AiM  Vol.2 No.4 , December 2012
Description of a Putative Oligosaccharyl:S-Layer Protein Transferase from the Tyrosine O-Glycosylation System of Paenibacillus alvei CCM 2051T
ABSTRACT

Surface (S)-layer proteins are model systems for studying protein glycosylation in bacteria and simultaneously hold promises for the design of novel, glyco-functionalized modules for nanobiotechnology due to their 2D self-assembly capability. Understanding the mechanism governing S-layer glycan biosynthesis in the Gram-positive bacterium Paenibacillus alvei CCM 2051T is necessary for the tailored glyco-functionalization of its S-layer. Here, the putative oligosaccharyl:S-layer protein transferase WsfB from the P. alvei S-layer glycosylation gene locus is characterized. The enzyme is proposed to catalyze the final step of the glycosylation pathway, transferring the elongated S-layer glycan onto distinct tyrosine O-glycosylation sites. Genetic knock-out of WsfB is shown to abolish glycosylation of the S-layer protein SpaA but not that of other glycoproteins present in P. alvei CCM 2051T, confining its role to the S-layer glycosylation pathway. A transmembrane topology model of the 781-amino acid WsfB protein is inferred from activity measurements of green fluorescent protein and phosphatase A fused to defined truncations of WsfB. This model shows an overall number of 13 membrane spanning helices with the Wzy_C domain characteristic of O-oligosaccharyl:protein transferases (O-OTases) located in a central extra-cytoplasmic loop, which both compares well to the topology of OTases from Gram-negative bacteria. Mutations in the Wzy C motif resulted in loss of WsfB function evidenced in reconstitution experiments in P. alvei ΔWsfB cells. Attempts to use WsfB for transferring heterologous oligosaccharides to its native S-layer target protein in Escherichia coli CWG702 and Salmonella enterica SL3749, which should provide lipid-linked oligosaccharide substrates mimicking to some extent those of the natural host, were not successful, possibly due to the stringent function of WsfB. Concluding, WsfB has all features of a bacterial O-OTase, making it the most probable candidate for the oligosaccharyl:S-layer protein transferase of P. alvei, and a promising candidate for the first O-OTase reported in Gram-positives.



Cite this paper
R. Ristl, B. Janesch, J. Anzengruber, A. Forsthuber, J. Blaha, P. Messner and C. Schaffer, "Description of a Putative Oligosaccharyl:S-Layer Protein Transferase from the Tyrosine O-Glycosylation System of Paenibacillus alvei CCM 2051T," Advances in Microbiology, Vol. 2 No. 4, 2012, pp. 537-546. doi: 10.4236/aim.2012.24069.
References
[1]   C. M. Szymanski, D. H. Burr and P. Guerry, “Campylobacter Protein Glycosylation Affects Host Cell Interactions,” Infection and Immunity, Vol. 70, No. 4, 2002, pp. 2242-2244. doi:10.1128/IAI.70.4.2242-2244.2002

[2]   C. Lizak, Y. Y. Fan, T. C. Weber and M. Aebi, “N-Linked Glycosylation of Antibody Fragments in Escherichia coli,” Bioconjugate Chemistry, Vol. 22, No. 3, 2011, pp. 488-496. doi:10.1021/bc100511k

[3]   J. D. Valderrama-Rincon, A. C. Fisher, J. H. Merritt, Y. Y. Fan, C. A. Reading, K. Chhiba, C. Heiss, P. Azadi, M. Aebi and M. P. DeLisa, “An Engineered Eukaryotic Protein Glycosylation Pathway in Escherichia coli,” Nature Chemical Biology, Vol. 8, No. 5, 2012, pp. 434-436. doi:10.1038/nchembio.921

[4]   H. Nothaft and C. M. Szymanski, “Protein Glycosylation in Bacteria: Sweeter than Ever,” Nature Reviews in Microbiology, Vol. 8, No. 11, 2010, pp. 765-778. doi:10.1038/nrmicro2383

[5]   N. E. Scott, B. L. Parker, A. M. Connolly, J. Paulech, A. V. Edwards, B. Crossett, L. Falconer, D. Kolarich, S. P. Djordjevic, P. Hojrup, N. H. Packer, M. R. Larsen and S. J. Cordwell, “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,” Molecular Cell Proteomics, Vol. 10, No. 2, 2011, pp. M000031-MCP201. doi:10.1074/mcp.M000031-MCP201

[6]   A. Faridmoayer, M. A. Fentabil, D. C. Mills, J. S. Klassen and M. F. Feldman, “Functional Characterization of Bacterial Oligosaccharyl Transferases Involved in O-Linked Protein Glycosylation,” Journal of Bacteriology, Vol. 189, No. 22, 2007, pp. 8088-8098. doi:10.1128/JB.01318-07

[7]   M. D. Hartley, M. J. Morrison, F. E. Aas, B. Borud, M. Koomey and B. Imperiali, “Biochemical Characterization of the O-Linked Glycosylation Pathway in Neisseria gonorrhoeae Responsible for Biosynthesis of Protein Glycans Containing N,N’-Diacetylbacillosamine,” Biochemistry, Vol. 50, No. 22, 2011, pp. 4936-4948. doi:10.1021/bi2003372

[8]   C. Gebhart, M. V. Ielmini, B. Reiz, N. L. Price, F. E. Aas, M. Koomey and M. F. Feldman, “Characterization of Exogenous Bacterial Oligosaccharyl Transferases in Escherichia coli Reveals the Potential for O-Linked Protein Glycosylation in Vibrio cholerae and Burkholderia thailandensis,” Glycobiology, Vol. 22, No. 7, 2012, pp. 962-974. doi:10.1093/glycob/cws059

[9]   C. M. Fletcher, M. J. Coyne, O. F. Villa, M. Chatzidaki-Livanis and L. E. Comstock, “A General O-Glycosylation System Important to the Physiology of a Major Human Intestinal Symbiont,” Cell, Vol. 137, No. 2, 2009, pp. 321-331. doi:10.1016/j.cell.2009.02.041

[10]   G. Posch, M. Pabst, L. Brecker, F. Altmann, P. Messner and C. Sch?ffer, “Characterization and Scope of S-Layer Protein O-Glycosylation in Tannerella forsythia,” Journal of Biological Chemistry, Vol. 286, No. 44, 2011, pp. 38714-38724. doi:10.1074/jbc.M111.284893

[11]   R. Ristl, K. Steiner, K. Zarschler, S. Zayni, P. Messner and C. Sch?ffer, “The S-Layer Glycome: Adding to the Sugar Coat of Bacteria,” International Journal of Microbiology, Vol. 2011, No. 2011, Article ID: 127870.

[12]   K. Steiner, R. Novotny, D. B. Werz, K. Zarschler, P. H. Seeberger, A. Hofinger, P. Kosma, C. Sch?ffer and P. Messner, “Molecular Basis of S-Layer Glycoprotein Glycan Biosynthesis in Geobacillus stearothermophilus,” Journal of Biological Chemistry, Vol. 283, No. 30, 2008, pp. 21120-21133. doi:10.1074/jbc.M801833200

[13]   K. Zarschler, B. Janesch, S. Zayni, C. Schaffer and P. Messner, “Construction of a Gene Knockout System for Application in Paenibacillus alvei CCM 2051T, Exemplified by the S-Layer Glycan Biosynthesis Initiation Enzyme WsfP,” Applied and Environmental Microbiology, Vol. 75, No. 10, 2009, pp. 3077-3085. doi:10.1128/AEM.00087-09

[14]   C. Sch?ffer, T. Wugeditsch, H. K?hlig, A. Scheberl, S. Zayni and P. Messner, “The Surface Layer (S-Layer) Glycoprotein of Geobacillus stearothermophilus NRS 2004/ 3a. Analysis of Its Glycosylation,” Journal of Biological Chemistry, Vol. 277, No. 8, 2002, pp. 6230-6239. doi:10.1074/jbc.M108873200

[15]   P. Messner, R. Christian, C. Neuninger and G. Schulz, “Similarity of ‘Core’ Structures in Two Different Glycans of Tyrosine-Linked Eubacterial S-Layer Glycoproteins,” Journal of Bacteriology, Vol. 177, No. 8, 1995, pp. 2188-2193.

[16]   R. Novotny, A. Pfoestl, P. Messner and C. Sch?ffer, “Genetic Organization of Chromosomal S-Layer Glycan Biosynthesis Loci of Bacillaceae,” Glycoconjugate Journal, Vol. 20, No. 7-8, 2004, pp. 435-447.

[17]   K. Zarschler, B. Janesch, M. Pabst, F. Altmann, P. Messner and C. Schaffer, “Protein Tyrosine O-Glycosylation: A Rather Unexplored Prokaryotic Glycosylation System,” Glycobiology, Vol. 20, No. 6, 2010, pp. 787-798. doi:10.1093/glycob/cwq035

[18]   C. Smythe and P. Cohen, “The Discovery of Glycogenin and the Priming Mechanism for Glycogen Biogenesis,” European Journal of Biochemistry, Vol. 200, No. 3, 1991, pp. 625-631. doi:10.1111/j.1432-1033.1991.tb16225.x

[19]   P. Lu, K. J. Kramer, P. A. Seib, D. D. Mueller, R. Ahmed and T. L. Hopkins, “β-D-Glucopyranosyl-O-L-Tyrosine: Synthesis, Properties and Titre during Insect Development,” Insect Biochemistry, Vol. 12, No. 4, 1982, pp. 377-381. doi:10.1016/0020-1790(82)90034-8

[20]   B. Kainz, K. Steiner, M. M?ller, D. Pum, C. Sch?ffer, U. B. Sleytr and J. L. Toca-Herrera, “Absorption, Steady-State Fluorescence, Fluorescence Lifetime, and 2D Self-Assembly Properties of Engineered Fluorescent S-Layer Fusion Proteins of Geobacillus stearothermophilus NRS 2004/3a,” Biomacromolecules, Vol. 11, No. 1, 2010, pp. 207-214. doi:10.1021/bm901071b

[21]   C. Hart, B. Schulenberg, T. H. Steinberg, W. Y. Leung and W. F. Patton, “Detection of Glycoproteins in Polyacrylamide Gels and on Electroblots Using Pro-Q EM-ERALD 488 Dye, a Fluorescent Periodate Schiff-Base Stain,” Electrophoresis, Vol. 24, No. 4, 2003, pp. 588-598. doi:10.1002/elps.200390069

[22]   D. O. Daley, M. Rapp, E. Granseth, K. Melen, D. Drew and G. von Heijne, “Global Topology Analysis of the Escherichia coli Inner Membrane Proteome,” Science, Vol. 308, No. 5726, 2005, pp. 1321-1323. doi:10.1126/science.1109730

[23]   S. T. Islam, V. L. Taylor, M. Qi and J. S. Lam, “Membrane Topology Mapping of the O-Antigen Flippase (Wzx), Polymerase (Wzy), and Ligase (WaaL) from Pseudomonas aeruginosa PAO1 Reveals Novel Domain Architectures,” MBio, Vol. 1, No. 3, 2010, Article ID: e00189.

[24]   K. Zarschler, B. Janesch, B. Kainz, R. Ristl, P. Messner and C. Schaffer, “Cell Surface Display of Chimeric Glycoproteins via the S-Layer of Paenibacillus alvei,” Carbohydrate Research, Vol. 345, No. 10, 2010, pp. 1422-1431. doi:10.1016/j.carres.2010.04.010

[25]   G. E. Tusnady and I. Simon, “The HMMTOP Transmembrane Topology Prediction Server,” Bioinformatics, Vol. 17, No. 9, 2001, pp. 849-850. doi:10.1093/bioinformatics/17.9.849

[26]   G. L. Blatch and M. Lassle, “The Tetratricopeptide Repeat: A Structural Motif Mediating Protein-Protein Interactions,” Bioessays, Vol. 21, No. 11, 1999, pp. 932-939. doi:10.1002/(SICI)1521-1878(199911)21:11<932::AID-BIES5>3.0.CO;2-N

[27]   M. Qutyan, M. Paliotti and P. Castric, “PilO of Pseudomonas aeruginosa 1244: Subcellular Location and Domain Assignment,” Molecular Microbiology, Vol. 66, No. 6, 2007, pp. 1444-1458.

[28]   P. M. Power, K. L. Seib and M. P. Jennings, “Pilin Glycosylation in Neisseria meningitidis Occurs by a Similar Pathway to wzy-Dependent O-Antigen Biosynthesis in Escherichia coli,” Biochemical and Biophysical Research Communications, Vol. 347, No. 4, 2006, pp. 904-908. doi:10.1016/j.bbrc.2006.06.182

[29]   M. F. Feldman, M. Wacker, M. Hernandez, P. G. Hitchen, C. L. Marolda, M. Kowarik, H. R. Morris, A. Dell, M. A. Valvano and M. Aebi, “Engineering N-Linked Protein Glycosylation with Diverse O Antigen Lipopolysaccharide Structures in Escherichia coli,” Proceeding of the National Academy of Sciences USA, Vol. 102, No. 8, 2005, pp. 3016-3021.

[30]   A. Faridmoayer, M. A. Fentabil, M. F. Haurat, W. Yi, R. Woodward, P. G. Wang and M. F. Feldman, “Extreme Substrate Promiscuity of the Neisseria Oligosaccharyl Transferase Involved in Protein O-Glycosylation,” Journal of Biological Chemistry, Vol. 283, No. 50, 2008, pp. 34596-34604. doi:10.1074/jbc.M807113200

[31]   K. Steiner, A. Hanreich, B. Kainz, P. G. Hitchen, A. Dell, P. Messner and C. Sch?ffer, “Recombinant Glycans on an S-Layer Self-Assembly Protein: A New Dimension for Nanopatterned Biomaterials,” Small, Vol. 4, No. 10, 2008, pp. 1728-1740. doi:10.1002/smll.200701215

[32]   S. A. Brooks, “Appropriate Glycosylation of Recombinant Proteins for Human Use: Implications of Choice of Expression System,” Molecular Biotechnology, Vol. 28, No. 3, 2004, pp. 241-255. doi:10.1385/MB:28:3:241

[33]   J. Pandhal and P. C. Wright, “N-Linked Glycoengineering for Human Therapeutic Proteins in Bacteria,” Biotechnology Letters, Vol. 32, No. 9, 2010, pp. 1189-1198. doi:10.1007/s10529-010-0289-6

[34]   A. Dell, A. Galadari, F. Sastre and P. Hitchen, “Similarities and Differences in the Glycosylation Mechanisms in Prokaryotes and Eukaryotes,” International Journal of Microbiology, Vol. 2011, 2011, Article ID: 148178.

 
 
Top