Asparagine-linked glycosylation involves the sequential assembly of an oligosaccharide onto a polyisoprenyl donor, followed by the en bloc transfer of the glycan to particular asparagine residues within acceptor proteins. These N-linked glycans play a critical role in a wide variety of biological processes, such as protein folding, cellular targeting and motility, and the immune response. In the last decade, research in the field of N-linked glycosylation has achieved major advances, including the discovery of new carbohydrate modifications, the biochemical characterization of the enzymes involved in glycan assembly, and the biological impact of these glycans on target proteins. It is now firmly established that this enzyme-catalyzed modification occurs in all three domains of life. However, despite similarities in the overall logic of N-linked glycoprotein biosynthesis amongst the three kingdoms, the structures of the appended glycans are markedly different and thus influence the functions of elaborated proteins in various ways. Though nearly all eukaryotes produce the same nascent tetradecasaccharide (Glc3Man9GlcNAc2), heterogeneity is introduced into this glycan structure after transfer to protein through a complex series of glycosyl trimming and addition steps. In contrast, bacteria and archaea display diversity within their N-linked glycan structures through the use of unique monosaccharide building blocks during the assembly process. In this review, recent progress toward gaining a deeper biochemical understanding of this modification across all three kingdoms will be summarized. In addition, a brief overview of the role of N-linked glycosylation in viruses will also be presented.
Asparagine-linked glycosylation is a complex protein modification conserved among all three domains of life. Herein we report the in vitro analysis of N-linked glycosylation from the methanogenic archaeon Methanococcus voltae. Using a suite of synthetic and semisynthetic substrates, we show that AglK initiates N-linked glycosylation in M. voltae through the formation of α-linked dolichyl monophosphate N-acetylglucosamine (Dol-P-GlcNAc), which contrasts with the polyprenyl-diphosphate intermediates that feature in both eukaryotes and bacteria. Intriguingly, AglK exhibits high sequence homology to dolichyl-phosphate β-glucosyltransferases, including Alg5 in eukaryotes, suggesting a common evolutionary origin. The combined action of the first two enzymes, AglK and AglC, afforded an α-linked Dol-P-glycan that serves as a competent substrate for the archaeal oligosaccharyl transferase AglB. These studies provide the first biochemical evidence revealing that despite the apparent similarity of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life.
The B-band O-antigen of the lipopolysaccharide found in the opportunistic pathogen Pseudomonas aeruginosa PAO1 (serotype O5) comprises a repeating trisaccharide unit that is critical for virulence and protection from host defense systems. One of the carbohydrates in this repeating unit, the rare diacetylated aminuronic acid derivative 2,3-diacetamido-2,3-dideoxy-β-D-mannuronic acid (ManNAc(3NAc)A), is thought to be produced by five enzymes (WbpA, WbpB, WbpE, WbpD and WbpI) in a stepwise manner starting from UDP-GlcNAc. Although the genes responsible for the biosynthesis of this sugar are known, only two of the five encoded proteins (WbpA and WbpI) have been thoroughly investigated. In this report, we describe the cloning, overexpression, purification and biochemical characterization of the three central enzymes in this pathway, WbpB, WbpE, and WbpD. Using a combination of capillary electrophoresis, RP-HPLC and NMR spectroscopy, we show that WbpB and WbpE are a dehydrogenase/aminotransferase pair that converts UDP-GlcNAcA to UDP-GlcNAc(3NH 2 )A in a coupled reaction via a unique NAD + recycling pathway. In addition, we confirm that WbpD catalyzes the acetylation of UDP-GlcNAc(3NH 2 )A to give UDP-GlcNAc (3NAc)A. Notably, WbpA, WbpB, WbpE, WbpD and WbpI can be combined in vitro to generate UDP-ManNAc(3NAc)A in a single reaction vessel, thereby providing supplies of this complex glycosyl donor for future studies of LPS assembly. This work completes the biochemical characterization of the enzymes in this pathway and provides novel targets for potential therapeutics to combat infections with drug resistant P. aeruginosa strains.The gram-negative pathogen Pseudomonas aeruginosa is a versatile organism responsible for infection in immunocompromised individuals (1). It is a major source of hospital-acquired pneumonia and bacteremia, causes severe inflammation and pulmonary failure in cystic fibrosis patients, and has emerged as a serious public health threat (2-5). Effective treatment of P. aeruginosa infection has proved challenging due to the strong inherent resistance of the organism to traditional antibiotics and the increasing emergence of multi-drug resistant strains (6-8). While several vaccines for P. aeruginosa have been described, none have thus far achieved clinical success (9). † This work was supported by a grant from National Institutes of Health (GM039334 to B.I.). *To whom correspondence should be addressed: Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139. Phone: (617) 253-1838; Fax: (617) 452-2419; Email: E-mail: imper@mit.edu. Table of constructs and oligonucleotides used in this study, as well as full 1 H, 13 C and COSY NMR spectra of UDP-GlcNAc(3NH 2 )A and UDP-GlcNAc(3NAc)A. This material is available free of charge via the Internet at http://pubs.acs.org. SUPPORTING INFORMATION AVAILABLE NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 June 16. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manusc...
Polyprenyl phosphates, including undecaprenyl phosphate and dolichyl phosphate, are essential intermediates in several important biochemical pathways including N-linked protein glycosylation in eukaryotes and prokaryotes and prokaryotic cell wall biosynthesis. Herein, we describe the evaluation of three potential undecaprenol kinases as agents for the chemoenzymatic synthesis of polyprenyl phosphates. Target enzymes were expressed in crude cell envelope fractions and quantified via the use of luminescent lanthanide-binding tags (LBTs). The Streptococcus mutans diacylglycerol kinase (DGK) was shown to be a very useful agent for polyprenol phosphorylation using ATP as the phosphoryl transfer agent. In addition, the S. mutans DGK can be coupled with two Campylobacter jejuni glycosyltransferases involved in N-linked glycosylation to efficiently biosynthesize the undecaprenyl pyrophosphate-linked disaccharide needed for studies of PglB, the C. jejuni oligosaccharyl transferase.
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