GlcNAc Residues Research Articles

Combining High-Energy C-Trap Dissociation and Electron Transfer Dissociation for Protein O-GlcNAc Modification Site Assignment

Mass spectrometry-based studies of proteins that are post-translationally modified by O-linked β-N-acetylglucosamine (O-GlcNAc) are challenged in effectively identifying the sites of modification while simultaneously sequencing the peptides. Here we tested the hypothesis that a combination of high-energy C-trap dissociation (HCD) and electron transfer dissociation (ETD) could specifically target the O-GlcNAc modified peptides and elucidate the amino acid sequence while preserving the attached GlcNAc residue for accurate site assignment. By taking advantage of the recently characterized O-GlcNAc-specific IgG monoclonal antibodies and the combination of HCD and ETD fragmentation techniques, O-GlcNAc modified proteins were enriched from HEK293T cells and subsequently characterized using the LTQ Orbitrap Velos ETD (Thermo Fisher Scientific) mass spectrometer. In our data set, 83 sites of O-GlcNAc modification are reported with high confidence confirming that the HCD/ETD combined approach is amenable to the detection and site assignment of O-GlcNAc modified peptides. Realizing HCD triggered ETD fragmentation on a linear ion trap/Orbitrap platform for more in-depth analysis and application of this technique to other post-translationally modified proteins are currently underway. Furthermore, this report illustrates that the O-GlcNAc transferase appears to demonstrate promiscuity with regards to the hydroxyl-containing amino acid modified in short stretches of primary sequence of the glycosylated polypeptides.

Comparison of hFSH Glycosylation by Electrospray Ionization Mass Spectrometry.

FSH is a highly heterogenous glycoprotein hormone, which possesses 4 N-glycosylation sites, each decorated with a family of glycans. Glycosylation heterogeneity is usually evaluated by fractionation methods that separate on the basis of negative charge. From the pattern of isoform isoelectric points, differences in glycosylation have been inferred. For example, because different isoform patterns were encountered when pituitary extract, serum samples, and urine samples were fractionated by chromatofocusing then evaluated by FSH RIA, glycosylation of FSH from all three sources was assumed to differ. Recent advances in mass spectrometry now permit direct comparison of FSH glycans. In the present study, we compared FSH glycosylation in human FSH preparations derived from pituitary glands, postmenopausal urine, and recombinant hFSH expressed in the rat GH3 cell line. The glycan populations released from reduced, carboxymethylated hFSH preparations obtained from the pituitary and from postmenopausal urine were virtually identical. The major glycans were identical, the major differences occurred in relatively minor glycan populations, including a 35% decrease in sulfated glycans, 27% decrease in glycans with a bisecting GlcNAc residue, and a 55% decrease in fucosylated glycans. The great similarity in pituitary and urinary hFSH glycan populations is a highly significant finding as it the absence of major changes in glycan popultations between pituitary and urinary FSH means that serum FSH glycans must also be the same. Therefore, examination of urinary hFSH glycosylation is directly relevant to serum hFSH glycosylation. We also compared glycosylation of tetra-glycosylated hFSH and two di-glycosylated hFSH preparations. The di-glycosylated hFSH derived from FSH preparations possessed a glycan population that was highly similar to that from tetra-glycosylated hFSH. Glycans from both glycoform preparations were very similar to pituitary hFSH, except the sulfated glycan populations were greater in the glycoform preparations. Glycosylation of di-glycosylated hFSH isolated from hLH preparations was strikingly different from all other hFSH preparations. The glycans were largely high mannose and in combinantion with their abundance, it could be argued that these represent an intracellular form. Glycosylation of recombinant hFSH revealed the absence of tetra-antennary glycans, which comprised 15% of pituitary hFSH glycans. Moreover, recombinant hFSH triantennary glycans possessed a third branch linked beta1-6 to the alpha1-6 mannose residue, which is due to the action of GlcNAc transferase V. In pituitary this branch is linked beta1-4 to the alpha1-3 mannose residue, as a consequence of GlcNAc transferase IV. This suggested the absence of GlcNAc transferase IV in rat GH3 cells. However, RT PCR found evidence for expression of two isoforms of this enzyme, while failing to detect GlcNAc transferase V. In order to make a more native-like recombinant hFSH, GH3 cells will have to be engineered to express the more human pituitary-like glycosyltransferase population. Supported by NIH grant P01 AG029531. (poster)

Structure elucidation of the O-antigen of Salmonella enterica O51 and its structural and genetic relation to the O-antigen of Escherichia coli O23

The O-polysaccharide (O-antigen) of Salmonella enterica O51 was isolated by mild acid degradation of the lipopolysaccharide and its structure was established using sugar analysis and NMR spectroscopy. The O-antigen of Escherichia coli O23, whose structure was elucidated earlier, possesses a similar structure and differs only in the presence of an additional lateral α-D-Glcp residue at position 6 of the GlcNAc residue in the main chain. Sequencing of the O-antigen gene clusters of S. enterica O51 and E. coli O23 revealed the same genes with a high-level similarity. By comparison with opened gene databases, all genes expected for the synthesis of the common structure of the two O-antigens were assigned functions. It is suggested that the gene clusters of both bacteria originated from a common ancestor, whereas the O-antigen modification in E. coli O23, which, most probably, is induced by prophage genes outside the gene cluster, could be introduced after the species divergence.

The N-glycan Glycoprotein Deglycosylation Complex (Gpd) from Capnocytophaga canimorsus Deglycosylates Human IgG

C. canimorsus 5 has the capacity to grow at the expenses of glycan moieties from host cells N-glycoproteins. Here, we show that C. canimorsus 5 also has the capacity to deglycosylate human IgG and we analyze the deglycosylation mechanism. We show that deglycosylation is achieved by a large complex spanning the outer membrane and consisting of the Gpd proteins and sialidase SiaC. GpdD, -G, -E and -F are surface-exposed outer membrane lipoproteins. GpdDEF could contribute to the binding of glycoproteins at the bacterial surface while GpdG is a endo-β-N-acetylglucosaminidase cleaving the N-linked oligosaccharide after the first N-linked GlcNAc residue. GpdC, resembling a TonB-dependent OM transporter is presumed to import the oligosaccharide into the periplasm after its cleavage from the glycoprotein. The terminal sialic acid residue of the oligosaccharide is then removed by SiaC, a periplasm-exposed lipoprotein in direct contact with GpdC. Finally, most likely degradation of the oligosaccharide proceeds sequentially from the desialylated non reducing end by the action of periplasmic exoglycosidases, including β-galactosidases, β-N-Acetylhexosaminidases and α-mannosidases.

Glycan structures of the structural subunit (HtH1) of Haliotis tuberculata hemocyanin

The oligosaccharide structures of the structural subunit HtH1 of Haliotis tuberculata hemocyanin (HtH) were studied by mass spectral sequence analysis of the glycans. The proposed structures are based on MALDI-TOF-MS data before and after treatment with the specific exoglycosidases β1-3,4,6-galactosidase and α1-6(>2,3,4) fucosidase followed by sequence analysis via electrospray ionization MS/MS-spectra. In total, 15 glycans were identified as a highly heterogeneous group of structures. As in most molluscan hemocyanins, the glycans of HtH1 contain a terminal MeHex, but more interestingly, a novel structural motif was observed: MeHex[Fuc(α1-3)-]GlcNAc, including thus MeHex and (α1-3)-Fuc residues being linked to an internal GlcNAc residue. While the functional unit (FU) c (HtH1-c) is completely lacking any potential glycosylation site, FU-h possesses a second exposed sugar attachment site between beta-strands 8 and 9 within the beta sandwich domain compared to the other FUs. The glycosylation pattern/sites show a high degree of conservation. In FU-h two prominent potential glycosylation sites can be detected. The finding that HtH1 is not able to form multidecameric structures in vivo could be explained by the presence of the exposed glycan on the surface of FU-h.

A HighlyN-Glycosylated Chitin Deacetylase Derived from a Novel Strain ofMortierellasp. DY-52

Chitin deacetylase (CDA), the enzyme that catalyzes the hydrolysis of acetamido groups of GlcNAc in chitin, was purified from culture filtrate of the fungus Mortierella sp. DY-52 and characterized. The extracellular enzyme is likely to be a highly N-glycosylated protein with a pI of 4.2-4.8. Its apparent molecular weight was determined to be about 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 67 kDa by size-exclusion chromatography. The enzyme had an optimum pH of 6.0 and an optimum temperature of 60 °C. Enzyme activity was slightly inhibited by 1-10 mM Co(2+) and strongly inhibited by 10 mM Cu(2+). It required at least two GlcNAc residues for catalysis. When (GlcNAc)(6) was used as substrate, K(m) and V(max) were determined to be 1.1 mM and 54.6 µmol min(-1) respectively.

Two gene clusters co‐ordinate for a functional N‐acetylglucosamine catabolic pathway in Vibrio cholerae

Pathogenic microorganisms like Vibrio cholerae are capable of adapting to diverse living conditions, especially when they transit from their environmental reservoirs to human host. V. cholerae attaches to N-acetylglucosamine (GlcNAc) residues in glycoproteins and lipids present in the intestinal epithelium and chitinous surface of zoo-phytoplanktons in the aquatic environment for its survival and colonization. GlcNAc utilization thus appears to be important for the pathogen to reach sufficient titres in the intestine for producing clinical symptoms of cholera. We report here the involvement of a second cluster of genes working in combination with the classical genes of GlcNAc catabolism, suggesting the occurrence of a novel variant of the process of biochemical conversion of GlcNAc to Fructose-6-phosphate as has been described in other organisms. Colonization was severely attenuated in mutants that were incapable of utilizing GlcNAc. It was also shown that N-acetylglucosamine specific repressor (NagC) performs a dual role - while the classical GlcNAc catabolic genes are under its negative control, the genes belonging to the second cluster are positively regulated by it. Further application of tandem affinity purification to NagC revealed its interaction with a novel partner. Our results provide a genetic program that probably enables V. cholerae to successfully utilize amino - sugars and also highlights a new mode of transcriptional regulation, not described in this organism.

Large-scale assignment of N-glycosylation sites using complementary enzymatic deglycosylation

Endoglycosidase is a class of glycosidases that specifically cleaves the glycosidic bond between two proximal residues of GlcNAc in the pentasaccharide core of N-glycan, leaving the innermost GlcNAc still attached to its parent protein, which provides a different diagnostic maker for N-glycosylation site assignment. This study aims to validate the use of endoglycosidase for high throughput N-glycosylation analysis. An endoglycosidase of Endo H and the conventional PNGase F were employed, with a similar accessible procedure, for large-scale assignment of N-glycosylation sites and then N-glycoproteome for rat liver tissue. ConA affinity chromatography was used to enrich selectively high-mannose and hybrid glycopeptides before enzymatic deglycosylation. As a result, a total of 1063 unique N-glycosites were identified by nano liquid chromatography tandem mass spectrometry, of which 53.0% were unknown in the Swiss-Prot database and 47.1% could be assigned only by either of the methods, confirmed the possibility of large-scale glycoproteomics by use of endoglycosidase. In addition, 11 glycosites were assigned with core-fucosylation by Endo H. A comparison between the two enzymatic deglycosylation methods was also investigated. Briefly, Endo H provides a more confident assignment but a smaller dataset compared with PNGase F, showing the complementary nature of the two N-glycosite assignment methods.

High-mannose N-glycan-specific lectin from the red alga Kappaphycus striatum (Carrageenophyte)

From a fresh sample (1 kg) of cultivated red alga Kappaphycus striatum, three isolectins, KSA-1 (15.1 mg), KSA-2 (58.0 mg) and KSA-3 (6.9 mg), were isolated by a combination of extraction with aqueous ethanol, ethanol precipitation, and ion exchange chromatography. Isolated KSAs were monomeric proteins of about 28 kDa having identical 20 N-terminal amino acid sequences to each other. Their hemagglutination activities were not inhibited by monosaccharides, but inhibited by glycoproteins bearing high-mannose N-glycans. In a binding experiment with pyridylaminated oligosaccharides by centrifugal ultrafiltration-HPLC assay, the isolectin KSA-2 was exclusively bound to high-mannose type N-glycans, but not to other glycans. Including complex types and a pentasaccharide core of N-glycans, indicating that it recognized branched oligomannosides. The binding activity of KSA-2 was slightly different among high-mannose N-glycans examined, indicating that the lectin has a higher affinity for those having the exposed (α1–3) Man in the D2 arm. On the other hand, KSA-2 did not bind to a free oligomannose that is a constituent of the branched oligomannosides, implying that the portion of the core GlcNAc residue(s) of the N-glycans is also essential for binding. Thus, KSA-2 appears to recognize the extended carbohydrate structure with a minimal length of a tetrasaccharide, Man(α1–3)Man(α1–6)Man(β1–4)GlcNAc. This study indicates that K. striatum, which has extensively been cultivated as a source of carrageenan, is a good source of a valuable lectin(s) that is strictly specific for high-mannose N-glycans.

Expression of β-1,4-galactosyltransferase and suppression of β- N-acetylglucosaminidase to aid synthesis of complex N-glycans in insect Drosophila S2 cells

Previously, we have shown that simple paucimannosidic N-glycan structures in insect Drosophila S2 cells arise mainly because of β- N-acetylglucosaminidase (GlcNAcase) action. Thus, in an earlier report, we suppressed GlcNAcase activity and clearly demonstrated that more complex N-glycans with two terminal N-acetylglucosamine (GlcNAc) residues were then synthesized. In the present work, we investigated the synergistic effects of β-1,4-galactosyltransferase (GalT) expression and GlcNAcase suppression on N-glycan patterns. We found that the N-glycan pattern of human erythropoietin secreted by engineered S2 cells expressing GalT but not GlcNAcase was complete, even in small portion, except for sialylation; the N-glycan structures had two terminal galactose (Gal) residues. When GalT was expressed but GlcNAcase was not inhibited, N-glycan with GlcNAc and Gal at only one branch end was synthesized. Therefore, it will be possible to express a complete functional human glycoprotein in engineered Drosophila S2 cells by suppressing GlcNAcase and co-expressing additional glycosyltransferases of N-glycosylation pathway.

Structural characterization of complex O-linked glycans from insect-derived material

Although insects are among the most diverse groups of the animal kingdom and may be found in nearly all environments, one can observe an obvious lack of structural data on their glycosylation ability. Hymenoptera is the second largest of all insect orders with more than 110.000 identified species and includes the most famous examples of social insects’ species such as wasps, bees and ants. In this report, the structural variety of O-glycans has been studied in two Hymenoptera species. In a previous study, we showed that major O-glycans from common wasp ( Vespula germanica) salivary mucins correspond to T and Tn antigen, eventually substituted by phosphoethanolamine or phosphate groups. More detailed structural analysis performed by mass spectrometry revealed numerous minor O-glycan structures bearing Gal, GlcNAc, GalNAc and Fuc residues. Thus, in order to investigate glycosylation diversity in insects, we used common wasp nest ( V. germanica) and hornet nest ( Vespa cabro) as starting materials. These materials were submitted to reductive β-elimination and the released oligosaccharide–alditols further fractionated by multidimensional HPLC. Tandem mass spectrometry analyses combined with NMR data revealed the presence of various families of complex O-glycans differing accordingly to both core structures and external motifs. Glycans from wasp were characterized by the presence of core types 1 and 2, Lewis X and internal Gal-Gal motifs. We also observed unusual O-glycans containing a reducing GalNAc unit directly substituted by a fucose residue. In contrast, hornet O-glycans appeared as a rather homogeneous family of core 1 type O-glycans extended by galactose oligomers.

Synthesis of mucin O-glycan core structures as their p-nitro- and p-aminophenyl glycosides

For the investigation of glycosidases, and for the construction of glycan arrays the p-nitrophenyl- and p-aminophenyl glycosides of mucin O-glycan core structures 1–7 and the 2,6-ST-antigen have been chemically synthesized using d-galactose as a precursor for GalNAc residues. GlcNAc residues have partly been introduced using a 4,6-di-O-benzoyl-2,3-N,O-oxazolidinone-protected donor, which allowed deprotection of the formed di- and tri-saccharides in one step using sodium methoxide.

Negative Ion CID Fragmentation of O-linked Oligosaccharide Aldoses—Charge Induced and Charge Remote Fragmentation

Collision induced dissociation (CID) fragmentation was compared between reducing and reduced sulfated, sialylated, and neutral O-linked oligosaccharides. It was found that fragmentation of the [M - H](-) ions of aldoses with acidic residues gave unique Z-fragmentation of the reducing end GalNAc containing the acidic C-6 branch, where the entire C-3 branch was lost. This fragmentation pathway, which is not seen in the alditols, showed that the process involved charge remote fragmentation catalyzed by a reducing end acidic anomeric proton. With structures containing sialic acid on both the C-3 and C-6 branch, the [M - H](-) ions were dominated by the loss of sialic acid. This fragmentation pathway was also pronounced in the [M - 2H](2-) ions revealing both the C-6 Z-fragment plus its complementary C-3 C-fragment in addition to glycosidic and cross ring fragmentation. This generation of the Z/C-fragment pairs from GalNAc showed that the charges were not participating in their generation. Fragmentation of neutral aldoses showed pronounced Z-fragmentation believed to be generated by proton migration from the C-6 branch to the negatively charged GalNAc residue followed by charge remote fragmentation similar to the acidic oligosaccharides. In addition, A-type fragments generated by charge induced fragmentation of neutral oligosaccharides were observed when the charge migrated from C-1 of the GalNAc to the GlcNAc residue followed by rearrangement to accommodate the (0,2)A-fragmentation. LC-MS also showed that O-linked aldoses existed as interchangeable α/β pyranose anomers, in addition to a third isomer (25% of the total free aldose) believed to be the furanose form.

Secondary cell wall polysaccharides from Bacillus cereus strains G9241, 03BB87 and 03BB102 causing fatal pneumonia share similar glycosyl structures with the polysaccharides from Bacillus anthracis

Secondary cell wall polysaccharides (SCWPs) are important structural components of the Bacillus cell wall and contribute to the array of antigens presented by these organisms in both spore and vegetative forms. We previously found that antisera raised to Bacillus anthracis spore preparations cross-reacted with SCWPs isolated from several strains of pathogenic B. cereus, but did not react with other phylogenetically related but nonpathogenic Bacilli, suggesting that the SCWP from B. anthracis and pathogenic B. cereus strains share specific structural features. In this study, SCWPs from three strains of B. cereus causing severe or fatal pneumonia (G9241, 03BB87 and 03BB102) were isolated and subjected to structural analysis and their structures were compared to SCWPs from B. anthracis. Complete structural analysis was performed for the B. cereus G9241 SCWP using NMR spectroscopy, mass spectrometry and derivatization methods. The analyses show that SCWPs from B. cereus G9241 has a glycosyl backbone identical to that of B. anthracis SCWP, consisting of multiple trisaccharide repeats of: →6)-α-d-GlcpNAc-(1→4)-β-d-ManpNAc-(1→4)-β-d-GlcpNAc-(1→. Both the B. anthracis and pathogenic B. cereus SCWPs are highly substituted at all GlcNAc residues with α- and β-Gal residues, however, only the SCWPs from B. cereus G9241 and 03BB87 carry an additional α-Gal substitution at O-3 of ManNAc residues, a feature lacking in the B. anthracis SCWPs. Both the B. anthracis and B. cereus SCWPs are pyruvylated, with an approximate molecular mass of ≈12,000Da. The implications of these findings regarding pathogenicity and cell wall structure are discussed.

Isothermal Titration Calorimetric and Computational Studies on the Binding of Chitooligosaccharides to Pumpkin (Cucurbita maxima) Phloem Exudate Lectin

The interaction of chitooligosaccharides [(GlcNAc)(2-6)] with pumpkin phloem exudate lectin (PPL) was investigated by isothermal titration calorimetry and computational methods. The dimeric PPL binds to (GlcNAc)(3-5) with binding constants of 1.26-1.53 × 10(5) M(-1) at 25 °C, whereas chitobiose exhibits approximately 66-fold lower affinity. Interestingly, chitohexaose shows nearly 40-fold higher affinity than chitopentaose with a binding constant of 6.16 × 10(6) M(-1). The binding stoichiometry decreases with an increase in the oligosaccharide size from 2.26 for chitobiose to 1.08 for chitohexaose. The binding reaction was essentially enthalpy driven with negative entropic contribution, suggesting that hydrogen bonds and van der Waals' interactions are the main factors that stabilize PPL-saccharide association. The three-dimensional structure of PPL was predicted by homology modeling, and binding of chitooligosaccharides was investigated by molecular docking and molecular dynamics simulations, which showed that the protein binding pocket can accommodate up to three GlcNAc residues, whereas additional residues in chitotetraose and chitopentaose did not exhibit any interactions with the binding pocket. Docking studies with chitohexaose indicated that the two triose units of the molecule could interact with different protein binding sites, suggesting formation of higher order complexes by the higher oligomers of GlcNAc by their simultaneous interaction with two protein molecules.

Different site-specific N-glycan types in wheat ( Triticum aestivum L.) PAP phytase

Phytase activity in grain is essential to make phosphate available to cell metabolism, and in food and feed. Cereals contain the purple acid phosphatase type of phytases (PAPhy). Mature wheat grain is dominated by TaPAPhy_a which, in the present work, has been characterized by extensive peptide and glycopeptide sequencing by mass spectrometry. Seven N-linked glycosylation sites were found. Three of these sites were dominated by variant forms of the XylMan 3FucGlcNAc 2, i.e. the HRP-type of glycan. Complex-type glycans with one or two additional GlcNAc were observed, however in trace amounts only. At four sites the glycan consisted of a single GlcNAc residue. The mature protein is ca. 500 residues in size and appears to be truncated at the N- and C-termini.

N-Glycans of Phaeodactylum tricornutum Diatom and Functional Characterization of Its N-Acetylglucosaminyltransferase I Enzyme

N-glycosylation, a major co- and post-translational event in the synthesis of proteins in eukaryotes, is unknown in aquatic photosynthetic microalgae. In this paper, we describe the N-glycosylation pathway in the diatom Phaeodactylum tricornutum. Bio-informatic analysis of its genome revealed the presence of a complete set of sequences potentially encoding for proteins involved in the synthesis of the lipid-linked Glc(3)Man(9)GlcNAc(2)-PP-dolichol N-glycan, some subunits of the oligosaccharyltransferase complex, as well as endoplasmic reticulum glucosidases and chaperones required for protein quality control and, finally, the α-mannosidase I involved in the trimming of the N-glycan precursor into Man-5 N-glycan. Moreover, one N-acetylglucosaminyltransferase I, a Golgi glycosyltransferase that initiates the synthesis of complex type N-glycans, was predicted in the P. tricornutum genome. We demonstrated that this gene encodes for an active N-acetylglucosaminyltransferase I, which is able to restore complex type N-glycans maturation in the Chinese hamster ovary Lec1 mutant, defective in its endogeneous N-acetylglucosaminyltransferase I. Consistent with these data, the structural analyses of N-linked glycans demonstrated that P. tricornutum proteins carry mainly high mannose type N-glycans ranging from Man-5 to Man-9. Although representing a minor glycan population, paucimannose N-glycans were also detected, suggesting the occurrence of an N-acetylglucosaminyltransferase I-dependent maturation of N-glycans in this diatom.

Wnt/β-Catenin Signaling Down-regulates N-Acetylglucosaminyltransferase III Expression: THE IMPLICATIONS OF TWO MUTUALLY EXCLUSIVE PATHWAYS FOR REGULATION

In previous studies, we reported that N-acetylglucosaminyltransferase III (GnT-III) activity and the enzyme product, bisected N-glycans, both were induced in cells cultured under dense conditions in an E-cadherin-dependent manner (Iijima, J., Zhao, Y., Isaji, T., Kameyama, A., Nakaya, S., Wang, X., Ihara, H., Cheng, X., Nakagawa, T., Miyoshi, E., Kondo, A., Narimatsu, H., Taniguchi, N., and Gu, J. (2006) J. Biol. Chem. 281, 13038-13046). Furthermore, we found that α-catenin, a component of the E-cadherin-catenin complex, was also required for this induction (Akama, R., Sato, Y., Kariya, Y., Isaji, T., Fukuda, T., Lu, L., Taniguchi, N., Ozawa, M., and Gu, J. (2008) Proteomics 8, 3221-3228). To further explore the molecular mechanism of this regulation, the roles of β-catenin, an essential molecule in both cadherin-mediated cell adhesion and canonical Wnt signaling, were investigated. Unexpectedly, shRNA knockdown of β-catenin resulted in a dramatic increase in GnT-III expression and its product, the bisected N-glycans, which was confirmed by RT-PCR and GnT-III activity and by E4-PHA lectin blot analysis. The induction of GnT-III expression increased bisecting GlcNAc residues on β1 integrin, which led to down-regulation of integrin-mediated cell adhesion and cell migration. Immunostaining showed that nuclear localization of β-catenin was greatly suppressed; intriguingly, the knockdown of β-catenin in the nuclei was more effective than that in cell-cell contacts in the knockdown cells, which was also confirmed by Western blot analysis. Stimulation of the Wnt signaling pathway by the addition of exogenous Wnt3a or BIO, a GSK-3β inhibitor, consistently and significantly inhibited GnT-III expression and its products. Conversely, the inhibition of β-catenin translocation into the nuclei increased GnT-III activation. Taken together, the results of the present study are the first to clearly demonstrate that GnT-III expression may be precisely regulated by the interplay of E-cadherin-catenin complex-mediated cell-cell adhesion and Wnt/β-catenin signaling, which are both crucial in the process of epithelial-mesenchymal transitions in physiological and pathological conditions.

Water-Soluble Substrates of the Peptidoglycan-Modifying Enzyme O-Acetylpeptidoglycan Esterase (Ape1) from Neisseria gonorrheae

Peptidoglycan is the component of the bacterial cell wall that is essential for maintaining the shape and rigidity of the cell. As such, its polymeric structure, consisting of alternating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), is also a target for the action of host defense enzymes, such as lysozymes. Many bacteria have developed methods of masking their cell wall from these environmental dangers through the addition of aglycon moieties that prevent recognition or sterically hinder the degradative action of exogenous enzymes that would otherwise prove detrimental to the cell. Peptidoglycan acetyl-transferases (Pat's) and O-acetylpeptidoglycan esterases (Ape's) are the enzymes responsible for the controlled addition and removal of acetate onto the C-6 hydroxyl group of MurNAc residues in peptidoglycan. Studies on Ape1, an O-acetylpeptidoglycan esterase found in Neisseria gonorrheae, have suggested that this enzyme is essential for bacterial viability and thus presents an attractive target for antibacterial design. Previous studies on Ape1 have been hindered by the fact that Ape1's natural substrate is an insoluble polymer. In this paper we outline the design, synthesis, and testing of the water-soluble di- and monosaccharide substrate analogues 1 and 2. Both 1 and 2 serve as substrates of Ape1 with k(cat)/K(M) values of (5.1 ± 1.7) × 10(3) M(-1) s(-1) and (3.1 ± 0.8) × 10(3) M(-1) s(-1), respectively. It was determined that the substitution of the GlcNAc residue in compound 1 with an O-benzyl group in compound 2 did not significantly decrease the enzyme's affinity for the monosaccharide. These findings are important as they demonstrate that the catalytic prowess of Ape1 is not dependent on its binding to a polymeric substrate. This ensures that small molecule transition state/intermediate analogues can also capture the transition state binding energy of Ape1 and potentially serve as potent inhibitors. The synthetic route to compounds 1 and 2 could readily be modified to allow for the installation of a wide variety of functional groups at the MurNAc C-6 position in both the mono- and disaccharide scaffolds. This will serve as a general method for the construction of Ape1 substrates and inhibitors.

N-glycosylation of zona glycoproteins during meiotic maturation is involved in sperm-zona pellucida interactions of porcine oocytes

The objective was to determine whether N-glycosylation of zona pellucida (ZP) glycoproteins occurred during meiotic maturation of porcine oocytes, and whether this had a role in fertilization. In the first of three experiments, carbohydrate residues in the ZP of in vitro matured porcine oocytes were blocked with various lectins and the influence of such blocking on sperm-ZP interactions was studied. The second experiment used a lectin-binding assay to determine whether the number of GlcNAc residues in ZP was changed by N-glycosylation during in vitro maturation (IVM) of porcine oocytes. The last experiment determined the effects of tunicamycin, a specific N-glycosylation inhibitor, for various intervals during IVM, on sperm-ZP interactions in porcine oocytes. The primary findings were that: 1) N-glycosylation of GlcNAc residues in porcine ZP occurred during the first 24 h of IVM; and 2) such glycosylation was indispensible for sperm-ZP interactions, e.g., number of sperm bound to ZP, acrosome-reacted sperm, sperm penetration rate, and level of polyspermy (P < 0.05). However, blocking N-glycosylation by tunicamycin treatment during IVM did not adversely influence the progression of oocytes to meiotic metaphase II and male pronucleus formation, indicating that this glycosylation was involved only in the initial stages of fertilization. We inferred that the increase in terminal GlcNAc residues in ZP glycoprotein through new N-glycosylation during the first 24 h of meiotic maturation played a critical role in porcine ZP acquiring the capacity to accept sperm.