Structural and functional glycosphingolipidomics by glycoblotting with aminooxy-functionalized gold nanoparticle

Glycosphingolipids (GSLs) synthesized in Golgi apparatus by sequential transfer of sugar residues to a ceramide lipid anchor are ubiquitously distributing on vertebrate plasma membranes. Standardized method allowing for high throughput structural profiling and functional characterization of living cell surface GSLs is of growing importance because they function as crucial signal transduction molecules in various processes of dynamic cellular recognitions. However, methods are not available for amplification of GSLs, while the genomic scale PCR amplification permits large-scale mammalian proteomic analysis.　Here we communicate such an approach to a novel "omics", namely glycosphingolipidomics based on the glycoblotting method. The method, which involves selective ozonolysis of the C-C double bond in ceramide moiety and subsequent enrichment of generated GSL-aldehydes by chemical ligation using aminooxy-functionalized gold nanoparticle (aoGNP) should be of widespread utility for identifying and characterizing whole GSLs present in the living cell surfaces. The present protocol using glycoblotting permitted MALDI-TOFMS-based high throughput structural profiling of mouse brain gangliosides such as GM1, GD1a/GD1b, and GT1b for adult or GD3 in case for embryonic mouse. When mouse melanoma B16 cells were subjected to this protocol, it was demonstrated that gangliosides enriched from the plasma membranes are only GM3 bearing microheteogeneity in the structure of N-acyl chain. Surface plasmon resonance analysis revealed that aoGNP displaying whole GSLs blotted from mouse B16 melanoma cell surfaces can be used directly for monitoring specific interaction with self-assembled monolayer (SAM) of Gg3Cer (gangliotriaosylceramide). Our results indicate that GSL-selective enrichment onto aoGNP from living cell surfaces allows for rapid reconstruction of plasma membrane models mimicking intact GSL-microdomain feasible for further structural and functional characterization

Determination of etoposide serum concentrations in small pediatric samples by an improved method of reversed-phase high-performance liquid chromatography.

Several specific assays have been developed for the measurement of etoposide in biological fluids. As large samples are required for high sensitivity, these systems are not appropriate for a pediatric practice. In the present study, however, an improved method for the determination of serum levels of the anticancer drug etoposide was developed, using high-performance liquid chromatography with fixed-wavelength ultraviolet detection. Etoposide was extracted from serum using dichloromethane. The efficiency of extraction from serum was 85.7 +/- 7.7% for etoposide and 81.1 +/- 8.4% for diphenylhydantoin, the internal standard. The serum concentrations of etoposide were measured in 0.2-ml serum samples. The lower limit of detection was 50 ng/ml. Each measurement was completed within 5 min. The linear quantitation range for etoposide was 0.05-50 microg/ml. This assay presents an alternative method for routine measurement of serum levels of etoposide in the pediatric oncology setting.</p

Fast epitope mapping for the anti-MUC1 monoclonal antibody by combining a one-bead-one-glycopeptide library and a microarray platform

Anti-MUC1 monoclonal antibodies (mAbs) are powerful tools that can be used to recognize cancer-related MUC1 molecules, the O-glycosylation status of which is believed to affect binding affinity. We demonstrate the feasibility of using a rapid screening methodology to elucidate those effects. The approach involves i)"one-bead-one-compound"-based preparation of bilayer resins carrying glycopeptides on the shell and mass-tag tripeptides coding O-glycan patterns in the core, ii)on-resin screening with an anti-MUC1 mAb, iii)separating positive resins by utilizing secondary antibody conjugation with magnetic beads, and (iv)decoding the mass-tag that is detached from the positive resins pool by using mass spectrometric analysis. We tested a small library consisting of 27 MUC1 glycopeptides with different O-glycosylations against anti-MUC1 mAb clone VU-3C6. Qualitative mass-tag analysis showed that increasing the number of glycans leads to an increase in the binding affinity. Six glycopeptides selected from the library were validated by using a microarray-based assay. Our screening provides valuable information on O-glycosylations of epitopes leading to high affinity with mAb

Microwave Effect for Glycosylation Promoted by Solid Super Acid in Supercritical Carbon Dioxide

The effects of microwave irradiation (2.45 GHz, 200 W) on glycosylation promoted by a solid super acid in supercritical carbon dioxide was investigated with particular attention paid to the structure of the acceptor substrate. Because of the symmetrical structure and high diffusive property of supercritical carbon dioxide, microwave irradiation did not alter the temperature of the reaction solution, but enhanced reaction yield when aliphatic acceptors are employed. Interestingly, the use of a phenolic acceptor under the same reaction conditions did not show these promoting effects due to microwave irradiation. In the case of aliphatic diol acceptors, the yield seemed to be dependent on the symmetrical properties of the acceptors. The results suggest that microwave irradiation do not affect the reactivity of the donor nor promoter independently. We conclude that the effect of acceptor structure on glycosylation yield is due to electric delocalization of hydroxyl group and dielectrically symmetric structure of whole molecule

An efficient protocol for the solid-phase synthesis of glycopeptides under microwave irradiation

A standardized and smooth protocol for solid-phase glycopeptides synthesis under microwave irradiation was developed. Double activation system was proved to allow for highly efficient coupling of Tn-Ser/Thr and bulky core 2-Ser/Thr derivatives. Versatility and robustness of the present strategy was demonstrated by constructing a Mucine-1 (MUC1) fragment and glycosylated fragments of tau protein. The success of this approach relies on the combination of microwave energy, a resin consisting totally of polyethylene glycol, a low excess of sugar amino acid and the "double activation" method. © 2012 The Royal Society of Chemistry

Functional network of glycan-related molecules: Glyco-Net in Glycoconjugate Data Bank

<p>Abstract</p> <p>Background</p> <p>Glycans are involved in a wide range of biological process, and they play an essential role in functions such as cell differentiation, cell adhesion, pathogen-host recognition, toxin-receptor interactions, signal transduction, cancer metastasis, and immune responses. Elucidating pathways related to post-translational modifications (PTMs) such as glycosylation are of growing importance in post-genome science and technology. Graphical networks describing the relationships among glycan-related molecules, including genes, proteins, lipids and various biological events are considered extremely valuable and convenient tools for the systematic investigation of PTMs. However, there is no database which dynamically draws functional networks related to glycans.</p> <p>Description</p> <p>We have created a database called Glyco-Net <url>http://www.glycoconjugate.jp/functions/</url>, with many binary relationships among glycan-related molecules. Using search results, we can dynamically draw figures of the functional relationships among these components with nodes and arrows. A certain molecule or event corresponds to a node in the network figures, and the relationship between the molecule and the event are indicated by arrows. Since all components are treated equally, an arrow is also a node.</p> <p>Conclusions</p> <p>In this paper, we describe our new database, Glyco-Net, which is the first database to dynamically show networks of the functional profiles of glycan related molecules. The graphical networks will assist in the understanding of the role of the PTMs. In addition, since various kinds of bio-objects such as genes, proteins, and inhibitors are equally treated in Glyco-Net, we can obtain a large amount of information on the PTMs.</p

Explosive nucleosynthesis in the neutrino-driven aspherical supernova explosion of a non-rotating 15M⊙M_{\odot} star with solar metallicity

We investigate explosive nucleosynthesis in a non-rotating 15$M_\odot$ star with solar metallicity that explodes by a neutrino-heating supernova (SN) mechanism aided by both standing accretion shock instability (SASI) and convection. To trigger explosions in our two-dimensional hydrodynamic simulations, we approximate the neutrino transport with a simple light-bulb scheme and systematically change the neutrino fluxes emitted from the protoneutron star. By a post-processing calculation, we evaluate abundances and masses of the SN ejecta for nuclei with the mass number $\le 70$ employing a large nuclear reaction network. Aspherical abundance distributions, which are observed in nearby core-collapse SN remnants, are obtained for the non-rotating spherically-symmetric progenitor, due to the growth of low-mode SASI. Abundance pattern of the supernova ejecta is similar to that of the solar system for models whose masses ranges (0.4-0.5) \Ms of the ejecta from the inner region (\le 10,000\km) of the precollapse core. For the models, the explosion energies and the \nuc{Ni}{56} masses are $\simeq 10^{51} \rm erg$ and (0.05-0.06) \Ms, respectively; their estimated baryonic masses of the neutron star are comparable to the ones observed in neutron-star binaries. These findings may have little uncertainty because most of the ejecta is composed by matter that is heated via the shock wave and has relatively definite abundances. The abundance ratios for Ne, Mg, Si and Fe observed in Cygnus loop are well reproduced with the SN ejecta from an inner region of the 15\Ms progenitor.Comment: 15 pages, 1 table, 17 figures, accepted for publication in Astrophyscal Journa

Altering the Modular Architecture of Galectins Affects its Binding with Synthetic a-Dystroglycan O-Mannosylated Core M1 Glycoconjugates In situ

The multifunctionality of galectins helps regulate a broad range of fundamental cellular processes via cis-binding and trans-bridging activities and has gained widespread attention with respect to the importance of the natural specificity/selectivity of this lectin family to its glycoconjugate receptors. Combining galectin (Gal)-1, -3, -4, and -9 variant test panels, achieved via rational protein engineering, and a synthetic a-dystroglycan (DG) O-Mannosylated core M1 glycopeptide library, a detailed comparative analysis was performed, utilizing microarray experiments to delineate the design-functionality relationships within this lectin family. Enhancement of prototype Gal-1 and chimera-type Gal-3 cis-binding toward the prepared ligands is possible by transforming these lectins into tandem-repeat type and prototypes, respectively. Furthermore, Gal-1 variants demonstrated improved trans-bridging capabilities between core M1 a-DG glycopeptides and laminins in microarray, suggesting the possible translational applications of these galectin variants in the treatment of some forms of a-dystroglycanopathy