Role of UDP-Glycosyltransferase (ugt) Genes in Detoxification and Glycosylation of 1-Hydroxyphenazine (1-HP) in Caenorhabditis elegans.

Caenorhabditis elegans is a useful model organism to study the xenobiotic detoxification pathways of various natural and synthetic toxins, but the mechanisms of phase II detoxification are understudied. 1-Hydroxyphenazine (1-HP), a toxin produced by the bacterium Pseudomonas aeruginosa, kills C. elegans. We previously showed that C. elegans detoxifies 1-HP by adding one, two, or three glucose molecules in N2 worms. Our current study evaluates the roles that some UDP-glycosyltransferase (ugt) genes play in 1-HP detoxification. We show that ugt-23 and ugt-49 knockout mutants are more sensitive to 1-HP than reference strains N2 or PD1074. Our data also show that ugt-23 knockout mutants produce reduced amounts of the trisaccharide sugars, while the ugt-49 knockout mutants produce reduced amounts of all 1-HP derivatives except for the glucopyranosyl product compared to the reference strains. We characterized the structure of the trisaccharide sugar phenazines made by C. elegans and showed that one of the sugar modifications contains an N-acetylglucosamine (GlcNAc) in place of glucose. This implies broad specificity regarding UGT function and the role of genes other than ogt-1 in adding GlcNAc, at least in small-molecule detoxification.

Activation of Ammonia by a Carbene-Stabilized Dithiolene Zwitterion.

A carbene-stabilized dithiolene zwitterion (3) activates ammonia, affording 4• and 5, through both single-electron transfer (SET) and hydrogen atom transfer (HAT). Reaction products were characterized spectroscopically and by single-crystal X-ray diffraction. The mechanism of the formation of 4• and 5 was probed by experimental and computational methods. This discovery is the first example of metal-free ammonia activation via HAT.

A terminal α3-galactose modification regulates an E3 ubiquitin ligase subunit in Toxoplasma gondii.

Skp1, a subunit of E3 Skp1/Cullin-1/F-box protein ubiquitin ligases, is modified by a prolyl hydroxylase that mediates O2 regulation of the social amoeba Dictyostelium and the parasite Toxoplasma gondii The full effect of hydroxylation requires modification of the hydroxyproline by a pentasaccharide that, in Dictyostelium, influences Skp1 structure to favor assembly of Skp1/F-box protein subcomplexes. In Toxoplasma, the presence of a contrasting penultimate sugar assembled by a different glycosyltransferase enables testing of the conformational control model. To define the final sugar and its linkage, here we identified the glycosyltransferase that completes the glycan and found that it is closely related to glycogenin, an enzyme that may prime glycogen synthesis in yeast and animals. However, the Toxoplasma enzyme catalyzes formation of a Galα1,3Glcα linkage rather than the Glcα1,4Glcα linkage formed by glycogenin. Kinetic and crystallographic experiments showed that the glycosyltransferase Gat1 is specific for Skp1 in Toxoplasma and also in another protist, the crop pathogen Pythium ultimum The fifth sugar is important for glycan function as indicated by the slow-growth phenotype of gat1Δ parasites. Computational analyses indicated that, despite the sequence difference, the Toxoplasma glycan still assumes an ordered conformation that controls Skp1 structure and revealed the importance of nonpolar packing interactions of the fifth sugar. The substitution of glycosyltransferases in Toxoplasma and Pythium by an unrelated bifunctional enzyme that assembles a distinct but structurally compatible glycan in Dictyostelium is a remarkable case of convergent evolution, which emphasizes the importance of the terminal α-galactose and establishes the phylogenetic breadth of Skp1 glycoregulation.

Carbene-Stabilized Dithiolene (L0 ) Zwitterions.

A series of reactions between Lewis bases and an imidazole-based dithione dimer (1) has been investigated. Both cyclic(alkyl)(amino)carbene (CAAC) (2) and N-heterocyclic carbene (NHC) (4), in addition to N-heterocyclic silylene (NHSi) (6), demonstrate the capability to cleave the sulphur-sulphur bonds in 1, giving carbene-stabilized dithiolene (L0 ) zwitterions (3 and 5) and a spirocyclic silicon-dithiolene compound (7), respectively. The bonding nature of 3, 5, and 7 are probed by both experimental and theoretical methods.

HNK-1 sulfotransferase modulates α-dystroglycan glycosylation by 3-O-sulfation of glucuronic acid on matriglycan.

Mutations in multiple genes required for proper O-mannosylation of α-dystroglycan are causal for congenital/limb-girdle muscular dystrophies and abnormal brain development in mammals. Previously, we and others further elucidated the functional O-mannose glycan structure that is terminated by matriglycan, [(-GlcA-ß3-Xyl-α3-)n]. This repeating disaccharide serves as a receptor for proteins in the extracellular matrix. Here, we demonstrate in vitro that HNK-1 sulfotransferase (HNK-1ST/carbohydrate sulfotransferase) sulfates terminal glucuronyl residues of matriglycan at the 3-hydroxyl and prevents further matriglycan polymerization by the LARGE1 glycosyltransferase. While α-dystroglycan isolated from mouse heart and kidney is susceptible to exoglycosidase digestion of matriglycan, the functional, lower molecular weight α-dystroglycan detected in brain, where HNK-1ST expression is elevated, is resistant. Removal of the sulfate cap by a sulfatase facilitated dual-glycosidase digestion. Our data strongly support a tissue specific mechanism in which HNK-1ST regulates polymer length by competing with LARGE for the 3-position on the nonreducing GlcA of matriglycan.

Trypanosoma cruzi 13C-labeled O-Glycan standards for mass spectrometry.

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease, a debilitating condition that affects over 10 million humans in the American continents. In addition to its traditional mode of human entry via the "kissing bug" in endemic areas, the infection can also be spread in non-endemic countries through blood transfusion, organ transplantation, eating food contaminated with the parasites, and from mother to fetus. Previous NMR-based studies established that the parasite expresses a variety of strain-specific and developmentally-regulated O-glycans that may contribute to virulence. In this report, we describe five synthetic O-glycan analytical standards and show their potential to enable a more facile analysis of native O-glycan isomers based on mass spectrometry.

O2 sensing-associated glycosylation exposes the F-box-combining site of the Dictyostelium Skp1 subunit in E3 ubiquitin ligases.

Skp1 is a conserved protein linking cullin-1 to F-box proteins in SCF (Skp1/Cullin-1/F-box protein) E3 ubiquitin ligases, which modify protein substrates with polyubiquitin chains that typically target them for 26S proteasome-mediated degradation. In Dictyostelium (a social amoeba), Toxoplasma gondii (the agent for human toxoplasmosis), and other protists, Skp1 is regulated by a unique pentasaccharide attached to hydroxylated Pro-143 within its C-terminal F-box-binding domain. Prolyl hydroxylation of Skp1 contributes to O2-dependent Dictyostelium development, but full glycosylation at that position is required for optimal O2 sensing. Previous studies have shown that the glycan promotes organization of the F-box-binding region in Skp1 and aids in Skp1's association with F-box proteins. Here, NMR and MS approaches were used to determine the glycan structure, and then a combination of NMR and molecular dynamics simulations were employed to characterize the impact of the glycan on the conformation and motions of the intrinsically flexible F-box-binding domain of Skp1. Molecular dynamics trajectories of glycosylated Skp1 whose calculated monosaccharide relaxation kinetics and rotational correlation times agreed with the NMR data indicated that the glycan interacts with the loop connecting two α-helices of the F-box-combining site. In these trajectories, the helices separated from one another to create a more accessible and dynamic F-box interface. These results offer an unprecedented view of how a glycan modification influences a disordered region of a full-length protein. The increased sampling of an open Skp1 conformation can explain how glycosylation enhances interactions with F-box proteins in cells.

Characterization of a cytoplasmic glucosyltransferase that extends the core trisaccharide of the Toxoplasma Skp1 E3 ubiquitin ligase subunit.

Skp1 is a subunit of the SCF (Skp1/Cullin 1/F-box protein) class of E3 ubiquitin ligases that are important for eukaryotic protein degradation. Unlike its animal counterparts, Skp1 from Toxoplasma gondii is hydroxylated by an O2-dependent prolyl-4-hydroxylase (PhyA), and the resulting hydroxyproline can subsequently be modified by a five-sugar chain. A similar modification is found in the social amoeba Dictyostelium, where it regulates SCF assembly and O2-dependent development. Homologous glycosyltransferases assemble a similar core trisaccharide in both organisms, and a bifunctional α-galactosyltransferase from CAZy family GT77 mediates the addition of the final two sugars in Dictyostelium, generating Galα1, 3Galα1,3Fucα1,2Galß1,3GlcNAcα1-. Here, we found that Toxoplasma utilizes a cytoplasmic glycosyltransferase from an ancient clade of CAZy family GT32 to catalyze transfer of the fourth sugar. Catalytically active Glt1 was required for the addition of the terminal disaccharide in cells, and cytosolic extracts catalyzed transfer of [3H]glucose from UDP-[3H]glucose to the trisaccharide form of Skp1 in a glt1-dependent fashion. Recombinant Glt1 catalyzed the same reaction, confirming that it directly mediates Skp1 glucosylation, and NMR demonstrated formation of a Glcα1,3Fuc linkage. Recombinant Glt1 strongly preferred the full core trisaccharide attached to Skp1 and labeled only Skp1 in glt1Δ extracts, suggesting specificity for Skp1. glt1-knock-out parasites exhibited a growth defect not rescued by catalytically inactive Glt1, indicating that the glycan acts in concert with the first enzyme in the pathway, PhyA, in cells. A genomic bioinformatics survey suggested that Glt1 belongs to the ancestral Skp1 glycosylation pathway in protists and evolved separately from related Golgi-resident GT32 glycosyltransferases.

Spin Diffusion Editing for Structural Fingerprints of Therapeutic Antibodies.

The growing importance of biologics and biosimilars as therapeutic and diagnostic agents is giving rise to new demands for analytical methodology that can quickly and accurately assess the chemical and physical state of protein-based products. A particular challenge exists in physical characterization where the proper fold and extent of disorder of a protein is a major concern. The ability of NMR to reflect structural and dynamic properties of proteins is well recognized, but sensitivity limitations and high levels of interference from excipients in typical biologic formulations have prevented widespread applications to quality assessment. Here we demonstrate applicability of a simple one-dimensional proton NMR method that exploits enhanced spin diffusion among protons in well-structured areas of a protein. We show that it is possible to reduce excipient signals and allow focus on structural characteristics of the protein. Additional decomposition of the resulting spectra based on rotating frame spin relaxation allows separate examination of components from aggregates and disordered regions. Application to a comparison of two different monoclonal antibodies and to detection of partial pH denaturation of a monoclonal antibody illustrates the procedure.

Impact of sulfation pattern on the conformation and dynamics of sulfated fucan oligosaccharides as revealed by NMR and MD.

Sulfated fucans from sea urchin egg jelly express well-defined chemical structures that vary with species. This species specificity regulates the sperm acrosome reaction, a critical step to assure intra-specific fertilization. In addition, these polysaccharides are involved in other biological activities such as anticoagulation. Although sulfation patterns are relevant to the levels of response in both activities, conformation and dynamics of these glycans are also contributing factors. However, data about these features of sulfated fucans are very rare. To address this, we have employed nuclear magnetic resonance experiments combined with molecular dynamics on structurally defined oligosaccharides derived from two sulfated fucans. The results have indicated that the oligosaccharides are flexible in solution. Ring conformation of their composing units displays just the (1)C4 chair configuration. In a particular octasaccharide, composed of two tetrasaccharide sequences, inter-residual hydrogen bonds play a role to decrease dynamics in these repeating units. Conversely, the linking disaccharide [-3)-α-L-Fucp-2(OSO3(-))-(1-3)-α-L-Fucp-4(OCO3(-))-(1-] located right between the two tetrasaccharide units has amplified motions suggested to be promoted by electrostatic repulsion of sulfates on opposite sides of the central glycosidic bond. This conjunction of information about conformation and dynamics of sulfated fucan oligosaccharides provides new insights to explain how these glycans behave free in solution and influenced by sulfation patterns. It may also serve for future studies concerning structure-function relationship of sulfated fucans, especially those involving sea urchin fertilization and anticoagulation.

Kinetics of Neuraminidase Action on Glycoproteins by 1D and 2D NMR.

The surfaces of mammalian cells are coated with complex carbohydrates, many terminated with a negatively charged N-acetylneuraminic acid residue. This motif is specifically targeted by pathogens, including influenza viruses and many pathogenic bacteria, to gain entry into the cell. A necessary step in the influenza virus life cycle is the release of viral particles from the cell surface; this is achieved by cleaving N-acetylneuraminic acid from cell surface glycans with a virally-produced neuraminidase. Here we present a laboratory exercise to model this process using a glycoprotein as a glycan carrier and using real time nuclear magnetic resonance (NMR) spectroscopy to monitor N-acetylneuraminic acid release as catalyzed by neuraminidase. A time-resolved two dimensional data processing technique, statistical total correlation spectroscopy (STOCSY), enhances the resolution of the complicated 1D glycoprotein spectrum and isolates characteristic peaks corresponding to substrates and products. This exercise is relatively straightforward and leads students through a wide range of biologically and chemically relevant procedures, including use of NMR spectroscopy, enzymology and data processing techniques.

Using molecular dynamics trajectories to predict nuclear spin relaxation behaviour in large spin systems.

Molecular dynamics (MD) trajectories provide useful insights into molecular structure and dynamics. However, questions persist about the quantitative accuracy of those insights. Experimental NMR spin relaxation rates can be used as tests, but only if relaxation superoperators can be efficiently computed from MD trajectories - no mean feat for the quantum Liouville space formalism where matrix dimensions quadruple with each added spin 1/2. Here we report a module for the Spinach software framework that computes Bloch-Redfield-Wangsness relaxation superoperators (including non-secular terms and cross-correlations) from MD trajectories. Predicted initial slopes of nuclear Overhauser effects for sucrose trajectories using advanced water models and a force field optimised for glycans are within 25% of experimental values.

A combined computational-experimental approach to define the structural origin of antibody recognition of sialyl-Tn, a tumor-associated carbohydrate antigen.

Anti-carbohydrate monoclonal antibodies (mAbs) hold great promise as cancer therapeutics and diagnostics. However, their specificity can be mixed, and detailed characterization is problematic, because antibody-glycan complexes are challenging to crystallize. Here, we developed a generalizable approach employing high-throughput techniques for characterizing the structure and specificity of such mAbs, and applied it to the mAb TKH2 developed against the tumor-associated carbohydrate antigen sialyl-Tn (STn). The mAb specificity was defined by apparent KD values determined by quantitative glycan microarray screening. Key residues in the antibody combining site were identified by site-directed mutagenesis, and the glycan-antigen contact surface was defined using saturation transfer difference NMR (STD-NMR). These features were then employed as metrics for selecting the optimal 3D-model of the antibody-glycan complex, out of thousands plausible options generated by automated docking and molecular dynamics simulation. STn-specificity was further validated by computationally screening of the selected antibody 3D-model against the human sialyl-Tn-glycome. This computational-experimental approach would allow rational design of potent antibodies targeting carbohydrates.

MYC Controls Human Pluripotent Stem Cell Fate Decisions through Regulation of Metabolic Flux.

As human pluripotent stem cells (hPSCs) exit pluripotency, they are thought to switch from a glycolytic mode of energy generation to one more dependent on oxidative phosphorylation. Here we show that, although metabolic switching occurs during early mesoderm and endoderm differentiation, high glycolytic flux is maintained and, in fact, essential during early ectoderm specification. The elevated glycolysis observed in hPSCs requires elevated MYC/MYCN activity. Metabolic switching during endodermal and mesodermal differentiation coincides with a reduction in MYC/MYCN and can be reversed by ectopically restoring MYC activity. During early ectodermal differentiation, sustained MYCN activity maintains the transcription of "switch" genes that are rate-limiting for metabolic activity and lineage commitment. Our work, therefore, shows that metabolic switching is lineage-specific and not a required step for exit of pluripotency in hPSCs and identifies MYC and MYCN as developmental regulators that couple metabolism to pluripotency and cell fate determination.

Primary structure of the 2-O-methyl-alpha-L-fucose-containing side chain of the pectic polysaccharide, rhamnogalacturonan II.

A 2-O-methylfucosyl-containing heptasaccharide was released from red wine rhamnogalacturonan II (RG-II) by acid hydrolysis of the glycosidic linkage of the aceryl acid residue (AceA) and purified to homogeneity by size-exclusion and high-performance anion-exchange chromatographies. The primary structure of the heptasaccharide was determined by glycosyl-residue and glycosyl-linkage composition analyses, ESIMS, and by 1H and 13C NMR spectroscopy. The NMR data indicated that the pyranose ring of the 2,3-linked L-arabinosyl residue is conformationally flexible. The L-Arap residue was confirmed to be alpha-linked by NMR analysis of a tetraglycosyl-glycerol fragment, [alpha-L-Arap-(1-->4)-beta-D-Galp-(1-->2)-alpha-L-AcefA-(1-->3)-beta-L-Rhap-(1-->3)-Gro], generated by Smith degradation of RG-II. Our data together with the results of a previous study,(1) establish that the 2-O-Me Fuc-containing nonasaccharide side chain of wine RG-II has the structure (Api [triple bond] apiose): [see structure]. Data are presented to show that in Arabidopsis RG-II the predominant 2-O-MeFuc-containing side chain is a mono-O-acetylated heptasaccharide that lacks the non-reducing terminal beta-L-Araf and the alpha-L-Rhap residue attached to the O-3 of Arap, both of which are present on the wine nonasaccharide.

Intracellular pyruvate flux in the methane-producing archaeon Methanococcus maripaludis.

During growth of the methanogenic archaeon Methanococcus maripaludis on alanine as the sole nitrogen source under H(2)/CO(2), alanine was incorporated into amino acids derived from pyruvate including leucine, isoleucine, and valine. Thus, growth with alanine was an efficient means of labeling intracellular pools of pyruvate in this lithotroph. Cells were grown with 18% [U-(13)C]alanine, and the distribution of the isotope in the branched-chain amino acids was determined by (13)C-NMR. Carbons derived from pyruvate contained 14.5% (13)C, indicating that most of the cellular pyruvate was obtained from alanine. In contrast, carbons derived from acetyl-CoA contained only 3-5% (13)C, indicating that only small amounts of acetyl-CoA were formed from pyruvate. Thus, autotrophic acetyl-CoA biosynthesis continued even in the presence of an organic carbon source. Moreover, the labeling of acetyl-CoA was lower than would be predicted if pyruvate was a C-1 donor for acetyl-CoA biosynthesis. Carbon derived from the C-1 of acetyl-CoA contained less (13)C than carbon derived from the C-2 of acetyl-CoA, and this difference was attributed to the acetyl-CoA:CO(2) exchange activity of acetyl-CoA synthase. No enrichment was detected for the C-1 of valine, which was derived from the C-1 of pyruvate. This result was attributed to the pyruvate:CO(2) exchange activity of pyruvate oxidoreductase and may have important implications for isotope tracer studies utilizing pyruvate. Lastly, these results demonstrate that the breakdown of pyruvate by methanococci is very limited even under conditions where it is the sole nitrogen and major carbon source.