Metabolic Inhibition of Sialyl-Lewis X Biosynthesis by 5-Thiofucose Remodels the Cell Surface and Impairs Selectin-Mediated Cell Adhesion

Sialyl-Lewis X (sLe^X) is a tetrasaccharide that serves as a ligand for the set of cell adhesion proteins known as selectins. This interaction enables adhesion of leukocytes and cancer cells to endothelial cells within capillaries, resulting in their extravasation into tissues. The last step in sLe^X biosynthesis is the α1,3-fucosyltrasferase (FUT)-catalyzed transfer of an L-fucose residue to carbohydrate acceptors. Impairing FUT activity compromises leukocyte homing to sites of inflammation and renders cancer cells less malignant. Inhibition of FUTs is, consequently, of great interest, but efforts to generate glycosyltransferase inhibitors, including FUT inhibitors, has proven challenging. Here we describe a metabolic engineering strategy to inhibit the biosynthesis of sLe^X in cancer cells using peracetylated 5-thio-L-fucose (5T-Fuc). We show that 5T-Fuc is taken up by cancer cells and then converted into a sugar nucleotide analog, GDP-5T-Fuc, that blocks FUT activity and limits sLe^X presentation on HepG2 cells with an EC₅₀ in the low micromolar range. GDP-5T-Fuc itself does not get transferred by either FUT3 or FUT7 at a measurable rate. We further demonstrate that treatment of cells with 5T-Fuc impaired their adhesive properties to immobilized adhesion molecules and human endothelial cells. 5T-Fuc, therefore, is a useful probe that can be used to modulate sLe^X levels in cells to evaluate the consequences of inhibiting FUT-mediated sLe^X formation. These data also reveal the utility of using sugar analogues that lead to formation of donor substrate analogues within cells as a general approach to blocking glycosyltransferases in cells.     

Effect of N-linked glycosylation on glycopeptide and glycoprotein structure

Asparagine-linked glycosylation is an enzyme-catalyzed, co-translational protein modification reaction that has the capacity to influence either the protein folding process or the stability of the native glycoprotein conjugate. Advances in both glycoconjugate chemical synthesis and glycoprotein expression methods have increased the availability of these once elusive biopolymers. The application of spectroscopic methods to these proteins has begun to illuminate the various ways in which the saccharide affects the structure, function and stability of the proteins.     

A molecular chaperone mediates a two-protein enzyme complex and glycosylation of serine-rich streptococcal adhesins

Serine-rich repeat glycoproteins identified from streptococci and staphylococci are important for bacterial adhesion and biofilm formation. Two putative glycosyltransferases, Gtf1 and Gtf2, from Streptococcus parasanguinis form a two-protein enzyme complex that is required for glycosylation of a serine-rich repeat adhesin, Fap1. Gtf1 is a glycosyltransferase; however, the function of Gtf2 is unknown. Here, we demonstrate that Gtf2 enhances the enzymatic activity of Gtf1 by its chaperone-like property. Gtf2 interacted with Gtf1, mediated the subcellular localization of Gtf1, and stabilized Gtf1. Deletion of invariable amino acid residues in a conserved domain of unknown function (DUF1975) at the N terminus of Gtf2 had a greater impact on Fap1 glycosylation than deletion of the C-terminal non-DUF1975 residues. The DUF1975 deletions concurrently reduced the interaction between Gtf1 and Gtf2, altered the subcellular localization of Gtf1, and destabilized Gtf1, suggesting that DUF1975 is crucial for the chaperone activity of Gtf2. Homologous GtfA and GtfB from Streptococcus agalactiae rescued the glycosylation defect in the gtf1gtf2 mutant; like Gtf2, GtfB also possesses chaperone-like activity. Taken together, our studies suggest that Gtf2 and its homologs possess the conserved molecular chaperone activity that mediates protein glycosylation of bacterial adhesins.     

Synthesis,glycosylation and rapid secretion of a glycoprotein by Achlya a primitive eucaryote

Achlya ambisexualis, a water mold, secretes several glycoproteins during exponential growth. Among these is a major protein of 39 000 daltons (protein A-39) which is secreted very rapidly. Protein A-39 is detected among the soluble cellular proteins labeled for 5 min. However, after longer labeling times, an additional 95 000 dalton glycoprotein was immunoprecipitated from among the cytoplasmic proteins by antiserum against protein A-39. This antiserum reacted with a single 37 000 dalton protein from the in vitro translation products of poly(A)-containing RNA in a wheat germ cell-free system which is cleaved to a faster moving component in the presence of dog pancreatic membranes. Immunoprecipitated, chain-completion products of polysomes also show a 37 000 dalton peptide which does not bind to lectins, indicating absence of co-translational cleavage and glycosylation.Tunicamycin inhibits the appearance of the 95 000 dalton protein. Several immunoprecipitable proteins, including protein A-39, having sizes identical to the secretory proteins accumulate in the cytoplasm in the presence of this inhibitor. A short pulse with [³H]glucosamine followed by a chase showed that incorporation in protein A-39 increases while that in 95 000 dalton protein is decreasing. These results suggest that the 95 000 dalton glycoprotein may serve as a glycosyl donor to secretory protein A-39.     

Recent progress in chemical synthesis of bacterial surface glycans

With the continuing advancement of carbohydrate chemical synthesis, bacterial glycomes have become increasingly attractive and accessible synthetic targets. Although bacteria also produce carbohydrate-containing secondary metabolites, our review here will cover recent chemical synthetic efforts on bacterial surface glycans. The obtained compounds are excellent candidates for the development of improved structurally defined glycoconjugate vaccines to combat bacterial infections. They are also important probes for investigating glycan–protein interactions. Glycosylation strategies applied for the formation of some challenging glycosidic bonds of various uncommon sugars in a number of recently synthesized bacterial surface glycans are highlighted.     

Plant O-Hydroxyproline Arabinogalactans Are Composed of Repeating Trigalactosyl Subunits with Short Bifurcated Side Chains

Classical arabinogalactan proteins partially defined by type II O-Hyp-linked arabinogalactans (Hyp-AGs) are structural components of the plant extracellular matrix. Recently we described the structure of a small Hyp-AG putatively based on repetitive trigalactosyl subunits and suggested that AGs are less complex and varied than generally supposed. Here we describe three additional AGs with similar subunits. The Hyp-AGs were isolated from two different arabinogalactan protein fusion glycoproteins expressed in tobacco cells; that is, a 22-residue Hyp-AG and a 20-residue Hyp-AG, both isolated from interferon α2b-(Ser-Hyp)₂₀, and a 14-residue Hyp-AG isolated from (Ala-Hyp)₅₁-green fluorescent protein. We used NMR spectroscopy to establish the molecular structure of these Hyp-AGs, which share common features: (i) a galactan main chain composed of two 1→3 β-linked trigalactosyl blocks linked by a β-1→6 bond; (ii) bifurcated side chains with Ara, Rha, GlcUA, and a Gal 6-linked to Gal-1 and Gal-2 of the main-chain trigalactosyl repeats; (iii) a common side chain structure composed of up to six residues, the largest consisting of an α-l-Araf-(1→5)-α-l-Araf-(1→3)-α-l-Araf-(1→3- unit and an α-l-Rhap-(1→4)-β-d-GlcUAp-(1→6)-unit, both linked to Gal. The conformational ensemble obtained by using nuclear Overhauser effect data in structure calculations revealed a galactan main chain with a reverse turn involving the β-1→6 link between the trigalactosyl blocks, yielding a moderately compact structure stabilized by H-bonds.     

Advances in the biotechnological glycosylation of valuable flavonoids

The natural flavonoids, especially their glycosides, are the most abundant polyphenols in foods and have diverse bioactivities. The biotransformation of flavonoid aglycones into their glycosides is vital in flavonoid biosynthesis. The main biological strategies that have been used to achieve flavonoid glycosylation in the laboratory involve metabolic pathway engineering and microbial biotransformation. In this review, we summarize the existing knowledge on the production and biotransformation of flavonoid glycosides using biotechnology, as well as the impact of glycosylation on flavonoid bioactivity. Uridine diphosphate glycosyltransferases play key roles in decorating flavonoids with sugars. Modern metabolic engineering and proteomic tools have been used in an integrated fashion to generate numerous structurally diverse flavonoid glycosides. In vitro, enzymatic glycosylation tends to preferentially generate flavonoid 3- and 7-O-glucosides; microorganisms typically convert flavonoids into their 7-O-glycosides and will produce 3-O-glycosides if supplied with flavonoid substrates having a hydroxyl group at the C-3 position. In general, O-glycosylation reduces flavonoid bioactivity. However, C-glycosylation can enhance some of the benefits of flavonoids on human health, including their antioxidant and anti-diabetic potential.     

A new synthesis of alpha-arbutin via Lewis acid catalyzed selective glycosylation of tetra-O-benzyl-alpha-D-glucopyranosyl trichloroacetimidate with hydroquinone

alpha-Arbutin has huge application potentials in the cosmetic industry, as its inhibitory effect on human tyrosinase is stronger than that of its naturally occurring anomer arbutin (4-hydroxyphenyl beta-D-glucopyranoside). Enzymatic synthesis was preferred for alpha-arbutin previously, and now a new chemical synthesis is reported. The reaction of tetra-O-benzyl-alpha-D-glucopyranosyl trichloroacetimidate, as glycosyl donor, with hydroquinone was initiated by catalytic amounts of trimethylsilyl trifluoromethanesulfonate (TMSOTf), resulting in 4-hydroxyphenyl 2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranoside with high stereoselectivity and yield, and then to alpha-arbutin quantitatively after deprotection.     

Improvement of dolichol-linked oligosaccharide biosynthesis by the squalene synthase inhibitor zaragozic acid

The majority of congenital disorders of glycosylation (CDG) are caused by defects of dolichol (Dol)-linked oligosaccharide assembly, which lead to under-occupancy of N-glycosylation sites. Most mutations encountered in CDG are hypomorphic, thus leaving residual activity to the affected biosynthetic enzymes. We hypothesized that increased cellular levels of Dol-linked substrates might compensate for the low biosynthetic activity and thereby improve the output of protein N-glycosylation in CDG. To this end, we investigated the potential of the squalene synthase inhibitor zaragozic acid A to redirect the flow of the polyisoprene pathway toward Dol by lowering cholesterol biosynthesis. The addition of zaragozic acid A to CDG fibroblasts with a Dol-P-Man synthase defect led to the formation of longer Dol-P species and to increased Dol-P-Man levels. This treatment was shown to decrease the pathologic accumulation of incomplete Dol pyrophosphate-GlcNAc(2)Man(5) in Dol-P-Man synthase-deficient fibroblasts. Zaragozic acid A treatment also decreased the amount of truncated protein N-linked oligosaccharides in these CDG fibroblasts. The increased cellular levels of Dol-P-Man and possibly the decreased cholesterol levels in zaragozic acid A-treated cells also led to increased availability of the glycosylphosphatidylinositol anchor as shown by the elevated cell-surface expression of the CD59 protein. This study shows that manipulation of the cellular Dol pool, as achieved by zaragozic acid A addition, may represent a valuable approach to improve N-linked glycosylation in CDG cells.     

A convergent strategy for the preparation of N-glycan core di-, tri-, and pentasaccharide thioaldoses for the site-specific glycosylation of peptides and proteins bearing free cysteines

Mammalian glycoprotein biosynthesis produces heterogeneous ranges of proteins that possess the same peptide backbone but differ in the nature and site of glycosylation. This feature has frustrated efforts to develop therapeutic glycoproteins as well as the elucidation of biological functions of individual glycoforms. We have developed an attractive approach to well-defined glycoforms of glycoproteins by oxidative coupling of thioaldoses to cysteine-containing peptides and proteins to give disulfide-linked neoglycoconjugates. To this end, the chemical synthesis di-, tri-, and pentasaccharide N-glycan thioaldoses was undertaken. A convergent approach was used for the preparation of the pentasaccharide containing a 'synthetically difficult' beta-mannoside linkage. This linkage was installed by forming initially the corresponding beta-glucoside-containing pentasaccharide, followed by inversion of configuration at C-2. This approach exploited a levulonyl ester at C-2 of a glucosyl donor, which directed the coupling to give the beta-glucoside exclusively and could be removed selectively using hydrazine acetate without affecting other base-labile functionalities. The resulting alcohol was converted into a triflate, which was displaced by tri-n-butylammonium acetate to give a beta-mannosidic linkage. The trisaccharide N-glycan was prepared in a similar manner. Thioaldoses were prepared by displacing the peracetylated alpha-glycosyl chlorides with thioacetate to give the peracetylated beta-thioacetates, which upon saponification gave the desired compounds. The incubation of molar excesses of chitobiose thioaldose with cysteine-containing glutathione and BSA resulted in the site-specific formation of a disulfide-linked neoglycopeptide and neoglycoprotein, respectively.     

Simple, automated, high resolution mass spectrometry method to determine the disulfide bond and glycosylation patterns of a complex protein: subgroup A avian sarcoma and leukosis virus envelope glycoprotein

Enveloped viruses must fuse the viral and cellular membranes to enter the cell. Understanding how viral fusion proteins mediate entry will provide valuable information for antiviral intervention to combat associated disease. The avian sarcoma and leukosis virus envelope glycoproteins, trimers composed of surface (SU) and transmembrane heterodimers, break the fusion process into several steps. First, interactions between SU and a cell surface receptor at neutral pH trigger an initial conformational change in the viral glycoprotein trimer followed by exposure to low pH enabling additional conformational changes to complete the fusion of the viral and cellular membranes. Here, we describe the structural characterization of the extracellular region of the subgroup A avian sarcoma and leukosis viruses envelope glycoproteins, SUATM129 produced in chicken DF-1 cells. We developed a simple, automated method for acquiring high resolution mass spectrometry data using electron capture dissociation conditions that preferentially cleave the disulfide bond more readily than the peptide backbone amide bonds that enabled the identification of disulfide-linked peptides. Seven of nine disulfide bonds were definitively assigned; the remaining two bonds were assigned to an adjacent pair of cysteine residues. The first cysteine of surface and the last cysteine of the transmembrane form a disulfide bond linking the heterodimer. The surface glycoprotein contains a free cysteine at residue 38 previously reported to be critical for virus entry. Eleven of 13 possible SUATM129 N-linked glycosylation sites were modified with carbohydrate. This study demonstrates the utility of this simple yet powerful method for assigning disulfide bonds in a complex glycoprotein.     

Alpha1,6-fucosyltransferase-deficient mice exhibit multiple behavioral abnormalities associated with a schizophrenia-like phenotype: importance of the balance between the dopamine and serotonin systems

Previously, we reported that α1,6-fucosyltransferase (Fut8)-deficient (Fut8(-/-)) mice exhibit emphysema-like changes in the lung and severe growth retardation due to dysregulation of TGF-β1 and EGF receptors and to abnormal integrin activation, respectively. To study the role of α1,6-fucosylation in brain tissue where Fut8 is highly expressed, we examined Fut8(-/-) mice using a combination of neurological and behavioral tests. Fut8(-/-) mice exhibited multiple behavioral abnormalities consistent with a schizophrenia-like phenotype. Fut8(-/-) mice displayed increased locomotion compared with wild-type (Fut8(+/+)) and heterozygous (Fut8(+/-)) mice. In particular, Fut8(-/-) mice showed strenuous hopping behavior in a novel environment. Working memory performance was impaired in Fut8(-/-) mice as evidenced by the Y-maze tests. Furthermore, Fut8(-/-) mice showed prepulse inhibition (PPI) deficiency. Intriguingly, although there was no significant difference between Fut8(+/+) and Fut8(+/-) mice in the PPI test under normal conditions, Fut8(+/-) mice showed impaired PPI after exposure to a restraint stress. This result suggests that reduced expression of Fut8 is a plausible cause of schizophrenia and related disorders. The levels of serotonin metabolites were significantly decreased in both the striatum and nucleus accumbens of the Fut8(-/-) mice. Likewise, treatment with haloperidol, which is an antipsychotic drug that antagonizes dopaminergic and serotonergic receptors, significantly reduced hopping behaviors. The present study is the first to clearly demonstrate that α1,6-fucosylation plays an important role in the brain, and that it might be related to schizophrenia-like behaviors. Thus, the results of the present study provide new insights into the underlying mechanisms responsible for schizophrenia and related disorders.     

Recent advances on the synthesis of N-linked glycoprotein for the elucidation of glycan functions

Glycoproteins play roles in many biological events, while, the glycan structure-function relationship has remained to be studied. In order to understand glycan function, homogeneous glycoproteins have been synthesized. This review introduced recent progress of their synthetic approaches.     

Development of new glycosylation methodologies for the synthesis of archaeal-derived glycolipid adjuvants

To commercialize the production of glycolipid adjuvants, their synthesis needs to be both robust and inexpensive. Herein we describe a semi-synthetic approach where the lipid acceptor is derived from the biomass of the archaeon Halobacterium salinarum, and the glycosyl donors are chemically synthesized. This work presents some preliminary results using the promoter system N-iodosuccinimide (NIS) and a stable 0.25 M solution of boron trifluoride-trifluoroethanol (BF₃·TFE₂) in dichloromethane. This promoter system allows for the use of peracetyl alkyl(aryl)thioglycosides that are available in high yield from inexpensive disaccharide starting materials by promoting clean glycosylation reactions from which pure product can be easily isolated. Conventional glycosylation using NIS-silver trifluoromethanesulfonate (AgOTf) leads to extensive acetyl transfer to the archaeol acceptor and numerous byproducts that make purification complicated. As part of preliminary structure-adjuvant activity studies, we describe the one-pot synthesis of a gentiobiose β-Glcp-(1→6)-Glcp-SEt donor with an O-2 benzoyl group, which can be used to prepare a disaccharide attached to archaeol in 85% overall yield, and the related glycolipid trisaccharide β-Glcp-(1→6)-β-Glcp-(1→6)-β-Glcp-(1→O)-archaeol. The synthesis of the isomeric β-Glcp-(1→6)-α-Glcp-(1→O)-archaeol featuring a >10:1 α/β α-selective glycosylation using the promoter system N-phenylselenylphthalimide-trifluoromethanesulfonic acid (TfOH) is also presented, along with the adjuvant properties of the corresponding archaeosomes (liposomes comprised entirely of combinations of isoprenoid archaeal-like lipids). These new vaccine formulations extend previous observations that glycolipids are integral to the activation of MHC type I pathways via CD8⁺ antigen-specific T-cells. The β-Glcp-(1→6)-β-Glcp-(1→6)-β-Glcp-(1→O)-archaeol trisaccharide is shown to be more active than the Glcp-(1→6)-β-Glcp-(1→O)-archaeol disaccharide.     

A stereoselective 1,2-cis glycosylation toward the synthesis of a novel N-linked glycan from the Gram-negative bacterium, Campylobacter jejuni

It has been shown that certain prokaryotes, such as Campylobacter jejuni, have asparagine (Asn)-linked glycoproteins. However, the structures of their glycans are distinct from those of eukaryotic origin. They consist of a bacillosamine residue linked to Asn, an alpha-(1-->4)-GalpNAc repeat, and a branching beta-Glcp residue. In this paper, we describe a strategy for the stereoselective construction of the alpha-(1-->4)-GalpNAc repeat of a C. jejuni N-glycan, utilizing a pentafluoropropionyl (PFP) group as a temporary protective group of the C-4 OH group of the GalpN donor. The strategy was applied to the synthesis of the hexasaccharide alpha-GalpNAc-(1-->4)-alpha-GalpNAc-(1-->4)-[beta-Glcp-(1-->3)]-alpha-GalpNAc(1-->4)-alpha-GalpNAc-(1-->4)-GalpNAc.     

Identification of glycoprotein receptors within the human salivary proteome for the lectin‐like BabA and SabA adhesins of Helicobacter pylori by fluorescence‐based 2‐D bacterial overlay

Because gastric infection by Helicobacter pylori takes place via the oral route, possible interactions of this bacterium with human salivary proteins could occur. By using modified 1‐ and 2‐D bacterial overlay, binding of H. pylori adhesins BabA and SabA to the whole range of salivary proteins was explored. Bound salivary receptor molecules were identified by MALDI‐MS and by comparison to previously established proteome maps of whole and glandular salivas. By use of adhesin‐deficient mutants, binding of H. pylori to MUC7 and gp‐340 could be linked to the SabA and BabA adhesins, respectively, whereas binding to MUC5B was associated with both adhesins. Binding of H. pylori to the proline‐rich glycoprotein was newly detected and assigned to BabA adhesin whereas the SabA adhesin was found to mediate binding to newly detected receptor molecules, including carbonic anhydrase VI, secretory component, heavy chain of secretory IgA1, parotid secretory protein and zinc‐α₂‐glycoprotein. Some of these salivary glycoproteins are known to act as scavenger molecules or are involved in innate immunity whereas others might come to modify the pathogenetic properties of this organism. In general, this 2‐D bacterial overlay technique represents a useful supplement in adhesion studies of bacteria with complex protein mixtures.     

Evidence for the glycoprotein nature of the crystalline cell wall surface layer of Bacillus stearothermophilus strain NRS2004/3a

The surface layer of Bacillus stearothermophilus strain NRS2004/3a was isolated and chemically characterized. The results of these initial studies lead to the conclusion that the cell surface protein is glycosylated.     

Early murine T-lymphocyte activation is accompanied by a switch from N-Glycolyl- to N-acetyl-neuraminic acid and generation of ligands for siglec-E

It is well established that murine T-lymphocyte activation is accompanied by major changes in cell-surface sialylation, potentially influencing interactions with sialic acid-binding immunoglobulin-like lectins (siglecs). In the present study, we analyzed early activation of murine CD4+ and CD8+ T-lymphocytes at 24 h. We observed a striking and selective up-regulation in the binding of a recombinant soluble form of siglec-E, an inhibitory siglec, which is expressed on several myeloid cell types including antigen-presenting dendritic cells. In contrast, much lower levels of T cell binding were observed with other siglecs, including sialoadhesin, CD22, and siglec-F and the plant lectins Maackia amurensis leukoagglutinin and Sambucus nigra agglutinin. By mass spectrometry, the sialic acid content of 24-h-activated CD4+ and CD8+ T-lymphocytes exhibited an increased proportion of N-acetyl-neuraminic acid (NeuAc) to N-glycolyl-neuraminic acid (NeuGc) in N-glycans. Reduced levels of NeuGc on the surface of activated T cells were demonstrated using an antibody specific for NeuGc and the expression levels of the gene encoding NeuAc- to NeuGc-converting enzyme, CMP-NeuAc hydroxylase, were also reduced. Siglec-E bound a wide range of sialylated structures in glycan arrays, had a preference for NeuAc versus NeuGc-terminated sequences and could recognize a set of sialoglycoproteins that included CD45, in lysates from activated T-lymphocytes. Collectively, these results show that early in T cell activation, glycan remodelling involves a switch from NeuGc- to NeuAc-terminating oligosaccharides on cell surface glycoproteins. This is associated with a strong up-regulation of siglec-E ligands, which may be important in promoting cellular interactions between early activated T-lymphocytes and myeloid cells expressing this inhibitory receptor.