General Tolerance of Galactosyltransferases toward UDP-galactosamine Expands Their Synthetic Capability.

Accessing large numbers of structurally diverse glycans and derivatives is essential to functional glycomics. We showed a general tolerance of galactosyltransferases toward uridine-diphosphate-galactosamine (UDP-GalN), which is not a commonly used sugar nucleotide donor. The property was harnessed to develop a two-step chemoenzymatic strategy for facile synthesis of novel and divergent N-acetylgalactosamine (GalNAc)-glycosides and derivatives in preparative scales. The discovery and the application of the new property of existing glycosyltransferases expand their catalytic capabilities in generating novel carbohydrate linkages, thus prompting the synthesis of diverse glycans and glycoconjugates for biological studies.

Effects of macromolecular crowding on the folding and aggregation of glycosylated MUC5AC.

OBJECTIVE: To investigate the effects of macromolecular crowding on the folding and aggregation of MUC5AC with different levels of glycosylation during refolding. METHODS: Part 1:An in vitro catalytic reaction comprising the ppGalNAc T2 enzyme, uridine-5'-diphospho-N-galactosamine (UDP-GalNAc) and an 11-amino acid peptide substrate, was used to assess the enzyme activity of the ppGalNAc T2 enzyme in macromolecular crowding environment respectively with bovine serum albumin (BSA), polyethylene glycol (PEG2000), Dextran70 and Ficoll70 at different concentration and temperature. Part 2: The recombinant MUC5AC was expressed in HEK293 cells and purified by nickel column chromatography. The purified protein was treated with PNGase F, and the degree of glycosylation was analyzed by SDS-PAGE. Macromolecular crowding was simulated using PEG2000 at the concentrations of 50, 100, and 200 g/L. Deglycosylated-MUC5AC (d-MUC5AC) and glycosylated MUC5AC (g-MUC5AC) were denatured by GdnHCl and renatured by dilution in a refolding buffer. Protein aggregation was monitored continuously by absorbance reading at 488 nm using a UV spectrophotometer at 25 °C. The refolded proteins were centrifuged, the protein concentration of the supernatant was measured, and refolding yield in different refolding buffers was determined. RESULTS: Enzyme activityof ppGalNAc T2 was observed to increase with increasing crowding agent concentration, with highest enzyme activity at 200 g/L. Compared with the group in the absence of crowding reagent, the refolding yield of g-MUC5AC and d-MUC5AC were reduced significantly in the presence of different concentrations of PEG2000 (200, 100, and 50 g/L). Compared with the dilute solution, aggregation increased significantly in the presence of PEG2000, especially at 200 g/L. Moreover, in the crowded reagent with the same concentration, the refolding yield of d-MUC5AC was higher than that of g-MUC5AC, whereas the degree of aggregation of d-MUC5AC was lower than that of g-MUC5AC. CONCLUSION: The crowded intracellular environment reduces the refolding rate of MUC5AC and strongly induces the misfolding and aggregation of glycosylated MUC5AC.

O-GlcNAc Site Mapping by Using a Combination of Chemoenzymatic Labeling, Copper-Free Click Chemistry, Reductive Cleavage, and Electron-Transfer Dissociation Mass Spectrometry.

As a dynamic post-translational modification, O-linked ß- N-acetylglucosamine ( O-GlcNAc) modification (i.e., O-GlcNAcylation) of proteins regulates many biological processes involving cellular metabolism and signaling. However, O-GlcNAc site mapping, a prerequisite for site-specific functional characterization, has been a challenge since its discovery. Herein we present a novel method for O-GlcNAc enrichment and site mapping. In this method, the O-GlcNAc moiety on peptides was labeled with UDP-GalNAz followed by copper-free azide-alkyne cycloaddition with a multifunctional reagent bearing a terminal cyclooctyne, a disulfide bridge, and a biotin handle. The tagged peptides were then released from NeutrAvidin beads upon reductant treatment, alkylated with (3-acrylamidopropyl)trimethylammonium chloride, and subjected to electron-transfer dissociation mass spectrometry analysis. After validation by using standard synthetic peptide gCTD and model protein α-crystallin, such an approach was applied to the site mapping of overexpressed TGF-ß-activated kinase 1/MAP3K7 binding protein 2 (TAB2), with four O-GlcNAc sites unambiguously identified. Our method provides a promising tool for the site-specific characterization of O-GlcNAcylation of important proteins.

Systematic identification of the protein substrates of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-T1/T2/T3 using a human proteome microarray.

O-GalNAc glycosylation is the initial step of the mucin-type O-glycosylation. In humans, it is catalyzed by a family of 20 homologous UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). So far, there is very limited information on their protein substrate specificities. In this study, we developed an on-chip ppGalNAc-Ts assay that could rapidly and systematically identify the protein substrates of each ppGalNAc-T. In detail, we utilized a human proteome microarray as the protein substrates and UDP-GalNAz as the nucleotide sugar donor for click chemistry detection. From a total of 16 368 human proteins, we identified 570 potential substrates of ppGalNAc-T1, T2, and T3. Among them, 128 substrates were overlapped, while the rest were isoform specific. Further cluster analysis of these substrates showed that the substrates of ppGalNAc-T1 had a closer phylogenetic relationship with that of ppGalNAc-T3 compared with ppGalNAc-T2, which was consistent with the topology of the phylogenetic tree of these ppGalNAc-Ts. Taken together, our microarray-based enzymatic assay comprehensively reveals the substrate profile of the ppGalNAc-T1, T2, and T3, which not only provides a plausible explanation for their partial functional redundancy as reported, but clearly implies some specialized roles of each enzyme in different biological processes.

Direct Intracellular Delivery of Cell-Impermeable Probes of Protein Glycosylation by Using Nanostraws.

Bioorthogonal chemistry is an effective tool for elucidating metabolic pathways and measuring cellular activity, yet its use is currently limited by the difficulty of getting probes past the cell membrane and into the cytoplasm, especially if more complex probes are desired. Here we present a simple and minimally perturbative technique to deliver functional probes of glycosylation into cells by using a nanostructured "nanostraw" delivery system. Nanostraws provide direct intracellular access to cells through fluid conduits that remain small enough to minimize cell perturbation. First, we demonstrate that our platform can deliver an unmodified azidosugar, N-azidoacetylmannosamine, into cells with similar effectiveness to a chemical modification strategy (peracetylation). We then show that the nanostraw platform enables direct delivery of an azidosugar modified with a charged uridine diphosphate group (UDP) that prevents intracellular penetration, thereby bypassing multiple enzymatic processing steps. By effectively removing the requirement for cell permeability from the probe, the nanostraws expand the toolbox of bioorthogonal probes that can be used to study biological processes on a single, easy-to-use platform.

Base-modified donor analogues reveal novel dynamic features of a glycosyltransferase.

Glycosyltransferases (GTs) are enzymes that are involved, as Nature's "glycosylation reagents," in many fundamental biological processes including cell adhesion and blood group biosynthesis. Although of similar importance to that of other large enzyme families such as protein kinases and proteases, the undisputed potential of GTs for chemical biology and drug discovery has remained largely unrealized to date. This is due, at least in part, to a relative lack of GT inhibitors and tool compounds for structural, mechanistic, and cellular studies. In this study, we have used a novel class of GT donor analogues to obtain new structural and enzymological information for a representative blood group GT. These analogues interfere with the folding of an internal loop and the C terminus, which are essential for catalysis. Our experiments have led to the discovery of an entirely new active site folding mode for this enzyme family, which can be targeted in inhibitor development, similar to the DFG motif in protein kinases. Taken together, our results provide new insights into substrate binding, dynamics, and utilization in this important enzyme family, which can very likely be harnessed for the rational development of new GT inhibitors and probes.

UDP-Gal/UDP-GlcNAc chimeric transporter complements mutation defect in mammalian cells deficient in UDP-Gal transporter.

The role of UDP-galactose transporter (UGT; SLC35A2) and UDP-N-acetylglucosamine transporter (NGT; SLC35A3) in glycosylation of macromolecules may be coupled and either of the transporters may partially replace the function played by its partner. The aim of this study was to construct chimeric transporters composed of the N-terminal portion of human NGT and the C-terminal portion of human UGT1 or UGT2 (NGT-UGT1 or NGT-UGT2, respectively), as well as of the N-terminal portion of UGT and C-terminal portion of NGT (UGT-NGT), overexpress them stably in UGT-deficient CHO-Lec8 and MDCK-RCA(r) cells, and characterize their involvement in glycosylation. Two chimeric proteins, NGT-UGT1 and NGT-UGT2, did not overexpress properly. In contrast, UGT-NGT chimeric protein was successfully overexpressed and localized properly to the Golgi apparatus. UGT-NGT chimeric transporter delivered UDP-Gal to the Golgi vesicles of UGT-deficient cells, which resulted in the increased content of galactosylated structures to such an extent that the wild-type phenotypes were completely restored. Our data further support our hypothesis that UGT and NGT cooperate in the UDP-Gal delivery for glycosyltransferases located in the Golgi apparatus. In a wider context, the results gained in this study add to our understanding of glycosylation, one of the basic posttranslational modifications, which affects the majority of macromolecules.

Crystal structure of Helicobacter pylori pseudaminic acid biosynthesis N-acetyltransferase PseH: implications for substrate specificity and catalysis.

Helicobacter pylori infection is the common cause of gastroduodenal diseases linked to a higher risk of the development of gastric cancer. Persistent infection requires functional flagella that are heavily glycosylated with 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (pseudaminic acid). Pseudaminic acid biosynthesis protein H (PseH) catalyzes the third step in its biosynthetic pathway, producing UDP-2,4-diacetamido-2,4,6-trideoxy-ß-L-altropyranose. It belongs to the GCN5-related N-acetyltransferase (GNAT) superfamily. The crystal structure of the PseH complex with cofactor acetyl-CoA has been determined at 2.3 Å resolution. This is the first crystal structure of the GNAT superfamily member with specificity to UDP-4-amino-4,6-dideoxy-ß-L-AltNAc. PseH is a homodimer in the crystal, each subunit of which has a central twisted ß-sheet flanked by five α-helices and is structurally homologous to those of other GNAT superfamily enzymes. Interestingly, PseH is more similar to the GNAT enzymes that utilize amino acid sulfamoyl adenosine or protein as a substrate than a different GNAT-superfamily bacterial nucleotide-sugar N-acetyltransferase of the known structure, WecD. Analysis of the complex of PseH with acetyl-CoA revealed the location of the cofactor-binding site between the splayed strands ß4 and ß5. The structure of PseH, together with the conservation of the active-site general acid among GNAT superfamily transferases, are consistent with a common catalytic mechanism for this enzyme that involves direct acetyl transfer from AcCoA without an acetylated enzyme intermediate. Based on structural homology with microcin C7 acetyltransferase MccE and WecD, the Michaelis complex can be modeled. The model suggests that the nucleotide- and 4-amino-4,6-dideoxy-ß-L-AltNAc-binding pockets form extensive interactions with the substrate and are thus the most significant determinants of substrate specificity. A hydrophobic pocket accommodating the 6'-methyl group of the altrose dictates preference to the methyl over the hydroxyl group and thus to contributes to substrate specificity of PseH.

The effects of galactosamine on UTP levels in the livers of young, adult and old rats.

Galactosamine (GalN), a well-known hepatotoxin that depletes the cellular pool of uracil nucleotides, was previously shown to have greater impact on the inhibition of protein synthesis in hepatocytes of old rats as compared with young animals (Kmiec 1994, Ann. N.Y. Ac. Sci. 717, 216-225). In the present study we compared the effects of GalN on the nucleotide content (measured by ion-exchange HPLC) in the livers of young (4 months), adult (12 months), and old (24-26 months old) rats two hours after its intraperitoneal administration. UTP content of the livers of old control rats was significantly lower (by 28%) than that of young animals. GalN administration decreased the UTP content in the livers of young, adult and old rats by, respectively, 55%, 65% and 89%, and increased the content of UDP-sugars by 189%, 175% and 305%. The hepatic content of ATP, ADP, AMP, NAD, GTP except CTP did not differ significantly among the age groups of rats studied, and was not changed by GalN treatment. The content of CTP was significantly higher in old rats (P < 0.03) upon GalN treatment. The lower hepatic content of UTP may partially explain the increased sensitivity of hepatocytes and livers of old rats to the action of galactosamine, and possibly to other hepatotoxic compounds that decrease transcription in the liver.

Syntheses of unnatural N-substituted UDP-galactosamines as alternative substrates for N-acetylgalactosaminyl transferases.

UDP-GalNAc analogues with slight modifications in the 2-acetamido group of the GalNAc moiety are prepared in order to study their role in the mechanism of the N-acetylgalactosaminyl transferase mediated glycosylation step. The analogues with N-propionyl-, N-butyryl- and N-bromoacetyl-groups were synthesized, utilizing Khorana's morpholidate coupling method starting from D-galactosaminyl-1-phosphate after selective N-acylation of its amino group with the appropriate N-acyloxysuccinimides. Furthermore, in addition to UDP-galactosamine its 2-azido analogue has been efficiently prepared involving a metal catalyzed diazo transfer reaction.

Synthesis of uridine 5'-(2-acetamido-2,4-dideoxy-4-fluoro-alpha-D-galactopyranosyl) diphosphate and uridine 5'-(2-acetamido-2,6-dideoxy-6-fluoro-alpha-D-glucopyranosyl) diphosphate.

Benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-alpha-D-glucopyranoside was converted into its 4-O-(methylsulfonyl) derivative (2) by treatment with methanesulfonyl chloride in pyridine. Displacement of the methylsulfonyloxy group of 2 with fluoride ion afforded benzyl 2-acetamido-3,6-di-O-benzyl-2,4-dideoxy-4-fluoro-alpha-D-galactopyranosi de, which on hydrogenolysis, followed by acetylation, furnished 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-4-fluoro-D-galactopyranose. Treatment of this and of 2-acetamido-1,3,4-tri-O-acetyl-2,6-dideoxy-6-fluoro-D-glucopyranose with trimethylsilyl trifluoromethanesulfonate in 1,2-dichloroethane at approximately 50 degrees afforded the 4-deoxy-4-fluoro- or the 6-deoxy-6-fluoro-oxazolines (5) and (11), respectively. Reaction of 5 and 11 with dibenzyl phosphate in 1,2-dichloroethane produced the alpha-linked dibenzyl phosphate derivatives 6 and 12, respectively. Catalytic hydrogenation of 6 provided 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-4-fluoro-alpha-D-galactopyranosy l phosphate (7), and that of 12 gave 2-acetamido-3,4-di-O-acetyl-2,6-dideoxy-6-fluoro-alpha-D-glucopyranosyl phosphate (13). Coupling of 7 and 13 with uridine 5'-monophosphomorpholidate in dry pyridine at approximately 37 degrees, followed by O-deacetylation, furnished the title compounds, respectively, isolated and characterized as their respective dilithium salts.

Enzymatic route to preparative-scale synthesis of UDP-GlcNAc/GalNAc, their analogues and GDP-fucose.

Enzymatic synthesis using glycosyltransferases is a powerful approach to building polysaccharides with high efficiency and selectivity. Sugar nucleotides are fundamental donor molecules in enzymatic glycosylation reactions by Leloir-type glycosyltransferases. The applications of these donors are restricted by their limited availability. In this protocol, N-acetylglucosamine (GlcNAc)/N-acetylgalactosamine (GalNAc) are phosphorylated by N-acetylhexosamine 1-kinase (NahK) and subsequently pyrophosphorylated by N-acetylglucosamine uridyltransferase (GlmU) to give UDP-GlcNAc/GalNAc. Other UDP-GlcNAc/GalNAc analogues can also be prepared depending on the tolerance of these enzymes to the modified sugar substrates. Starting from L-fucose, GDP-fucose is constructed by one bifunctional enzyme L-fucose pyrophosphorylase (FKP) via two reactions.

Biochemical characterization of the Campylobacter jejuni Cj1294, a novel UDP-4-keto-6-deoxy-GlcNAc aminotransferase that generates UDP-4-amino-4,6-dideoxy-GalNAc.

Campylobacter jejuni produces multiple glycoproteins whose glycans contain 4-amino 6-deoxy sugars or their derivatives, such as diacetamidobacillosamine or pseudaminic acid. Because the proteoglycans contribute to bacterial virulence and their constitutive sugars are not commonly found in humans, inhibitors developed against the enzymes that are responsible for their biosynthesis could be novel therapeutic targets to fight this important food-borne pathogen. The biosynthesis of diacetamidobacillosamine is anticipated to involve a sugar nucleotide C6 dehydratase, a C4 aminotransferase and an acetyltransferase. We have identified a set of genes (cj1293, cj1294, and cj1298) potentially encoding one of each enzymatic activity, and demonstrated earlier that Cj1293 was a UDP-GlcNAc-specific C6 dehydratase. Others have shown that Cj1293 was involved in protein glycosylation. Here, we report on our investigation of the potential activity of Cj1294 as a sugar nucleotide C4 aminotransferase. Our biochemical characterization of overexpressed and purified protein shows that Cj1294 is a pyridoxal phosphate-dependent aminotransferase specific for UDP-4-keto-6-deoxy-GlcNAc that uses preferentially glutamic acid as an amino donor. A detailed physicokinetic study of Cj1294 was performed to determine the K(m) of 1.28 +/- 0.2 mm and k(cat) of 11.5 +/- 1.3 min(-1). Also, two residues essential for protein stability and activity, Arg(228) and Lys(181), respectively, were identified by site-directed mutagenesis. Finally, we demonstrated by NMR analysis of purified reaction product that Cj1294 produces UDP-4-amino-4,6-dideoxy-GalNAc. These results indicate that Cj1294 is involved in the biosynthesis of diacetamidofucosamine, a C4 epimer of diacetamidobacillosamine not yet described in C. jejuni proteoglycans, suggesting that the composition of C. jejuni proteoglycans is more variable than anticipated.

Occurrence in Bacillus megaterium of uridine 5'-diphospho-N-acetylgalactosamine and uridine 5'-diphosphogalactosamine, intermediates in the biosynthesis of galactosamine-6-phosphate polymer.

We isolated two galactosamine derivatives from Bacillus megaterium sporulating cells by lectin affinity chromatography followed by DEAE-Sephadex A-25 chromatography. From chemical analyses and measurements of these compounds, it was determined that one was uridine 5'-diphospho-N-acetylgalactosamine and that the other was uridine 5'-diphosphogalactosamine. They appeared in the middle stage of sporulation and disappeared during the period when galactosamine-6-phosphate is deposited on the forespore surface. These results suggest that uridine 5'-diphospho-N-acetylgalactosamine and uridine 5'-diphosphogalactosamine are intermediates in the biosynthesis of the galactosamine-6-phosphate polymer, a backbone structure of the exosporium.

Probing glycosyltransferase activities with the Staudinger ligation.

The development of rapid screening methods for probing glycosyltransferase activities is essential for advancing the field of glycobiology. While assays for specific glycosyltransferases exist, there is no generalizable method that can be applied across the enzyme superfamily. Herein we describe a novel glycosyltransferase assay that exploits their unnatural substrate tolerance and the unique chemical reactivity of the azide. We applied this "azido-ELISA" to the family of polypeptide alpha-N-acetylgalactosaminyltransferases (ppGalNAcTs), all of which were able to transfer N-azidoacetylgalactosamine (GalNAz) from the unnatural nucleotide sugar donor UDP-GalNAz. The azide was detected and quantified by Staudinger ligation with a phosphine probe in a microtiter plate format. This approach should be applicable to any glycosyltransferase or group-transfer enzyme that tolerates unnatural azido substrates.

Improved high-performance liquid chromatographic method for N-acetylgalactosamine-4-sulfate sulfatase (arylsulfatase B) activity determination using uridine diphospho-N-acetylgalactosamine-4-sulfate.

UDP-N-acetylgalactosamine-4-sulfate (UDP-GalNAc-4-S) was isolated from hen oviduct (isthmus) with a yield of 31 mumol per 100 g of wet tissue and used for arylsulfatase B (ASB) activity determination. Two HPLC methods of separation and quantitation of the reaction product were described: (1) an original gradient elution method which makes it possible to determine the reaction product when only partially purified ASB was used and additional uridine derivatives were formed during incubation; (2) an improved, fast isocratic elution method which may be used in the case of purified ASB preparations, devoid of other nucleotide hydrolysing enzymes. For both methods the detection limit was 0.1 nmol of product with standard error of determination < or = 3%. Using the gradient elution method we have found that UDP-GalNAc-4-S was hydrolysed by bovine arylsulfatase B1 most efficiently at pH 5.0 and concentration 0.5 mM with K(m) = 85 microM.

A sulfotransferase-sulfatase system in avian oviduct which catalyzes a conversion of UDP-N-acetylgalactosamine 4-sulfate to the 6-sulfate isomer.

Magnum from quail oviduct was subfractionated to yield epithelium and tubular glands. The in vitro enzymatic activities involved in sulfated sugar nucleotide biosynthesis were assayed in these isolated tissues. The results demonstrated that the activities necessary for a series of reactions, UDP-N-acetylgalactosamine----UDP-N-acetylgalactosamine 4-sulfate----UDP-N-acetylgalactosamine 4,6- bisulfate ----UDP-N-acetylgalactosamine 6-sulfate, are located predominantly in the tubular gland. Both time course and pulse-chase studies with [35S]sulfate gave results that were consistent with this reaction scheme. A microsomal preparation from the magnum was shown to be capable of labeling all three sulfate sugar nucleotides with [35S]sulfate upon incubation with UDP-N-acetylgalactosamine and 3'- phosphoadenylyl [35S]sulfate. Again, their relative labeling rates were in the order necessary to allow for a synthesis of sulfated sugar nucleotides in the sequence described above. Furthermore, incubation of the microsomal preparation with UDP-N-[14C]acetylgalactosamine 4-sulfate and 3'- phosphoadenylyl sulfate resulted in the formation of UDP-N-[14C]acetylgalactosamine 6-sulfate. Also shown was the existence in the microsomal preparation of a sulfatase specific for the sulfate at position 4 of UDP-N-acetylgalactosamine 4,6- bisulfate . The results, together with those obtained in previous investigations, suggest that the tubular gland of quail oviduct contains a microsomal multienzyme system which catalyzes a series of sulfation and desulfation of N-acetylgalactosamine residues at the nonreducing terminal position of either sugar nucleotides or polysaccharide chains.

Synthesis and biological evaluation of new UDP-GalNAc analogues for the study of polypeptide-alpha-GalNAc-transferases.

A series of three O-methylated UDP-GalNAc analogues have been synthesised using a divergent strategy from a 3,6-di-O-pivaloyl GlcNAc derivative. The biological activity of these probes toward polypeptide-alpha-GalNAc-transferase T1 has been investigated. This study shows that this glycosyltransferase exhibits a very high substrate specificity.

Synthesis of aryl azide derivatives of UDP-GlcNAc and UDP-GalNAc and their use for the affinity labeling of glycosyltransferases and the UDP-HexNAc pyrophosphorylase.

The chemical synthesis and utilization of two photoaffinity analogs, 125I-labeled 5-[3-(p-azidosalicylamido)-1-propenyl]-UDP-GlcNAc and -UDP-GalNAc, is described. Starting with either UDP-GlcNAc or UDP-GalNAc, the synthesis involved the preparation of the 5-mercuri-UDP-HexNAc and then attachment of an allylamine to the 5 position to give 5-(3-amino)allyl-UDP-HexNAc. This was followed by acylation with N-hydroxysuccinimide p-aminosalicylic acid to form the final product, i.e., 5-[3-(p-azidosalicylamido)-1-propenyl]-UDP-GlcNAc or UDP-GalNAc. These products could then be iodinated with chloramine T to give the 125I-derivatives. Both the UDP-GlcNAc and the UDP-GalNAc derivatives reacted in a concentration-dependent manner with a highly purified UDP-HexNAc pyrophosphorylase, and both specifically labeled the subunit(s) of this protein. The labeling of the protein by the UDP-GlcNAc derivative was inhibited in dose-dependent fashion by either unlabeled UDP-GlcNAc or unlabeled UDP-GalNAc. Likewise, labeling with the UDP-GalNAc probe was blocked by either UDP-GlcNAc or UDP-GalNAc. The UDP-GlcNAc probe also specifically labeled a partially purified preparation of GlcNAc transferase I.