Glycoproteomics and glycomics investigation of membrane N-glycosylproteins from human colon carcinoma cells

Aberrant glycosylation of proteins is known to profoundly affect cellular adhesion or motility of tumoral cells. In this study, we used HT-29 human colon epithelial cancer cells as a cellular model of cancer progression, as they can either proliferate or differentiate into enterocyte phenotype. A glycoproteomic approach based on Con A lectin-affinity chromatography, SDS-PAGE and MS analysis, allowed the identification of membrane N-glycoproteins from Triton X-100-solubilized proteins from membrane preparation. Among them, 65% were membrane proteins, and 45% were known to be N-glycosylated, such as alpha chains integrin and dipeptidyl isomerase IV. By lectin-blot analysis, significant changes of alpha-2,3- and alpha-2,6-sialylation of membrane glycoproteins were observed between proliferating and differentiated HT-29 cells. From these results, nano-LC-MS/MS analysis of the tryptic digests of the corresponding bands was performed and led to the identification of several transmembrane glycoproteins, like members of the solute carrier family and adhesion proteins. Finally, we compared N-glycans profiles and monosaccharide composition of proliferating and enterocyte-like HT-29 cells using MALDI-MS and GC-MS analyses of permethylated derivatives. This glycomic approach allowed to underscore significant changes in N-glycans structure, in particular the expression of atypical N-acetylglucosamine (GlcNAc)-ended N-glycans in enterocyte-like HT-29 cells.     

Extraction of leukemia specific glycan motifs in humans by computational glycomics

There have been almost no standard methods for conducting computational analyses on glycan structures in comparison to DNA and proteins. In this paper, we present a novel method for extracting functional motifs from glycan structures using the KEGG/GLYCAN database. First, we developed a new similarity measure for comparing glycan structures taking into account the characteristic mechanisms of glycan biosynthesis, and we tested its ability to classify glycans of different blood components in the framework of support vector machines (SVMs). The results show that our method can successfully classify glycans from four types of human blood components: leukemic cells, erythrocyte, serum, and plasma. Next, we extracted characteristic functional motifs of glycans considered to be specific to each blood component. We predicted the substructure alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-D-GlcpNAc as a leukemia specific glycan motif. Based on the fact that the Agrocybe cylindracea galectin (ACG) specifically binds to the same substructure, we conducted an experiment using cell agglutination assay and confirmed that this fungal lectin specifically recognized human leukemic cells.     

Targeted glycomics by selected reaction monitoring for highly sensitive glycan compositional analysis

The development of glycomics increasingly requires the detection and quantification of large numbers of glycans, which is only partially achieved by current glycomics approaches. Taking advantage of selected reaction monitoring to enhance both sensitivity and selectivity, we report here a strategy termed targeted glycomics that enables highly sensitive and consistent identification and quantification of diverse glycans across multiple samples at the same time. In this proof-of-principle study, we validated the method by analyzing global N-glycans expressed in different systems: single proteins, cancer cells, and serum samples. A dynamic range of three orders of magnitude was obtained for the detection of all five glycans released from ribonuclease B. The limit of detection of 80 attomole for Man(9) GlcNAc(2) demonstrated the excellent sensitivity of the method. The capability of the strategy to identify diverse glycans was demonstrated by identification and detection of 162 different glycans and isomers from pancreatic cancer cells. The sensitivity of the method was illustrated further by the ability to detect eight glycans from 250 cancer cells and five glycans released from 100 cancer cells. In serum obtained from rabbits fed control diet or diet enriched with 2% cholesterol, differences to 42 glycans were accurately measured and this indicates that this strategy might find use in studies of biomarker discovery and validation.     

Multiplexed analytical glycomics: rapid and confident IgG N-glycan structural elucidation

N-glycans attached to the C(H)2 domains of the Fc or the antigen binding regions of IgG play an important role in stabilizing and modulating antibody activity. Exhaustive elucidation of 32 IgG N-glycans using a combination of weak anion exchange enrichment and exoglycosidase array digestion with subsequent profiling exceeded 48 h. Pursuing increased throughput and associated structural annotation confidence, we compared the 1.7 μm hydrophilic interaction phase for UPLC with CE-LIF for the rapid and comprehensive characterization of N-glycans released from healthy human serum polyclonal IgG. Combination of the data individually generated using each technique demonstrated that complete structural annotation was possible within a total analysis time of 20 min due to the advantageous orthogonality of the separation mechanisms. The parallel use of both analytical techniques provides a powerful platform for rapid and comprehensive analysis of IgG N-glycosylation present on therapeutic antibodies or on antibodies of biomedical or pathological significance.     

Concept, strategy and realization of lectin-based glycan profiling

Lectins are a diverse group of carbohydrate-binding proteins. Each lectin has its own specificity profile. It is believed that lectins exist in all living organisms that produce glycans. From a practical viewpoint, lectins have been used extensively in biochemical fields including proteomics due to their usefulness as detection and enrichment tools for specific glycans. Nevertheless, they have often been underestimated as probes, especially compared with antibodies, because of their low affinity and broad specificity. However, together with the concept of glycomics, such properties of lectins are now considered to be suitable for the task of 'profiling' in order to cover a wider range of ligands. Recently there has been rapid movement in the field of proteomics aimed at the investigation of glycan-related biomarkers. This is partly because of limitations of the present approach of simply following changes in protein-level expression, without paying sufficient attention to the fact and effects of glycosylation. The trend is reflected in the frequent use of lectins in the contexts of glycoprotein enrichment and glycan profiling. However, there are many aspects to be considered in using lectins, which differ considerably from antibodies. In this article, the author, as a developer of two unique methodologies, frontal affinity chromatography (FAC) and the lectin microarray, describes critical points concerning the use of lectins, together with the concept, strategy and means to achieve advances in these emerging glycan profiling technologies.     

Optimized extraction of glycosaminoglycans from normal and osteoarthritic cartilage for glycomics profiling

Articular cartilage is a highly specialized smooth connective tissue whose proper functioning depends on the maintenance of an extracellular matrix consisting of an integrated assembly of collagens, glycoproteins, proteoglycans (PG), and glycosaminoglycans. Isomeric chondroitin sulfate glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work introduces a novel glycosaminoglycan extraction method for the quantification of mixtures of chondroitin sulfate oligosaccharides from intact cartilage tissue for mass spectral analysis. Glycosaminoglycans were extracted from intact cartilage samples using a combination of ethanol precipitation and enzymatic release followed by reversed-phase and strong anion exchange solid-phase extraction steps. Extracted chondroitin sulfate glycosaminoglycans were partially depolymerized using chondroitinases, labeled with 2-anthranilic acid-d(4) (2-AA) and subjected to size exclusion chromatography with online electrospray ionization mass spectrometric detection in the negative ion mode. The method presented herein enabled simultaneous determination of sulfate position and uronic acid epimerization in juvenile bovine and adult human cartilage samples. The method was applied to a series of 13 adult human cartilage explants. Standard deviation of the mean for the measurements was 1.6 on average. Coefficients of variation were approximately 4% for all compositions of 40% or greater. These results show that the new method has sufficient accuracy to allow determination of topographical distribution of glycoforms in connective tissue.     

Exploring the glycan repertoire of genetically modified mice by isolation and profiling of the major glycan classes and nano-NMR analysis of glycan mixtures

The production of mice with genetic alterations in glycosyltransferases has highlighted the need to isolate and study complex mixtures of the major classes of oligosaccharides (glycans) from intact tissues. We have found that nano-NMR spectroscopy of whole mixtures of N- and O-glycans can complement HPLC profiling methods for elucidating structural details. Working toward obtaining such glycan mixtures from mouse tissues, we decided to develop an approach to isolate not only N- and O-glycans, but also to separate out glycosphingolipids, glycosaminoglycans and glycosylphosphatidylinositol anchors. We describe here a comprehensive Glycan Isolation Protocol that is based primarily upon the physicochemical characteristics of the molecules, and requires only commonly available reagents and equipment. Using radiolabeled internal tracers, we show that recovery of each major class of glycans is as good or better than with conventional approaches for isolating individual classes, and that cross-contamination is minimal. The recovered glycans are of sufficient purity to provide a "glycoprofile" of a cell type or tissue. We applied this approach to compare the N- and O-glycans from wild type mouse tissues with those from mice genetically deficient in glycosyltransferases. N- and O-glycan mixtures from organs of mice deficient in ST6Gal-I (CMP-Sia:Galbeta1-4GlcNAc alpha2-6 sialyltransferase) were studied by the nano-NMR spectroscopy approach, showing no detectable alpha2-6-linked sialic acids. Thus, ST6Gal-I is likely responsible for generating most or all of these residues in normal mice. Similar studies indicate that this linkage is very rare in ganglioside glycans, even in wild-type tissues. In mice deficient in GalNAcT-8 (UDP-GalNAc:polypeptide O-Ser/Thr GalNAc transferase 8), HPLC profiling indicates that O-glycans persist in the thymus in large amounts, without a major change in overall profile, suggesting that other enzymes can synthesize the GalNAc-O-Ser/Thr linkage in this tissue. These results demonstrate the applicability of nano-NMR spectroscopy to complex glycan mixtures, as well as the versatility of the Glycan Isolation Protocol, which makes possible the concurrent examination of multiple glycan classes from intact vertebrate tissues.     

Shotgun glycomics: a microarray strategy for functional glycomics

Major challenges of glycomics are to characterize a glycome and identify functional glycans as ligands for glycan-binding proteins (GBPs). To address these issues we developed a general strategy termed shotgun glycomics. We focus on glycosphingolipids (GSLs), a class of glycoconjugates that is challenging to study, recognized by toxins, antibodies and GBPs. We derivatized GSLs extracted from cells with a heterobifunctional fluorescent tag suitable for covalent immobilization. We separated fluorescent GSLs by multidimensional chromatography, quantified them and coupled them to glass slides to create GSL shotgun microarrays. Then we interrogated the microarrays with cholera toxin, antibodies and sera from individuals with Lyme disease to identify biologically relevant GSLs that we subsequently characterized by mass spectrometry. Shotgun glycomics incorporating GSLs and potentially glycoprotein-derived glycans is an approach for accessing the complex glycomes of animal cells and is a strategy for focusing structural analyses on functionally important glycans.     

Evolution and structural dynamics of bacterial glycan binding adhesins

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A combined strategy for glycan profiling: a model study with pyridylaminated oligosaccharides

Structural glycomics plays a fundamental role in glycoscience and glycotechnology. In this paper, a novel strategy for the structural characterization of glycans is described, in which MS2 analysis involving a LIFT-TOF/TOF procedure is combined with frontal affinity chromatography (FAC). As model compounds, 20 neutral pyridylaminated (PA) oligosaccharides were chosen, which included four groups of structural isomers differing in sequence, linkage, position, or branching features. By depicting significant diagnostic ions on MS2, most of the analyzed oligosaccharides were successfully differentiated, while two pairs of linkage isomers, i.e., LNT/LNnT, and LNH/LNnH were not. For subsequent analysis by FAC, 14 lectins showing significant affinity to either LNT (type 1) or LNnT (type 2) were screened, and a galectin from the marine sponge Geodia cydonium (GC1) and a plant seed lectin from Ricinus communis (RCA-I) were used for determination of type 1 and 2 chains, respectively. With these specific probes, both of the isomeric pairs were unambiguously differentiated. Furthermore, a pair of triantennary, asparagine-linked oligosaccharide isomers could also be successfully differentiated. Thus, the combination of MS2 and FAC is a practical alternative for the structural characterization of complex glycans.     

Glycoproteomics: Past, present and future

This invited paper reviews the study of protein glycosylation, commonly known as glycoproteomics, beginning with the origins of the subject area in the early 1970s shortly after mass spectrometry was first applied to protein sequencing. We go on to describe current analytical approaches to glycoproteomic analyses, with exemplar projects presented in the form of the complex story of human glycodelin and the characterisation of blood group H eptitopes on the O-glycans of gp273 from Unio elongatulus. Finally, we present an update on the latest progress in the field of automated and semi-automated interpretation and annotation of these data in the form of GlycoWorkBench, a powerful informatics tool that provides valuable assistance in unravelling the complexities of glycoproteomic studies.     

Consequences of glycan truncation on Fc structural integrity

Effective characterization of protein-based therapeutic candidates such as monoclonal antibodies (mAbs) is important to facilitate their successful progression from early discovery and development stages to marketing approval. One challenge relevant to biopharmaceutical development is, understanding how the stability of a protein is affected by the presence of an attached oligosaccharide, termed a glycan. To explore the utility of molecular dynamics simulations as a complementary technique to currently available experimental methods, the Fc fragment was employed as a model system to improve our understanding of protein stabilization by glycan attachment. Long molecular dynamics simulations were performed on three Fc glycoform variants modeled using the crystal structure of a human IgG1 mAb. Two of these three glycoform variants have their glycan carbohydrates partially or completely removed. Structural differences among the glycoform variants during simulations suggest that glycan truncation and/or removal can cause quaternary structural deformation of the Fc as a result of the loss or disruption of a significant number of inter-glycan contacts that are not formed in the human IgG1 crystal structure, but do form during simulations described here. Glycan truncation/removal can also increase the tertiary structural deformation of C_H2 domains, demonstrating the importance of specific carbohydrates toward stabilizing individual C_H2 domains. At elevated temperatures, glycan truncation can also differentially affect structural deformation in locations (Helix-1 and Helix-2) that are far from the oligosaccharide attachment point. Deformation of these helices, which form part of the FcRn, could affect binding if these regions are unable to refold after temperature normalization. During elevated temperature simulations of the deglycosylated variant, C_H2 domains collapsed onto C_H3 domains. Observations from these glycan truncation/removal simulations have improved our understanding on how glycan composition can affect mAb stability.     

O-glycan profiling of serum glycan for potential renal cancer biomarkers

Serum was obtained from 25 male renal cell carcinoma (RCC) patients and 21 healthy males. O-glycans were released by a β-elimination reaction and purified by graphitized carbon cartridge solid phase extraction, then profiled by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. After noise removal and peak alignment, 1372 peaks were extracted from 200000 data points. Feature peaks were analyzed by calculation of differential sensitivity and specificity. The combination of two feature peaks was chosen as a biomarker and could clearly differentiate RCC and normal samples in our study group.     

Advancing glycomics: implementation strategies at the consortium for functional glycomics

Glycomics-an integrated approach to study structure-function relationships of complex carbohydrates (or glycans)-is an emerging field in this age of post-genomics. Realizing the importance of glycomics, many large scale research initiatives have been established to generate novel resources and technologies to advance glycomics. These initiatives are generating and cataloging diverse data sets necessitating the development of bioinformatic platforms to acquire, integrate, and disseminate these data sets in a meaningful fashion. With the consortium for functional glycomics (CFG) as the model system, this review discusses databases and the bioinformatics platform developed by this consortium to advance glycomics.     

Two categories of mammalian galactose-binding receptors distinguished by glycan array profiling

Profiling of the four known galactose-binding receptors in the C-type lectin family has been undertaken in parallel on a glycan array. The results are generally consistent with those of previous assays using various different formats, but they provide a direct comparison of the properties of the four receptors, revealing that they fall into two distinct groups. The major subunit of the rat asialoglycoprotein receptor and the rat Kupffer cell receptor show similar broad preferences for GalNAc-terminated glycans, while the rat macrophage galactose lectin and the human scavenger receptor C-type lectin (SRCL) bind more restricted sets of glycans. Both of these receptors bind to Lewis x-type structures, but the macrophage galactose lectin also interacts strongly with biantennary galactose- and GalNAc-terminated glycans. Although the similar glycan-binding profiles for the asialoglycoprotein receptor and the Kupffer cell receptor might suggest that these receptors are functionally redundant, analysis of fibroblasts transfected with full-length Kupffer cell receptor reveals that they fail to endocytose glycosylated ligand.     

糖生物学和糖组学

糖生物学和糖组学,虽然都是研究糖类在机体中的作用,但是,它们分别有不同的视角。糖生物学是沿袭了糖化学、糖生物化学发展而来,着重研究糖类和蛋白质的相互作用;而糖组学则源于基因组学,以基因编码糖基转移酶为起始,由这些酶得到糖组,进而开展糖组的研究。     

Application of lectin microarray to crude samples: differential glycan profiling of lec mutants

We recently developed a novel system for lectin microarray based on the evanescent-field fluorescence-detection principle, by which even weak lectin-oligosaccharide interactions are detectable without a washing procedure. For its practical application, cell glycan analysis was performed for Chinese hamster ovary (CHO) cells and their glycan profile was compared with those of their glycosylation-defective Lec mutants. Each of the cell surface extracts gave a significantly different profile from that of the parental CHO cells in a manner reflecting denoted biosynthetic features. Hence, the developed lectin microarray system is considered to be fully applicable for differential glycan profiling of crude samples.