University of Groningen Exploring and exploiting bacterial protein glycosylation systems Yakovlieva, Liubov
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(2) . CHAPTER 1 General Introduction . . .
(3) CHAPTER 1. Protein glycosylation is one of the most abundant and diverse post-translational modifications in nature. During this process select protein amino acid side chains are covalently decorated with sugar moieties (glycans).1 This process is catalyzed by a special family of enzymes, so-called protein glycosyltransferases (GTs) (Figure 1A).1 Formation of glycoproteins is non-templated and is instead driven by spatiotemporal factors, such as co-occurrence of protein GTs and their sugar donor and protein acceptor substrates.2 The two major forms of protein glycosylation typically take place either on the side chains of asparagine (N-linked) in N-X-S/T recognition sequons (XPro) (Figure 1B), or of serine/threonine (O-linked) (Figure 1C).3 The structural complexity and diversity of glycoproteins stem from several factors, including i) the site of modification; ii) the composition and branching of the glycan; and iii) microheterogeneity, which is the simultaneous presence of multiple glycoforms of the same protein.. Figure 1. A: General scheme of protein glycosylation. B: N-linked glycoprotein. C: Olinked glycoprotein. Hexagon = carbohydrate or glycan.. The importance of protein glycosylation is in expanding the repertoire of functions encoded in the genome. The presence of carbohydrate moieties confers various properties onto the protein, including stability, solubility, transport, tagging for degradation, and recognition, which influences all the downstream processes that the glycoprotein is involved in.1 This, in turn, 6. .
(4) GENERAL INTRODUCTION diversifies the roles and functions of the single gene-encoded protein and expands the accessible cell proteome. Conventional protein N-glycosylation is generally a well-conserved process across all domains of life (Eukaryotes, Prokaryotes and Archaea).4,5 The typical N-glycoprotein biosynthesis in eukaryotes is depicted in Figure 2. The process starts in the cytoplasm with the synthesis of a lipid-linked oligosaccharide by the action of several glycosyltransferases.5 The lipid-linked glycan is subsequently flipped to the luminal side, where it is eventually transferred en bloc to the acceptor protein by a membrane-associated Noligosaccharryltransferase (OST).4,5 Interestingly, whereas eukaryotic glycans are generally composed of a limited range of monosaccharides, bacterial glycoproteins are more diverse and frequently feature bacteria-specific, rare sugars (e.g. bacillosamine, pseudaminic acid, rhamnose).6 Importantly, surface glycoproteins of bacterial pathogens are implicated in virulence and promoting survival in the host via motility (flagella), adhesion (pili, adhesin proteins), immune system evasion, biofilm formation, amongst others.7. Figure 2. Conventional N-glycosylation pathway (eukaryotic protein glycosylation).. Furthermore, despite commonalities in N-protein glycosylation, novel protein glycosylation systems have been identified in bacteria that challenge the established view of classic protein N-glycosylation.8 A selection of the most . 7.
(5) CHAPTER 1 notable examples is depicted in Figure 3. These unusual protein glycosyltransferases are soluble cytoplasmic enzymes and transfer simple monosaccharides to their acceptor proteins: glucose (HMW1C, Figure 3A)9, Nacetylglucosamine (NleB, Figure 3B),10 and rhamnose (EarP, Figure 3C).11,12. Figure 3. Examples of atypical bacterial protein N-glycosylation systems. A: Asparagine hyperglucosylation of Haemophilus influenzae adhesin by cytoplasmic Nglycosyltransferase HMW1C. B: Arginine GlcNAcylation of host death domains by bacterial effector proteins NleB and SseK. C: Arginine rhamnosylation of elongation factor P (EF-P) by the EarP rhamnosyltransferase.. Unconventional protein glycosylation systems unique to bacteria are emerging as attractive antibiotic targets. Unravelling the mechanism of these unusual glycosyltransferases is a first step towards the rational design of novel inhibitors.. 8. .
(6) GENERAL INTRODUCTION. Thesis outline The aim of the work described in this thesis was to elucidate specific mechanistic aspects of two unusual bacterial N-glycosyltransferases and to develop novel methods to study glycoproteins. In Chapter 2 the process of processivity, which is a fascinating mechanistic feature of enzymes, is reviewed in the context of bacterial glycosyltransferases. A comprehensive overview of various examples is given, as well as a section dedicated to available methods designed to study processivity. Consequently, in Chapter 3 the processivity of bacterial Nglycosyltransferases (NGTs) is investigated. Interestingly, the mechanism of NGT-catalyzed adhesin hyperglycosylation was designated as semiprocessive with an initial processive phase and a subsequent switch to a product inhibitioninduced mechanism with distributive character. Processivity was found to be driven by the proximity of glycosylation sites, the increased affinity for partially modified substrates and the accessible active site of NGT. The NGT-adhesin system was used in combination with unnatural UDP-azidoglucose donors in Chapter 4 to generate azidoglucose-labeled HMW1ct adhesin fragments. By performing in vitro reaction screening, optimal conditions were identified that allowed incorporation of several azidoglucose moieties in the HMW1ct protein. The reactivity of various UDP-azidoglucoses used in the study was further evaluated with docking and MD simulations. Azidoglucose-labeled HMW1ct was isolated for future ELISA-based studies with antibodies from multiple sclerosis patients’ sera. The NGT-HMW1ct system was further explored in Chapter 5 where the oligomeric states of ApNGT, HiNGT and HMW1ct were determined. Based on the results of several techniques to study multimeric proteins (native PAGE, SEC, SEC-MALLS) the oligomeric state of ApNGT was determined to be a dimer, whereas HiNGT was designated as a trimer. The main oligomeric state of HMW1ct was a monomer, although a small degree of multimerization was also observed. In Chapter 6 a proof-of-concept method to oxidize glucopeptides is described. An organometallic Pd catalyst was used to perform chemoselective oxidation of the glucose moiety of a select panel of glycopeptides. The resulting keto-group was shown to be successfully functionalized to the oxime. The described methodology of oxidation-oxime formation was subsequently extended to tryptic glucopeptides of HMW1ct. In Chapter 7 the substrate recognition of the EarP rhamnosyltransferase from Pseudomonas aeruginosa was investigated. By screening cyclic peptide . 9.
(7) CHAPTER 1 mimics of various length and amino acid sequence a cyclic 11mer peptide was identified as the optimal substrate. Detailed NMR studies revealed that the minimal recognition epitope for arginine rhamnosylation features a β-hairpin secondary structure, which is a rare recognition element in N-protein glycosylation. Finally, Chapter 8 contains a summary of the work performed in this Thesis, as well as an outlook on some future directions for the research described here.. 10. .
(8) GENERAL INTRODUCTION.
(9) 1. 2.. Varki, A. Essentials of Glycobiology. 2017. Moreman, K. W.; Tiemeyer, M.; Nairn, A. V. Vertebrate protein glycosylation: diversity, synthesis and function. Nature 2012, 13, 448-462. 3. Spiro, R. Protein glycosylation: nature, distribution, enzymatic formation, and disease implication of glycopeptides bonds. Glycobiology 2002, 12, 43R-56R. 4. Schwarz, F.; Aebi, M. Mechanisms and principles of N-linked protein glycosylation. Curr. Opin. Struct. Biol. 2011, 21, 576–582. 5. Dell, A.; Galadari, A.; Sastre, F; Hitchen, P. Similarities and Differences in the Glycosylation Mechanisms in Prokaryotes and Eukaryotes. Int. J. Microbiol. 2010, 148178. 6. Tytgat, H. L. P.; Lebeer, S. The Sweet Tooth of Bacteria: Common Themes in Bacterial Glycoconjugates. Microbiol. Mol. Biol. Rev. 2014, 78, 372–417. 7. Schmidt, M. A.; Riley, L. W.; Benz, I. Sweet new world: glycoproteins in bacterial pathogens. Trends Microbiol. 2003, 11, 554-561. 8. Nothaft, H.; Szymanski, C. M. New discoveries in bacterial N-glycosylation to expand the synthetic biology toolbox. Curr. Opin. Chem. Biol. 2019, 53, 16–24. 9. Grass, S.; Lichti, C. F.; Townsend, R. R.; Gross, J.; St. Geme, III, J. W.; The Haemophilus influenzae HMW1C protein is a glycosyltransferase that transfers hexose residues to asparagine sites in the HMW1 adhesin. PLoS Pathog. 2010, 6, e1000919. 10. Li, S.; Zhang, L.; Yao, Q.; Li, L.; Dong, N.; Rong, J.; Gao, W.; Ding, X.; Sun, L.; Chen, X.; Chen, S.; Shao, F. Pathogen blocks host death receptor signalling by arginine GlcNAcylation of death domains. Nature 2013, 501, 242–246. 11. Lassak, J.; Keilhauer, E. C.; Fürst, M.; Wuichet, K.; Gödeke, J.; Starosta, A. L.; Chen, J. - M.; Søgaard-Andersen, L.; Rohr, J.; Wilson, D. N.; Häussler, S.; Mann, M.; Jung, K. Argininerhamnosylation as new strategy to activate translation elongation factor P. Nat. Chem. Biol. 2015, 11, 266-270. 12. Rajkovic, A.; Erickson, S.; Witzky, A.; Branson, O. E.; Seo, J.; Gafken, P. R.; Frietas, M. A.; Whitelegge, J. P.; Faull, K. F.; Navarre, W.; Darwin, A. J.; Ibba, M. Cyclic rhamnosylated elongation factor P establishes antibiotic resistance in Pseudomonas aeruginosa. mBio 2015, 6, e00823-15.. . 11.
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