Identification of novel O-GlcNAc transferase substrates using yeast cells expressing OGT.

O-GlcNAc modification mediated by O-GlcNAc transferase (OGT) is a reversible protein modification in which O-GlcNAc moieties are attached to target proteins in the cytosol, nucleus, and mitochondria. O-GlcNAc moieties attached to proteins can be removed by O-GlcNAcase (OGA). The addition of an O-GlcNAc moiety can influence several aspects of protein function, and aberrant O-GlcNAc modification is linked to a number of diseases. While OGT and OGA are conserved across eukaryotic cells, yeasts lack these enzymes. Previously, we reported that protein O-GlcNAc modification occurred in the budding yeast Saccharomyces cerevisiae when OGT was ectopically expressed. Because yeast cells lack OGA, O-GlcNAc moieties are stably attached to target proteins. Thus, the yeast system may be useful for finding novel OST substrates. By proteomic analysis, we identified 468 O-GlcNAcylated proteins in yeast cells expressing human OGT. Among these proteins, 13 have human orthologues that show more than 30% identity to their corresponding yeast orthologue, and possible glycosylation residues are conserved in these human orthologues. In addition, the orthologues have not been reported as substrates of OGT. We verified that some of these human orthologues are O-GlcNAcylated in cultured human cells. These proteins include an ubiquitin-conjugating enzyme, UBE2D1, and an eRF3-similar protein, HBS1L. Thus, the yeast system would be useful to find previously unknown O-GlcNAcylated proteins and regulatory mechanisms.

Shape-function insights into bifunctional O-GlcNActransferase of Listeria monocytogenes EGD-e.

O-GlcNAcylation is an important post-translational modification of proteins. O-GlcNAcylated proteins have crucial roles in several cellular contexts both in eukaryotes and bacteria. O-GlcNActransferase (OGT) is the enzyme instrumental in O-GlcNAcylation of proteins. OGT is conserved across eukaryotes. The first bacterial OGT discovered is GmaR in Listeria monocytogenes. GmaR is a GT-2 family bifunctional protein that catalyzes glycosylation of the flagellin protein FlaA and controls transcription of flagellar motility genes in a temperature-dependent manner. Here, we provide methods for heterologous expression and purification of recombinant GmaR and FlaA, in vivo/in vitro glycosylation assays, analysis of the molecular form of recombinant GmaR and detailed enzyme kinetics. We study the structure and functional dynamics of GmaR. Using solution small-angle X-ray scattering and molecular modeling, we show that GmaR adopts an extended shape with two distinctly spaced structural units in the presence of cofactor Mg2+ and with donor UDP-GlcNAc and cofactor combined. Comparisons of restored structures revealed that in-solution binding of Mg2+ ions brings about shape rearrangements and induces structural-rigidity in hyper-variable regions at the N-terminus of GmaR protein. Taking function and shape data together, we describe that Mg2+ binding enables GmaR to adopt a shape that can bind the substrate. The manuscript provides the first 3D solution structure of a bacterial OGT of GT-2 family and detailed biochemical characterization of GmaR to facilitate its future applications.

Heterologous expression, purification and characterization of human ß-1,2-N-acetylglucosaminyltransferase II using a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system.

ß-1,2-N-Acetylglucosaminyltransferase II (GnTII, EC 2.4.1.143) is a Golgi-localized type II transmembrane enzyme that catalyzes the transfer of N-acetylglucosamine to the 6-arm of the trimanosyl core of N-glycans, an essential step in the conversion of oligomannose-type to complex-type N-glycans. Despite its physiological importance, there have been only a few reports on the heterologous expression and structure-function relationship of this enzyme. Here, we constructed a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system and expressed human GnTII (hGnTII) lacking the N-terminal cytosolic tail and transmembrane region. The recombinant hGnTII was purified from silkworm larval hemolymph in two steps by using tandem affinity purification tags, with a yield of approximately 120 µg from 10 mL hemolymph, and exhibited glycosyltransferase activity and strict substrate specificity. The enzyme was found to be N-glycosylated by the enzymatic cleavage of glycans, while hGnTII expressed in insect cells had not been reported to be glycosylated. Although insects typically produce pauci-mannosidic-type glycans, the structure of N-glycans in the recombinant hGnTII was suggested to be of the complex type, and the removal of the glycans did not affect the enzymatic activity.

Discovery of a Low Toxicity O-GlcNAc Transferase (OGT) Inhibitor by Structure-based Virtual Screening of Natural Products.

O-GlcNAc transferase (OGT) plays an important role in regulating numerous cellular processes through reversible post-translational modification of nuclear and cytoplasmic proteins. However, the function of O-GlcNAcylation is still not well understood. Cell permeable OGT inhibitors are needed to manipulate O-GlcNAcylation levels and clarify the regulatory mechanism of this modification. Here, we report a specific natural-product OGT inhibitor (L01), which was identified from a structure-based virtual screening analysis. L01 inhibited O-GlcNAcylation both in vitro and in cells without significantly altering cell surface glycans. Molecular dynamics and site-directed mutagenesis indicated a new binding mechanism in which L01 could interact with Asn557 near the UDP binding pocket of OGT. This residue may contribute to the specificity of L01. Furthermore, as a specific OGT inhibitor, L01 produced low toxicity in cellular and zebrafish models. The identification of L01 validates structure-based virtual screening approaches for the discovery of OGT inhibitors. L01 can also serve as a chemical tool to further characterize O-GlcNAcylation functions or a new molecular core for structure-activity relationship studies to optimize the biochemical potencies.

MreB and MurG as scaffolds for the cytoplasmic steps of peptidoglycan biosynthesis.

Peptidoglycan is a major determinant of cell shape in bacteria, and its biosynthesis involves the concerted action of cytoplasmic, membrane-associated and periplasmic enzymes. Within the cytoplasm, Mur enzymes catalyse the first steps leading to peptidoglycan precursor biosynthesis, and have been suggested as being part of a multicomponent complex that could also involve the transglycosylase MurG and the cytoskeletal protein MreB. In order to initialize the characterization of a potential Mur interaction network, we purified MurD, MurE, MurF, MurG and MreB from Thermotoga maritima and characterized their interactions using membrane blotting and surface plasmon resonance. MurD, MurE and MurF all recognize MurG and MreB, but not each other, while the two latter proteins interact. In addition, we solved the crystal structures of MurD, MurE and MurF, which indicate that their C-termini display high conformational flexibilities. The differences in Mur conformations could be important parameters for the stability of an intracytoplasmic murein biosynthesis complex.

Enzymatic characterization of recombinant enzymes of O-GlcNAc cycling.

The dynamic addition of O-GlcNAc to target proteins is now recognized as a major signaling paradigm impacting phosphorylation, protein turnover, gene expression, and other posttranslational modifications influencing epigenetics. Here we describe the production of and methods for assay of the recombinant enzymes of O-GlcNAc cycling: O-linked GlcNAc Transferase (OGT) and O-GlcNAcase (OGA).

N-acetylglucosaminyltransferase (GnT) assays using fluorescent oligosaccharide acceptor substrates: GnT-III, IV, V, and IX (GnT-Vb).

Determining glycosyltransferase activities gives a clue for better understanding an underlying mechanism for glycomic alterations of carrier molecules. N-glycan branch formation is concertedly regulated by cooperative and competitive activities of N-acetylglucosaminyltransferases (GnTs). Here, we describe methods for large scale preparation of the oligosaccharide acceptor substrate, fluorescence-labeling of oligosaccharides by pyridylamination, quality control, and reversed phase HPLC-based measurement of GnT activities including GnT-III, IV, V, and IX.

A method for determination of UDP-GlcNAc: GlcNAcß1-6(GlcNAcß1-2)Manα1-R [GlcNAc to Man] ß1-4N-acetylglucosaminyltransferase VI activity.

To characterize and purify glycosyltransferases, it is essential to establish a simple and sensitive assay method. Here, we describe a method for determination of the activity of GnT VI (UDP-GlcNAc: GlcNAcß1-6(GlcNAcß1-2)Manα1-R [GlcNAc to Man] ß1-4N-acetylglucosaminyltransferase VI) using a fluorescently labeled oligosaccharide.

Tet proteins connect the O-linked N-acetylglucosamine transferase Ogt to chromatin in embryonic stem cells.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (Ogt) activity is essential for embryonic stem cell (ESC) viability and mouse development. Ogt is present both in the cytoplasm and the nucleus of different cell types and catalyzes serine and threonine glycosylation. We have characterized the biochemical features of nuclear Ogt and identified the ten-eleven translocation (TET) proteins Tet1 and Tet2 as stable partners of Ogt in the nucleus of ESCs. We show at a genome-wide level that Ogt preferentially associates with Tet1 to genes promoters in close proximity of CpG-rich transcription start sites. These regions are characterized by low levels of DNA modification, suggesting a link between Tet1 and Ogt activities in regulating CpG island methylation. Finally, we show that Tet1 is required for binding of Ogt to chromatin affecting Tet1 activity. Taken together, our data characterize how O-GlcNAcylation is recruited to chromatin and interacts with the activity of 5-methylcytosine hydroxylases.

The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2.

In recent years, sugars with a unique chemical handle have been used to detect and elucidate the function of glycoconjugates. Such chemical handles have generally been part of an N-acetyl moiety of a sugar. We have previously developed several applications using the single mutant Y289L-ß1,4-galactosyltransferase I (Y289L-ß4Gal-T1) and the wild-type polypeptide-α-GalNAc-T enzymes with UDP-C2-keto-Gal. Here, we describe for the first time that the GlcNAc-transferring enzymes-R228K-Y289L-ß4Gal-T1 mutant enzyme, the wild-type human ß1,3-N-acetylglucosaminyltransferase-2 and human Maniac Fringe-can also transfer the GlcNAc analog C2-keto-Glc molecule from UDP-C2-keto-Glc to their respective acceptor substrates. Although the R228K-Y289L-ß4Gal-T1 mutant enzyme transfers the donor sugar substrate GlcNAc or its analog C2-keto-Glc only to its natural acceptor substrate, GlcNAc, it does not transfer to its analog C2-keto-Glc. Thus, these observations suggest that the GlcNAc-transferring glycosyltransferases can generally accommodate a chemical handle in the N-acetyl-binding cavity of the donor sugar substrate, but not in the N-acetyl-binding cavity of the acceptor sugar.

Purification and characterization of an active N-acetylglucosaminyltransferase enzyme complex from Streptococci.

A new family of bacterial serine-rich repeat glycoproteins can function as adhesins required for biofilm formation and pathogenesis in streptococci and staphylococci. Biogenesis of these proteins depends on a gene cluster coding for glycosyltransferases and accessory secretion proteins. Previous studies show that Fap1, a member of this family from Streptococcus parasanguinis, can be glycosylated by a protein glycosylation complex in a recombinant heterogeneous host. Here we report a tandem affinity purification (TAP) approach used to isolate and study protein complexes from native streptococci. This method demonstrated that a putative glycosyltransferase (Gtf2), which is essential for Fap1 glycosylation, readily copurified with another glycosyltransferase (Gtf1) from native S. parasanguinis. This result and the similar isolation of a homologous two-protein complex from Streptococcus pneumoniae indicate the biological relevance of the complexes to the glycosylation in streptococci. Furthermore, novel N-acetylglucosaminyltransferase activity was discovered for the complexes. Optimal activity required heterodimer formation and appears to represent a novel type of glycosylation.

Purification and characterization of the lipid A disaccharide synthase (LpxB) from Escherichia coli, a peripheral membrane protein.

Escherichia coli LpxB, an inverting glycosyl transferase of the GT-B superfamily and a member of CAZy database family 19, catalyzes the fifth step of lipid A biosynthesis: UDP-2,3-diacylglucosamine + 2,3-diacylglucosamine 1-phosphate --> 2',3'-diacylglucosamine-(beta,1'-6)-2,3-diacylglucosamine 1-phosphate + UDP. LpxB is a target for the development of new antibiotics, but no member of family 19, which consists entirely of LpxB orthologues, has been characterized mechanistically or structurally. Here, we have purified E. coli and Haemophilus influenzae LpxB to near homogeneity on a 10-100 mg scale using protease-cleavable His(10)-tagged constructs. E. coli LpxB activity is dependent upon the bulk surface concentration of its substrates in a mixed micelle assay system, suggesting that catalysis occurs at the membrane interface. E. coli LpxB (M(r) approximately 43 kDa) sediments with membranes at low salt concentrations but is largely solubilized with buffers of high ionic strength. It purifies with 1.6-3.5 mol of phospholipid/mol of LpxB polypeptide. Transmission electron microscopy reveals the accumulation of aberrant intracellular membranes when LpxB is overexpressed. Mutagenesis of LpxB identified two conserved residues, D89A and R201A, for which no residual catalytic activity was detected. Our results provide a rational starting point for structural studies.

Functional conservation of the lipid II biosynthesis pathway in the cell wall-less bacteria Chlamydia and Wolbachia: why is lipid II needed?

Cell division and cell wall biosynthesis in prokaryotes are driven by partially overlapping multiprotein machineries whose activities are tightly controlled and co-ordinated. So far, a number of protein components have been identified and acknowledged as essential for both fundamental cellular processes. Genes for enzymes of both machineries have been found in the genomes of the cell wall-less genera Chlamydia and Wolbachia, raising questions as to the functionality of the lipid II biosynthesis pathway and reasons for its conservation. We provide evidence on three levels that the lipid II biosynthesis pathway is indeed functional and essential in both genera: (i) fosfomycin, an inhibitor of MurA, catalysing the initial reaction in lipid II biosynthesis, has a detrimental effect on growth of Wolbachia cells; (ii) isolated cytoplasmic membranes from Wolbachia synthesize lipid II ex vivo; and (iii) recombinant MraY and MurG from Chlamydia and Wolbachia exhibit in vitro activity, synthesizing lipid I and lipid II respectively. We discuss the hypothesis that the necessity for maintaining lipid II biosynthesis in cell wall-lacking bacteria reflects an essential role of the precursor in prokaryotic cell division. Our results also indicate that the lipid II pathway may be exploited as an antibacterial target for chlamydial and filarial infections.

The Aspergillus nidulans pigP gene encodes a subunit of GPI-N-acetylglucosaminyltransferase which influences filamentation and protein secretion.

Glycosylphosphatidylinositol (GPI) anchoring is the main mechanism allowing proper localization of secretory proteins in cell membranes. We have isolated an Aspergillus nidulans homolog of the human PIG-P gene, which encodes a subunit of acetylglucosaminyltransferase (GPI-GnT)-an enzyme involved in the synthesis of GPI anchors. A. nidulans pigP mutants have significantly decreased GPI synthesis. On solid media they show strong growth retardation (the "button" phenotype) while in liquid minimal media they show overall good growth but with hyperbranched and bulbous hyphae with impaired septation. Furthermore, the pigP strains, in contrast to the wild-type, abundantly secrete a 33-kDa alkaline serine protease (ALP) into the liquid medium.

Expression and characterization of human N-acetylglucosaminyltransferases and alpha2,3-sialyltransferase in insect cells for in vitro glycosylation of recombinant erythropoietin.

The glycans linked to the insect cell-derived glycoproteins are known to differ from those expressed mammalian cells, partly because of the low level or lack of glycosyltransferase activities. GnT II, GnT IV, GnT V, and ST3Gal IV, which play important roles in the synthesis of tetraantennary-type complex glycan structures in mammalian cells, were overexpressed in Trichoplusia ni cells by using a baculovirus expression vector. The glycosyltransferases, expressed as a fusion form with the IgG-binding domain, were secreted into the culture media and purified using IgG sepharose resin. The enzyme assay, performed using pyridylaminated-sugar chain as an acceptor, indicated that the purified glycosyltransferases retained their enzyme activities. Human erythropoietin expressed in T. ni cells (rhEPO) was subjected to in vitro glycosylation by using recombinant glycosyltransferases and was converted into complex-type glycan with terminal sialic acid. The presence of N-acetylglucosamine, galactose, and sialic acid on the rhEPO moiety was detected by a lectin blot analysis, and the addition of galactose and sialic acid to rhEPO was confirmed by autoradiography using UDP-14C-Gal and CMP-14C-Sia as donors. The in vitro glycosylated rhEPO was injected into mice, and the number of reticulocytes among the red blood cells was counted using FACS. A significant increase in the number of reticulocytes was not observed in the mice injected with in vitro glycosylated rhEPO as compared with those injected with rhEPO.

The biosynthesis of peptidoglycan lipid-linked intermediates.

The biosynthesis of bacterial cell wall peptidoglycan is a complex process involving many different steps taking place in the cytoplasm (synthesis of the nucleotide precursors) and on the inner and outer sides of the cytoplasmic membrane (assembly and polymerization of the disaccharide-peptide monomer unit, respectively). This review summarizes the current knowledge on the membrane steps leading to the formation of the lipid II intermediate, i.e. the substrate of the polymerization reactions. It makes the point on past and recent data that have significantly contributed to the understanding of the biosynthesis of undecaprenyl phosphate, the carrier lipid required for the anchoring of the peptidoglycan hydrophilic units in the membrane, and to the characterization of the MraY and MurG enzymes which catalyze the successive transfers of the N-acetylmuramoyl-peptide and N-acetylglucosamine moieties onto the carrier lipid, respectively. Enzyme inhibitors and antibacterial compounds interfering with these essential metabolic steps and interesting targets are presented.

Role of the amino acid invariants in the active site of MurG as evaluated by site-directed mutagenesis.

To evaluate their role in the active site of the MurG enzyme from Escherichia coli, 13 residues conserved in the sequences of 73 MurG orthologues were submitted to site-directed mutagenesis. All these residues lay within, or close to, the active site of MurG as defined by its tridimensional structure [Ha et al., Prot. Sci. 9 (2000) 1045-1052, and Hu et al., Proc. Natl. Acad. Sci. USA 100 (2003) 845-849]. Thirteen mutants proteins, in which residues T15, H18, Y105, H124, E125, N127, N134, S191, N198, R260, E268, Q288 or N291 have been replaced by alanine, were obtained as the C-terminal His-tagged forms. The effects of the mutations on the activity were checked: (i) by functional complementation of an E. coli murG mutant strain by the mutated genes; and (ii) by the determination of the steady-state kinetic parameters of the purified proteins. Most mutations resulted in an important loss of activity and, in the case of N134A, in the production of a highly unstable protein. The results correlated with the assigned or putative functions of the residues based on the tridimensional structure.

Construction of a cysteine protease deficient Bombyx mori multiple nucleopolyhedrovirus bacmid and its application to improve expression of a fusion protein.

The bacmid system of BmMNPV with cysteine protease gene deletion (CPD-BmMNPV bacmid) was constructed using the lambda recombination system. The protease activities of Bombyx mori cells and silkworm larvae infected with this CPD-BmMNPV bacmid were reduced by 94% and 85%, respectively. By using this system, a GFP(uv)-beta1,3-N-acetylglucosaminyltransferase 2 (GFP(uv)-beta3GnT2) fusion protein was successfully expressed in silkworm larvae with less protein degradation and without larvae liquefaction; beta3GnT activity improved 30%. This CPD-BmMNPV bacmid system provides rapid protein production in silkworms and can be used for the production of recombinant eukaryotic proteins without proteolytic degradation.

Characterization of specific donor binding to alpha1,4-N-acetylhexosaminyltransferase EXTL2 using isothermal titration calorimetry.

Glycosyltransferases encompass one of the largest families of enzymes found in nature. Their principle function is to catalyze the transfer of activated donor-sugar molecules to various acceptor substrates. The molecular basis that governs this specific transfer reaction, such as how a given transferase determines donor-sugar specificity, remains to be elucidated. Human alpha1,4-N-acetylhexosaminyltransferase (EXTL2) transfers N-acetylglucosamine and N-acetylgalactosamine but does not transfer glucose or galactose. Isothermal titration calorimetry (ITC) is a powerful technique used to characterize various binding reactions, including both protein-ligand and protein-protein interactions. ITC provides the binding stoichiometry, affinity, and the thermodynamic parameters free energy (DeltaG), enthalpy (DeltaH), and entropy (DeltaS) of these binding interactions. This chapter describes our ITC study demonstrating the two-step mechanism that regulates the specific binding of N-acetylhexosamines to EXTL2.

X-ray crystal structure determination of mammalian glycosyltransferases.

The vast majority of mammalian glycosyltransferases are endoplasmic reticulum (ER) and Golgi resident type II membrane proteins. As such, producing large quantities of properly folded and active enzymes for X-ray crystallographic analysis is a challenge. Described here are the methods that we have developed to facilitate the structural characterization of these enzymes. The approach involves the production of a soluble Protein A-tagged form of the catalytic domain in a mammalian cell expression system. Production is scaled up in a perfusion-fed bioreactor with media flow rates of 3-5 liters/day. Expression levels are typically in the 1- to 4-mg/liter range and a simple and efficient purification method based on immunoglobulin G (IgG)-Sepharose affinity chromatography has been developed. Our approach to delimiting the catalytic domain and deglycosylating it when necessary is also discussed. Finally, we describe the selenomethionine labeling protocol used in our X-ray crystal structure determination of leukocyte-type Core 2 beta1,6-N-acetylglucosaminyltransferase.