N-linked protein glycosylation in a bacterial system.
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.
Nothaft
Harald
H
Department of Biological Sciences, Alberta Ingenuity Centre for Carbohydrate Science, University of Alberta, Edmonton, Alberta, Canada.
Liu
Xin
X
McNally
David J
DJ
Szymanski
Christine M
CM
D001671:Biochemistry / Q000379:methods; D002241:Carbohydrates / Q000737:chemistry; D002809:Chondroitin Sulfates / Q000378:metabolism*; D003001:Cloning, Molecular; D019075:Electrophoresis, Capillary / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D054794:Glycomics / Q000379:methods*; D006031:Glycosylation; D008055:Lipids / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D008967:Molecular Biology / Q000379:methods; D009844:Oligosaccharides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry
D002241:Carbohydrates; D008055:Lipids; D009844:Oligosaccharides; D011134:Polysaccharides; D002809:Chondroitin Sulfates