Increased efficiency of Campylobacter jejuni N-oligosaccharyltransferase PglB by structure-guided engineering

Conjugate vaccines belong to the most efficient preventive measures against life-threatening bacterial infections. Functional expression of N-oligosaccharyltransferase (N-OST) PglB of Campylobacter jejuni in Escherichia coli enables a simplified production of glycoconjugate vaccines in prokaryotic cells. Polysaccharide antigens of pathogenic bacteria can be covalently coupled to immunogenic acceptor proteins bearing engineered glycosylation sites. Transfer efficiency of PglBCj is low for certain heterologous polysaccharide substrates. In this study, we increased glycosylation rates for Salmonella enterica sv. Typhimurium LT2 O antigen (which lacks N-acetyl sugars) and Staphylococcus aureus CP5 polysaccharides by structure-guided engineering of PglB. A three-dimensional homology model of membrane-associated PglBCj, docked to the natural C. jejuni N-glycan attached to the acceptor peptide, was used to identify potential sugar-interacting residues as targets for mutagenesis. Saturation mutagenesis of an active site residue yielded the enhancing mutation N311V, which facilitated fivefold to 11-fold increased in vivo glycosylation rates as determined by glycoprotein-specific ELISA. Further rounds of in vitro evolution led to a triple mutant S80R-Q287P-N311V enabling a yield improvement of S. enterica LT2 glycoconjugates by a factor of 16. Our results demonstrate that bacterial N-OST can be tailored to specific polysaccharide substrates by structure-guided protein engineering.

[1]  Uwe T Bornscheuer,et al.  Natural Diversity to Guide Focused Directed Evolution , 2010, Chembiochem : a European journal of chemical biology.

[2]  Z. Jaradat,et al.  Production and characterization of monoclonal antibodies against the O-5 antigen of Salmonella typhimurium lipopolysaccharide , 1996, Applied and environmental microbiology.

[3]  Marco Biasini,et al.  SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information , 2014, Nucleic Acids Res..

[4]  J. Shiloach,et al.  Safety and Immunogenicity of ImprovedShigella O-Specific Polysaccharide-Protein Conjugate Vaccines in Adults in Israel , 2001, Infection and Immunity.

[5]  Simon J North,et al.  N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. , 2002, Science.

[6]  L. Thöny-Meyer,et al.  Production of glycoprotein vaccines in Escherichia coli , 2010, Microbial cell factories.

[7]  Hyeon Joo,et al.  OPM database and PPM web server: resources for positioning of proteins in membranes , 2011, Nucleic Acids Res..

[8]  M. Aebi,et al.  N-Linked glycosylation of antibody fragments in Escherichia coli. , 2011, Bioconjugate chemistry.

[9]  Johannes Söding,et al.  Fast and accurate automatic structure prediction with HHpred , 2009, Proteins.

[10]  W. Stemmer.  Rapid evolution of a protein in vitro by DNA shuffling , 1994, Nature.

[11]  L. Johnson,et al.  Enzymatic catalysis in crystals of Escherichia coli maltodextrin phosphorylase. , 2002, Journal of molecular biology.

[12]  Yosephine Gumulya,et al.  Iterative saturation mutagenesis accelerates laboratory evolution of enzyme stereoselectivity: rigorous comparison with traditional methods. , 2010, Journal of the American Chemical Society.

[13]  M. Wacker,et al.  Engineering, conjugation, and immunogenicity assessment of Escherichia coli O121 O antigen for its potential use as a typhoid vaccine component , 2013, Glycoconjugate Journal.

[14]  A. Razmpour,et al.  Comparison of pneumococcal conjugate polysaccharide and free polysaccharide vaccines in elderly adults: conjugate vaccine elicits improved antibacterial immune responses and immunological memory. , 2008, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[15]  Dan S. Tawfik,et al.  Directed evolution of sulfotransferases and paraoxonases by ancestral libraries. , 2011, Journal of molecular biology.

[16]  Barbara Imperiali,et al.  The expanding horizons of asparagine-linked glycosylation. , 2011, Biochemistry.

[17]  Nancy L Price,et al.  Production of a recombinant vaccine candidate against Burkholderia pseudomallei exploiting the bacterial N-glycosylation machinery , 2014, Front. Microbiol..

[18]  H. Nothaft,et al.  Protein glycosylation in bacteria: sweeter than ever , 2010, Nature Reviews Microbiology.

[19]  Marco Biasini,et al.  Assessing the local structural quality of transmembrane protein models using statistical potentials (QMEANBrane) , 2014, Bioinform..

[20]  Jeremy R. Greenwood,et al.  Epik: a software program for pKa prediction and protonation state generation for drug-like molecules , 2007, J. Comput. Aided Mol. Des..

[21]  B. Wren,et al.  Exploitation of bacterial N-linked glycosylation to develop a novel recombinant glycoconjugate vaccine against Francisella tularensis , 2013, Open Biology.

[22]  F. Arnold,et al.  How enzymes adapt: lessons from directed evolution , 2001, Trends in biochemical sciences.

[23]  Marcie B. Jaffee,et al.  Exploiting topological constraints to reveal buried sequence motifs in the membrane-bound N-linked oligosaccharyl transferases. , 2011, Biochemistry.

[24]  G. Patrick,et al.  Sweeter Than Ever , 2007 .

[25]  András Fiser,et al.  Comparative protein structure modeling by combining multiple templates and optimizing sequence-to-structure alignments , 2007, Bioinform..

[26]  Manfred T Reetz,et al.  Addressing the Numbers Problem in Directed Evolution , 2008, Chembiochem : a European journal of chemical biology.

[27]  N. Khardori.  Comparison of Pneumococcal Conjugate Polysaccharide and Free Polysaccharide Vaccines in Elderly Adults: Conjugate Vaccine Elicits Improved Antibacterial Immune Responses and Immunological Memory , 2008 .

[28]  John H. Grate,et al.  A green-by-design biocatalytic process for atorvastatin intermediate , 2010 .

[29]  B. Imperiali,et al.  Substrate specificity of the glycosyl donor for oligosaccharyl transferase. , 2001, The Journal of organic chemistry.

[30]  Silvio C. E. Tosatto,et al.  Global and local model quality estimation at CASP8 using the scoring functions QMEAN and QMEANclust , 2009, Proteins.

[31]  Anne A. Ollis,et al.  Engineered oligosaccharyltransferases with greatly relaxed acceptor-site specificity. , 2014, Nature chemical biology.

[32]  Manfred T. Reetz,et al.  Creation of Enantioselective Biocatalysts for Organic Chemistry by In Vitro Evolution , 1997 .

[33]  I. Jónsdóttir,et al.  Neonatal immune response and serum bactericidal activity induced by a meningococcal conjugate vaccine is enhanced by LT-K63 and CpG2006. , 2008, Vaccine.

[34]  J. Sternon,et al.  [Conjugate vaccines]. , 2002, Journal de pharmacie de Belgique.

[35]  S. Matsumoto,et al.  Crystal structures of an archaeal oligosaccharyltransferase provide insights into the catalytic cycle of N-linked protein glycosylation , 2013, Proceedings of the National Academy of Sciences.

[36]  R. Byrd,et al.  Structure of the type 5 capsular polysaccharide of Staphylococcus aureus. , 1990, Carbohydrate research.

[37]  R. Dwek,et al.  Glycobiology , 2018, Biochimie.

[38]  C. Czibener,et al.  Exploiting the Campylobacter jejuni protein glycosylation system for glycoengineering vaccines and diagnostic tools directed against brucellosis , 2012, Microbial Cell Factories.

[39]  Markus Aebi,et al.  Relaxed acceptor site specificity of bacterial oligosaccharyltransferase in vivo. , 2011, Glycobiology.

[40]  Yang Zhang,et al.  Automated protein structure modeling in CASP9 by I‐TASSER pipeline combined with QUARK‐based ab initio folding and FG‐MD‐based structure refinement , 2011, Proteins.

[41]  G. Lipowsky,et al.  Prevention of Staphylococcus aureus infections by glycoprotein vaccines synthesized in Escherichia coli. , 2014, The Journal of infectious diseases.

[42]  P. Reeves,et al.  Relationships among the O-Antigen Gene Clusters ofSalmonella enterica Groups B, D1, D2, and D3 , 1998, Journal of bacteriology.

[43]  M. Wacker,et al.  Structural insights from random mutagenesis of Campylobacter jejuni oligosaccharyltransferase PglB , 2012, BMC Biotechnology.

[44]  M. Aebi,et al.  Substrate specificity of bacterial oligosaccharyltransferase suggests a common transfer mechanism for the bacterial and eukaryotic systems. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[45]  B. Wren,et al.  Characterization of the Structurally Diverse N-Linked Glycans of Campylobacter Species , 2012, Journal of bacteriology.

[46]  J. Reymond,et al.  A Catalytically Essential Motif in External Loop 5 of the Bacterial Oligosaccharyltransferase PglB* , 2013, The Journal of Biological Chemistry.

[47]  M. Aebi,et al.  Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[48]  Markus Aebi,et al.  Definition of the bacterial N‐glycosylation site consensus sequence , 2006, The EMBO journal.

[49]  Keith A. Powell,et al.  Directed Evolution and Biocatalysis. , 2001, Angewandte Chemie.

[50]  Markus Aebi,et al.  X-ray structure of a bacterial oligosaccharyltransferase , 2011, Nature.

[51]  P. Anderson.  Antibody Responses to Haemophilus influenzae Type b and Diphtheria Toxin Induced by Conjugates of Oligosaccharides of the Type b Capsule with the Nontoxic Protein CRM197 , 1983, Infection and immunity.

[52]  Andreas S Bommarius,et al.  Status of protein engineering for biocatalysts: how to design an industrially useful biocatalyst. , 2011, Current opinion in chemical biology.

[53]  Lai-Xi Wang,et al.  A combined method for producing homogeneous glycoproteins with eukaryotic N-glycosylation , 2010, Nature chemical biology.

[54]  Johannes Söding,et al.  The HHpred interactive server for protein homology detection and structure prediction , 2005, Nucleic Acids Res..