O-GlcNAcylation of carbohydrate-responsive element-binding protein (ChREBP) is believed as an important modulator of ChREBP activities, however little direct evidence of O-GlcNAcylation on ChREBP and ...no exact O-GlcNAcylation sites have been reported so far. Here, we validate O-GlcNAcylation on ChREBP in cell-free coupled transcription/translation system and in cells by chemoenzymatic and metabolic labeling, respectively. Moreover, for the first time, we identify O-GlcNAcylation on Ser614 in the C-terminus of ChREBP by mass spectrometry and validate two important sites, Thr517 and Ser839 for O-GlcNAcylation and their function via molecular and chemical biological method. Under high glucose conditions, Ser514 phosphorylation enhances ChREBP O-GlcNAcylation, maintaining the transcriptional activity of ChREBP; Ser839 O-GlcNAcylation is essential for Mlx-heterodimerization and DNA-binding activity enhancement, consequently inducing transcriptional activity. Ser839 O-GlcNAcylation is also crucial for ChREBP nuclear export partially by strengthening interactions with CRM1 and 14-3-3. This work is a detailed study of ChREBP O-GlcNAcylation and highlights the biological consequences of the site-specific O-GlcNAcylation dynamics of ChREBP.
An efficient protocol for the O-sialylation using thiosialoside donors under visible light photocatalysis was developed. Thiosialosides were activated under the irradiation with blue light in the ...presence of Ru(bpy)3(PF6)2 as photocatalyst, Umemoto’s reagent as CF3 radical source and Cu(OTf)2 as an additive in acetonitrile/dichloromethane at −30 °C, and the subsequent reaction with glycosyl acceptors generally produced the desired sialosides in good to excellent yields with the satisfactory α-selectivity.
Even though a vaccine that targets tumor-associated carbohydrate antigens on epithelial carcinoma cells presents an attractive therapeutic approach, relatively poor immunogenicity limits its ...development. In this study, we investigated the immunological activity of a fluoro-substituted Sialyl-Tn (F-STn) analogue coupled to the non-toxic cross-reactive material of diphtheria toxin197 (CRM197). Our results indicate that F-STn-CRM197 promotes a greater immunogenicity than non-fluorinated STn-CRM197. In the presence or absence of adjuvant, F-STn-CRM197 remarkably enhances both cellular and humoral immunity against STn by increasing antigen-specific lymphocyte proliferation and inducing a mixed Th1/Th2 response leading to production of IFN-γ and IL-4 cytokines, as well as STn-specific antibodies. Furthermore, antisera produced from F-STn-CRM197 immunization significantly recognizes STn-positive tumor cells and increases cancer cell lysis induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) pathways. Our data suggest that this F-STn vaccine may be useful for cancer immunotherapy and possibly for prophylactic prevention of cancer.
Terminal α-2,6-sialylation of
N
-glycans is a humanized glycosylation that affects the properties and efficacy of therapeutic glycoproteins. Fc di-sialylation (a biantennary
N
-glycan with two ...α-2,6-linked sialic acids) of IgG antibodies imparts them with enhanced anti-inflammatory activity and other roles. However, the microheterogeneity of
N
-glycoforms presents a challenge for therapeutic development. Therefore, controlled sialylation has drawn considerable attention, but direct access to well-defined di-sialylated antibodies remains limited. Herein, a one-pot three-enzyme protocol was developed by engineering a bacterial sialyltransferase to facilitate the modification of therapeutic antibodies with
N
-acetylneuraminic acid or its derivatives towards optimized glycosylation. To overcome the low proficiency of bacterial sialyltransferase in antibody remodeling, the
Photobacterium
sp.
JT-ISH-224
α-2,6-sialyltransferase (Psp2,6ST) was genetically engineered by terminal truncation and site-directed mutagenesis based on its protein crystal structure. With the optimized reaction conditions and using activity-based screening of various Psp2,6ST variants, a truncated mutant Psp2,6ST (111-511)-His
6
A235M/A366G was shown to effectively improve the catalytic efficiency of antibody di-sialylation. Herceptin and the donor substrate promiscuity allow the introduction of bioorthogonal modifications of
N
-acetylneuraminic acid into antibodies for site-specific conjugation. 2-AB hydrophilic interaction chromatography analysis of the released
N
-glycans and intact mass characterization confirmed the high di-sialylation of Herceptin
via
the optimized one-pot three-enzyme reaction. This study established a versatile enzymatic approach for producing highly di-sialylated IgG antibodies. It provides new insights into engineering bacterial sialyltransferase for adaptation to the enzymatic glycoengineering of therapeutic antibodies and the glycosite-specific conjugation of antibodies.
A one-pot three-enzyme protocol was developed by engineering a bacterial sialyltransferase to facilitate the modification of therapeutic antibodies with
N
-acetylneuraminic acid or its derivatives towards optimized glycosylation.
The C-glycosylation of C-nucleophiles including allyltrimethylsilane, silyl enol ethers and phenols with N-(glycosyloxy)acetamides as glycosyl donors has been realized. This protocol provides a ...convenient and practical route for the synthesis of alkyl C-glycosides and aryl 2-deoxy-β-C-glycosides under mild reaction conditions.
In the new transition-state based sialyltransferase inhibitors, an amide group was placed at the corresponding C-2 position of CMP-sialic acid to mimic the geometry and charge distribution in the ...transition state, and simple aromatic or aliphatic rings were used instead of the sialic acid moiety. All synthetic compounds exhibited excellent α(2-6)-sialyltransferase inhibition, resulting in up to a 2600-fold higher affinity for the enzyme than CMP-Neu5Ac, suggesting that amide is a key element for simulating transition-state features.
Abnormal glycosylation is a hallmark of tumor development, and tumor-associated carbohydrate antigens are potential immune targets for tumor therapy. Tumor-associated extracellular microvesicles are ...subcellular vesicles released from cell membranes that have immunogenicity similar to that of precursor cells. However, unmodified tumor-derived microvesicles have weaknesses, such as low immunogenicity, poor biostability, and short half-life in vivo. For the first time, we herein generated extracellular microvesicles containing modified tumor-associated carbohydrate antigens by constructing a cell line with highly expressed antigen-processing enzymes utilizing fluorine-modified monosaccharide substrates via a metabolic oligosaccharide engineering strategy. The microvesicles were applied to tumor immunity, achieving enhanced immunoprophylaxis and immunotherapy effects. Furthermore, the mechanisms of antitumor immunity were explored. Our findings may provide new insights into the adhibition of suitably modified extracellular microvesicles and the development of more effective carbohydrate-based anticancer vaccines.
The multifaceted biological importance of carbohydrates has made them very popular targets in modern synthetic chemistry. Great progress has been made in stereoselective glycosylation methods and the ...construction of complex oligosaccharides. This Focus Review highlights the versatile applications of preactivation strategies in stereoselective glycosylation and oligosaccharide synthesis. Recent advances in synthetic methods, such as 4,6‐O‐benzylidene‐acetal‐directed β‐mannosylation, the 1,2‐cis‐glycosylation mediated by a chiral auxiliary, the use of an oxazolidinone or carbonate protecting group, and glycosylation with participating additives, are described. Various examples of sequential glycosylation based on preactivation protocols for efficient oligosaccharide assembly are also summarized.
Pre‐emptive strike: Preactivation has become an indispensable strategy for stereoselective glycosylation and efficient oligosaccharide synthesis. In this Focus Review, recent advances in synthetic methods, such as the directed β‐mannosylation, 1,2‐cis‐glycosylation mediated by a chiral auxiliary, the use of protecting groups (PGs), and glycosylation with participating additives, are described. Preactivation‐based oligosaccharide assembly is also summarized.
A glycosyl coupling reaction via photoinduced direct activation of thioglycosides and subsequent O-glycosylation in the absence of photosensitizer was developed for the first time. This reaction ...underwent a selectively homolytic cleavage of a C–S bond to generate a glycosyl radical, which was oxidized to an oxacarbenium ion by Cu(OTf)2, and a sequential O-glycosylation. A wide range of glycosides were synthesized in moderate to excellent yield using sugars, amino acids, or cholesterol as the acceptors.
Iterative One-Pot Synthesis of Oligosaccharides Huang, Xuefei; Huang, Lijun; Wang, Haisheng ...
Angewandte Chemie (International ed.),
October 4, 2004, Volume:
43, Issue:
39
Journal Article
Peer reviewed
Straight to the point! Preactivation of a p‐tolyl thioglycoside donor, followed by sequential addition of p‐tolyl thioglycosyl acceptors in one reaction flask allowed rapid syntheses of ...oligosaccharides independent of anomeric reactivities of donors and acceptors (see scheme). This strategy greatly streamlines the assembly of oligosaccharides.
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