Functional analysis of the Helicobacter pullorum N-linked protein glycosylation system

Abstract N-linked protein glycosylation systems operate in species from all three domains of life. The model bacterial N-linked glycosylation system from Campylobacter jejuni is encoded by pgl genes present at a single chromosomal locus. This gene cluster includes the pglB oligosaccharyltransferase responsible for transfer of glycan from lipid carrier to protein. Although all genomes from species of the Campylobacter genus contain a pgl locus, among the related Helicobacter genus only three evolutionarily related species (H. pullorum, H. canadensis and H. winghamensis) potentially encode N-linked protein glycosylation systems. Helicobacter putative pgl genes are scattered in five chromosomal loci and include two putative oligosaccharyltransferase-encoding pglB genes per genome. We have previously demonstrated the in vitro N-linked glycosylation activity of H. pullorum resulting in transfer of a pentasaccharide to a peptide at asparagine within the sequon (D/E)XNXS/T. In this study, we identified the first H. pullorum N-linked glycoprotein, termed HgpA. Production of histidine-tagged HgpA in the background of insertional knockout mutants of H. pullorum pgl/wbp genes followed by analysis of HgpA glycan structures demonstrated the role of individual gene products in the PglB1-dependent N-linked protein glycosylation pathway. Glycopeptide purification by zwitterionic-hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry identified six glycosites from five H. pullorum proteins, which was consistent with proteins reactive with a polyclonal antiserum generated against glycosylated HgpA. This study demonstrates functioning of a H. pullorum N-linked general protein glycosylation system.

[1]  D. Maskell,et al.  The N-linking glycosylation system from Actinobacillus pleuropneumoniae is required for adhesion and has potential use in glycoengineering , 2017, Open Biology.

[2]  Hyunjoong Kim,et al.  Functional Analysis I , 2017 .

[3]  B. Wren,et al.  Functional analysis of N-linking oligosaccharyl transferase enzymes encoded by deep-sea vent proteobacteria. , 2016, Glycobiology.

[4]  D. Kohda,et al.  Comparative Analysis of Archaeal Lipid-linked Oligosaccharides That Serve as Oligosaccharide Donors for Asn Glycosylation* , 2016, The Journal of Biological Chemistry.

[5]  Nichollas E. Scott,et al.  Comparative proteomics and glycoproteomics reveal increased N-linked glycosylation and relaxed sequon specificity in Campylobacter jejuni NCTC11168 O. , 2014, Journal of proteome research.

[6]  C. Sihlbom,et al.  A novel O-linked glycan modulates Campylobacter jejuni major outer membrane protein-mediated adhesion to human histo-blood group antigens and chicken colonization , 2014, Open Biology.

[7]  M. Aebi,et al.  Molecular Analysis of an Alternative N-Glycosylation Machinery by Functional Transfer from Actinobacillus pleuropneumoniae to Escherichia coli* , 2013, The Journal of Biological Chemistry.

[8]  Z. Guan,et al.  Two Distinct N-Glycosylation Pathways Process the Haloferax volcanii S-Layer Glycoprotein upon Changes in Environmental Salinity , 2013, mBio.

[9]  J. Reymond,et al.  Unexpected reactivity and mechanism of carboxamide activation in bacterial N-linked protein glycosylation , 2013, Nature Communications.

[10]  U. Gophna,et al.  Phylogenetic- and genome-derived insight into the evolution of N-glycosylation in Archaea. , 2013, Molecular phylogenetics and evolution.

[11]  J. Eichler.  Extreme sweetness: protein glycosylation in archaea , 2013, Nature Reviews Microbiology.

[12]  Jean-Louis Reymond,et al.  Mechanism of Bacterial Oligosaccharyltransferase , 2013, The Journal of Biological Chemistry.

[13]  Nichollas E. Scott,et al.  Diversity in the Protein N-Glycosylation Pathways Within the Campylobacter Genus* , 2012, Molecular & Cellular Proteomics.

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

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

[16]  M. Aebi,et al.  Mechanisms and principles of N-linked protein glycosylation. , 2011, Current opinion in structural biology.

[17]  F. Kawai,et al.  Structural Insights into the Glycosyltransferase Activity of the Actinobacillus pleuropneumoniae HMW1C-like Protein* , 2011, The Journal of Biological Chemistry.

[18]  M. Aebi,et al.  Cytoplasmic N-Glycosyltransferase of Actinobacillus pleuropneumoniae Is an Inverting Enzyme and Recognizes the NX(S/T) Consensus Sequence* , 2011, The Journal of Biological Chemistry.

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

[20]  M. V. Ielmini,et al.  Desulfovibrio desulfuricans PglB homolog possesses oligosaccharyltransferase activity with relaxed glycan specificity and distinct protein acceptor sequence requirements. , 2011, Glycobiology.

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

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

[23]  J. S. St. Geme,et al.  The Actinobacillus pleuropneumoniae HMW1C-Like Glycosyltransferase Mediates N-Linked Glycosylation of the Haemophilus influenzae HMW1 Adhesin , 2010, PloS one.

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

[25]  Jerry Eichler,et al.  Protein glycosylation in Archaea: sweet and extreme. , 2010, Glycobiology.

[26]  P. Hitchen,et al.  Modification of the Campylobacter jejuni flagellin glycan by the product of the Cj1295 homopolymeric-tract-containing gene , 2010, Microbiology.

[27]  P. Hitchen,et al.  Characterization of N-Linked Protein Glycosylation in Helicobacter pullorum , 2010, Journal of bacteriology.

[28]  E. Vinogradov,et al.  Biosynthesis of uronamide sugars in Pseudomonas aeruginosa O6 and Escherichia coli O121 O antigens. , 2010, Environmental microbiology.

[29]  Cheryl F. Lichti,et al.  The Haemophilus influenzae HMW1C Protein Is a Glycosyltransferase That Transfers Hexose Residues to Asparagine Sites in the HMW1 Adhesin , 2010, PLoS pathogens.

[30]  Nichollas E. Scott,et al.  Simultaneous Glycan-Peptide Characterization Using Hydrophilic Interaction Chromatography and Parallel Fragmentation by CID, Higher Energy Collisional Dissociation, and Electron Transfer Dissociation MS Applied to the N-Linked Glycoproteome of Campylobacter jejuni* , 2010, Molecular & Cellular Proteomics.

[31]  Nichollas E. Scott,et al.  Mass spectrometric characterization of the surface-associated 42 kDa lipoprotein JlpA as a glycosylated antigen in strains of Campylobacter jejuni. , 2009, Journal of proteome research.

[32]  G. Barton,et al.  Distinct donor and acceptor specificities of Trypanosoma brucei oligosaccharyltransferases , 2009, The EMBO journal.

[33]  J. Gross,et al.  The Haemophilus influenzae HMW1 Adhesin Is a Glycoprotein with an Unusual N-Linked Carbohydrate Modification* , 2008, Journal of Biological Chemistry.

[34]  F. Gamarro,et al.  All in one: Leishmania major STT3 proteins substitute for the whole oligosaccharyltransferase complex in Saccharomyces cerevisiae. , 2008, Molecular biology of the cell.

[35]  B. Imperiali,et al.  From peptide to protein: comparative analysis of the substrate specificity of N-linked glycosylation in C. jejuni. , 2007, Biochemistry.

[36]  B. Imperiali,et al.  In vitro biosynthesis of UDP-N,N'-diacetylbacillosamine by enzymes of the Campylobacter jejuni general protein glycosylation system. , 2006, Biochemistry.

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

[38]  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.

[39]  B. Imperiali,et al.  Direct biochemical evidence for the utilization of UDP-bacillosamine by PglC, an essential glycosyl-1-phosphate transferase in the Campylobacter jejuni N-linked glycosylation pathway. , 2006, Biochemistry.

[40]  Cristina Alaimo,et al.  Two distinct but interchangeable mechanisms for flipping of lipid‐linked oligosaccharides , 2006, The EMBO journal.

[41]  B. Imperiali,et al.  Chemoenzymatic synthesis of glycopeptides with PglB, a bacterial oligosaccharyl transferase from Campylobacter jejuni. , 2005, Chemistry & biology.

[42]  B. Imperiali,et al.  In vitro assembly of the undecaprenylpyrophosphate-linked heptasaccharide for prokaryotic N-linked glycosylation. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Anne Dell,et al.  Functional analysis of the Campylobacter jejuni N‐linked protein glycosylation pathway , 2005, Molecular microbiology.

[44]  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.

[45]  M. Apicella,et al.  The Haemophilus influenzae HMW1 adhesin is glycosylated in a process that requires HMW1C and phosphoglucomutase, an enzyme involved in lipooligosaccharide biosynthesis , 2003, Molecular microbiology.

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

[47]  Binding and Catalysis , 2001 .

[48]  C. Szymanski,et al.  Evidence for a system of general protein glycosylation in Campylobacter jejuni , 1999, Molecular microbiology.

[49]  J. Ketley,et al.  7.7 Genetic Manipulation of Enteric Campylobacter Species , 1998 .

[50]  D. Geyl,et al.  In Vitro Biosynthesis of , 1991 .