title: Structural insights from random mutagenesis of Campylobacter jejuni oligosaccharyltransferase PglB.
abstract: BACKGROUND
  Protein glycosylation is of fundamental importance in many biological systems. The discovery of N-glycosylation in bacteria and the functional expression of the N-oligosaccharyltransferase PglB of Campylobacter jejuni in Escherichia coli enabled the production of engineered glycoproteins and the study of the underlying molecular mechanisms. A particularly promising application for protein glycosylation in recombinant bacteria is the production of potent conjugate vaccines where polysaccharide antigens of pathogenic bacteria are covalently bound to immunogenic carrier proteins.
  
  
  RESULTS
  In this study capsular polysaccharides of the clinically relevant pathogen Staphylococcus aureus serotype 5 (CP5) were expressed in Escherichia coli and linked in vivo to a detoxified version of Pseudomonas aeruginosa exotoxin (EPA). We investigated which amino acids of the periplasmic domain of PglB are crucial for the glycosylation reaction using a newly established 96-well screening system enabling the relative quantification of glycoproteins by enzyme-linked immunosorbent assay. A random mutant library was generated by error-prone PCR and screened for inactivating amino acid substitutions. In addition to 15 inactive variants with amino acid changes within the previously known, strictly conserved WWDYG motif of N-oligosaccharyltransferases, 8 inactivating mutations mapped to a flexible loop in close vicinity of the amide nitrogen atom of the acceptor asparagine as revealed in the crystal structure of the homologous enzyme C. lari PglB. The importance of the conserved loop residue H479 for glycosylation was confirmed by site directed mutagenesis, while a change to alanine of the adjacent, non-conserved L480 had no effect. In addition, we investigated functional requirements in the so-called MIV motif of bacterial N-oligosaccharyltransferases. Amino acid residues I571 and V575, which had been postulated to interact with the acceptor peptide, were subjected to cassette saturation mutagenesis. With the exception of I571C only hydrophobic residues were found in active variants. Variant I571V performed equally well as the wild type, cysteine at the same position reduced glycoprotein yield slightly, while a change to phenylalanine reduced activity by a factor of three.
  
  
  CONCLUSIONS
  This study provides novel structure-function relationships for the periplasmic domain of the Campylobacter jejuni N-oligosaccharyltransferase PglB and describes procedures for generating and screening oligosaccharyltransferase mutant libraries in an engineered E. coli system.
authors.affiliation: Empa, Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Biomaterials, CH-9014, St, Gallen, Switzerland.
authors.forename: Julian
authors.initials: J
authors.lastname: Ihssen
authors.forename: Michael
authors.initials: M
authors.lastname: Kowarik
authors.forename: Luzia
authors.initials: L
authors.lastname: Wiesli
authors.forename: Renate
authors.initials: R
authors.lastname: Reiss
authors.forename: Michael
authors.initials: M
authors.lastname: Wacker
authors.forename: Linda
authors.initials: L
authors.lastname: Thöny-Meyer
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016123:Campylobacter jejuni / Q000201:enzymology*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D006031:Glycosylation; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase