Desulfovibrio desulfuricans PglB homolog possesses oligosaccharyltransferase activity with relaxed glycan specificity and distinct protein acceptor sequence requirements.

Oligosaccharyltransferases (OTases) are responsible for the transfer of carbohydrates from lipid carriers to acceptor proteins and are present in all domains of life. In bacteria, the most studied member of this family is PglB from Campylobacter jejuni (PglB(Cj)). This enzyme is functional in Escherichia coli and, contrary to its eukaryotic counterparts, has the ability to transfer a variety of oligo- and polysaccharides to protein carriers in vivo. Phylogenetic analysis revealed that in the delta proteobacteria Desulfovibrio sp., the PglB homolog is more closely related to eukaryotic and archaeal OTases than to its Campylobacter counterparts. Genetic analysis revealed the presence of a putative operon that might encode all enzymes required for N-glycosylation in Desulfovibrio desulfuricans. D. desulfuricans PglB (PglB(Dd)) was cloned and successfully expressed in E. coli, and its activity was confirmed by transferring the C. jejuni heptasaccharide onto the model protein acceptor AcrA. In contrast to PglB(Cj), which adds two glycan chains to AcrA, a single oligosaccharide was attached to the protein by PglB(Dd). Site-directed mutagenesis of the five putative N-X-S/T glycosylation sites in AcrA and mass spectrometry analysis showed that PglB(Dd) does not recognize the "conventional bacterial glycosylation sequon" consisting of the sequence D/E-X(1)-N-X(2)-S/T (where X(1) and X(2) are any amino acid except proline), and instead used a different site for the attachment of the oligosaccharide than PglB(Cj.). Furthermore, PglB(Dd) exhibited relaxed glycan specificity, being able to transfer mono- and polysaccharides to AcrA. Our analysis constitutes the first characterization of an OTase from delta-proteobacteria involved in N-linked protein glycosylation.

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

[2]  M. Koomey,et al.  Genetic, Structural, and Antigenic Analyses of Glycan Diversity in the O-Linked Protein Glycosylation Systems of Human Neisseria Species , 2010, Journal of bacteriology.

[3]  M. Feldman,et al.  Helicobacter pylori Lipopolysaccharide Is Synthesized via a Novel Pathway with an Evolutionary Connection to Protein N-Glycosylation , 2010, PLoS pathogens.

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

[5]  Daisuke Kohda,et al.  Comparative Structural Biology of Eubacterial and Archaeal Oligosaccharyltransferases* , 2009, The Journal of Biological Chemistry.

[6]  R. Glockshuber,et al.  Oxidoreductase activity of oligosaccharyltransferase subunits Ost3p and Ost6p defines site-specific glycosylation efficiency , 2009, Proceedings of the National Academy of Sciences.

[7]  B. Wren,et al.  N-linked glycosylation in bacteria: an unexpected application. , 2009, Future microbiology.

[8]  W. Yi,et al.  Extreme Substrate Promiscuity of the Neisseria Oligosaccharyl Transferase Involved in Protein O-Glycosylation* , 2008, Journal of Biological Chemistry.

[9]  C. Szymanski,et al.  Affinity-capture tandem mass spectrometric characterization of polyprenyl-linked oligosaccharides: tool to study protein N-glycosylation pathways. , 2008, Analytical chemistry.

[10]  K. Jarrell,et al.  Sweet to the extreme: protein glycosylation in Archaea , 2008, Molecular microbiology.

[11]  J. Klassen,et al.  Functional Characterization of Bacterial Oligosaccharyltransferases Involved in O-Linked Protein Glycosylation , 2007, Journal of bacteriology.

[12]  M. Koomey,et al.  Neisseria gonorrhoeae O-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure , 2007, Molecular microbiology.

[13]  M. Romão,et al.  Crystal structure of the 16 heme cytochrome from Desulfovibrio gigas: a glycosylated protein in a sulphate-reducing bacterium. , 2007, Journal of molecular biology.

[14]  Benjamin L Schulz,et al.  N-Linked Glycosylation of Folded Proteins by the Bacterial Oligosaccharyltransferase , 2006, Science.

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

[16]  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.

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

[18]  Mihai Nita-Lazar,et al.  The N-X-S/T consensus sequence is required but not sufficient for bacterial N-linked protein glycosylation. , 2005, Glycobiology.

[19]  C. Szymanski,et al.  Protein glycosylation in bacterial mucosal pathogens , 2005, Nature Reviews Microbiology.

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

[21]  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.

[22]  G. Bourenkov,et al.  Crystallization and preliminary X-ray diffraction analysis of the 16-haem cytochrome of Desulfovibrio gigas. , 2004, Acta crystallographica. Section D, Biological crystallography.

[23]  Rekha Seshadri,et al.  The genome sequence of the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough , 2004, Nature Biotechnology.

[24]  V. Peraino,et al.  Desulfovibrio desulfuricans Bacteremia and Review of Human Desulfovibrio Infections , 2003, Journal of Clinical Microbiology.

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

[26]  C. Szymanski,et al.  Structure of the N-Linked Glycan Present on Multiple Glycoproteins in the Gram-negative Bacterium, Campylobacter jejuni * , 2002, The Journal of Biological Chemistry.

[27]  F. Guerlesquin,et al.  Escherichia coli is able to produce heterologous tetraheme cytochrome c(3) when the ccm genes are co-expressed. , 2000, Biochimica et biophysica acta.

[28]  M. Valvano,et al.  Genetic organization of the O7-specific lipopolysaccharide biosynthesis cluster of Escherichia coli VW187 (O7:K1). , 1999, Microbiology.

[29]  T. Ogawa,et al.  Isolation and Characterization of Rhamnose-binding Lectins from Eggs of Steelhead Trout (Oncorhynchus mykiss) Homologous to Low Density Lipoprotein Receptor Superfamily* , 1998, The Journal of Biological Chemistry.

[30]  B. Imperiali,et al.  Asparagine-linked glycosylation: specificity and function of oligosaccharyl transferase. , 1995, Bioorganic & medicinal chemistry.

[31]  W. Hamilton.  Bioenergetics of sulphate-reducing bacteria in relation to their environmental impact , 2004, Biodegradation.

[32]  Hamilton Wa.  Bioenergetics of sulphate-reducing bacteria in relation to their environmental impact , 1998 .

[33]  D. Dykxhoorn,et al.  A set of compatible tac promoter expression vectors. , 1996, Gene.