.8 Extraction of nucleotides and nucleotide sugars .................................................................. 21 2.8.1 Quenching fungal cells and extraction of polar metabolites ......................................... 21 2.8.2 Capillary electrophoresis with diode array detection .................................................... 21 2.8.3 Analysis of the ATP standard using mass spectrometry ................................................. 22 2.9 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and protein identification by mass spectrometry ........................................................................................... 22 2.10 N-glycan and O-glycan analysis of secreted proteins .......................................................... 23 i 2.10.1 Protein immobilisation and N-glycan/O-glycan release .............................................. 23 2.10.2 Mass spectrometric analysis of released glycans ........................................................ 24 2.10.3 Data Analysis ................................................................................................................ 25 Chapter 3 Results ............................................................................................................................ 26 3.1 Physiological studies ............................................................................................................. 26 3.1.1 Biolog assay for carbon utilisation ................................................................................. 26 3.1.2 Mycelial growth and pH of shake flask cultures with glucose or lactose as sole carbon source ...................................................................................................................................... 27 3.1.3 Protein secretion and CBHI activity in the culture supernatants ................................... 29 3.2 Analysis of nucleotides and nucleotide sugars ..................................................................... 30 3.2.1 CE-UV method development for metabolite analysis. ................................................... 30 3.2.2 Comparison of nucleotide–sugar and nucleotide pools in RUT-C30 grown on glucose and lactose .............................................................................................................................. 32 3.3 N-glycan and O-glycan analysis of secreted proteins ........................................................... 33 Chapter 4 Discussion ....................................................................................................................... 41 4.1 The value of Biolog assays for determining carbon source utilisation of T. reesei ............... 41 4.2 Growth phases of T. reesei under different carbon sources ................................................. 42 4.3 Secreted protein concentration and CBHI activity show similar but not identical trends over the cultivation period .................................................................................................................. 44 4.4 Analysis of nucleotides and nucleotide sugars ..................................................................... 44 4.4.1 Preparation of samples for CE-UV analysis .................................................................... 44 4.4.2 Nucleotide and nucleotide sugar analysis ...................................................................... 45 4.5 N-glycan and O-glycan profiles of secreted protein.............................................................. 47 4.5.1 The relative abundance of N-glycans in glucose and lactose cultures........................... 47 4.5.2 Phosphorylation of N-glycans ........................................................................................ 48 4.5.3 The identification of extended O-glycan compositions ................................................. 49 4.5.4 Identification of HexA in O-glycans of T. reesei ............................................................. 49 4.5.5 Role of negatively charged glycans in T. reesei and yeast ............................................. 50 Chapter 5 Summary and future directions ..................................................................................... 51 Chapter 6 References ........................................................................................................................ a Chapter 7 Supplementary Material ................................................................................................... i 7.1 Biolog........................................................................................................................................ i 7.2 Capillary electrophoresis ......................................................................................................... k 7.3 N-glycan and O-glycan MS/MS structures table .................................................................... m 7.4 Annotated MS/MS of O-glycans .............................................................................................. o 7.5 Identification of CBHI as a major constituent of total secreted protein .................................. r ii Declaration This thesis is a presentation of my original research work carried out as part of the Master of Research program at Macquarie University. Wherever contributions of others are involved, every effort is made to indicate this clearly, with due reference to the literature, and acknowledgement of personal assistance or advice. The thesis is formatted according to Master of Research guidelines and the journal Glycobiology. Chris Ashwood iii Acknowledgements This thesis would look