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Site-selective chemoenzymatic construction of synthetic glycoproteins using endoglycosidases
Combined chemical tagging followed by Endo-A catalysed elongation allows access to homogeneous, elaborated glycoproteins. A survey of different linkages and sugars demonstrated not only that unnatural linkages can be tolerated but they can provide insight into the scope of Endo-A transglycosylation activity. S-linked GlcNAc-glycoproteins are useful substrates for Endo-A extensions and display enhanced stability to hydrolysis at exposed sites. O-CH 2-triazole-linked GlcNAc-glycoproteins derived from azidohomoalanine-tagged protein precursors were found to be optimal at sterically demanding sites. © The Royal Society of Chemistry.
Structures of DPAGT1 Explain Glycosylation Disease Mechanisms and Advance TB Antibiotic Design.
Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic "lipid-altered" tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.
Dissecting tunicamycin biosynthesis by genome mining: Cloning and heterologous expression of a minimal gene cluster
Tunicamycin nucleoside antibiotics were the first known to target the formation of peptidoglycan precursor lipid I in bacterial cell wall biosynthesis. They have also been used extensively as inhibitors of protein N-glycosylation in eukaryotes, blocking the biogenesis of early intermediate dolichyl-pyrophosphoryl-N-acetylglucosamine. Despite their unusual structures and useful activities, little is known about their biosynthesis. Here we report identification of the tunicamycin biosynthetic genes in Streptomyces chartreusis following genome sequencing and a chemically-guided strategy for in silico genome mining that allowed rapid identification and unification of an operon fractured across contigs. Heterologous expression established a likely minimal gene set necessary for antibiotic production, from which a detailed metabolic pathway for tunicamycin biosynthesis is proposed. These studies unlock a comprehensive and unusual toolbox of biosynthetic machinery with which to create variants of this important natural product, allowing possible improved understanding of the mode of action and facilitating future redesign. We anticipate that these results will enable the generation of altered specific inhibitors of diverse carbohydrate-processing enzymes, including improved targeting of lipid I biosynthesis. © 2010 The Royal Society of Chemistry.
Diversification in substrate usage by glutathione synthetases from soya bean (Glycine max), wheat (Triticum aestivum) and maize (Zea mays).
Unlike animals which accumulate glutathione (gamma-glutamyl-L-cysteinyl-glycine) alone as their major thiol antioxidant, several crops synthesize alternative forms of glutathione by varying the carboxy residue. The molecular basis of this variation is not well understood, but the substrate specificity of the respective GSs (glutathione synthetases) has been implicated. To investigate their substrate tolerance, five GS-like cDNAs have been cloned from plants that can accumulate alternative forms of glutathione, notably soya bean [hGSH (homoglutathione or gamma-glutamyl-L-cysteinyl-beta-alanine)], wheat (hydroxymethylglutathione or gamma-glutamyl-L-cysteinyl-serine) and maize (gamma-Glu-Cys-Glu). The respective recombinant GSs were then assayed for the incorporation of differing C-termini into gamma-Glu-Cys. The soya bean enzyme primarily incorporated beta-alanine to form hGSH, whereas the GS enzymes from cereals preferentially catalysed the formation of glutathione. However, when assayed with other substrates, several GSs and one wheat enzyme in particular were able to synthesize a diverse range of glutathione variants by incorporating unusual C-terminal moieties including D-serine, non-natural amino acids and alpha-amino alcohols. Our results suggest that plant GSs are capable of producing a diverse range of glutathione homologues depending on the availability of the acyl acceptor.
Surface plasmon resonance imaging of glycoarrays identifies novel and unnatural carbohydrate-based ligands for potential ricin sensor development
Carbohydrate microarrays provide access to high through-put analysis of protein-carbohydrate interactions. Herein we demonstrate the use of SPR imaging (SPRi) of glycoarrays to assess the ligand specificity of the reputedly galactose-specific plant lectin RCA120 (Ricinus communis agglutinin 120), a surrogate for the bioterrorism agent ricin. Glycoarray studies identified RCA120 ligands based on galactose substituted at the 6-position with sialic acid. These observations, which were confirmed by saturation transfer difference (STD) NMR spectroscopy studies, inspired the synthesis of non-natural 6-substituted galactose derivatives, which were shown to have ∼3-4 fold enhanced binding to RCA120 with respect to the unsubstituted compound. These novel unnatural galactosides, which are chemically and biologically more robust than their natural glycan counterparts, represent new potential ligands for the development of carbohydrate-based ricin sensors. © The Royal Society of Chemistry 2011.
Methods for converting cysteine to dehydroalanine on peptides and proteins
Dehydroalanine is a synthetic precursor to a wide array of protein modifications. We describe multiple methods for the chemical conversion of cysteine to dehydroalanine on peptides and proteins. The scope and limitations of these methods were investigated with attention paid to side reactions, scale, and aqueous- and bio-compatibility. The most general method investigated - a bis-alkylation-elimination of cysteine to dehydroalanine - was applied successfully to multiple proteins and enabled the site-selective synthesis of a glycosylated antibody. © The Royal Society of Chemistry 2011.
Isotopic hydration of cellobiose: vibrational spectroscopy and dynamical simulations.
The conformation and structural dynamics of cellobiose, one of the fundamental building blocks in nature, its C4' epimer, lactose, and their microhydrated complexes, isolated in the gas phase, have been explored through a combination of experiment and theory. Their structures at low temperature have been determined through double resonance, IR-UV vibrational spectroscopy conducted under molecular beam conditions, substituting D(2)O for H(2)O to separate isotopically, the carbohydrate (OH) bands from the hydration (OD) bands. Car-Parrinello (CP2K) simulations, employing dispersion corrected density functional potentials and conducted "on-the-fly" from ∼20 to ∼300 K, have been used to explore the consequences of raising the temperature. Comparisons between the experimental data, anharmonic vibrational self-consistent field calculations based upon ab initio potentials, and the CP2K simulations have established the role of anharmonicity; the reliability of classical molecular dynamics predictions of the vibrational spectra of carbohydrates and the accuracy of the dispersion corrected (BLYP-D) force fields employed; the structural consequences of increasing hydration; and the dynamical consequences of increasing temperature. The isolated and hydrated cellobiose and lactose units both present remarkably rigid structures: their glycosidic linkages adopt a "cis" (anti-ϕ and syn-ψ) conformation bound by inter-ring hydrogen bonds. This conformation is maintained when the temperature is increased to ∼300 K and it continues to be maintained when the cellobiose (or lactose) unit is hydrated by one or two explicitly bound water molecules. Despite individual fluctuations in the intra- and intermolecular hydrogen bonding pattern and some local structural motions, the water molecules remain locally bound and the isolated carbohydrates remain trapped within the cis potential well. The Car-Parrinello dynamical simulations do not suggest any accessible pathway to the trans conformations that are formed in aqueous solution and are widespread in nature.
Studying glycobiology at the single-molecule level
Attempts to elucidate the roles of carbohydrate-associated structures in biology have led to the distinct field of glycobiology research. The focus of this field has been in understanding the evolution, biosynthesis and interactions of glycans, both individually and as components of larger biomolecules. However, as most approaches for studying glycans (including mass spectrometry and various binding assays) use ensemble measurements, they lack the precision required to uncover the discrete roles of glycoconjugates, which are often heterogeneous, in biomolecular processes. Single-molecule techniques can examine individual events within challenging mixtures, and they are beginning to be applied to glycobiology. For example, single-molecule force spectroscopy (SMFS) by atomic force microscopy (AFM) has enabled the molecular interactions of sugars to be studied, single-molecule fluorescence microscopy and spectroscopy have led to insight into the role of sugars in biological processes and nanopores have revealed interactions between polysaccharides and their transporters. Thus, single-molecule technology is becoming a valuable tool in glycoscience.
Heavy water hydration of mannose: The anomeric effect in solvation, laid bare
The presence and consequences of the anomeric effect have been explored and directly exposed, through an investigation of the vibrational spectroscopy of the doubly and triply hydrated a and b anomers of phenyl D-mannopyranoside, (PhMan) isolated under molecular beam conditions in the gas phase. The experiments have been aided by the simple trick of substituting D2O for H2O, which has the advantage of isotopically isolating the carbohydrate (OH) bands from the water (OD) bands. Recording the double resonance, IR-UV ion dip spectra of the hydrated complexes, a- and b-PhMan·(D2O)2,3 in a series of 'proof of principle' experiments, revealed that these heavy water molecules engage the key endocyclic oxygen atom, O5, allowing the anomeric effect to be probed through a combination of vibrational spectroscopy and quantum chemical calculations. Importantly, in the dihydrates, both anomers adopt the same conformation and the two water molecules occupy the same template. One of them acts as a remarkably sensitive reporter, able to sense and expose subtle stereoelectronic changes through the resulting changes in its hydrogen-bonded interaction with the substrate. © The Royal Society of Chemistry 2011.
A triply divergent reagent for glycoprotein synthesis
Chemical synthesis of glycoproteins through three different strategies, from the same reagent, is herein described. The flexibility of this system, which allows the comparison of different linkage motifs between the same glycan and protein, is shown by one-step diversification of a GlcNAc-ylating reagent and its application in both site-specific and non-site-specific glycoprotein synthesis to create different conjugates from common representative protein scaffolds.
A triply divergent reagent for glycoprotein synthesis
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Chemical synthesis of glycoproteins through three different strategies, from the same reagent, is herein described. The flexibility of this system, which allows the comparison of different linkage motifs between the same glycan and protein, is shown by one-step diversification of a GlcNAc-ylating reagent and its application in both site-specific and non-site-specific glycoprotein synthesis to create different conjugates from common representative protein scaffolds.
QuaNCAT: Quantitating proteome dynamics in primary cells
Here we demonstrate quantitation of stimuli-induced proteome dynamics in primary cells by combining the power of bio-orthogonal noncanonical amino acid tagging (BONCAT) and stable-isotope labeling of amino acids in cell culture (SILAC). In conjunction with nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS), quantitative noncanonical amino acid tagging (QuaNCAT) allowed us to monitor the early expression changes of >600 proteins in primary resting T cells subjected to activation stimuli. © 2013 Nature America, Inc. All rights reserved.
Designing logical codon reassignment - Expanding the chemistry in biology.
Over the last decade, the ability to genetically encode unnatural amino acids (UAAs) has evolved rapidly. The programmed incorporation of UAAs into recombinant proteins relies on the reassignment or suppression of canonical codons with an amino-acyl tRNA synthetase/tRNA (aaRS/tRNA) pair, selective for the UAA of choice. In order to achieve selective incorporation, the aaRS should be selective for the designed tRNA and UAA over the endogenous amino acids and tRNAs. Enhanced selectivity has been achieved by transferring an aaRS/tRNA pair from another kingdom to the organism of interest, and subsequent aaRS evolution to acquire enhanced selectivity for the desired UAA. Today, over 150 non-canonical amino acids have been incorporated using such methods. This enables the introduction of a large variety of structures into proteins, in organisms ranging from prokaryote, yeast and mammalian cells lines to whole animals, enabling the study of protein function at a level that could not previously be achieved. While most research to date has focused on the suppression of 'non-sense' codons, recent developments are beginning to open up the possibility of quadruplet codon decoding and the more selective reassignment of sense codons, offering a potentially powerful tool for incorporating multiple amino acids. Here, we aim to provide a focused review of methods for UAA incorporation with an emphasis in particular on the different tRNA synthetase/tRNA pairs exploited or developed, focusing upon the different UAA structures that have been incorporated and the logic behind the design and future creation of such systems. Our hope is that this will help rationalize the design of systems for incorporation of unexplored unnatural amino acids, as well as novel applications for those already known.
Sugar-protein hybrids for biomedical applications
This chapter gives an overview of the preparation and applications of biomedically relevant synthetic glycoproteins. The crucial role of natural sugar-protein conjugates in nature makes synthetic variants an interesting instrument to better understand the complex processes in which they are involved. Glycoproteins participate in communication events, like those resulting in inflammation, fertilization, or host immune response. In addition, carbohydrates are able to stabilize the tertiary structure of proteins by inducing more compact structures. Glycosylation is one of the most multifaceted posttranslational modifications of proteins. The chapter examines the different methods for the preparation of relevant synthetic glycoproteins largely where the linkage between the proteic part and the saccharidic moiety is unnatural. It focuses on the application of these synthetic hybrids in the field of biomedicine through their use as vaccine candidates, anti-infective agents, or drug delivery systems, among others.
Vignette: Extending the Application of Metathesis in Chemical Biology - The Development of Site-Selective Peptide and Protein Modifications
Olefin metathesis (OM) is one of the most versatile methods used to create carbon-carbon bonds in molecules. The synthetic strategies that it offers have enabled the construction of biologically active natural products. OM is exploited in various ways in chemical biology. Advances in aqueous metathesis, including the development of water-soluble metathesis catalysts and use of organic co-solvents or surfactants to aid solubility of conventional metathesis catalysts, have laid the groundwork enabling OM on proteins together with earlier work carried out on amino acids and peptides. This chapter describes the laboratory research efforts toward enabling the application of OM to site-selective protein modification. The cross-metathesis (CM) of unsaturated amino acids with allyl alcohol is analyzed. The enhanced reactivity of allyl sulfides (and other allyl chalcogenides) in aqueous metathesis has enabled OM on protein surfaces, and there are increasing numbers of applications in chemical biology and synthetic chemistry.
Dissecting the reaction of Phase II metabolites of ibuprofen and other NSAIDS with human plasma protein
Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most widely used drugs on the market. Whilst they are considered safe, several NSAIDs have been withdrawn from the market as a result of adverse drug reactions. NSAIDs are extensively metabolised to their 1-β-O-acyl glucuronides (AGs), and the risk of NSAID AGs covalently modifying biomacromolecules such as proteins or DNA, leading to immune responses and cellular dysfunction constitutes a major concern in drug discovery and development. The assessment of the degree of protein modification and potential toxicity of individual NSAID AGs is therefore of importance in both drug monitoring and development. Herein, we report the covalent reaction of 1-β-O-acyl glucuronides of ibuprofen and several NSAID analogues with human serum albumin (HSA) protein in vitro under concentrations encountered in therapy. Stable transacylation and glycosylation adducts are formed; the observed protein product ratios can be rationalised by the degree of α-substitution in the acyl group. Structure-based protein reactivity correlations of AGs, such as these, may prove a useful tool in distinguishing between carboxylic acid-containing drugs of similar structure that ultimately prove beneficial (e.g., ibuprofen) from those that prove toxic (e.g., ibufenac). © 2014 the Partner Organisations.