Structural Analysis Of Glycans Research Articles

GENETICS OF RESPONSE TO COGNITIVE BEHAVIOR THERAPY IN ADULTS WITH MAJOR DEPRESSION

Glycodelin is a major glycoprotein expressed in reproductive tissues, like secretory and decidualized endometrium. It has several reproduction related functions that are dependent on specific glycosylation, but it has also been found to drive differentiation of endometrial carcinoma cells toward a less malignant phenotype. Here we aimed to elucidate whether the glycosylation and function of glycodelin is altered in endometrial carcinoma as compared with a normal endometrium. We carried out glycan structure analysis of glycodelin expressed in HEC-1B human endometrial carcinoma cells (HEC-1B Gd) by mass spectrometry glycomics strategies. Glycans of HEC-1B Gd were found to comprise a typical mixture of high-mannose, hybrid, and complex-type N-glycans, often containing undecorated LacNAc (Galβ1–4GlcNAc) antennae. However, several differences, as compared with previously reported glycan structures of normal human decidualized endometrium-derived glycodelin isoform, glycodelin-A (GdA), were also found. These included a lower level of sialylation and more abundant poly-LacNAc antennae, some of which are fucosylated. This allowed us to select lectins that showed different binding to these classes of glycodelin. Despite the differences in glycosylation between HEC-1B Gd and GdA, both showed similar inhibitory activity on trophoblast cell invasion and peripheral blood mononuclear cell proliferation. For the detection of cancer associated glycodelin, we established a novel in situ proximity-ligation based histochemical staining method using a specific glycodelin antibody and UEAI lectin. We found that the UEAI reactive glycodelin was abundant in endometrial carcinoma, but virtually absent in normal endometrial tissue even when glycodelin was strongly expressed. In conclusion, we established a histochemical staining method for the detection of endometrial carcinoma-associated glycodelin and showed that this specific glycodelin is exclusively expressed in cancer, not in normal endometrium. Similar methods can be used for studies of other glycoproteins.

High-Throughput Automated Micro-permethylation for Glycan Structure Analysis.

Permethylation is an essential technique for the detailed structural characterization of glycans by mass spectrometry. However, it requires specialized skills and equipment and is labor-intensive. While this limits glycan analysis to a handful of experts, the increasing awareness of the tremendous importance of glycans in physiological processes of health and disease has drastically raised the demand for detailed structural characterization of glycans. We have developed a simple microplate permethylation method that relies upon solid phase extraction using C18 tips to purify the permethylated glycans. The procedure is easy to perform, making it accessible to nonexperts, and fast, promising to accelerate glycan research. A 96-well plate of released glycan samples can be permethylated in less than an hour. The procedure can be carried out without discontinuous steps in an automatic pipet and integrated seamlessly with glycan release and mass spectrometry.

A Lifetime of Adventures in Glycobiology.

My initial research experience involved studying how bacteria synthesize nucleotide sugars, the donors for the formation of cell wall polysaccharides. During this time, I became aware that mammalian cells also have a surface coat of sugars and was intrigued as to whether these sugars might be arranged in specific sequences that function as information molecules in biologic processes. Thus began a long journey that has taken me from glycan structural analysis and determination of plant lectin-binding preferences to the biosynthesis of Asn-linked oligosaccharides and the mannose 6-phosphate (Man-6-P) lysosomal enzyme targeting pathway. The Man-6-P system represents an early example of a glycan serving as an information molecule in a fundamental cellular function. The remarkable advances in the field of glycobiology since I entered have uncovered scores of additional examples of oligosaccharide-lectin interactions mediating critical biologic processes. It has been a rewarding experience to participate in the efforts that have established a central role for glycans in biology.

Functional analysis of the Helicobacter pullorum N-linked protein glycosylation system.

N-linked protein glycosylation systems operate in species from all three domains of life. The model bacterial N-linked glycosylation system from Campylobacter jejuni is encoded by pgl genes present at a single chromosomal locus. This gene cluster includes the pglB oligosaccharyltransferase responsible for transfer of glycan from lipid carrier to protein. Although all genomes from species of the Campylobacter genus contain a pgl locus, among the related Helicobacter genus only three evolutionarily related species (H. pullorum, H. canadensis and H. winghamensis) potentially encode N-linked protein glycosylation systems. Helicobacter putative pgl genes are scattered in five chromosomal loci and include two putative oligosaccharyltransferase-encoding pglB genes per genome. We have previously demonstrated the in vitro N-linked glycosylation activity of H. pullorum resulting in transfer of a pentasaccharide to a peptide at asparagine within the sequon (D/E)XNXS/T. In this study, we identified the first H. pullorum N-linked glycoprotein, termed HgpA. Production of histidine-tagged HgpA in the background of insertional knockout mutants of H. pullorum pgl/wbp genes followed by analysis of HgpA glycan structures demonstrated the role of individual gene products in the PglB1-dependent N-linked protein glycosylation pathway. Glycopeptide purification by zwitterionic-hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry identified six glycosites from five H. pullorum proteins, which was consistent with proteins reactive with a polyclonal antiserum generated against glycosylated HgpA. This study demonstrates functioning of a H. pullorum N-linked general protein glycosylation system.

Gas-Phase Hydrogen/Deuterium Exchange for Distinguishing Isomeric Carbohydrate Ions.

The structural diversity of carbohydrates presents a major challenge for glycobiology and the analysis of glycoconjugates. Mass spectrometry has become a primary tool for glycan analysis thanks to its speed and sensitivity, but the information content regarding the glycan structure of protonated glycoconjugates is hindered by the inability to differentiate linkage and stereoisomers. Here, we examine a variety of protonated carbohydrate structures by gas-phase hydrogen/deuterium exchange (HDX) to discover that the exchange rates are distinct for isomeric carbohydrates with even subtle structural differences. By incorporating an internal exchange standard, HDX could effectively distinguish all linkage and stereoisomers that were examined and presents a mass spectrometry-based approach for glycan structural analysis with immense potential.

Differentiation of Glycan Diversity with Serial Affinity Column Set (SACS)

Targeted glycoproteomics is an effective way to discover disease-associated glycoproteins in proteomics and serial affinity chromatography (SAC) using lectin and glycan-targeting antibodies shows glycan diversity on the captured glycoproteins. This study suggests a way to determine glycan heterogeneity and structural analysis on the post-translationally modified proteins through serial affinity column set (SACS) using four Lycopersicon esculentum lectin (LEL) columns. The great advantage of this method is that it differentiates between glycoproteins on the basis of their binding affinity. Through this study, some proteins were identified to have glycoforms with different affinity on a single glycoprotein. It will be particularly useful in determining biomarkers in which the disease-specific feature is a unique glycan, or a group of glycans.

Atomic visualization of a flipped-back conformation of bisected glycans bound to specific lectins.

Glycans normally exist as a dynamic equilibrium of several conformations. A fundamental question concerns how such molecules bind lectins despite disadvantageous entropic loss upon binding. Bisected glycan, a glycan possessing bisecting N-acetylglucosamine (GlcNAc), is potentially a good model for investigating conformational dynamics and glycan-lectin interactions, owing to the unique ability of this sugar residue to alter conformer populations and thus modulate the biological activities. Here we analyzed bisected glycan in complex with two unrelated lectins, Calsepa and PHA-E. The crystal structures of the two complexes show a conspicuous flipped back glycan structure (designated ‘back-fold’ conformation), and solution NMR analysis also provides evidence of ‘back-fold’ glycan structure. Indeed, statistical conformational analysis of available bisected and non-bisected glycan structures suggests that bisecting GlcNAc restricts the conformations of branched structures. Restriction of glycan flexibility by certain sugar residues may be more common than previously thought and impinges on the mechanism of glycoform-dependent biological functions.

Eradicating mass spectrometric glycan rearrangement by utilizing free radicals.

Mass spectrometric glycan rearrangement is problematic because it provides misleading structural information. Here we report on a new reagent, a methylated free radical activated glycan sequencing reagent (Me-FRAGS), which combines a free radical precursor with a methylated pyridine moiety that can be coupled to the reducing terminus of glycans. The collisional activation of Me-FRAGS-derivatized glycans generates a nascent free radical that concurrently induces abundant glycosidic bond and cross-ring cleavage without the need for subsequent activation. The product ions resulting from glycan rearrangement, including internal residue loss and multiple external residue losses, are precluded. Glycan structures can be easily assembled and visualized using a radical driven glycan deconstruction diagram (R-DECON diagram). The presence and location of N-acetylated saccharide units and branch sites can be identified from the characteristic dissociation patterns observed only at these locations. The mechanisms of dissociation are investigated and discussed. This Me-FRAGS based mass spectrometric approach creates a new blueprint for glycan structure analysis.

Sugars Interfere or Glycomics in the Field of Cancer Biomarkers

Glycomics is concerned with detection and characterization of glycans present in biological samples. It is well-known that glycan structures impart high degree of structural diversity to biomolecules and thus add wide -ranging biological functions, such as cellular recognition, adhesion or involvement in cellular signaling pathways. They substantially participate in oncogenesis, e. g. in phases of invasion, metastasis and angiogenesis. Therefore, analysis of glycan structures in tumor tissues or body liquids is a promising tool for searching for potential tumor biomarkers essential for an early diagnosis of the neoplastic disease. The presented review describes the process of glycosylation and the origination of N and O glycans, presenting examples of glycan profiling in pancreatic, prostate and ovarian cancer.

Online Effective Identification of Glycopeptide Using Liquid Chromatography Combined with Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS)*

The detailed glycan structural analysis of glycoprotein is amenable to glycopeptide enrichment. Here, we develop a simple, effective and economical approach to enrich glycopeptides from proteolytically digested peptide mixtures by chromatographic column packed with graphite carbon and activated charcoal (G/A-column). Glycopeptide from ovalbumin was efficiently enriched by homemade G/A-column using liquid chromatography and the structure of glycopeptide was obtained by tandem mass spectrometry using Fourier transform ion cyclotron resonance mass spectrometry. The results in this study demonstrate that G/A-column can be used to enrich N-glycolpeptides and be benefit for online identification of glycopeptide using LC-MS.

Structural Characterization by Multistage Mass Spectrometry (MSn) of Human Milk Glycans Recognized by Human Rotaviruses

We have shown that recombinant forms of VP8* domains of the human rotavirus outer capsid spike protein VP4 from human neonatal strains (N155(G10P[11]) and RV3(G3P[6]) and a bovine strain (B223) recognize unique glycans within the repertoire of human milk glycans. The accompanying study by Yu et al.(2), describes a human milk glycan shotgun glycan microarray that led to the identification of 32 specific glycans in the human milk tagged glycan library that were recognized by these human rotaviruses. These microarray analyses also provided a variety of metadata about the recognized glycan structures compiled from anti-glycan antibody and lectin binding before and after specific glycosidase digestions, along with compositional information from mass analysis by matrix-assisted laser desorption ionization-mass spectrometry. To deduce glycan sequence and utilize information predicted by analyses of metadata from each glycan, 28 of the glycan targets were retrieved from the tagged glycan library for detailed sequencing using sequential disassembly of glycans by ion-trap mass spectrometry. Our aim is to obtain a deeper structural understanding of these key glycans using an orthogonal approach for structural confirmation in a single ion trap mass spectrometer. This sequential ion disassembly strategy details the complexities of linkage and branching in multiple compositions, several of which contained isomeric mixtures including several novel structures. The application of this approach exploits both library matching with standard materials and de novo approaches. This combination together with the metadata generated from lectin and antibody-binding data before and after glycosidase digestions provide a heretofore-unavailable level of analytical detail to glycan structure analysis. The results of these studies showed that, among the 28 glycan targets analyzed, 27 unique structures were identified, and 23 of the human milk glycans recognized by human rotaviruses represent novel structures not previously described as glycans in human milk. The functional glycomics analysis of human milk glycans provides significant insight into the repertoire of glycans comprising the human milk metaglycome.

Highlights 2014 on glycoscience; glycosyltransferases and glycobiomarkers.

The field of glycobiology has continued to grow and mature, establishing the role of glycans in a myriad of biological processes. Many new functions of glycans have emerged from studies on different eukaryotic and prokaryotic organisms. The development of new analytical tools and techniques have fuelled the generation of a lot of information on the structure of glycans, factors determining their structure and their derangement in several pathological conditions. One of the major themes of the recently held International Symposium on Carbohydrates in Bangalore, India was glycoconjugates and glycobiology, in which scientists working at the forefront of research in these areas reported their findings on different aspects of glycans in physiology and pathology. Selected contributions from these sessions are presented in this special issue of the journal. These contributions comprise short critical reviews or original research papers focusing on (a) glycosyl transferases in physiology and pathology (b) glycobiomarkers and (c) structural analysis of glycans. Critical information embedded within the oligosaccharides on the glycoconjugates as represented by the structural features of the glycans are important in specific biological processes however alterations in glycan structures occur during development of diseases such as cancer. Glycosyl transferases, which constitute a large family of enzymes displaying exquisite specificity for both glycosyl donor and the acceptor, are largely responsible for glycosyl transfer reactions that generate a diversity of glycan structures on glycoconjugates. Many substances including natural compounds are known to affect the activity of these enzymes, making them potential drug targets. At least six articles in the ensuing pages describe the biological importance and features of glycosyl transferases. These include data relating to (a) resiliency to mutagenicity of glycosyltransferases involved in bloodgroup antigen synthesis (b) role of O-linked glycans and protein –O-glycosylation in protein folding (c) microbial cell wall biosynthesis as well as formation of GPI anchors (d) characterisation of glycosyltransferases from new sources (e) glycosyl transferasebased assays to demonstrate glycan changes in cancer tissues. A number of changes can occur in glycan expression during the development of pathological conditions. Identification and validation of these glycan changes in disease conditions make them useful surrogate markers for the disease or drug response. A strategy for developing glycobiomarkers for disease has been described in one of the contributions while another paper reports the potential of 9-O-acylated sialic acids in differentiating normal haematopoietic stem cells from leukemic stem cells. Further, changes in cell surface proteoglycans in tumor cells and the potential of cell surface molecules such as receptor for advanced glycation end-products in recognising such molecules and development of glycomimetics based on these, have been discussed. P. R. Sudhakaran (*) Department of Computational Biology and Bioinformatics, University of Kerala, Karyavattom, Trivandrum, Kerala, India e-mail: prslab@gmail.com

Glycosylation analysis of Chinese hamster ovary produced glycoproteins

Glycosylation is a critical quality attribute of biotherapeutics produced from mammalian cells. Small changes in the glycan structures may have profound effects on the efficacy and functions of the glycoprotein, such as fucosylation of the IgG glycan reducing the ability of the IgG to bind to the FcϒRIIIA receptor. Chinese hamster ovary produced glycoprotein biotherapeutics may contain antigenic glycan structures that must be defined and monitored. Therefore, structural analysis of glycans is a key component in the development and production of glycosylated products. We discuss the current techniques available for glycan, glycopeptide and whole glycoprotein analysis. We emphasize recent analytic developments in high-throughput automated methods, as well as, further refinement of methods for separation and identification of isomers and potentially antigenic structures.

Characterization of putative glycosylphosphatidylinositol-anchoring motifs for surface display in the methylotrophic yeast Hansenula polymorpha.

Bioinformatic analysis of the genome of the methylotrophic yeast Hansenula polymorpha revealed 39 putative glycosylphosphatidylinositol-anchored proteins (GPI-proteins). Notably, dibasic motifs in the proximal ω-site, that has been reported as a plasma membrane retention signal in Saccharomyces cerevisiae GPI-proteins, were not found in any of the predicted GPI-proteins of H. polymorpha. To evaluate the in silico prediction, C-terminal peptides of 40 amino acids derived from ten H. polymorpha GPI-proteins were fused to the Aspergillus saitoi α-1,2-mannosidase (msdS). Cell wall fraction analysis showed that nine of the ten msdS-GPI fusion proteins were mostly localized at the cell wall. Surface expression of functional msdS was further confirmed by in vitro enzyme activity assay and by glycan structure analysis of cell wall mannoproteins. The recombinant H. polymorpha strains expressing surface-displayed msdS have the potential as useful hosts to produce glycoproteins with decreased mannosylation.

Integrated Glycoproteomics Demonstrates Fucosylated Serum Paraoxonase 1 Alterations in Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an aggressive type of lung cancer, and the detection of SCLCs at an early stage is necessary for successful therapy and for improving cancer survival rates. Fucosylation is one of the most common glycosylation-based modifications. Increased levels of fucosylation have been reported in a number of pathological conditions, including cancers. In this study, we aimed to identify and validate the aberrant and selective fucosylated glycoproteins in the sera of patients with SCLC. Fucosylated glycoproteins were enriched by the Aleuria aurantia lectin column after serum albumin and IgG depletion. In a narrowed down and comparative data analysis of both label-free proteomics and isobaric peptide-tagging chemistry iTRAQ approaches, the fucosylated glycoproteins were identified as up- or down-regulated in the sera of limited disease and extensive disease stage patients with SCLC. Verification was performed by multiple reaction monitoring-mass spectrometry to select reliable markers. Four fucosylated proteins, APCS, C9, SERPINA4, and PON1, were selected and subsequently validated by hybrid A. aurantia lectin ELISA (HLE) and Western blotting. Compared with Western blotting, the HLE analysis of these four proteins produced more optimal diagnostic values for SCLC. The PON1 protein levels were significantly reduced in the sera of patients with SCLC, whereas the fucosylation levels of PON1 were significantly increased. Fucosylated PON1 exhibited an area under curve of 0.91 for the extensive disease stage by HLE, whereas the PON1 protein levels produced an area under curve of 0.82 by Western blot. The glycan structural analysis of PON1 by MS/MS identified a biantennary fucosylated glycan modification consisting of a core + 2HexNAc + 1Fuc at increased levels in the sera of patients with SCLC. In addition, the PON1 levels were decreased in the sera of the Lewis lung carcinoma lung cancer mouse model that we examined. Our data suggest that fucosylated protein biomarkers, such as PON1, and their fucosylation levels and patterns can serve as diagnostic and prognostic serological markers for SCLC.

Biomimetic Reagents for the Selective Free Radical and Acid–Base Chemistry of Glycans: Application to Glycan Structure Determination by Mass Spectrometry

Nature excels at breaking down glycans into their components, typically via enzymatic acid-base catalysis to achieve selective cleavage of the glycosidic bond. Noting the importance of proton transfer in the active site of many of these enzymes, we describe a sequestered proton reagent for acid-catalyzed glycan sequencing (PRAGS) that derivatizes the reducing terminus of glycans with a pyridine moiety possessing moderate proton affinity. Gas-phase collisional activation of PRAGS-derivatized glycans predominately generates C1-O glycosidic bond cleavages retaining the charge on the reducing terminus. The resulting systematic PRAGS-directed deconstruction of the glycan can be analyzed to extract glycan composition and sequence. Glycans are also highly susceptible to dissociation by free radicals, mainly reactive oxygen species, which inspired our development of a free radical activated glycan sequencing (FRAGS) reagent, which combines a free radical precursor with a pyridine moiety that can be coupled to the reducing terminus of target glycans. Collisional activation of FRAGS-derivatized glycans generates a free radical that reacts to yield abundant cross-ring cleavages, glycosidic bond cleavages, and combinations of these types of cleavages with retention of charge at the reducing terminus. Branched sites are identified with the FRAGS reagent by the specific fragmentation patterns that are observed only at these locations. Mechanisms of dissociation as well as application of the reagents for both linear and highly branched glycan structure analysis are investigated and discussed. The approach developed here for glycan structure analysis offers unique advantages compared to earlier studies employing mass spectrometry for this purpose.

Unraveling the Glyco-Puzzle: Glycan Structure Identification by Capillary Electrophoresis

State-of-the-art high-resolution separation techniques play an important role in the full structural elucidation of glycans. Capillary electrophoresis (CE) offers a rapid yet simple method for exhaustive carbohydrate profiling. CE is a versatile analytical platform, which can be operated in several separation modes, simply by altering separation conditions during operation. For in-depth glycan structural analysis, CE has also gained significantly from the additional resolution introduced by complementary and orthogonal separation techniques such as ion exchange or hydrophilic interaction chromatography. Commercially available mass spectrometry (MS) interfaces have not only brought this information-rich detection technique within reach, but CE also represents an expedient highly efficient separation inlet for MS, capable of separating isobaric oligosaccharide isomers prior to MS detection and MS/MS fragmentation based identification. This Perspective gives a sophisticated impression of the versatility of capillary electrophoresis for deep structural elucidation of carbohydrates derived from glycoproteins of biomedical interest. Different separation modes for the analysis of both charged and neutral glycans, such as influencing electroosmotic flow, using complexation/interaction based secondary equilibria, and the use of charged and neutral labels are compared. The merits of introducing orthogonal and complementary techniques, such as exoglycosidase digestion arrays, analytical/preparative chromatography and mass spectrometric detection, extending the dynamic range and resolution of CE are all thoroughly discussed.

A Lectin-Based Glycomic Approach to Identify Characteristic Features of Xenopus Embryogenesis

Cell surface glycans show dynamic changes during cell differentiation. Several glycans are useful biomarkers of tumors, stem cells, and embryogenesis. Glycomic studies have been performed using liquid chromatography and mass spectrometry, which are powerful tools for glycan structural analysis but are difficult to use for small sample sizes. Recently, a lectin microarray system was developed for profiling cell surface glycome changes to terminal carbohydrate chains and branch types, using sample sizes of a few micrograms. In this study, we used the lectin microarray system for the first time to investigate stage-specific glycomes in Xenopus laevis embryos. Unsupervised cluster analysis of lectin microarray data indicated that glycan profiles changed sequentially during development. Nine lectin probes showed significantly different signals between early and the late-stage embryos: 4 showed higher signals in the early stages, and 5 exhibited higher signals in the late stages. The gene expression profiles of relevant glycosyltransferase genes support the lectin microarray data. Therefore, we have shown that lectin microarray is an effective tool for high-throughput glycan analysis in Xenopus embryogenesis, allowing glycan profiling of early embryos and small biopsy specimens.

Alteration of the Serum N-glycome of Mice Locally Exposed to High Doses of Ionizing Radiation

Exposure of the skin to ionizing radiation leads to characteristic reactions that will often turn into a pathophysiological process called the cutaneous radiation syndrome. The study of this disorder is crucial to finding diagnostic and prognostic bioindicators of local radiation exposure or radiation effects. It is known that irradiation alters the serum proteome content and potentially post-translationally modifies serum proteins. In this study, we investigated whether localized irradiation of the skin alters the serum glycome. Two-dimensional differential in-gel electrophoresis of serum proteins from a man and from mice exposed to ionizing radiation showed that potential post-translational modification changes occurred following irradiation. Using a large-scale quantitative mass-spectrometry-based glycomic approach, we performed a global analysis of glycan structures of serum proteins from non-irradiated and locally irradiated mice exposed to high doses of γ-rays (20, 40, and 80 Gy). Non-supervised descriptive statistical analyses (principal component analysis) using quantitative glycan structure data allowed us to discriminate between uninjured/slightly injured animals and animals that developed severe lesions. Decisional statistics showed that several glycan families were down-regulated whereas others increased, and that particular structures were statistically significantly changed in the serum of locally irradiated mice. The observed increases in multiantennary N-glycans and in outer branch fucosylation and sialylation were associated with the up-regulation of genes involved in glycosylation in the liver, which is the main producer of serum proteins, and with an increase in the key proinflammatory serum cytokines IL-1β, IL-6, and TNFα, which can regulate the expression of glycosylation genes. Our results suggest for the first time a role of serum protein glycosylation in response to irradiation. These protein-associated glycan structure changes might signal radiation exposure or effects.

Strategies for the profiling, characterisation and detailed structural analysis of N-linked oligosaccharides

Many post-translational modifications, including glycosylation, are pivotal for the structural integrity, location and functional activity of glycoproteins. Sub-populations of proteins that are relocated or functionally changed by such modifications can change resting proteins into active ones, mediating specific effector functions, as in the case of monoclonal antibodies. To ensure safe and efficacious drugs it is essential to employ appropriate robust, quantitative analytical strategies that can (i) perform detailed glycan structural analysis, (ii) characterise specific subsets of glycans to assess known critical features of therapeutic activities (iii) rapidly profile glycan pools for at-line monitoring or high level batch to batch screening. Here we focus on these aspects of glycan analysis, showing how state-of-the-art technologies are required at all stages during the production of recombinant glycotherapeutics. These data can provide insights into processing pathways and suggest markers for intervention at critical control points in bioprocessing and also critical decision points in disease and drug monitoring in patients. Importantly, these tools are now enabling the first glycome/genome studies in large populations, allowing the integration of glycomics into other 'omics platforms in a systems biology context.