Mass Spectral Similarity Networking and Gas-Phase Fragmentation Reactions in the Structural Analysis of Flavonoid Glycoconjugates.

Flavonoids represent an important class of natural products with a central role in plant physiology and human health. Their accurate annotation using untargeted mass spectrometry analysis still relies on differentiating similar chemical scaffolds through spectral matching to reference library spectra. In this work, we combined molecular network analysis with rules for fragment reactions and chemotaxonomy to enhance the annotation of similar flavonoid glyconjugates. Molecular network topology progressively propagated the flavonoid chemical functionalization according to collision-induced dissociation (CID) reactions, as the following chemical attributes: aglycone nature, saccharide type and number, and presence of methoxy substituents. This structure-based distribution across the spectral networks revealed the chemical composition of flavonoids across intra- and interspecies and guided the putatively assignment of 64 isomers and isobars in the Chrysobalanaceae plant species, most of which are not accurately annotated by automated untargeted MS2 matching. These proof of concept results demonstrate how molecular networking progressively grouped structurally related molecules according to their product ion scans, abundances, and ratios. The approach can be extrapolated to other classes of metabolites sharing similar structures and diagnostic fragments from tandem mass spectrometry.

Sugar Coating the Envelope: Glycoconjugates for Microbe-Host Crosstalk.

Tremendous progress has been made on mapping the mainly bacterial members of the human intestinal microbiota. Knowledge on what is out there, or rather what is inside, needs to be complemented with insight on how these bacteria interact with their biotic environment. Bacterial glycoconjugates, that is, the collection of all glycan-modified molecules, are ideal modulators of such interactions. Their enormous versatility and diversity results in a species-specific glycan barcode, providing a range of ligands for host interaction. Recent reports on the functional importance of glycosylation of important bacterial ligands in beneficial and pathogenic species underpin this. Glycoconjugates, and glycoproteins in particular, are an underappreciated, potentially crucial, factor in understanding bacteria-host interactions of old friends and foes.

Functional network of glycan-related molecules: glyco-net in glycoconjugate data bank.

BACKGROUND: 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. DESCRIPTION: We have created a database called Glyco-Net http://www.glycoconjugate.jp/functions/, 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. CONCLUSIONS: 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.

Characterization of glycoconjugate antigens in mouse embryonic neural precursor cells.

Neuronal and glial cells organizing the central nervous system (CNS) are generated from common neural precursor cells (NPCs) during neural development. However, the expression of cell-surface glycoconjugates that are crucial for determining the properties and biological function of these cells at different stages of development has not been clearly defined. In this study, we investigated the expression of several stage-specific glycoconjugate antigens, including several b-series gangliosides GD3, 9-O-acetyl GD3, GT1b and GQ1b, stage-specific embryonic antigen-1 (SSEA-1) and HNK-1, in mouse embryonic NPCs employing immunocytochemistry and flow cytometry. In addition, several of these antigens were positively identified by chemical means for the first time. We further showed that the SSEA-1 immunoreactivity was contributed by both glycoprotein and glycolipid antigens, and that of HNK-1 was contributed only by glycoproteins. Functionally, SSEA-1 may participate in migration of the cells from neurospheres in an NPC cell culture system, and the effect could be repressed by anti-SSEA-1 antibody. Based on this observation, we identified beta1 integrin as one of the SSEA-1 carrier glycoproteins. Our data thus provide insights into the functional role of certain glycoconjugate antigens in NPCs during neural development.

Chemical and biological strategies for engineering cell surface glycosylation.

Oligosaccharides play a crucial role in many of the recognition, signaling, and adhesion events that take place at the surface of cells. Abnormalities in the synthesis or presentation of these carbohydrates can lead to misfolded and inactive proteins, as well as to several debilitating disease states. However, their diverse structures, which are the key to their function, have hampered studies by biologists and chemists alike. This review presents an overview of techniques for examining and manipulating cell surface oligosaccharides through genetic, enzymatic, and chemical strategies.

Lectin-histochemical detection of degenerative glycoconjugate deposits in human brain.

Several lectins were used to study the localization of glycoconjugates in brain of elderly people and patients with Alzheimer type dementia (ATD) and Down's syndrome (DS). Five kinds of degenerated or deposited materials stained clearly by lectins specific to GalNAC, Gal, Fuc, and/or Man were recognized much in ATD and DS, less in elderly peoples, in addition to the binding of the lectins to neurons. (i) Round shape deposits called corpora amylacea (CA) which consisted of various sizes of round material, existed mainly on the surface of cerebral cortex and some in white matter of the brain. They were colored by Alcian blue (AB), Aldehyde fucsin (AF) and periodic acid shiff (PAS) and weakly by Hematoxylin (H), but not by Eosin (B). They showed clear reactivity with lectins specific to GalNAC, Gal, Fuc and Gal-GalNAC. (ii) Amorphous and variform amyloid deposits existed around blood vessels in the white matter were stained by thioflavin and lectins specific to GalNAC, Gal and Fuc, but not with Man specific lectins and PAS, AB, AF and HE. (iii) Another kind of amyloid deposits which showed a similar characteristic to the previous one and were recognized mainly in white matter and independent blood vessels. These deposits were stained by thioflavin but not by PAS, AB, AF and HE and showed good reactivity with lectins specific to GalNAC, Gal, Fuc, Gal-GalNAC, Gal-GIcNAc and Man. The reactivity with lectins specific to Gal, Fuc, and Man was seen in senile plaques (iv) and neurofibrillary tangles (v). Although at present we are unable to explain the origin of these deposits, it is clear from this study that the glycoconjugates form an integral part of the degeneration in the brain. The lectin staining with GS-I is useful in the forensic pathology to diagnose brain disorders at postmortem examination, since these lectin were able to detect five types of degeneration changes and/or deposits.

Strategies for glycoconjugate analysis.

Glycoconjugates such as glycoproteins and glycosphingolipids exhibit multiple functions in biological systems. A correlation of functional features with defined structural parameters, however, presupposes detailed information on the glycoconjugates' carbohydrate moieties including the type of linkage of the oligosaccharide chain(s), monosaccharide composition as well as sequence, linkage positions and anomeric configurations of individual sugar monomers. Chemical and biochemical analyses are often impeded by the limited amounts of sample available and the vast structural heterogeneity of glycoconjugate glycans, thus requiring highly sensitive and efficient methods for detection, separation and structural elucidation of these carbohydrates. The aim of the article is to present suitable strategies for structural characterization of glycoconjugate glycans and to briefly review some of the techniques commonly used in this field.

Ultrastructural localization and semiquantitative analysis of glycoconjugates in the tectorial membrane.

The tectorial membrane of the gerbil cochlea was analyzed with lectin-gold cytochemical methods for demonstrating and characterizing glycoconjugates (GCs) in situ. Binding of lectins from Limax flavus (LFA), Lens culinaris (LCA), Datura stramonium (DSA), Ricinus communis (RCA I), Ulex europeus (UEA I) and Phaseolus vulgaris (PHA L) was assayed semiquantitatively on ultrathin sections. Binding occurred throughout the tectorial membrane with all lectins except UEA I but the labelling density with a given lectin differed among substructures. The cover net disclosed the highest level of GC with four lectins whereas the fibrous layer revealed the lowest level. DSA, LCA and PHA L demonstrated considerable similarity between the cover net and the marginal band in content of GC with N-linked oligosaccharide. The cover net differed from the marginal band, however, in containing more RCA I reactive GC with terminal lactosamine. Hensen's stripe, with which inner hair cell stereocilia are thought to interact, differed from other substructures in containing the highest level of PHA L-reactive traintennate N-linked chains and except for the basal layer the lowest concentration of GC with terminal lactosamine. Fucosylated GC detectable with UEA I-gold was present at low levels in all substructures except the cover net and marginal band. Distribution of GCs in the fibrous layer and less consistently in the cover net differed between limbal and middle zones. The differences observed here in the carbohydrate composition among substructures in the tectorial membrane support and extend previous cytochemical observations and imply a role for different classes of GCs in determining the biophysical and physiological properties of the tectorial membrane.