Strict Stereocontrol by 2,4-<i>O</i>-Di-<i>tert</i>-butylsilylene Group on β-Glucuronylations

Strict β-controlled glucuronylations without classical neighboring-group participation were achieved by the assistance of a 2,4-<i>O</i>-di-<i>tert</i>-butylsilylene group. Comparison of activation conditions and conformational analysis indicated that the strict β-selectivity was achieved by steric hindrance of the 2,4-<i>O</i>-di-<i>tert</i>-butylsilylene group and not by complex glycosyl intermediates

Comparative Glycomics of Fat Globule Membrane Glycoconjugates from Buffalo (<i>Bubalus bubalis</i>) Milk and Colostrum

The health-promoting effects of milk fat globule membrane (MFGM) glycoconjugates has attracted curiosity especially with regard to the challenges encountered to unravel the glycan complexities of MFGM glycoproteins and glycosphingolipids. In this context, we characterized glycans present in buffalo milk and colostrum fat globule membranes by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis by adopting chemoselective glycoblotting technique. Unlike human and bovine MFGM glycoproteins, the variations were obvious with respect to their number, size, heterogeneity, and abundance among the samples analyzed. Among N-linked glycans, mono-, di-, and trisialyl glycans were apparent in colostrum, while MFGM predominantly contained mono- and disialyl glycans, in addition to neutral and high-mannose glycoforms. The structural assignments of major glycans were confirmed by TOF/TOF analysis. Core 1 O-glycans were more common in both samples, and the major glycosphingolipids were GM3 and GD3 irrespective of the samples analyzed. The colostrum N-glycans, being effective antibacterials against human pathogens, established the structure–function relationship of oligosaccharides in early milk in providing innate protection to the newborn

Chemical Synthesis Demonstrates That Dynamic O‑Glycosylation Regulates the Folding and Functional Conformation of a Pivotal EGF12 Domain of the Human NOTCH1 Receptor

The interaction of the human NOTCH1 receptor and its ligands is a crucial step in initiating the intracellular signal transductions, in which O-glycosylation of the extracellular EGF-like domain strongly affects multiple aspects of cell differentiation, development, and cancer biology. However, consequences of biosynthetic O-glycosylation processes in the endoplasmic reticulum (ER) and Golgi on the folding of EGF domains remain unclear. Synthetic human NOTCH1 EGF12 modules allow for new insight into the crucial roles of O-glycosylation in the folding and conformation of this pivotal domain. Here, we show for the first time that predominant O-glucosylation at Ser458 facilitates proper folding of the EGF12 domain in the presence of calcium ion, while the nonglycosylated linear EGF12 peptide affords large amounts of misfolded products (>50%) during <i>in vitro</i> oxidative folding. Strikingly, O-fucosylation at Thr466 prior to O-glucosylation at Ser458 totally impedes folding of EGF12 independent of calcium ion, whereas modification of the Fucα1→ moiety with β-linked GlcNAc dramatically enhances folding efficiency. In addition, we elicit that extension of the Glcβ1→ moiety with xyloses is a negative-regulation mechanism in the folding of EGF12 when synthesis of a trisaccharide (Xylα1→3­Xylα1→3­Glcβ1→) dominates over the posttranslational modification at Thr466. Comprehensive nuclear magnetic resonance studies of correctly folded EGF12 modules demonstrate that noncovalently bonded bridges between sugars and peptide moieties, namely sugar bridges, contribute independently to the stabilization of the antiparallel β-sheet in the ligand-binding region. Our results provide evidence that the dynamic O-glycosylation status of the EGF12 domain elaborated in the ER and Golgi strongly affects folding and trafficking of the human NOTCH1 receptor

Synthetic Human NOTCH1 EGF Modules Unraveled Molecular Mechanisms for the Structural and Functional Roles of Calcium Ions and <i>O</i>‑Glycans in the Ligand-Binding Region

The Notch signaling pathway is an evolutionarily highly conserved mechanism that operates across multicellular organisms and is critical for cell-fate decisions during development and homeostasis in most tissues. Notch signaling is modified by posttranslational glycosylations of the Notch extracellular EGF-like domain. To evaluate the structural and functional roles of various glycoforms at multiple EGF domains in the human Notch transmembrane receptor, we established a universal method for the construction of NOTCH1 EGF modules displaying the desired <i>O</i>-glycans at the designated glycosylation sites. The versatility of this strategy was demonstrated by the rapid and highly efficient synthesis of NOTCH1 EGF12 concurrently having a β-d-glucopyranose-initiated glycan (Xylα1→3Xylα1→3Glcβ1→) at Ser458 and α-l-fucopyranose-initiated glycan (Neu5Acα2→3Galβ1→4GlcNAcβ1→3Fucα1→) at Thr466. The efficiency of the proper folding of the glycosylated EGF12 was markedly enhanced in the presence of 5 mM CaCl₂. A nuclear magnetic resonance study revealed the existence of strong nuclear Overhauser effects between key sugar moieties and neighboring amino acid residues, indicating that both <i>O</i>-glycans contribute independently to the intramolecular stabilization of the antiparallel β-sheet structure in the ligand-binding region of EGF12. A preliminary test using synthetic human NOTCH1 EGF modules showed significant inhibitory effects on the proliferation and adhesiveness of human breast cancer cell line MCF-7 and lung adenocarcinoma epithelial cell line A549, demonstrating for the first time evidence that exogenously applied synthetic EGF modules have the ability to interact with intrinsic Notch ligands on the surface of cancer cells

Convergent Solid-Phase Synthesis of Macromolecular MUC1 Models Truly Mimicking Serum Glycoprotein Biomarkers of Interstitial Lung Diseases

Synthetic macromolecular MUC1 glycopeptides have been used to unravel molecular mechanisms in antibody recognition of disease-specific epitopes. We have established a novel synthetic strategy for MUC1 tandem repeats having complex O-glycosylation states at each repeating unit based on convergent solid-phase fragment condensation under microwave irradiation. We have accomplished the synthesis of 77 amino acid MUC1 glycopeptides (MW = 12 759) having three major antigenic O-glycoforms [Tn, core 1 (T), and core 2 structures] at 10 designated positions out of 19 potential O-glycosylation sites. We demonstrate that the macromolecular MUC1 glycopeptide displaying the essential glycopeptidic neoepitope Pro-Asp-Thr­(sialyl-T)-Arg-Pro-Ala-Pro at two different tandem repeats is an excellent serum MUC1 model showing ideal stoichiometric binding with anti-KL6/MUC1 antibody in the sandwich ELISA to quantify human serum KL6/MUC1 levels as a critical biomarker of interstitial lung diseases

Rapid Endolysosomal Escape and Controlled Intracellular Trafficking of Cell Surface Mimetic Quantum-Dots-Anchored Peptides and Glycopeptides

A novel strategy for the development of a high performance nanoparticules platform was established by means of cell surface mimetic quantum-dots (QDs)-anchored peptides/glycopeptides, which was developed as a model system for nanoparticle-based drug delivery (NDD) vehicles with defined functions helping the specific intracellular trafficking after initial endocytosis. In this paper, we proposed a standardized protocol for the preparation of multifunctional QDs that allows for efficient cellular uptake and rapid escaping from the endolysosomal system and subsequent cytoplasmic molecular delivery to the target cellular compartment. Chemoselective ligation of the ketone-functionalized hexahistidine derivative facilitated both efficient endocytic entry and rapid endolysosomal escape of the aminooxy/phosphorylcholine self-assembled monolayer-coated QDs (AO/PCSAM-QDs) to the cytosol in various cell lines such as human normal and cancer cells, while modifications of these QDs with cell-penetrating arginine-rich peptides showed poor cellular uptake and induced self-aggregation of AO/PCSAM-QDs. Combined use of hexahistidylated AO/PCSAM-QDs with serglycine-like glycopeptides, namely synthetic proteoglycan initiators (PGIs), elicited the entry and controlled intracellular trafficking, Golgi localization, and also excretion of these nanoparticles, which suggested that the present approach would provide an ideal platform for the design of high performance NDD systems

Glycopeptides as Targets for Dendritic Cells: Exploring MUC1 Glycopeptides Binding Profile toward Macrophage Galactose-Type Lectin (MGL) Orthologs

The macrophage galactose-type lectin (MGL) recognizes glycan moieties exposed by pathogens and malignant cells. Particularly, mucin-1 (MUC1) glycoprotein presents an altered glycosylation in several cancers. To estimate the ability of distinct MGL orthologs to recognize aberrant glycan cores in mucins, we applied evanescent-field detection to a versatile MUC1-like glycopeptide microarray platform. Here, as binding was sequence-dependent, we demonstrated that not only sugars but also peptide region impact the recognition of murine MGL1 (mMGL1). In addition, we observed for all three MGL orthologs that divalent glycan presentation increased the binding. To assess the utility of the glycopeptide binders of the MGL orthologs for MGL targeting, we performed uptake assays with fluorescein-MUC1 using murine dendritic cells. A diglycosylated MUC1 peptide was preferentially internalized in an MGL-dependent fashion, thus showing the utility for divalent MGL targeting. These findings may be relevant to a rational design of antitumor vaccines targeting dendritic cells via MGL

Large-Scale Glycomics of Livestock: Discovery of Highly Sensitive Serum Biomarkers Indicating an Environmental Stress Affecting Immune Responses and Productivity of Holstein Dairy Cows

Because various stresses strongly influence the food productivity of livestock, biomarkers to indicate unmeasurable environmental stress in domestic animals are of increasing importance. Thermal comfort is one of the basic principles of dairy cow welfare that enhances productivity. To discover sensitive biomarkers that monitor such environmental stresses in dairy cows, we herein performed, for the first time, large-scale glycomics on 336 lactating Holstein cow serum samples over 9 months between February and October. Glycoblotting combined with MALDI-TOF/MS and DMB/HPLC allowed for comprehensive glycomics of whole serum glycoproteins. The results obtained revealed seasonal alterations in serum <i>N</i>-glycan levels and their structural characteristics, such as an increase in high-mannose type <i>N</i>-glycans in spring, the occurrence of di/triantennary complex type <i>N</i>-glycans terminating with two or three Neu5Gc residues in summer and autumn, and <i>N</i>-glycans in winter dominantly displaying Neu5Ac. A multivariate analysis revealed a correlation between the serum expression levels of these season-specific glycoforms and productivity

Generation of Novel Anti-MUC1 Monoclonal Antibodies with Designed Carbohydrate Specificities Using MUC1 Glycopeptide Library

Numerous anti-mucin 1 (anti-MUC1) antibodies that recognize <i>O</i>-glycan core structures have already been developed. However, most of them show low specificities toward <i>O</i>-glycan structures and/or low affinity toward a monovalent epitope. In this study, using an MUC1 glycopeptide library, we established two novel anti-MUC1 monoclonal antibodies (1B2 and 12D10) with designed carbohydrate specificities. Compared with previously reported anti-MUC1 antibodies, 1B2 and 12D10 showed quite different features regarding their specificities, affinities, and reactivity profiles to various cell lines. Both antibodies recognized specific <i>O</i>-glycan structures at the PDT*R motif (the asterisk represents an <i>O</i>-glycosylation site). 1B2 recognized <i>O</i>-glycans with an unsubstituted <i>O</i>-6 position of the GalNAc residue (Tn, T, and 23ST), whereas 12D10 recognized Neu5Ac at the same position (STn, 26ST, and dST). Neither of them bound to glycopeptides with core 2 <i>O</i>-glycans that have GlcNAc at the <i>O</i>-6 position of the GalNAc residue. Furthermore, 1B2 and 12D10 showed a strong binding to not only native MUC1 but also 20-mer glycopeptide with a monovalent epitope. These anti-MUC1 antibodies should thus become powerful tools for biological studies on MUC1 <i>O</i>-glycan structures. Furthermore, the strategy of using glycopeptide libraries should enable the development of novel antibodies with predesigned <i>O</i>-glycan specificities

Swainsonine reduces 5-fluorouracil tolerance in the multistage resistance of colorectal cancer cell lines-4

<p><b>Copyright information:</b></p><p>Taken from "Swainsonine reduces 5-fluorouracil tolerance in the multistage resistance of colorectal cancer cell lines"</p><p>http://www.molecular-cancer.com/content/6/1/58</p><p>Molecular Cancer 2007;6():58-58.</p><p>Published online 21 Sep 2007</p><p>PMCID:PMC2071919.</p><p></p>s of oligosaccharide structures described in the reference [34]