A Unified Strategy for the Synthesis of Mucin Cores 1–4 Saccharides and the Assembled Multivalent Glycopeptides

By displaying different O‐glycans in a multivalent mode, mucin and mucin‐like glycoproteins are involved in a plethora of protein binding events. The understanding of the roles of the glycans and the identification of potential glycan binding proteins are major challenges. To enable future binding studies of mucin glycan and glycopeptide probes, a method that gives flexible and efficient access to all common mucin core‐glycosylated amino acids was developed. Based on a convergent synthesis strategy starting from a shared early stage intermediate by differentiation in the glycoside acceptor reactivity, a common disaccharide building block allows for the creation of extended glycosylated amino acids carrying the mucin type‐2 cores 1–4 saccharides. Formation of a phenyl‐sulfenyl‐N‐Troc (Troc=trichloroethoxycarbonyl) byproduct during N‐iodosuccinimide‐promoted thioglycoside couplings was further characterized and a new methodology for the removal of the Troc group is described. The obtained glycosylated 9‐fluorenylmethoxycarbonyl (Fmoc)‐protected amino acid building blocks are incorporated into peptides for multivalent glycan display.     

Application of Lectin Microarrays for Biomarker Discovery

Many proteins in living organisms are glycosylated. As their glycan patterns exhibit protein-, cell-, and tissue-specific heterogeneity, changes in the glycosylation levels could serve as useful indicators of various pathological and physiological states. Thus, the identification of glycoprotein biomarkers from specific changes in the glycan profiles of glycoproteins is a trending field. Lectin microarrays provide a new glycan analysis platform, which enables rapid and sensitive analysis of complex glycans without requiring the release of glycans from the protein. Recent developments in lectin microarray technology enable high-throughput analysis of glycans in complex biological samples. In this review, we will discuss the basic concepts and recent progress in lectin microarray technology, the application of lectin microarrays in biomarker discovery, and the challenges and future development of this technology. Given the tremendous technical advancements that have been made, lectin microarrays will become an indispensable tool for the discovery of glycoprotein biomarkers.     

Double‐layer poly(vinyl alcohol)‐coated capillary for highly sensitive and stable capillary electrophoresis and capillary electrophoresis with mass spectrometry glycan analysis

Glycosylation plays an important role in protein conformations and functions as well as many biological activities. Capillary electrophoresis combined with various detection methods provided remarkable developments for high‐sensitivity glycan profiling. The coating of the capillary is needed for highly polar molecules from complex biosamples. A poly(vinyl alcohol)‐coated capillary is commonly utilized in the capillary electrophoresis separation of saccharides sample due to the high‐hydrophilicity properties. A modified facile coating workflow was carried out to acquire a novel multiple‐layer poly(vinyl alcohol)‐coated capillary for highly sensitive and stable analysis of glycans. The migration time fluctuation was used as index in the optimization of layers and a double layer was finally chosen, considering both the effects and simplicity in fabrication. With migration time relative standard deviation less than 1% and theoretical plates kept stable during 100 consecutive separations, the method was presented to be suitable for the analysis of glycosylation with wide linear dynamic range and good reproducibility. The glycan profiling of enzymatically released N‐glycans from human serum was obtained by the presented capillary electrophoresis method combined with mass spectrometry detection with acceptable results.     

Absolute Quantitation of Glycosylation Site Occupancy Using Isotopically Labeled Standards and LC-MS

N-linked glycans are required to maintain appropriate biological functions on proteins. Underglycosylation leads to many diseases in plants and animals; therefore, characterizing the extent of glycosylation on proteins is an important step in understanding, diagnosing, and treating diseases. To determine the glycosylation site occupancy, protein N-glycosidase F (PNGase F) is typically used to detach the glycan from the protein, during which the formerly glycosylated asparagine undergoes deamidation to become an aspartic acid. By comparing the abundance of the resulting peptide containing aspartic acid against the one containing non-glycosylated asparagine, the glycosylation site occupancy can be evaluated. However, this approach can give inaccurate results when spontaneous chemical deamidation of the non-glycosylated asparagine occurs. To overcome this limitation, we developed a new method to measure the glycosylation site occupancy that does not rely on converting glycosylated peptides to their deglycosylated forms. Specifically, the overall protein concentration and the non-glycosylated portion of the protein are quantified simultaneously by using heavy isotope-labeled internal standards coupled with LC-MS analysis, and the extent of site occupancy is accurately determined. The efficacy of the method was demonstrated by quantifying the occupancy of a glycosylation site on bovine fetuin. The developed method is the first work that measures the glycosylation site occupancy without using PNGase F, and it can be done in parallel with glycopeptide analysis because the glycan remains intact throughout the workflow.

Analysis of a series of isomeric oligosaccharides by energy‐resolved mass spectrometry: a challenge on homobranched trisaccharides

Glycans exist as part of glycoproteins and glycolipids, which are involved in a variety of biological functions. The analysis of glycan structures, particularly that of structural isomers, is fundamentally important since isomeric glycans often show distinct functions; however, a method for their structural elucidation has not yet been established. Anomeric configurations, linkage positions and branching are the major factors in glycans and their alteration results in a large diversity of glycan structures. The analysis of vicinally substituted oligosaccharides is extremely difficult because the product ions formed in tandem mass spectrometry (MS/MS) often have the same m/z values. In our endeavor to address the issue, we analyzed a series of homo‐substituted trisaccharides consisting only of glucose by collision‐induced dissociation (CID), especially energy‐resolved mass spectrometry (ERMS). It was found that these structurally related glycans could be distinguished by taking advantage of differences in their activation energies in ERMS. Copyright © 2009 John Wiley & Sons, Ltd.     

Transduction of glycan-lectin binding using near-infrared fluorescent single-walled carbon nanotubes for glycan profiling

There is significant interest in developing new detection platforms for characterizing glycosylated proteins, despite the lack of easily synthesized model glycans or high affinity receptors for this analytical problem. In this work, we demonstrate a sensor array employing recombinant lectins as glycan recognition sites tethered via Histidine tags to Ni(2+) complexes that act as fluorescent quenchers for semiconducting single-walled carbon nanotubes (SWNTs) embedded in a chitosan hydrogel spot to measure binding kinetics of model glycans. We examine, as model glycans, both free and streptavidin-tethered biotinylated monosaccharides. Two higher-affined glycan-lectin pairs are explored: fucose (Fuc) to PA-IIL and N-acetylglucosamine (GlcNAc) to GafD. The dissociation constants (K(D)) for these pairs as free glycans (106 and 19 μM, respectively) and streptavidin-tethered (142 and 50 μM respectively) were found. The absolute detection limit for the current platform was found to be 2 μg of glycosylated protein or 100 ng of free glycan to 20 μg of lectin. Glycan detection (GlcNAc-streptavidin at 10 μM) is demonstrated at the single nanotube level as well by monitoring the fluorescence from individual SWNT sensors tethered to GafD lectin. Over a population of 1000 nanotubes, 289 of the SWNT sensors had signals strong enough to yield kinetic information (K(D) of 250 ± 10 μM). We are also able to identify the locations of "strong transducers" on the basis of dissociation constant (four sensors with K(D) < 10 μM) or overall signal modulation (eight sensors with >5% quench response). We report the key finding that the brightest SWNTs are not the best transducers of glycan binding. SWNTs ranging in intensity between 50 and 75% of the maximum show the greatest response. The ability to pinpoint strong-binding, single sensors is promising to build a nanoarray of glycan-lectin transducers as a high throughput method to profile glycans without protein labeling or glycan liberation pretreatment steps.     

Breaking the Limits in Analyzing Carbohydrate Recognition by NMR Spectroscopy: Resolving Branch‐Selective Interaction of a Tetra‐Antennary N‐Glycan with Lectins

The biological recognition of complex‐type N ‐glycans is part of many key physiological and pathological events. Despite their importance, the structural characterization of these events remains unsolved. The inherent flexibility of N ‐glycans hampers crystallization and the chemical equivalence of individual branches precludes their NMR characterization. By using a chemoenzymatically synthesized tetra‐antennary N ‐glycan conjugated to a lanthanide binding tag, the NMR signals under paramagnetic conditions discriminated all four N ‐acetyl lactosamine antennae with unprecedented resolution. The NMR data revealed the conformation of the N ‐glycan and permitted for the first time the direct identification of individual branches involved in the recognition by two N ‐acetyllactosamine‐binding lectins, Datura stramonium seed lectin (DSL) and Ricinus Communis agglutinin (RCA120).     

Comprehensive native glycan profiling with isomer separation and quantitation for the discovery of cancer biomarkers

Glycosylation is highly sensitive to the biochemical environment and has been implicated in many diseases including cancer. Glycan compositional profiling of human serum with mass spectrometry has already identified potential biomarkers for several types of cancer and diseases; however, composition alone does not fully describe glycan stereo- and regioisomeric diversity. The vast structural heterogeneity of glycans presents a formidable analytical challenge. We have developed a method to identify and quantify isomeric native glycans using nanoflow liquid chromatography (nano-LC)/mass spectrometry. A microfluidic chip packed with graphitized carbon was used to chromatographically separate the glycans. To determine the utility of this method for structure-specific biomarker discovery, we analyzed serum samples from two groups of prostate cancer patients with different prognoses. More than 300 N-glycan species (including isomeric structures) were identified, corresponding to over 100 N-glycan compositions. Statistical tests established significant differences in glycan abundances between patient groups. This method provides comprehensive, selective, and quantitative glycan profiling.     

Metabolic Labeling and Imaging of N‐Linked Glycans in Arabidopsis Thaliana

Molecular imaging of glycans has been actively pursued in animal systems for the past decades. However, visualization of plant glycans remains underdeveloped, despite that glycosylation is essential for the life cycle of plants. Metabolic glycan labeling in Arabidopsis thaliana by using N‐azidoacetylglucosamine (GlcNAz) as the chemical reporter is reported. GlcNAz is metabolized through the salvage pathway of N‐acetylglucosamine (GlcNAc) and incorporated into N‐linked glycans, and possibly intracellular O‐GlcNAc. Click‐labeling with fluorescent probes enables visualization of newly synthesized N‐linked glycans. N‐glycosylation in the root tissue was discovered to possess distinct distribution patterns in different developmental zones, suggesting that N‐glycosylation is regulated in a developmental stage‐dependent manner. This work shows the utility of metabolic glycan labeling in elucidating the function of N‐linked glycosylation in plants.     

Rational Design and Identification of a Non‐Peptidic Aggregation Inhibitor of Amyloid‐β Based on a Pharmacophore Motif Obtained from cyclo[‐Lys‐Leu‐Val‐Phe‐Phe‐]

Inhibition of pathogenic protein aggregation may be an important and straightforward therapeutic strategy for curing amyloid diseases. Small‐molecule aggregation inhibitors of Alzheimer’s amyloid‐β (Aβ) are extremely scarce, however, and are mainly restricted to dye‐ and polyphenol‐type compounds that lack drug‐likeness. Based on the structure‐activity relationship of cyclic Aβ16–20 (cyclo‐[KLVFF]), we identified unique pharmacophore motifs comprising side‐chains of Leu², Val³, Phe⁴, and Phe⁵ residues without involvement of the backbone amide bonds to inhibit Aβ aggregation. This finding allowed us to design non‐peptidic, small‐molecule aggregation inhibitors that possess potent activity. These molecules are the first successful non‐peptidic, small‐molecule aggregation inhibitors of amyloids based on rational molecular design.     

Nucleophile‐Directed Stereocontrol Over Glycosylations Using Geminal‐Difluorinated Nucleophiles

The glycosylation reaction is the key transformation in oligosaccharide synthesis, but it is still difficult to control in many cases. Stereocontrol during cis‐glycosidic linkage formation relies almost exclusively on tuning the glycosylating agent or the reaction conditions. Herein, we use nucleophile‐directed stereocontrol to manipulate the stereoselectivity of glycosylation reactions. Placing two fluorine atoms in close proximity to the hydroxy group of an aliphatic amino alcohol lowers the oxygen nucleophilicity and reverses the stereoselectivity of glycosylations to preferentially form the desired cis‐glycosides with a broad set of substrates. This concept was applied to the design of a cis‐selective linker for automated glycan assembly. Fluorination of an amino alcohol linker does not impair glycan immobilization and lectin binding as illustrated by glycan microarray experiments. These fluorinated linkers enable the facile generation of α‐terminating synthetic glycans for the formation of glycoconjugates.     

How Post‐Translational Modifications Influence Amyloid Formation: A Systematic Study of Phosphorylation and Glycosylation in Model Peptides

A reciprocal relationship between phosphorylation and O‐glycosylation has been reported for many cellular processes and human diseases. The accumulated evidence points to the significant role these post‐translational modifications play in aggregation and fibril formation. Simplified peptide model systems provide a means for investigating the molecular changes associated with protein aggregation. In this study, by using an amyloid‐forming model peptide, we show that phosphorylation and glycosylation can affect folding and aggregation kinetics differently. Incorporation of phosphoserines, regardless of their quantity and position, turned out to be most efficient in preventing amyloid formation, whereas O‐glycosylation has a more subtle effect. The introduction of a single β‐galactose does not change the folding behavior of the model peptide, but does alter the aggregation kinetics in a site‐specific manner. The presence of multiple galactose residues has an effect similar to that of phosphorylation.     

Correlating Glycoforms of DC‐SIGN with Stability Using a Combination of Enzymatic Digestion and Ion Mobility Mass Spectrometry

The immune scavenger protein DC‐SIGN interacts with glycosylated proteins and has a putative role in facilitating viral infection. How these recognition events take place with different viruses is not clear and the effects of glycosylation on the folding and stability of DC‐SIGN have not been reported. Herein, we report the development and application of a mass‐spectrometry‐based approach to both uncover and characterise the effects of O‐glycans on the stability of DC‐SIGN. We first quantify the Core 1 and 2 O‐glycan structures on the carbohydrate recognition and extracellular domains of the protein using sequential exoglycosidase sequencing. Using ion mobility mass spectrometry, we show how specific O‐glycans, and/or single monosaccharide substitutions, alter both the overall collision cross section and the gas‐phase stability of the DC‐SIGN isoforms. We find that rather than the mass or length of glycoprotein modifications, the stability of DC‐SIGN is better correlated with the number of glycosylation sites.     

A Self‐Assembled Spherical Complex Displaying a Gangliosidic Glycan Cluster Capable of Interacting with Amyloidogenic Proteins

Physiological and pathological functions of glycans are promoted through their clustering effects as exemplified by a series of gangliosides, sialylated glycosphingolipids, which serve as acceptors for bacterial toxins and viruses. Furthermore, ganglioside GM1 clusters on neuronal cell membranes specifically interact with amyloidogenic proteins, triggering their conformational transitions and leading to neurodegeneration. Here we develop a self‐assembled spherical complex that displays a cluster of the GM1 pentasaccharide, and successfully demonstrate its ability to interact with amyloid β and α‐synuclein. Due to the lack of hydrophobic lipid moieties, which would stably trap these cohesive proteins or give rise to toxic aggregates, this artificial cluster enabled NMR spectroscopic characterization of the early encounter stage of protein interactions with its outer carbohydrate moieties, which were not observable with previous glycan clusters.     

Chemoenzymatic Assembly of Mammalian O‐Mannose Glycans

O‐Mannose glycans account up to 30 % of total O‐glycans in the brain. Previous synthesis and functional studies have only focused on the core M3 O‐mannose glycans of α‐dystroglycan, which are a causative factor for various muscular diseases. In this study, a highly efficient chemoenzymatic strategy was developed that enabled the first collective synthesis of 63 core M1 and core M2 O‐mannose glycans. This chemoenzymatic strategy features the gram‐scale chemical synthesis of five judiciously designed core structures, and the diversity‐oriented modification of the core structures with three enzyme modules to provide 58 complex O‐mannose glycans in a linear sequence that does not exceed four steps. The binding profiles of synthetic O‐mannose glycans with a panel of lectins, antibodies, and brain proteins were also explored by using a printed O‐mannose glycan array.     

Nanochaperone‐Based Strategies to Control Protein Aggregation Linked to Conformational Diseases

The generation of highly organized amyloid fibrils is associated with a wide range of conformational pathologies, including primarily neurodegenerative diseases. Such disorders are characterized by misfolded proteins that lose their normal physiological roles and acquire toxicity. Recent findings suggest that proteostasis network impairment may be one of the causes leading to the accumulation and spread of amyloids. These observations are certainly contributing to a new focus in anti‐amyloid drug design, whose efforts are so far being centered on single‐target approaches aimed at inhibiting amyloid aggregation. Chaperones, known to maintain proteostasis, hence represent interesting targets for the development of novel therapeutics owing to their potential protective role against protein misfolding diseases. In this minireview, research on nanoparticles that can either emulate or help molecular chaperones in recognizing and/or correcting protein misfolding is discussed. The nascent concept of “nanochaperone” may indeed set future directions towards the development of cost‐effective, disease‐modifying drugs to treat several currently fatal disorders.     

Chemical Synthesis of Asparagine‐Linked Archaeal N‐Glycan from Methanothermus fervidus

Several N‐linked glycoproteins have been identified in archaea and there is growing evidence that the N‐glycan is involved in survival and functioning of archaea in extreme conditions. Chemical synthesis of the archaeal N‐glycans represents a crucial step towards understanding the putative function of protein glycosylation in archaea. Herein the first total synthesis of the archaeal L ‐asparagine linked hexasaccharide from Methanothermus fervidus is reported using a highly convergent [3+3] glycosylation approach in high overall yields. The synthesis relies on efficient preparation of regioselectively protected thioglycoside building blocks for orthogonal glycosylations and late stage N‐aspartylation.     

Recognition of Complex Core‐Fucosylated N‐Glycans by a Mini Lectin

The mini fungal lectin PhoSL was recombinantly produced and characterized. Despite a length of only 40 amino acids, PhoSL exclusively recognizes N‐glycans with α1,6‐linked fucose. Core fucosylation influences the intrinsic properties and bioactivities of mammalian N‐glycoproteins and its level is linked to various cancers. Thus, PhoSL serves as a promising tool for glycoprofiling. Without structural precedence, the crystal structure was solved using the zinc anomalous signal, and revealed an interlaced trimer creating a novel protein fold termed β‐prism III. Three biantennary core‐fucosylated N‐glycan azides of 8 to 12 sugars were cocrystallized with PhoSL. The resulting highly resolved structures gave a detailed view on how the exclusive recognition of α1,6‐fucosylated N‐glycans by such a small protein occurs. This work also provided a protein consensus motif for the observed specificity as well as a glimpse into N‐glycan flexibility upon binding.     

Glycomics and glycoproteomics: Approaches to address isomeric separation of glycans and glycopeptides

Changes in the glycome of human proteins and cells are associated with the progression of multiple diseases such as Alzheimer's, diabetes mellitus, many types of cancer, and those caused by viruses. Consequently, several studies have shown essential modifications to the isomeric glycan moieties for diseases in different stages. However, the elucidation of extensive isomeric glycan profiles remains challenging because of the lack of analytical techniques with sufficient resolution power to separate all glycan and glycopeptide iso‐forms. Therefore, the development of sensitive and accurate approaches for the characterization of all the isomeric forms of glycans and glycopeptides is essential to tracking the progression of pathology in glycoprotein‐related diseases. This review describes the isomeric separation achievements reported in glycomics and glycoproteomics in the last decade. It focuses on the mass spectrometry–based analytical strategies, stationary phases, and derivatization techniques that have been developed to enhance the separation mechanisms in liquid chromatography systems and the detection capabilities of mass spectrometry systems.