N-linked Protein Glycosylation in Helicobacter Species A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences 2012 Alison Grace Wood Contents Abstract: ……………………………………………………………………………9 Declaration ................................................................................................................. 10 Chapter 1 .................................................................................................................... 15 General Introduction .................................................................................................. 15 1.0 Introduction ...................................................................................................... 16 1.1 Eukaryotic Protein Glycosylation .................................................................... 16 1.2 Protein Glycosylation in Archaea .................................................................... 19 1.3 O-linked Protein Glycosylation in Bacteria ..................................................... 20 1.4 Bacterial Cytoplasmic N-glycosylation Systems ............................................. 21 1.5 The C. jejuni N-linked Protein Glycosylation System ..................................... 22 1.6 The Bacterial OST and Amino Acid Sequon Specificity................................. 23 1.7 C. jejuni Glycosyltransferases and Assembly of the Heptasaccharide Glycan 26 1.8 Function of C. jejuni N-linked Glycoproteins.................................................. 28 1.9 Exploitating the C. jejuni N-linked Protein Glycosylation System ................. 29 1.10 Campylobacter N-linked Protein Glycosylation (pgl) Genes ........................ 32 1.11 The N-linked Protein Glycosylation System of W. succinogenes ................. 34 1.12 N-Linked Glycosylation Systems in Helicobacter Species ........................... 35 1.13 Summary ........................................................................................................ 38 1.14 Aims of This Study ........................................................................................ 39 Chapter 2 .................................................................................................................... 40 Materials and Methods ............................................................................................... 40 2.1 Bacterial Strains and Growth Conditions ......................................................... 41 2.2 Isolation of Plasmids and Genomic DNA ........................................................ 41 2.3 PCR Amplification ........................................................................................... 42 2.4 Reverse Transcriptase PCR .............................................................................. 42 2.5 Agarose Gel Electrophoresis ............................................................................ 42 2 2.6 DNA Manipulation .......................................................................................... 43 2.7 E. coli Transformation ..................................................................................... 43 2.7 DNA Sequencing. ............................................................................................ 44 2.8 Site Directed Mutagenesis................................................................................ 44 2.9 Helicobacter Transformation ........................................................................... 44 2.10 Purification of Glycoproteins from H. pullorum............................................ 45 2.11 SDS-PAGE and Western Blotting. ................................................................ 46 2.12 Mass Spectrometery for Characterisation of Proteins .................................... 47 2.13 Preparation of Bacterial Cell Membranes ...................................................... 48 2.14 Oligosaccharyltransferase Assay ................................................................... 48 2.15 Tricine SDS-PAGE ........................................................................................ 49 2.16 Purification of Biotinylated Peptide from the In vitro OST Assay ................ 49 Chapter 3 .................................................................................................................... 51 The Second Helicobacter pullorum Oligosaccharyltransferase (PglB2) is Neither Essential nor Required for N-linked Protein Glycosylation ...................................... 51 3.1 Background ...................................................................................................... 52 3.2 Results .............................................................................................................. 54 3.2.1 Construction of suicide plasmids for site specific integration of antibiotic resistance cassettes onto the H. pullorum chromosome. .................................... 54 3.2.2 Integration of antibiotic resistance cassettes onto the H. pullorum chromosome within the 23S rRNA gene. .......................................................... 56 3.2.3 Natural Transformation of H. pullorum NCTC 12824T with genomic DNA from H. pullorum 23::E and 23::Kn strains. ...................................................... 58 3.2.4 Expression of H. pullorum pglB1 and pglB2 genes .................................. 59 3.2.5 Insertional mutagenesis of H. pullorum pglB1 and pglB2 genes ............. 61 3.2.6 Role of PglB2 in the N-glycosylation of a small peptide in vitro ............. 64 3.2.7 The N-glycan structure of glycopeptide derived from the H. pullorum pglB2 mutant. ..................................................................................................... 66 3 3.3 Discussion ........................................................................................................ 69 Chapter 4 .................................................................................................................... 72 Identification of Putative H. pullorum Glycoproteins and Their Glycosylation in E. coli with the C. jejuni Heptasaccharide...................................................................... 72 4.1 Background ...................................................................................................... 73 4.2 Results .............................................................................................................. 75 4.2.1 Bioinformatic approach to predicting N-linked glycoproteins in Helicobacter species .......................................................................................... 75 4.2.2 Production of H. pullorum HgpA and Hp0114 putative glycoproteins in E. coli ...................................................................................................................... 78 4.2.3 Glycosylation of H. pullorum HgpA and Hp0114 in E. coli with a C. jejuni heptasaccharide glycan. ........................................................................... 80 4.2.4 Localisation of the site of glycosylation of Hp0114 ................................. 84 4.2.5 Amino acid site of glycosylation of H. pullorum HgpA ........................... 86 4.3 Discussion ........................................................................................................ 88 Chapter 5 .................................................................................................................... 91 Glycosylation of Hp0114 and HgpA in H. pullorum ................................................. 91 5.1 Background ...................................................................................................... 92 5.2 Results .............................................................................................................. 93 5.2.1 Production of hexa histidine tagged Hp0114 and HgpA proteins in H. pullorum ............................................................................................................. 93 5.2.3 Site of glycosylation of Hp0114 in H. pullorum ....................................... 98 5.2.4 Intact mass analysis of HgpA glycoprotein derived from H. pullorum .. 100 5.2.5 MALDI Mass Spectrometry of the HgpA glycoprotein derived from wild type H. pullorum. ............................................................................................. 103 5.3 Discussion ...................................................................................................... 110 Chapter 6 .................................................................................................................. 113 4 Characterization of the H. pullorum PglB1-Dependent N-Linked Protein Glycosylation System .............................................................................................. 113 6.1 Background .................................................................................................... 114 6.2 Results ............................................................................................................ 118 6.2.1 The effect of insertional mutagenesis of putative glycosyltransferase encoding genes on the electrophoretic mobility of H. pullorum HgpA protein .......................................................................................................................... 118 6.2.2 Intact mass analysis of HgpA derived from H. pullorum glycosyltransferase-encoding gene mutants. .................................................... 120 6.2.3 MALDI Mass Spectrometry of trypsin digested HgpA peptides from H. pullorum glycosyltransferase-encoding