Investigation of asparagine-linked glycosylation in archaeal and bacterial systems

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, February 2011.Vita. Cataloged from PDF version of thesis.Includes bibliographical references.Asparagine-linked protein glycosylation entails the stepwise assembly of an oligosaccharide onto a polyisoprenyl diphosphate carrier, followed by the en bloc transfer of the glycan onto acceptor proteins by oligosaccharyl transferase (OTase). It is now clear that this N-linked protein modification catalyzed by OTases is conserved in all three kingdoms of life. In contrast to eukaryotic OTases, which are multimeric complexes made up of several membrane-spanning proteins, bacterial and archaeal OTases instead appear to be composed of just a single membrane-bound subunit, offering a more tractable system for detailed biochemical characterization. Although significant progress has been made with the bacterial OTase PglB from Campylobacterjejuni, problems with low protein expression yields and poor stability have complicated in depth study. In order to identify a more suitable OTase candidate, a selection of archaeal OTases was screened for heterologous expression levels in Escherichia coli, and it was determined that the homolog from the methanogen Methanococcus voltae possessed the best protein expression profile, indicating a 100-fold improvement over PglB. In an effort to generate a substrate to probe the function of the M voltae OTase, we required a robust synthesis of the highly modified UDP-GlcNAc(3NAc)A and thus turned to the Wbp pathway from the opportunistic pathogen Pseudomonas aeruginosa. Early genetic studies suggested that P. aeruginosa produces UDP-GlcNAc(3NAc)A as a precursor to ManNAc(3NAc)A, a carbohydrate found in the O-antigen of the lipopolysaccharide of the organism. Using a combination of protein biochemistry and NMR spectroscopy, three enzymes (WbpB, WbpE, and WbpD) were confirmed to be responsible for the biosynthesis of UDPGlcNAc( 3NAc)A. It is shown that WbpB and WbpE are a dehydrogenase/aminotransferase pair that converts UDP-GlcNAcA to UDP-GIcNAc(3NH 2)A in a coupled reaction via a unique NAD* recycling pathway. In addition, the X-ray crystal structure of WbpE was solved in complex with its PLP cofactor and UDP-GlcNAc(3NH 2)A product as the external aldimine. With UDP-GlcNAc(3NAc)A in hand, preliminary steps towards completion of the desired dolichyl-linked substrate for the study of the M. voltae OTase are described. Finally, biochemical studies were undertaken in an attempt to inhibit the C. jejuni OTase, PglB. To this end, a panel of isosteric peptides was synthesized to identify possible PglB inhibitors. In addition, treatment of PglB with residue-specific alkylating agents coupled with site-directed mutagenesis revealed a key histidine residue that may play an important role in enzyme catalysis. Taken together, these studies offer insight into long-standing questions about the mechanism of oligosaccharide transfer.by Angelyn Larkin.Ph.D

Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesis

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, which contrasts with the polyprenyl diphosphate intermediates that feature in both eukaryotes and bacteria. Notably, AglK has 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 dolichyl monophosphate glycan that serves as a competent substrate for the archaeal oligosaccharyl transferase AglB. These studies provide what is to our knowledge 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.National Institutes of Health (U.S.) (Grant GM039334

The Expanding Horizons of Asparagine-Linked Glycosylation

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 past 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 determination of 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 among 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 it is transferred to the 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.National Institutes of Health (U.S.) (GM039334

Biosynthesis of UDP-GlcNAc(3NAc)A by WbpB, WbpE, and WbpD: Enzymes in the Wbp Pathway Responsible for O-antigen Assembly in Pseudomonas aeruginosa PAO1

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[subscript 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[subscript 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 lipopolysaccharide 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.National Institutes of Health (U.S.) (Grant GM039334

Structural Analysis of WbpE from Pseudomonas aeruginosa PAO1: A Nucleotide Sugar Aminotransferase Involved in O-antigen Assembly

In recent years, the opportunistic pathogen Pseudomonas aeruginosa has emerged as a major source of hospital-acquired infections. Effective treatment has proven increasingly difficult due to the spread of multidrug resistant strains and thus requires a deeper understanding of the biochemical mechanisms of pathogenicity. The central carbohydrate of the P. aeruginosa PAO1 (O5) B-band O-antigen, ManNAc(3NAc)A, has been shown to be critical for virulence and is produced in a stepwise manner by five enzymes in the Wbp pathway (WbpA, WbpB, WbpE, WbpD, and WbpI). Herein, we present the crystal structure of the aminotransferase WbpE from P. aeruginosa PAO1 in complex with the cofactor pyridoxal 5′-phosphate (PLP) and product UDP-GlcNAc(3NH2)A as the external aldimine at 1.9 Å resolution. We also report the structures of WbpE in complex with PMP alone as well as the PLP internal aldimine and show that the dimeric structure of WbpE observed in the crystal structure is confirmed by analytical ultracentrifugation. Analysis of these structures reveals that the active site of the enzyme is composed of residues from both subunits. In particular, we show that a key residue (Arg229), which has previously been implicated in direct interactions with the α-carboxylate moiety of α-ketoglutarate, is also uniquely positioned to bestow specificity for the 6′′-carboxyl group of GlcNAc(3NH2)A through a salt bridge. This finding is intriguing because while an analogous basic residue is present in WbpE homologues that do not process 6′′-carboxyl-modified saccharides, recent structural studies reveal that this side chain is retracted to accommodate a neutral C6′′ atom. This work represents the first structural analysis of a nucleotide sugar aminotransferase with a bound product modified at the C2′′, C3′′, and C6′′ positions and provides insight into a novel target for treatment of P. aeruginosa infection.National Institutes of Health (U.S.) (Grant GM039334