Search

Glycoblotting, high throughput method for N-glycan enrichment analysis based on the specific chemical ligation between aminooxy/hydrazide-polymers/solids and reducing N-glycans released from whole serum and cellular glycoproteins, was proved to be feasible for selective enrichment analysis of O-glycans of common (mucin) glycoproteins. We established a standard protocol of glycoblotting-based O-glycomics in combination with nonenzymatic chemical treatment to release reducing Oglycans predominantly from various glycoprotein samples. It was demonstrated that the nonreductive condition employing a simple ammonium salt, ammonium carbamate, made glycoblotting-based enrichment analysis of O-glycans possible without significant loss or unfavorable side reactions. A general workflow of glycoblotting using a hydrazide bead (BlotGlyco H), on-bead chemical manipulations, and subsequent mass spectrometry allowed for rapid O-glycomics of human milk osteopontin (OPN) and urinary MUC1 glycoproteins purified from healthy donors in a quantitative manner. It was revealed that structures of O-glycans in human milk OPN were varied with habitual fucosylation and N-acetyllactosamine units. It was also suggested that purified human urinary MUC1 was modified preferentially by sialylated O-glycans (94% of total) with 7:3 ratio of core 1 to core 2 type O-glycans. Versatility of the present strategy is evident because this method was proved to be suited for the enrichment analysis of general biological and clinical samples such as human serum and urine, cultured human cancer cells, and formalin-fixed paraffin-embedded tissue sections. It is our belief that the present protocols would greatly accelerate discovery of disease-relevant O-glycans as potential biomarkers. 10.1021/ac101599p

Changes in protein glycosylation profoundly affect protein function. To understand these effects of altered protein glycosylation, we urgently need high-throughput technologies to analyze glycan expression and glycan-protein interactions. Methods are not available for amplification of glycans; therefore, highly efficient sample preparation is a major issue. Here we present a novel strategy that allows flexible and sequential incorporation of various functional tags into oligosaccharides derived from biological samples in a practical manner. When combined with a chemoselective glycoblotting platform, our analysis enables us to complete sample preparation (from serum to released, purified, methyl-esterified, and labeled glycans) in 8 h from multiple serum samples (up to 96 samples) using a 96-well microplate format and a standard de-N-glycosylation protocol that requires reductive alkylation and tryptic digestion prior to PNGase F digestion to ensure maximal de-N-glycosylation efficiency. Using this technique, we quantitatively detected more than 120 glycans on human carcinoembryonic antigens for the first time. This approach was further developed to include a streamlined method of purification, chromatographic fractionation, and immobilization onto a solid support for interaction analysis. Since our approach enables rapid, flexible, and highly efficient tag conversion, it will contribute greatly to a variety of glycomic studies.

Glycosylation has an important role in regulating properties of proteins and is associated with many diseases. To examine the alteration of serum N-glycans in type 2 diabetes, we used the db/db mouse model. Serum N-glycans were fluorescence labeled and applied to HPLC. There were reproducible differences in N-glycan profiles between the db/db mouse model and the db/+ control. The structures of the oligosaccharides, which had changed in their amounts, were analyzed by a two-dimensional mapping method, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, and exoglycosidase digestion. Those analyses revealed an increase in the N-glycans possessing [alpha]1,6-fucose in the serum of db/db mice. The level of [alpha]1,6-fucosyltransferase mRNA was increased in the liver of the db/db mice. The ratio of a biantennary N-glycan with [alpha]1,6-fucose to that without [alpha]1,6-fucose in the liver tissue of the db/db mouse was increased relative to the db/+ control. Next, we analyzed the serum N-glycan profile in human subjects with type 2 diabetes and found an increased amount of a biantennary N-glycan that had an [alpha]1,6-fucose with a bisecting N-acetylglucosamine. In conclusion, the increase in [alpha]1,6-fucosylation is a striking change in the serum N-glycans of the db/db mice, whereas the change in the fucosylation in humans with type 2 diabetes was small, albeit statistically significant. It is likely that the change is caused, at least partially, by the increase in the ct 1,6-fucosyltransferase mRNA level in the liver. The increased [alpha]1,6-fucosylation may affect protein properties associated with the pathophysiology of type 2 diabetes. [alpha]1,6-fucosylation; [alpha]1,6-fucosyltransferase; liver

The matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS technique described to date has proven to be a convenient and rapid method for identification and characterizations of proteins. However, the general MALDI-TOF MS analysis of complex carbohydrates and glycopeptides still entails special consideration of ionization and the fragmentation characteristics of labile carbohydrate moieties. In this study, an efficient and practical method we termed the matrix-dependent selective fragmentation (MDSF) technique of MALDI-TOF/TOF MS, which allows highly sensitive and reliable fragmentation of oligosaccharides and N-glycopeptides. Results from application of the MDSF technique to TOF/TOF MS analysis demonstrated that in comparison to the conventional postsource decay up to 170 times more sensitive product ion peaks could be obtained. It was also suggested that MDSF generates desired structural information based on the controlled cleavage of the singly charged precursor ion with different electronic excited states made by this method. Ideal product ion peaks observed by MDSF in TOF/TOF MS facilitated structural characterization of complex oligosaccharide derivatives including unstable Neu5Ac and Fuc residues and N-glycopeptides.

Immunoglobulin G (IgG) has a conserved N-glycosylation site at Asn297 in the fragment crystallizable (Fc) region. Previous studies have shown that N-glycosylation of this site is a critical mediator of the antibody's effector functions, such as antibody-dependent cellular cytotoxicity. While the N -glycan structures attached to the IgG-Fc region are generally heterogenous, IgGs engineered to be homogenously glycosylated with functional N -glycans may improve the efficacy of antibodies. The major glycoforms of the N -glycans on the IgG-Fc region are bi-antennary complex-type N -glycans, while multibranched complex-type N -glycans are not typically found. However, IgGs with tri-antennary complex-type N -glycans have been generated using the N -glycan remodeling technique, suggesting that more branched N -glycans might be artificially attached. At present, little is known about the properties of these IgGs. In this study, IgGs with multibranched N -glycans on the Fc region were prepared by using a combination of the glycosynthase/oxazoline substrate-based N -glycan remodeling technique and successive reactions with glycosyltransferases. Among the IgGs produced by these methods, the largest N -glycan attached was a bisecting N -acetylglucosamine containing a sialylated penta-antennary structure. Concerning the Fc-mediated effector functions, the majority of IgGs with tri- and tetra-antennary N -glycans on their Fc region showed properties similar to IgGs with ordinary bi-antennary N -glycans. [ABSTRACT FROM AUTHOR]

A systematic synthesis and structural characterization of a versatile compound library of lactosaminoglycan derivatives based on the substrate specificity of LgtA and human [beta]1,4-galactosyltransferase ([beta]1,4-GalT) toward various synthetic glycosyl acceptors is described. The findings indicate that the sugar extension strategy could be optimally employed for the modification of lactosyl ceramide mimetic polymers to afford convenient precursors for the synthesis of various glycosphingolipids.

A practical synthesis of highly glycosylated MUC1 glycopeptides having core-2 type O-glycans is carried out by combined microwave-assisted solid-phase glycopeptide synthesis (MA-SPGS) irradiation and enzymatic modifications. The reactions have proceeded successfully in the presence of 0.2% Trinton X-100 to convert the chemically synthesized trisaccharide glycans to disialylated hexasaccharide.

A novel mechanism-based fluorescent labeling reagent, 5-acetamido-2- (4-N-5-dimethylaminonaphthalene-1-sulfonyl-2-difluoromethylphenyl)-3,5-dideoxy-D-glycero- alpha-D-ga-lacto-2-nonulopyranosonic acid (1) is reported, which becomes a unique irreversible inhibitor of vibrio cholerae neuraminidase (VCNA). VCNA plays a significant role in the pathogenesis of cholera by removing sialic acid residues from higher-order gangliosides to an unmasked GMI, the essential receptor for cholera toxin.

Despite the growing importance of mucin core O-glycosylation in many biological processes including the protection of epithelial cell surfaces, the immune response, cell adhesion, inflammation, and tumorigenesis/metastasis, the regulation mechanism and conformational significance of the multiple introduction of α-GalNAc residues by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs) remains unclear. Here we report an efficient approach by combining MS and NMR spectroscopy that allows for the identification of O-glycosylation site(s) and the effect of O-glycosylation on the peptide backbone structures during enzymatic mucin domain assembly by using an isoform UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-T2 (ppGalNAcT2) in vitro. An electron-capture dissociation device in a linear radio-frequency quadrupole ion trap (RFQ-ECD) combined with a time-of-flight (TOF) mass spectrometer was employed for the identification of Thr/Ser residues occupied by α-GalNAc branching among multiple and potential O-glycosylation sites in the tandem repeats of human mucin glycoproteins MUC4 (Thr-Ser-Ser-Ala-Ser-Thr-Gly-His-Ala-Thr-Pro-Leu-Pro-Val-Thr-Asp) and MUC5AC (Pro-Thr-Thr-Val-Gly-Ser-Thr-Thr-Val-Gly). In the present study, O-glycosylation was initiated specifically at Thr10 in naked MUC4 peptide and additional introduction of α-GalNAc proceeded preferentially but randomly at three other Thr residues to afford densely glycosylated MUC4 containing six α-GalNAc residues at Thr1, Ser2, Ser5, Thr6, Thr10, and Thr15. On the contrary, O-glycosylation of naked MUC5AC peptide occurred predominantly at consecutive Thr residues and led to MUC5AC with four α-GalNAc residues at Thr2, Thr3, Thr7, and Thr8. The solution structures determined by NMR spectroscopic studies elicited that the preferential introduction of α-GalNAc at Thr10 of MUC4 stabilizes specifically a β-like extended backbone structure at this area, whereas other synthetic models with a single α-GalNAc residue at Thr1, Thr6, or Thr15 did not exhibit any converged three-dimensional structure at the proximal peptide moiety. Such conformational impact on the underlying peptides was proved to be remarkable in the glycosylation at the consecutive Thr residues of MUC5AC., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)