Biochemical characterisation of an α1,4 galactosyltransferase from Neisseria weaveri for the synthesis of α1,4-linked galactosides.

The human cell surface trisaccharide motifs globotriose and P1 antigen play key roles in infections by pathogenic bacteria, which makes them important synthetic targets as antibacterial agents. Enzymatic strategies to install the terminal α1,4-galactosidic linkage are very attractive but have only been demonstrated for a limited set of analogues. Herein, a new bacterial α1,4 galactosyltransferase from N. weaveri was cloned and produced recombinantly in E. coli BL21 (DE3) cells, followed by investigation of its substrate specificity. We demonstrate that the enzyme can tolerate galactosamine (GalN) and also 6-deoxygalactose and 6-deoxy-6-fluorogalactose as donors, and lactose and N-acetyllactosamine as acceptors, leading directly to analogues of Gb3 and P1 that are valuable chemical probes and showcase how biocatalysis can provide fast access to a number of unnatural carbohydrate analogues.

Expression and purification of the full-length N-acetylgalactosaminyltransferase and galactosyltransferase from Campylobacter jejuni in Escherichia coli.

The successful enzymatic synthesis of various ganglioside-related oligosaccharides requires many available glycan-processing enzymes. However, the number of available glycan-processing enzymes remains limited. In this study, the full-length CgtA43456 (ß-(1→4)-N-acetylgalactosaminyltransferase) and CgtB11168 (ß-(1→3)-galactosyltransferase) were successfully produced from Escherichia coli through the optimization of E. coli-preferable codon usage, selection of E. coli strain, and use of the molecular chaperone GroEL-GroES (GroEL/ES). The CgtA43456 enzyme was produced as a soluble form in E. coli C41(DE3) co-expressed with codon-optimized CgtA43456 and GroEL/ES. However, soluble CgtB11168 was well expressed in E. coli C41(DE3) with only the codon-optimized CgtB11168. Rather, when co-expressed with GroEL/ES, total production of CgtB11168 was reduced. Using immobilized-metal affinity chromatography, the CgtA43456 and CgtB11168 proteins were obtained with approximately 75-78 % purity. The purified CgtA43456 showed a specific activity of 21 mU/mg using UDP-N-acetylgalactosamine and GM3 trisaccharide as donor and acceptor, respectively. The purified CgtB11168 catalyzed the transfer of galactose from UDP-Gal to GM2 tetrasaccharide with a specific activity of 16 mU/mg. We propose that they could be used as catalysts for enzymatic synthesis of GM1 ganglioside-related oligosaccharides.

Expression and Purification of DGD2, a Chloroplast Outer Membrane-Associated Glycosyltransferase for Galactolipid Synthesis.

Galactolipids are characteristic lipids of the photosynthetic membranes. They are highly enriched in the chloroplast and are present in photosystem structures. There are two major types of galactolipids, i.e., monogalactosyldiacylglycerol and digalactosyldiacylglycerol (DGDG) in chloroplastic membranes, which amount to ∼50 and ∼20 mol % of the total chloroplast lipids, respectively. Under phosphate-limiting conditions, the amount of DGDG increases dramatically for rescuing phosphate from phospholipids. In Arabidopsis thaliana, the gene digalactosyldiacylglycerol synthase 2 (DGD2) encodes a membrane-associated glycosyltransferase. The gene expression is highly responsive to phosphate starvation and is significantly upregulated in this case. To understand the molecular mechanism of DGD2, we established a protocol for DGD2 expression and purification in an Escherichia coli-based system. The work involved optimization of the expression condition and the purification protocol and a careful selection of buffer additives. It was found that a removal of around 70 C-terminal residues was necessary to produce a homogeneous monomeric protein sample with high purity, which was highly active. The purified sample was characterized by an activity assay for enzyme kinetics in which a range of membrane mimetics with different lipid compositions were used. The results demonstrate that DGD2 activity is stimulated by the presence of negatively charged lipids, which highlight the importance of the membrane environment in modulating the enzyme's activity. The study also paves way for future biophysical and structural studies of the enzyme.

The dimeric structure of wild-type human glycosyltransferase B4GalT1.

Most glycosyltransferases, including B4GalT1 (EC 2.4.1.38), are known to assemble into enzyme homomers and functionally relevant heteromers in vivo. However, it remains unclear why and how these enzymes interact at the molecular/atomic level. Here, we solved the crystal structure of the wild-type human B4GalT1 homodimer. We also show that B4GalT1 exists in a dynamic equilibrium between monomer and dimer, since a purified monomer reappears as a mixture of both and as we obtained crystal forms of the monomer and dimer assemblies in the same crystallization conditions. These two crystal forms revealed the unliganded B4GalT1 in both the open and the closed conformation of the Trp loop and the lid regions, responsible for donor and acceptor substrate binding, respectively. The present structures also show the lid region in full in an open conformation, as well as a new conformation for the GlcNAc acceptor loop (residues 272-288). The physiological relevance of the homodimer in the crystal was validated by targeted mutagenesis studies coupled with FRET assays. These showed that changing key catalytic amino acids impaired homomer formation in vivo. The wild-type human B4GalT1 structure also explains why the variant proteins used for crystallization in earlier studies failed to reveal the homodimers described in this study.

Isolation, purification, and characterization of AgUCGalT1, a galactosyltransferase involved in anthocyanin galactosylation in purple celery (Apium graveolens L.).

MAIN CONCLUSION: This study showed that a galactosyltransferase, AgUCGalT1, is involved in anthocyanin galactosylation in purple celery. Celery is a well-known vegetable because of its rich nutrients, low calories, and medicinal values. Its petioles and leaf blades are the main organs acting as nutrient sources. UDP-galactose: cyanidin 3-O-galactosyltransferase can transfer the galactosyl moiety from UDP-galactose to the 3-O-position of cyanidin through glycosylation. This process enhances the stability and water solubility of anthocyanins. In the present study, LC-MS data indicated that abundant cyanidin-based anthocyanins accumulated in the petioles of purple celery ('Nanxuan liuhe purple celery'). A gene encoding UDP-galactose: cyanidin 3-O-galactosyltransferase, namely AgUCGalT1, was isolated from purple celery and expressed in Escherichia coli BL21 (DE3). Sequence alignments revealed that the AgUCGalT1 protein contained a highly conserved putative secondary plant glycosyltransferase (PSPG) motif. The glycosylation product catalyzed by AgUCGalT1 was detected using UPLC equipment. The recombinant AgUCGalT1 had an optimal enzyme activity at 35 °C and pH 8.0, and showed highest enzyme activity toward cyanidin among the enzyme activities involving other substances, namely, peonidin, quercetin, and kaempferol. The expression levels of AgUCGalT1 were positively correlated with the total anthocyanin contents in purple and non-purple celery varieties. Crude enzymes extracted from purple celery exhibited glycosylation ability, whereas crude enzymes obtained from non-purple celery did not have this ability. This work provided evidence as a basis for investigations on the function of AgUCGalT1 in anthocyanin glycosylation in purple celery.

Methods for the Detection, Study, and Dynamic Profiling of O-GlcNAc Glycosylation.

The addition of O-linked ß-N-acetylglucosamine (O-GlcNAc) to serine/threonine residues of proteins is a ubiquitous posttranslational modification found in all multicellular organisms. Like phosphorylation, O-GlcNAc glycosylation (O-GlcNAcylation) is inducible and regulates a myriad of physiological and pathological processes. However, understanding the diverse functions of O-GlcNAcylation is often challenging due to the difficulty of detecting and quantifying the modification. Thus, robust methods to study O-GlcNAcylation are essential to elucidate its key roles in the regulation of individual proteins, complex cellular processes, and disease. In this chapter, we describe a set of chemoenzymatic labeling methods to (1) detect O-GlcNAcylation on proteins of interest, (2) monitor changes in both the total levels of O-GlcNAcylation and its stoichiometry on proteins of interest, and (3) enable mapping of O-GlcNAc to specific serine/threonine residues within proteins to facilitate functional studies. First, we outline a procedure for the expression and purification of a multiuse mutant galactosyltransferase enzyme (Y289L GalT). We then describe the use of Y289L GalT to modify O-GlcNAc residues with a functional handle, N-azidoacetylgalactosamine (GalNAz). Finally, we discuss several applications of the copper-catalyzed azide-alkyne cycloaddition "click" reaction to attach various alkyne-containing chemical probes to GalNAz and demonstrate how this functionalization of O-GlcNAc-modified proteins can be used to realize (1)-(3) above. Overall, these methods, which utilize commercially available reagents and standard protein analytical tools, will serve to advance our understanding of the diverse and important functions of O-GlcNAcylation.

Revisiting the expression and purification of MGD1, the major galactolipid synthase in Arabidopsis to establish a novel standard for biochemical and structural studies.

Monogalactosyldiacylglycerol, the major lipid of plants and algal plastids, is synthesized by MGDG synthases (MGD). MGDs belong to the large glycosyltransferase family. They catalyze the transfer of a galactose residue from the donor UDP-Gal to a 1,2-sn-diacylglycerol acceptor. MGDs are monotopic proteins localized in the plastid envelope and, as such, they are difficult to purify. This study re-examined previous purification procedures and aimed to set up a standard protocol for expression and purification of recombinant MGD1, addressing problems frequently encountered with the purification of glycosyltransferases, particularly protein aggregation, and enabling crystallization for structural studies. Briefly, His-tagged versions of MGD1 were expressed in Escherichia coli and purified by a two-step procedure, including immobilized metal affinity chromatography and size-exclusion chromatography. We demonstrated that E. coli is an appropriate host cell to produce a soluble and active form of MGD1. We also investigated the effects of various buffers and additives used during the purification and concentration steps on the biochemical behavior of the enzyme. The protocol we developed typically yields milligram quantities of pure and homogenous protein material and proved suitable for crystallization and biochemical studies. We also revisited the conditions for activity tests and effects of known positive effectors of MGD1 such as phosphatidic acid and phosphatidylglycerol.

New high-performance liquid chromatography assay for glycosyltransferases based on derivatization with anthranilic acid and fluorescence detection.

Assays were developed using the unique labeling chemistry of 2-aminobenzoic acid (2AA; anthranilic acid, AA) for measuring activities of both ß1-4 galactosyltransferase (GalT-1) and α2-6 sialyltransferase (ST-6) by high-performance liquid chromatography (HPLC) with fluorescence detection (Anumula KR. 2006. Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates. Anal Biochem. 350:1-23). N-Acetylglucosamine (GlcNAc) and N-acetyllactosamine were used as acceptors and uridine diphosphate (UDP)-galactose and cytidine monophosphate (CMP)-N-acetylneuraminic acid (NANA) as donors for GalT-1 and ST-6, respectively. Enzymatic products were labeled in situ with AA and were separated from the substrates on TSKgel Amide 80 column using normal-phase conditions. Enzyme units were determined from the peak areas by comparison with the concomitantly derivatized standards Gal-ß1-4GlcNAc and NANA-α2-6 Gal-ß1-4GlcNAc. Linearity (time and enzyme concentration), precision (intra- and interassay) and reproducibility for the assays were established. The assays were found to be useful in monitoring the enzyme activities during isolation and purification. The assays were highly sensitive and performed equal to or better than the traditional radioactive sugar-based measurements. The assay format can also be used for measuring the activity of other transferases, provided that the carbohydrate acceptors contain a reducing end for labeling. An assay for glycoprotein acceptors was developed using IgG. A short HPLC profiling method was developed for the separation of IgG glycans (biantennary G0, G1, G2, mono- and disialylated), which facilitated the determination of GalT-1 and ST-6 activities in a rapid manner. Furthermore, this profiling method should prove useful for monitoring the changes in IgG glycans in clinical settings.

Characterization and synthetic application of a novel beta1,3-galactosyltransferase from Escherichia coli O55:H7.

A beta1,3-galactosyltransferase (WbgO) was identified in Escherichia coli O55:H7. Its function was confirmed by radioactive activity assay and structure analysis of the disaccharide synthesized with the recombinant enzyme. WbgO requires a divalent metal ion, either Mn(2+) or Mg(2+), for its activity and is active between pH 6.0-8.0 with a pH optimum of 7.0. N-acetylglucosamine (GlcNAc) and oligosaccharides with GlcNAc at the non-reducing end were shown to be its preferred substrates and it can be used for the synthesis of type 1 glycan chains from these substrates. Together with a recombinant bacterial GlcNAc-transferase, benzyl beta-lacto-N-tetraoside was synthesized with the purified WbgO to demonstrate the synthetic utility of WbgO.

Thermodynamic insights into the structural basis governing the donor substrate recognition by human beta1,4-galactosyltransferase 7.

Human beta1,4-GalT (galactosyltransferase)7 is involved in the biosynthesis of the tetrasaccharide linker protein region (GlcAbeta1-->3Galbeta1-->3Galbeta1-->4Xylbeta1) (where GlcA is glucuronic acid and Xyl is xylose) of proteoglycans, by catalysing the transfer of Gal (galactose) from the uridine 5'-diphosphogalactose to a Xyl residue. This reaction is rate-limiting in glycosaminoglycan biosynthesis. In the present study, we established a large-scale production system of beta1,4-GalT7 fused with the maltose-binding protein to study substrate recognition. Calorimetric binding studies showed that the binding of the donor substrate UDP-Gal largely promoted binding of the acceptor substrate. To identify the structural basis governing substrate recognition, we used a fragment-based approach involving the artificial breakdown of the donor substrate into smaller fragments and characterization of their respective binding to the enzyme by isothermal titration calorimetry. The beta-phosphate, and to a lesser extent the alpha-phosphate, largely contributed to the binding energy. However, the uridine moiety was found to be essential for the optimal positioning of the donor substrate within the binding site. Unexpectedly, the contribution of the Gal moiety in substrate recognition was found to be negligible. Indeed, UDP-Gal, but also various UDP-sugars, could bind to beta1,4-GalT7. Surprisingly, in contrast with other GalTs, soluble beta1,4-GalT7 was able to transfer Glc (glucose), Xyl and, to a lesser extent GlcA and GlcNAc (N-acetyl glucosamine), to acceptor sugars, whereas UDP-Man (mannose) and UDP-GalNAc (N-acetyl galactosamine) were not substrates.

Characterization of a bacterial beta-1,3-galactosyltransferase with application in the synthesis of tumor-associated T-antigen mimics.

T-Antigen (Gal-beta1,3-GalNAc-alpha-O-Ser/Thr) is an important precursor of mucin-type O-glycans. T-Antigen is found to be closely associated with cancer progression and metastasis and has been used to develop carbohydrate-based anticancer vaccines. Enzymatic synthesis of T-antigen disaccharides have relied on the use of beta-1,3-galactosyltransferases recently cloned and characterized from several eukaryotic organisms. However, its application is limited by the difficulty of obtaining homogeneous enzymes and the strict substrate specificity of enzymes. Recently, a number of bacteria have been found to express carbohydrate structures that mimic host glycans. The corresponding glycosyltransferases have been exploited in the facile synthesis of a number of clinically important glycoconjugate mimics. In this study, we biochemically characterized a bacterial beta-1,3-galactosyltransferase (WbiP) from Escherichia coli O127, which expresses a T-antigen mimic in the lipopolysaccharide (LPS) structure. Substrate study showed that WbiP could readily glycosylate a series of N-acetylgalactosamine (GalNAc) analogues with alpha-substitutions at the reducing end, including glycosylated Ser and Thr (GalNAc-alpha-O-Ser/Thr), which illustrates the use of WbiP for the facile synthesis of T-antigens. Alignment of a group of putative bacterial beta-1,3-galactosyltransferases revealed the presence of two conserved DXD motifs, possibly suggesting a different functional role of each motif. Site-directed mutagenesis, enzyme kinetics as well as UDP-bead binding assays were carried out to investigate the role of each DXD motif in WbiP. The results suggest that 88DSD90 is critical in the binding of sugar donor UDP-Gal, whereas 174DYD176 may participate in the binding of the sugar acceptor. This study expands the scope of using bacterial glycosyltransferases as tools for in vitro synthesis of glycoconjugate mimics with clinical significance.

Chain elongation of pectic beta-(1-->4)-galactan by a partially purified galactosyltransferase from soybean (Glycine max Merr.) hypocotyls.

Pectin is one of the major cell wall polysaccharides found in dicotyledonous plants. We have solubilized and partially purified a beta-(1-->4)-galactosyltransferase (GalT) involved in the synthesis of the beta-(1-->4)-galactan side chains of pectin. The enzyme protein was almost completely solubilized by mixing a crude microsomal preparation of etiolated 6-day-old soybean (Glycine max Merr.) hypocotyls with a detergent, Triton X-100 (0.75%, w/v), in buffer. The solubilized enzyme was partially purified by ion-exchange chromatography. The crude membrane-bound GalT transferred Gal from UDP-Gal onto 2-aminobenzamide (AB)-derivatized beta-(1-->4)-galactoheptaose (Gal(7)-AB), leading to the formation of Gal(8-11)-AB by attachment of a series of one to four galactosyl residues; this is similar to what has previously been observed for 2-aminopyridine-derivatized beta-(1-->4)-galactooligomer acceptors (Konishi et al. in Planta 218:833-842, 2004). The partially purified GalT, by contrast, was able to transfer more than 25 galactosyl residues and elongated the chains to about Gal(35)-AB, thus almost reaching the length (43-47 Gal units) of native beta-(1-->4)-galactan side chains found in pectic polysaccharides from soybean cotyledons (Nakamura et al. in Biosci Biotechnol Biochem 66:1301-1313, 2002). Enzyme activity increased with increasing chain length of beta-(1-->4)-galactooligomers and reached maximal activity at heptaose, whereas galactooligomers higher than heptaose showed lower acceptor efficiency.

Fragment-based screening of the donor substrate specificity of human blood group B galactosyltransferase using saturation transfer difference NMR.

Saturation transfer difference NMR experiments on human blood group B alpha-(1,3)-galactosyltransferase (GTB) for the first time provide a comprehensive set of binding epitopes of donor substrate analogs in relation to the natural donor UDP-Gal. This study revealed that the enzyme binds several UDP-activated sugars, including UDP-Glc, UDP-GlcNAc, and UDP-GalNAc. In all cases, UDP is the dominant binding epitope. To identify the minimum requirements for specific binding, a detailed analysis utilizing a fragment-based approach was employed. The binding of donor substrate to GTB is essentially controlled by the base as a "molecular anchor." Uracil represents the smallest fragment that is recognized, whereas CDP, AMP, and GDP do not exhibit any significant binding affinity for the enzyme. The ribose and beta-phosphate moieties increase the affinity of the ligands, whereas the pyranose sugar apparently weakens the binding, although this part of the molecule controls the specificity of the enzyme. Accordingly, UDP represents the best binder. The binding affinities of UDP-Gal, UDP-Glc, and UMP are about the same, but lower than that of UDP. Furthermore, we observed that beta-D-galactose and alpha-D-galactose bind weakly to GTB. Whereas beta-D-galactose binds to the acceptor and donor sites, it is suggested that alpha-D-galactose occupies a third hitherto unknown binding pocket. Finally, our experiments revealed that modulation of enzymatic activity by metal ions critically depends on the total enzyme concentration, raising the question as to which of the bivalent metal cations Mg(2+) and Mn(2+) is more relevant under physiological conditions.

Identification of core 1 O-glycan T-synthase from Caenorhabditis elegans.

The common O-glycan core structure in animal glycoproteins is the core 1 disaccharide Galbeta1-3GalNAcalpha1-Ser/Thr, which is generated by the addition of Gal to GalNAcalpha1-Ser/Thr by core 1 UDP-alpha-galactose (UDP-Gal):GalNAcalpha1-Ser/Thr beta1,3-galactosyltransferase (core 1 beta3-Gal-T or T-synthase, EC2.4.1.122). Although O-glycans play important roles in vertebrates, much remains to be learned from model organisms such as the free-living nematode Caenorhabditis elegans, which offer many advantages in exploring O-glycan structure/function. Here, we report the cloning and enzymatic characterization of T-synthase from C. elegans (Ce-T-synthase). A putative C. elegans gene for T-synthase, C38H2.2, was identified in GenBank by a BlastP search using the human T-synthase protein sequence. The full-length cDNA for Ce-T-synthase, which was generated by polymerase chain reaction using a C. elegans cDNA library as the template, contains 1170 bp including the stop TAA. The cDNA encodes a protein of 389 amino acids with typical type II membrane topology and a remarkable 42.7% identity to the human T-synthase. Ce-T-synthase has seven Cys residues in the lumenal domain including six conserved Cys residues in all orthologs. The Ce-T-synthase has four potential N-glycosylation sequons, whereas the mammalian orthologs lack N-glycosylation sequons. Only one gene for Ce-T-synthase was identified in the genome-wide search, and it contains eight exons. Promoter analysis of the Ce-T-synthase using green fluorescent protein (GFP) constructs shows that the gene is expressed at all developmental stages and appears to be in all cells. Unexpectedly, only minimal activity was recovered in the recombinant, soluble Ce-T-synthase secreted from a wide variety of mammalian cell lines, whereas robust enzyme activity was recovered in the soluble Ce-T-synthase expressed in Hi-5 insect cells. Vertebrate T-synthase requires the molecular chaperone Cosmc, but our results show that Ce-T-synthase does not require Cosmc and might require invertebrate-specific factors for the formation of the optimally active enzyme. These results show that the Ce-T-synthase is a functional ortholog to the human T-synthase in generating core 1 O-glycans and open new avenues to explore O-glycan function in this model organism.

Expression, purification, and characterization of a galactofuranosyltransferase involved in Mycobacterium tuberculosis arabinogalactan biosynthesis.

The major structural component of the cell wall of Mycobacterium tuberculosis is a lipidated polysaccharide, the mycoyl-arabinogalactan-peptidoglycan (mAGP) complex. This glycoconjugate plays a key role in the survival of the organism, and thus, enzymes involved in its biosynthesis have attracted attention as sites for drug action. At the core of the mAGP is a galactan composed of D-galactofuranose residues attached via alternating beta-(1-->5) and beta-(1-->6) linkages. A single enzyme, glfT, has been shown to synthesize both glycosidic linkages. We report here the first high-level expression and purification of glfT by expression of the Rv3808c gene in Escherichia coli C41(DE3). Following a three-step purification procedure, 3-7 mg of protein of >95% purity was isolated from each liter of culture. We subsequently probed the substrate specificity of glfT by evaluating a panel of potential mono- and oligosaccharide substrates and demonstrated, for the first time, that trisaccharides are better substrates than disaccharides and that one disaccharide, in which the terminal D-galactofuranose residue is replaced with an L-arabinofuranose moiety, is a weak substrate. Kinetic characterization of the enzyme using four of the oligosaccharide acceptors gave K(m) values ranging from 204 microM to 1.7 mM. Through the use of NMR spectroscopy and mass spectrometry, we demonstrated that this recombinant enzyme, like the wild-type protein, is bifunctional and can synthesize both beta-(1-->6) and beta-(1-->5)-linkages in an alternating fashion. Access to purified glfT is expected to facilitate the development of high-throughput assays for the identification of inhibitors of the enzyme, which are potential antituberculosis agents.

Amino-acid substitution in the disordered loop of blood group B-glycosyltransferase enzyme causes weak B phenotype.

BACKGROUND: Few studies have investigated the reaction kinetics and interactions with nucleotide donor and acceptor substrates of mutant human ABO glycosyltransferases. Previous work identified a B(w) allele featuring a 556G>A polymorphism giving rise to a weak B phenotype. This polymorphism is predicted to cause a M186V amino-acid mutation within a highly conserved series of 16 amino acids present both in both blood group A- and blood group B-synthesizing enzymes. These residues are known as the disordered loop because their location cannot be determined in the crystal structure of the enzyme. Another patient has been identified with a 556G>A B(w) allele and the kinetics of the resulting mutant glycosyltransferase were studied. STUDY DESIGN AND METHODS: Serologic testing with murine and human reagents, amplification of the coding regions of exons 6 and 7, and DNA sequencing were performed with standard protocols. Enzyme kinetic studies utilized a model of human GTB M186V expressed in Escherichia coli with radiolabeled UDP-galactose and UDP-N-acetylgalactosamine as donor substrates and synthetic H-disaccharide as acceptor following standard protocols. RESULTS: The patient's red blood cells demonstrated a weak, but not mixed-field, B phenotype. Kinetic studies on the mutant enzyme revealed diminished activity (k(cat) = 0.15 per sec with UDP-galactose compared to 5.1 per sec for wild-type GTB) and elevated K(m) values for all substrates. CONCLUSION: This enzyme with a mutation in the disordered loop produces weak B antigen expression because of greatly decreased enzyme activity and reduced affinity for B-donor and acceptor substances.

The capsular polysaccharide biosynthesis of Streptococcus pneumoniae serotype 8: functional identification of the glycosyltransferase WciS (Cap8H).

CPS (capsular polysaccharide) is a major virulence factor in Streptococcus pneumoniae. Biosynthesis of CPS RU (repeat unit) proceeds by sequential transfer of sugar residues from the appropriate sugar donor to an activated lipid carrier by committed GTs (glycosyltransferases). While the nucleotide sequence of many cps loci is already known, the real substrate specificity of the hypothetical GTs, as well as the sequence of sugar addition is unclear. In the present paper, we report the biochemical characterization of one alpha-galactosyltransferase, WciS (Cap8H), a member of GT family 4. This enzyme is implicated in the tetrasaccharide RU biosynthetic pathway of Strep. pneumoniae CPS 8 ([-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Galp-(1-->4)-beta-D-GlcAp-(1-->4)-beta-D-Glcp-(1-->]n). Expression of WciS-His6 in Escherichia coli BL21 (DE3) strains or BL21 (DE3)/DeltagalU strain resulted in synthesis of a 39 kDa membrane-associated protein identified by N-terminal sequencing and recognized by anti-His6-tag antibody. This protein was capable of adding a galactose residue cellobiuronic acid [beta-D-GlcAp-(1-->4)-D-Glcp]-pyrophosphate-polyprenol from UDP-Gal. The newly added galactose residue is removed by alpha-galactosidase, indicating that WciS is a retaining GT. Our results suggest that WciS catalyses the addition of the third sugar residue of the CPS 8 RU. The recombinant WciS-His6 was solubilized and purified as a soluble multimer, opening the way for structural studies.

Unusual properties of the prespore-specific enzyme, UDPgalactose:polysaccharide galactosyl transferase, of Dictyostelium discoideum.

UDPgalactose:polysaccharide galactosyl-transferase is the enzyme that is specifically localized in prespore cells of Dictyostelium discoideum and its activity sharply changes in response to differentiation and dedifferentiation. To clarify the nature of this enzyme, we first developed an improved assay method for the enzyme, and by using this method, we partially purified the enzyme through DEAE-sepharose, phenyl-sepharose and ATP-sepharose chromatography. The apparent molecular mass of the enzyme was ca. 200 KDa (by non-denaturing polyacrylamide gel gradient analysis) and the isoelectric point was around pH 7. The enzyme exhibited a hitherto undescribed property, that is the reaction proceeds faster at 0 degrees C than at 21 degrees C, with a smaller K(m) value and an unchanged V(max) value. This low-temperature resistant property of the enzyme is consistent with the previous observation (Maeda 1984, J. Cell Sci. 69, 159-165) that prespore differentiation is favored at low temperatures. The reaction appears to proceed in a double displacement manner. ATP reversibly inhibited the enzyme with a K(i) value of 2 mM, suggesting the possibility that ATP regulates its activity in vivo.

Cloning, expression, and purification of the 27 kDa (MPT51, Rv3803c) protein of Mycobacterium tuberculosis.

A limited number of proteins of Mycobacterium tuberculosis have been characterized so far for their use as potential candidates for diagnosis and vaccine studies. This study was aimed at cloning, expression, and purification of a 27 kDa protein (otherwise known as the MPT51 or Rv3803c protein) of M. tuberculosis. The Rv3803c gene was PCR amplified using primers that contain specific restriction sites. The amplified product was inserted initially into pTOPO and then sub-cloned into pET15b and pET24d vectors, such that the recombinant protein is predicted to contain an N-terminal or a C-terminal histidine tag, respectively. The recombinant plasmids were introduced into Escherichia coli BL21 (DE3) and the recombinant proteins were purified from the cytosolic fractions of the E. coli sonicates by nickel-NTA chromatography. The purity, molecular mass, and the conformation of the proteins were determined by high performance liquid chromatography (HPLC), matrix assisted laser desorption-ionization-time-of-flight (MALDI-TOF), and circular dichroism (CD) studies, respectively. The purified proteins were found to be immunogenic and useful for immunodiagnostic studies of tuberculosis by enzyme linked immunosorbent assay (ELISA), with a sensitivity of 71% and specificity of 95%.

Refolding from denatured inclusion bodies, purification to homogeneity and simplified assay of MGDG synthases from land plants.

In plant cells, the synthesis of monogalactosyldiacylglycerol (MGDG) is catalyzed within plastid envelope membranes by MGD proteins. MGDG synthesis was also reported in apicomplexan parasites, a phylum of protists harbouring a plastid that proved essential for the parasite survival. MGD activity is therefore a potent target for herbicidal and anti-parasitic molecules. In this study, we describe a detailed in vitro refolding protocol for denatured recombinant MGD accumulated in inclusion bodies from transformed Escherichia coli. The refolding process was dependent on CHAPS detergent and lipids, such as diacylglycerol and phosphatidylglycerol, as well as bivalent metals. Owing to this refolding procedure, the recombinant MGD protein from spinach was purified to homogeneity, allowing a definite characterization of its non-processivity and an investigation of its dimerization using cross-linking reagents. Additionally, using the portion of recombinant enzyme that accumulates in an active form in bacterial membranes, we developed a miniature assay for high-throughput screening for inhibitors.