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Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro-heterogeneity) and evaluate the molar site occupancy (macro-heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N-glycans was chemically synthesised by solid-phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N-acetylglucosamine-linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well-defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI-IT, ESI-Q-TOF, MALDI-TOF, ESI/MALDI-FT-ICR-MS). Depending on the ion source/mass analyser, glycopeptides carrying complex-type N-glycans exhibited clearly lower signal strengths (10-50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano-ESI and medium-pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro-heterogeneity and macro-heterogeneity by label-free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics., (Copyright © 2013 John Wiley & Sons, Ltd.)

Carbohydrates are the basis for many therapeutic and diagnostic strategies, yet the full potential of glycans in medicine has not been realized. The study of the precise role of different carbohydrates, bound to either proteins or lipids, is hampered by difficulties in accessing pure, well-defined glycoconjugates. This Review highlights recent advances in glycobiology with a particular emphasis on oligosaccharide synthesis and conjugation techniques for the construction of homogeneous glycoconjugates. New methods for the study of protein-glycan interactions such as carbohydrate arrays and in vivo visualization of glycosylation pattern changes will also be addressed. The development of glycotherapeutics is just beginning, and much remains to be understood about the relationship between glycoconjugate structure and function. The emergence of novel tools will certainly facilitate and expand the use of carbohydrates in therapeutics and diagnostics.

The dendritic cell surface receptor DC-SIGN and the closely related endothelial cell receptor DC-SIGNR specifically recognize high mannose N-linked carbohydrates on viral pathogens. Previous studies have shown that these receptors bind the outer trimannose branch Manalpha1-3[Manalpha1-6]Manalpha present in high mannose structures. Although the trimannoside binds to DC-SIGN or DC-SIGNR more strongly than mannose, additional affinity enhancements are observed in the presence of one or more Manalpha1-2Manalpha moieties on the nonreducing termini of oligomannose structures. The molecular basis of this enhancement has been investigated by determining crystal structures of DC-SIGN bound to a synthetic six-mannose fragment of a high mannose N-linked oligosaccharide, Manalpha1-2Manalpha1-3[Manalpha1-2Manalpha1-6]Manalpha1-6Man and to the disaccharide Manalpha1-2Man. The structures reveal mixtures of two binding modes in each case. Each mode features typical C-type lectin binding at the principal Ca2+-binding site by one mannose residue. In addition, other sugar residues form contacts unique to each binding mode. These results suggest that the affinity enhancement displayed toward oligosaccharides decorated with the Manalpha1-2Manalpha structure is due in part to multiple binding modes at the primary Ca2+ site, which provide both additional contacts and a statistical (entropic) enhancement of binding.

Helicobacter pylori infects over half the world's population, but only 3% of those infected develop peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. In H. pylori, alpha-glucosyl cholesterol constitutes more than 25% of cell wall lipids, and it has been suggested that alpha-glucosyl cholesterol is essential for H. pylori viability. Here, we identified cholesterol alpha-glucosyltransferase (CHLalphaGcT) using an expression cloning strategy and showed that this enzyme is distinctively inhibited by mucin-type O-glycans similar to those present in deeper portions of the gastric mucosa. Moreover, inactivation of CHLalphaGcT by homologous recombination led to H. pylori lethality. These results indicate that H. pylori CHLalphaGcT is a unique enzyme targeted by a natural antibiotic mucin and constitutes an excellent therapeutic target to prevent H. pylori-induced peptic ulcer, gastric carcinoma, and MALT lymphoma.

The fundamental role of glycoconjugates in many biological processes is now well appreciated and has intensified the development of innovative and improved synthetic strategies. All areas of synthetic methodology have seen major advances and many complex, highly branched carbohydrates and glycoproteins have been prepared using solution- and/or solid-phase approaches. The development of an automated oligosaccharide synthesizer provides rapid access to biologically relevant compounds. These chemical approaches help to produce sufficient quantities of defined oligosaccharides for biological studies. Synthetic chemistry also supports an improved understanding of glycobiology and will eventually result in the discovery of new therapeutics.

Defining HIV envelope glycoprotein interactions with host factors or binding partners advances our understanding of the infectious process and provides a basis for the design of vaccines and agents that interfere with HIV entry. Here we employ carbohydrate and glycoprotein microarrays to analyze glycan-dependent gp120-protein interactions. In concert with new linking chemistries and synthetic methods, the carbohydrate arrays combine the advantages of microarray technology with the flexibility and precision afforded by organic synthesis. With these microarrays, we individually and competitively determined the binding profiles of five gp120 binding proteins, established the carbohydrate structural requirements for these interactions, and identified a potential strategy for HIV vaccine development.

[structure: see text] Described is the first automated solid-phase synthesis of the core N-linked pentasaccharide, common to all N-linked glycoproteins via stepwise assembly from mono- and disaccharide building blocks. The challenging beta-mannosidic linkage was incorporated by the inclusion of a disaccharide trichloroacetimidate. This automated synthesis provides rapid access to an oligosaccharide common to an entire class of glycoconjugates.

Glypiation is a common posttranslational modification of eukaryotic proteins involving the attachment of a glycosylphosphatidylinositol (GPI) glycolipid. GPIs contain a conserved phosphoglycan that is modified in a cell‐ and tissue‐specific manner. GPI complexity suggests roles in biological processes and effects on the attached protein, but the difficulties to get homogeneous material have hindered studies. We disclose a one‐pot intein‐mediated ligation (OPL) to obtain GPI‐anchored proteins. The strategy enables the glypiation of folded and denatured proteins with a natural linkage to the glycolipid. Using the strategy, glypiated eGFP, Thy1, and the Plasmodium berghei protein MSP119 were prepared. Glypiation did not alter the structure of eGFP and MSP119 proteins in solution, but it induced a strong pro‐inflammatory response in vitro. The strategy provides access to glypiated proteins to elucidate the activity of this modification and for use as vaccine candidates against parasitic infections. [ABSTRACT FROM AUTHOR]

Carbohydrates and glycoconjugates are involved in a number of biological processes including host-pathogen interactions, cell communication, proliferation and differentiation as well as the initiation of immune responses. However, access to synthetic oligosaccharide structures has been limited for long time since no standardized protocols for the synthesis of complex oligosaccharides from monomeric building block existed. Recent advances in glycochemistry and glycobiology have helped elucidate biological functions of carbohydrates. Novel methods such as automated synthesis of vaccine candidates and immunologically relevant carbohydrates have contributed to this success as well as high-throughput methods such as glycan arrays. This review illustrates the impact of carbohydrate synthesis on the design of efficient carbohydrate-based antimicrobial and anticancer vaccines. Furthermore, this review focuses on how immunological research can benefit from these novel tools provided by glycochemistry. [ABSTRACT FROM AUTHOR]

3-Deoxysugars are important constituents of complex carbohydrates. For example, 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) is an essential component of lipopolysaccharides in Gram-negative bacteria, 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (KDN) is widely found in carbohydrates of the bacterial cell wall and in lower vertebrates, and sialic acid is a common cap of mammalian glycoproteins. Although ready access to such sugars would benefit the creation of vaccine candidates, antibiotics and small-molecule drugs, their chemical synthesis is difficult. Here we present a simple chemoenzymatic method for preparing differentially protected 3-deoxysugar derivatives from readily available starting materials. It exploits the promiscuous aldolase activity of the enzyme macrophomate synthase (MPS) to add pyruvate enolate diastereoselectively to a wide range of structurally complex aldehydes. A short synthesis of KDN illustrates the utility of this approach. Enzyme promiscuity, which putatively fosters large functional leaps in natural evolution, has great promise as a source of synthetically useful catalytic transformations. [ABSTRACT FROM AUTHOR]

The authors discuss the paper by L. Meuris and colleagues regarding the use of an elegant method called GlycoDelete to reduce N-glycan heterogeneity of recombinant glycoproteins in mammalian cells. They mention that the authors performed manipulation to the glycosylation machinery in the Golgi apparatus. They state that the method may be feasible for the manipulation of enzymes in Golgi does not interfere with the quality mediated by N-glycan in the endoplasmic reticulum.

Explores the three main classes of repeating biopolymers and its roles in the cell. Link of small oligosaccharides with proteins and lipids to form glycoproteins and glycolipids; Creation of tools that are commonplace in genomic and proteomics; Role of specific carbohydrates in the infectious process of diseases

Glycomics and glycoproteomics have become indispensible tools in the study of glycoconjugates. Mass spectrometry based methods are standardly used to study the proteome and/or glycome and these approaches are capable of providing both, qualitative and quantitative information using top down techniques. The human immune system marks a particular area of interest for glycomics and glycoproteomics research since a large number of key proteins in innate and adaptive immunity are glycoproteins. In numerous examples, the crucial influence of glycosylation on critical steps such as receptor interaction and binding has been demonstrated. In this review, we focus on different glycomics and glycoproteomics approaches and their application for studying protein glycosylation in the immune system. [Copyright &y& Elsevier]

The study of protein–carbohydrate interactions is one central theme of glycomics research. The challenges encountered when investigating these interactions have resulted in an approach that studies saccharides through the enzymes that process them. Proteins and their function are often probed by manipulating the genes that encode them. Efforts in proteoglycomics exploring protein-binding properties and the enzymatic modification of carbohydrates have intensified, and synthetic tools, including activity- and affinity-based probes, have enhanced our understanding of the roles of carbohydrates in biology. [Copyright &y& Elsevier]

The Gram-positive bacterium Geobacillus stearothermophilus NRS 2004/3a possesses a cell wall containing an oblique surface layer (S-layer) composed of glycoprotein subunits. 0-Glycans with the structure [→2)-α-L-Rhap-(1→3)-β-L-Rhap-(1→2)-α-L-Rhap-(1→]n = 13-18 a 2-O-methyl group capping the terminal repeating unit at the nonreducing end and a L-Rhap- [(1→3)-α-L-Rhap]n = 1-2(1→3)- adaptor are linked via a β-D-Galp residue to distinct sites of the S-layer protein SgsE. S-layer glycan biosynthesis is encoded by a polycistronic slg (surface layer glycosylation) gene cluster. Four assigned glycosyltransferases named WsaC-WsaF, were investigated by a combined biochemical and NMR approach, starting from synthetic octyl-linked saccharide precursors. We demonstrate that three of the enzymes are rhamnosyl-transferases that are responsible for the transfer of L-rhamnose from a dTDP→-L-Rha precursor to the nascent S-layer glycan, catalyzing the formation of the α1,3- (WsaC and WsaD) and β1,2-linkages (WsaF) present in the adaptor saccharide and in the repeating units of the mature S-layer glycan, respectively. These enzymes work in concert with a multifunctional methylrhamnosyltransferase (WsaE). The N-terminal portion of WsaE is responsible for the S-adenosylmethionine-dependent methylation reaction of the terminal α1,3-linked L-rhamnose residue, and the central and C-terminal portions are involved in the transfer of L-rhamnose from dTDP-β-L-rhamnose to the adaptor saccharide to form the α1,2- and α1,3-linkages during S-layer glycan chain elongation, with the methylation and the glycosylation reactions occurring independently. Characterization of these enzymes thus reveals the complete molecular basis for S-layer glycan biosynthesis. [ABSTRACT FROM AUTHOR]