Abstract
N-Linked protein glycosylation is conserved throughout the three domains of life and influences protein function, stability, and protein complex formation. N-Linked glycosylation is an essential process in Eukaryotes; however, although N-glycosylation affects multiple cellular processes in Archaea and Bacteria, it is not needed for cell survival. Methods for the analyses of N-glycosylation in eukaryotes are well established, but comparable techniques for the analyses of the pathways in Bacteria and Archaea are needed. In this chapter we describe new methods for the detection and analyses of N-linked, and the recently discovered free oligosaccharides (fOS), from whole cell lysates of Campylobacter jejuni using non-specific pronase E digestion and permethylation followed by mass spectrometry. We also describe the expression and immunodetection of the model N-glycoprotein, AcrA, fused to a hexa-histidine tag to follow protein glycosylation in C. jejuni. This chapter concludes with the recent demonstration that high-resolution magic angle spinning NMR of intact bacterial cells provides a rapid, non-invasive method for analyzing fOS in C. jejuni in vivo. This combination of techniques provides a powerful tool for the exploration, quantification, and structural analyses of N-linked and free oligosaccharides in the bacterial system.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Helenius, A. and Aebi, M. (2001) Intracellular functions of N-linked glycans. Science 291, 2364–2369.
Abu-Qarn, M., Yurist-Doutsch, S., Giordano, A., Trauner, A., Morris, H. R., Hitchen, P., Medalia, O., Dell, A., and Eichler, J. (2007) Haloferax volcanii AglB and AglD are involved in N-glycosylation of the S-layer glycoprotein and proper assembly of the surface layer. J. Mol. Biol. 374, 1224–1236.
Chaban, B., Voisin, S., Kelly, J., Logan, S. M., and Jarrell, K. F. (2006) Identification of genes involved in the biosynthesis and attachment of Methanococcus voltae N-linked glycans: insight into N-linked glycosylation pathways in Archaea. Mol. Microbiol. 61, 259–268.
Yurist-Doutsch, S., Chaban, B., VanDyke, D. J., Jarrell, K. F., and Eichler, J. (2008) Sweet to the extreme: protein glycosylation in Archaea. Mol. Microbiol. 68, 1079–1084.
Wacker, M., Linton, D., Hitchen, P. G., Nita-Lazar, M., Haslam, S. M., North, S. J., Panico, M., Morris, H. R., Dell, A., Wren, B. W., and Aebi, M. (2002) N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 298, 1790–1793.
Szymanski, C. M., and Wren, B. W. (2005) Protein glycosylation in bacterial mucosal pathogens. Nat. Rev. Microbiol. 3, 225–237.
Nothaft, H., Amber, S., Aebi, M., and Szymanski, C. M. (2008) N-Linked Protein Glycosylation in Campylobacter. In: Campylobacter (3rd edn.) Nachamkin, I., Szymanski, C. M., and Blaser, M. J. (eds). Washington, DC: ASM Press, pp. 447–469.
Hendrixson, D. R. and DiRita, V. J. (2004) Identification of Campylobacter jejuni genes involved in commensal colonization of the chick gastrointestinal tract. Mol. Microbiol. 52, 471–484.
Larsen, J. C., Szymanski, C., and Guerry, P. (2004) N-linked protein glycosylation is required for full competence in Campylobacter jejuni 81-176. J. Bacteriol. 186, 6508–6514.
Szymanski, C. M., Goon, S., Allan, B., and Guerry, P. (2005) Protein glycosylation in Campylobacter. In: Campylobacter: Molecular and Cellular Biology. Ketley, J. and Konkel, M. (eds). Norfolk: Horizon Bioscience, pp. 259–273.
Szymanski, C. M., Logan, S. M., Linton, D., and Wren, B. W. (2003) Campylobacter – a tale of two protein glycosylation systems. Trends Microbiol. 11, 233–238.
Szymanski, C. M., Yao, R., Ewing, C. P., Trust, T. J., and Guerry, P. (1999) Evidence for a system of general protein glycosylation in Campylobacter jejuni. Mol. Microbiol. 32, 1022–1030.
Kowarik, M., Young, N. M., Numao, S., Schulz, B. L., Hug, I., Callewaert, N., Mills, D. C., Watson, D. C., Hernandez, M., Kelly, J. F., Wacker, M., and Aebi, M. (2006) Definition of the bacterial N-glycosylation site consensus sequence. EMBO J. 25, 1957–1966.
Young, N. M., Brisson, J. -R., Kelly, J., Watson, D. C., Tessier, L., Lanthier, P. H., Jarrell, H. C., Cadotte, N., St Michael, F., Aberg, E., and Szymanski, C. M. (2002) Structure of the N-linked glycan present on multiple glycoproteins in the Gram-negative bacterium, Campylobacter jejuni. J. Biol. Chem. 277, 42530–42539.
Alaimo, C., Catrein, I., Morf, L., Marolda, C. L., Callewaert, N., Valvano, M. A., Feldman, M. F., and Aebi, M. (2006) Two distinct but interchangeable mechanisms for flipping of lipid-linked oligosaccharides. EMBO J. 25, 967–976.
Liu, X., McNally, D. J., Nothaft, H., Szymanski, C. M., Brisson, J. -R., and Li, J. (2006) Mass spectrometry-based glycomics strategy for exploring N-linked glycosylation in eukaryotes and bacteria. Anal. Chem. 78, 6081–6087.
Abu-Qarn, M., Eichler, J., and Sharon, N. (2008) Not just for Eukarya anymore: protein glycosylation in Bacteria and Archaea. Curr. Opin. Struct. Biol. 18, 544–550.
Glover, K. J., Weerapana, E., and Imperiali, B. (2005) In vitro assembly of the undecaprenylpyrophosphate-linked heptasaccharide for prokaryotic N-linked glycosylation. Proc. Natl. Acad. Sci. U. S. A. 102, 14255–14259.
Weerapana, E., Glover, K. J., Chen, M. M., and Imperiali, B. (2005) Investigating bacterial N-linked glycosylation: synthesis and glycosyl acceptor activity of the undecaprenyl pyrophosphate-linked bacillosamine. J. Am. Chem. Soc. 127, 13766–13767.
Merry, T., Taverna, M., Tran, T., and Harvey, D. (2002) Analyses of glycans of recombinant glycoproteins. In: Cell Engineering. Al-Rubeai, M. (ed). Dordrecht: Springer, pp. 1–60.
Kelly, J., Jarrell, H., Millar, L., Tessier, L., Fiori, L. M., Lau, P. C., Allan, B., and Szymanski, C. M. (2006) Biosynthesis of the N-linked glycan in Campylobacter jejuni and addition onto protein through block transfer. J. Bacteriol. 188, 2427–2434.
Linton, D., Allan, E., Karlyshev, A. V., Cronshaw, A. D., and Wren, B. W. (2002) Identification of N-acetylgalactosamine-containing glycoproteins PEB3 and CgpA in Campylobacter jejuni. Mol. Microbiol. 43, 497–508.
Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual (3rd edn). Cold Spring Harbor Laboratory, New York.
Labigne-Roussel, A., Harel, J., and Tompkins, L. (1987) Gene transfer from Escherichia coli to Campylobacter species: development of shuttle vectors for genetic analysis of Campylobacter jejuni. J. Bacteriol. 169, 5320–5323.
St Michael, F., Szymanski, C. M., Li, J., Chan, K. H., Khieu, N. H., Larocque, S., Wakarchuk, W. W., Brisson, J. -R., and Monteiro, M. A. (2002) The structures of the lipooligosaccharide and capsule polysaccharide of Campylobacter jejuni genome sequenced strain NCTC 11168. Eur. J. Biochem. 269, 5119–5136.
Szymanski, C. M., Michael, F. S., Jarrell, H. C., Li, J., Gilbert, M., Larocque, S., Vinogradov, E., and Brisson, J. -R. (2003) Detection of conserved N-linked glycans and phase-variable lipooligosaccharides and capsules from campylobacter cells by mass spectrometry and high resolution magic angle spinning NMR spectroscopy. J. Biol. Chem. 278, 24509–24520.
Meiboom, S. and Gill, D. (1958) Modified spin-echo method for measuring nuclear relaxation times. Rev. Sci. Instrum. 29, 688–691.
Brisson, J. -R., Sue, S. C., Wu, W. G., McManus, G., Nghia, P. T., and Uhrin, D. (2002) NMR of carbohydrates: 1D homonuclear selective methods. In: NMR Spectroscopy of Glycoconjugates. Jimenez-Barbero, J. and Peters, T. (eds). Weinhem, Germany: Wiley-VCH, pp. 59–93.
UhrÃn, D. and Brisson, J. -R. (2000) Structure determination of microbial polysaccharides by high resolution NMR spectroscopy. In: NMR in Microbiology: Theory and Applications. Barbotin, J. N. and Portais, J. C. (eds). Wymondham, UK: Horizon Scientific Press, pp. 165–210.
McNally, D. J., Lamoureux, M., Li, J., Kelly, J., Brisson, J. -R., Szymanski, C. M., and Jarrell, H. C. (2006) HR-MAS NMR studies of 15N labeled cells confirm the structure of the O-methyl phosphoramidate CPS modification in Campylobacter jejuni and provide insight into its biosynthesis. Can. J. Chem. 84, 676–684.
McNally, D. J., Lamoureux, M. P., Karlyshev, A. V., Fiori, L. M., Li, J., Thacker, G., Coleman, R. A., Khieu, N. H., Wren, B. W., Brisson, J. -R., Jarrell, H. C., and Szymanski, C. M. (2007) Commonality and biosynthesis of the O-methyl phosphoramidate capsule modification in Campylobacter jejuni. J. Biol. Chem. 282, 28566–28576.
McNally, D. J., Jarrell, H. C., Khieu, N. H., Li, J., Vinogradov, E., Whitfield, D. M., Szymanski, C. M., and Brisson, J. -R. (2006) The HS:19 serostrain of Campylobacter jejuni has a hyaluronic acid-type capsular polysaccharide with a nonstoichiometric sorbose branch and O-methyl phosphoramidate group. Febs J. 273, 3975–3989.
McNally, D. J., Jarrell, H. C., Li, J., Khieu, N. H., Vinogradov, E., Szymanski, C. M., and Brisson, J. -R. (2005) The HS:1 serostrain of Campylobacter jejuni has a complex teichoic acid-like capsular polysaccharide with nonstoichiometric fructofuranose branches and O-methyl phosphoramidate groups. Febs J. 272, 4407–4422.
Guerry, P., Ewing, C. P., Schoenhofen, I. C., and Logan, S. M. (2007) Protein glycosylation in Campylobacter jejuni: partial suppression of pglF by mutation of pseC. J. Bacteriol. 189, 6731–6733.
Acknowledgments
We would like to dedicate this chapter to our fantastic mentors and colleagues at the National Research Council – Institute for Biological Sciences who are always pushing forward the boundaries in glycomics. In particular, for these studies, we thank Jean-Robert Brisson, Harold Jarrell and Jianjun Li. This work was funded by the NRC Genomics and Health Initiative.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press
About this protocol
Cite this protocol
Nothaft, H., Liu, X., McNally, D.J., Szymanski, C.M. (2010). N-Linked Protein Glycosylation in a Bacterial System. In: Li, J. (eds) Functional Glycomics. Methods in Molecular Biology, vol 600. Humana Press. https://doi.org/10.1007/978-1-60761-454-8_16
Download citation
DOI: https://doi.org/10.1007/978-1-60761-454-8_16
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-453-1
Online ISBN: 978-1-60761-454-8
eBook Packages: Springer Protocols