Special Issue "Challenges in Glycan, Glycoprotein and Proteoglycan Research"

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A special issue of Biomolecules (ISSN 2218-273X).

Deadline for manuscript submissions: closed (31 May 2015)

Special Issue Editor

Guest Editor
Prof. Dr. Hans Vliegenthart

Bijvoet Center, Division Bioorganic Chemistry, Utrecht University, Padualaan 8, NL 3584 CH Utrecht, The Netherlands
Website | E-Mail
Interests: carbohydrates; glycoconjugates; unsaturated fatty acids; lipoxygenase and related enzymes

Special Issue Information

Dear Colleagues,

In the past decades Glycoscience has evolved tremendously. The progress that has been made in the analytical and synthetic aspects is spectacular. The introduction of high resolution NMR spectroscopy and Mass spectrometry has enabled the unravelling of the structure of glycans derived from glycoconjugates like glycoproteins, proteoglycans and glycolipids as well as of oligo- and polysaccharides. Improvements in the synthetic procedures, e.g., the development of solid state synthesis and of oligosaccharide synthesizers have rendered the synthesis of complex glycans possible in a reasonable period of time. Intriguing are the roles glycans can play at the molecular level in a large diversity of biological processes like normal and abnormal cell growth and development, recognition and interaction phenomena, adhesion of pathogens.

The biological features imply that the borders with (micro)biology, medicine, immunology, vaccines etc. have definitively been crossed. Many new challenges are waiting in the field.

To illustrate for the readers of “Biomolecules” the importance of Glycoscience as a multidisciplinary field of research, this special issue is intended to show the various aspects, possibilities, applications and challenges of glycans and related compounds.

We look forward to reading your contributions,

Prof. Dr. Hans Vliegenthart
Guest Editor

Submission

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Keywords

  • Glycoscience
  • glycoproteins
  • proteoglycans
  • glycolipids
  • oligosaccharides
  • polysaccharides

Published Papers (27 papers)

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Research

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Open AccessArticle Functional Analysis of the Glucuronyltransferases GlcAT-P and GlcAT-S of Drosophila melanogaster: Distinct Activities towards the O-linked T-antigen
Biomolecules 2016, 6(1), 8; doi:10.3390/biom6010008
Received: 26 May 2015 / Revised: 21 October 2015 / Accepted: 22 November 2015 / Published: 6 January 2016
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Abstract
The Drosophila melanogaster glucuronyltransferases dGlcAT-S and dGlcAT-P were reported to be expressed ubiquitously and results of in vitro activity assays indicate a functional redundancy. We analyzed both transferases in vivo and in vitro and could show significant differences in their activity towards N
[...] Read more.
The Drosophila melanogaster glucuronyltransferases dGlcAT-S and dGlcAT-P were reported to be expressed ubiquitously and results of in vitro activity assays indicate a functional redundancy. We analyzed both transferases in vivo and in vitro and could show significant differences in their activity towards N-and O-glycoproteins in vivo. While GlcAT-P is able to use N-linked N-acetyllactosamine chains and the O-linked T-antigen as a substrate to form non-sulfated HNK1- (GlcAβ1-3Galβ1-4GlcNAcβ1-) and glucuronyl-T-antigens in vivo, GlcAT-S adds glucuronic acid only to N-linked chains, thereby synthesizing only the non-sulfated HNK1-antigen. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
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Open AccessArticle The Hinge Region of Bovine Zona Pellucida Glycoprotein ZP3 Is Involved in the Formation of the Sperm-Binding Active ZP3/ZP4 Complex
Biomolecules 2015, 5(4), 3339-3353; doi:10.3390/biom5043339
Received: 5 September 2015 / Revised: 31 October 2015 / Accepted: 16 November 2015 / Published: 23 November 2015
Cited by 2 | PDF Full-text (2596 KB) | HTML Full-text | XML Full-text
Abstract
The zona pellucida (ZP) surrounds the mammalian oocyte and mediates species-selective sperm-oocyte interactions. Bovine ZP consists of glycoproteins ZP2, ZP3, and ZP4. Neither ZP3 nor ZP4 alone shows inhibitory activity for the binding of sperm to the ZP; however, this activity is seen
[...] Read more.
The zona pellucida (ZP) surrounds the mammalian oocyte and mediates species-selective sperm-oocyte interactions. Bovine ZP consists of glycoproteins ZP2, ZP3, and ZP4. Neither ZP3 nor ZP4 alone shows inhibitory activity for the binding of sperm to the ZP; however, this activity is seen with the ZP3/ZP4 heterocomplex. Here, we constructed a series of bovine ZP3 mutants to identify the ZP4-binding site on ZP3. Each ZP3 mutant was co-expressed with ZP4 using a baculovirus-Sf9 cell expression system and examined for interaction with ZP4 as well as inhibitory activity for sperm-ZP binding. N-terminal fragment Arg-32 to Arg-160 of ZP3 interacted with ZP4 and inhibited sperm-ZP binding, whereas fragment Arg-32 to Thr-155 showed much weaker interaction with ZP4. Mutation of N-glycosylated Asn-146 to Asp in the N-terminal fragment Arg-32 to Glu-178 of ZP3 did not interrupt the interaction of this fragment with ZP4, but it did reduce the inhibitory activity of the complex for sperm-ZP binding. In contrast, mutation of N-glycosylated Asn-124 to Asp did not significantly reduce the activity. Taken together, these results suggest that one of the ZP4 binding sites exists in the flexible hinge region of ZP3 and that the N-glycosylation in this region is involved in the sperm binding. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
Open AccessArticle Fluorinated Carbohydrates as Lectin Ligands: 19F-Based Direct STD Monitoring for Detection of Anomeric Selectivity
Biomolecules 2015, 5(4), 3177-3192; doi:10.3390/biom5043177
Received: 9 June 2015 / Revised: 2 November 2015 / Accepted: 4 November 2015 / Published: 13 November 2015
Cited by 3 | PDF Full-text (769 KB) | HTML Full-text | XML Full-text
Abstract
The characterization of the binding of reducing carbohydrates present as mixtures of anomers in solution to a sugar recepor (lectin) poses severe difficulties. In this situation, NMR spectroscopy enables the observation of signals for each anomer in the mixture by applying approaches based
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The characterization of the binding of reducing carbohydrates present as mixtures of anomers in solution to a sugar recepor (lectin) poses severe difficulties. In this situation, NMR spectroscopy enables the observation of signals for each anomer in the mixture by applying approaches based on ligand observation. Saturation transfer difference (STD) NMR allows fast and efficient screening of compound mixtures for reactivity to a receptor. Owing to the exceptionally favorable properties of 19F in NMR spectroscopy and the often complex 1H spectra of carbohydrates, 19F-containing sugars have the potential to be turned into versatile sensors for recognition. Extending the recently established 1H → 1H STDre19F-NMR technique, we here demonstrate its applicability to measure anomeric selectivity of binding in a model system using the plant lectin concanavalin A (ConA) and 2-deoxy-2-fluoro-d-mannose. Indeed, it is also possible to account for the mutual inhibition between the anomers on binding to the lectin by means of a kinetic model. The monitoring of 19F-NMR signal perturbation disclosed the relative activities of the anomers in solution and thus enabled the calculation of their binding affinity towards ConA. The obtained data show a preference for the α anomer that increases with temperature. This experimental approach can be extended to others systems of biomedical interest by testing human lectins with suitably tailored glycan derivatives. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
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Open AccessArticle Engineering and Characterization of a Fluorescent Native-Like HIV-1 Envelope Glycoprotein Trimer
Biomolecules 2015, 5(4), 2919-2934; doi:10.3390/biom5042919
Received: 31 July 2015 / Revised: 13 October 2015 / Accepted: 15 October 2015 / Published: 23 October 2015
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Abstract
Generation of a stable, soluble mimic of the HIV-1 envelope glycoprotein (Env) trimer on the virion surface has been considered an important first step for developing a successful HIV-1 vaccine. Recently, a soluble native-like Env trimer (BG505 SOSIP.664) has been described. This protein
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Generation of a stable, soluble mimic of the HIV-1 envelope glycoprotein (Env) trimer on the virion surface has been considered an important first step for developing a successful HIV-1 vaccine. Recently, a soluble native-like Env trimer (BG505 SOSIP.664) has been described. This protein has facilitated major advances in the HIV-1 vaccine field, since it was the first Env immunogen that induced consistent neutralizing antibodies against a neutralization-resistant (tier 2) virus. Moreover, BG505 SOSIP.664 enabled elucidation of the atomic resolution structure of the Env trimer and facilitated the isolation and characterization of new broadly neutralizing antibodies against HIV-1. Here, we designed and characterized the BG505 SOSIP.664 trimer fused to fluorescent superfolder GFP (sfGFP), a GFP variant that allows efficient folding (BG505 SOSIP.664-sfGFP). Despite the presence of the sfGFP, the Env protein largely retained its morphology, antigenicity, glycan composition, and thermostability. In addition, we show that BG505 SOSIP.664-sfGFP can be used for fluorescence-based assays, such as flow cytometry. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
Open AccessArticle Global N-linked Glycosylation is Not Significantly Impaired in Myoblasts in Congenital Myasthenic Syndromes Caused by Defective Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT1)
Biomolecules 2015, 5(4), 2758-2781; doi:10.3390/biom5042758
Received: 16 June 2015 / Revised: 28 August 2015 / Accepted: 13 October 2015 / Published: 16 October 2015
Cited by 3 | PDF Full-text (12301 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosynthetic pathway. It transfers an amino group from glutamine to fructose-6-phosphate to yield glucosamine-6-phosphate, thus providing the precursor for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is an essential substrate for all
[...] Read more.
Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosynthetic pathway. It transfers an amino group from glutamine to fructose-6-phosphate to yield glucosamine-6-phosphate, thus providing the precursor for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is an essential substrate for all mammalian glycosylation biosynthetic pathways and N-glycan branching is especially sensitive to alterations in the concentration of this sugar nucleotide. It has been reported that GFPT1 mutations lead to a distinct sub-class of congenital myasthenic syndromes (CMS) termed “limb-girdle CMS with tubular aggregates”. CMS are hereditary neuromuscular transmission disorders in which neuromuscular junctions are impaired. To investigate whether alterations in protein glycosylation at the neuromuscular junction might be involved in this impairment, we have employed mass spectrometric strategies to study the N-glycomes of myoblasts and myotubes derived from two healthy controls, three GFPT1 patients, and four patients with other muscular diseases, namely CMS caused by mutations in DOK7, myopathy caused by mutations in MTND5, limb girdle muscular dystrophy type 2A (LGMD2A), and Pompe disease. A comparison of the relative abundances of bi-, tri-, and tetra-antennary N-glycans in each of the cell preparations revealed that all samples exhibited broadly similar levels of branching. Moreover, although some differences were observed in the relative abundances of some of the N-glycan constituents, these variations were modest and were not confined to the GFPT1 samples. Therefore, GFPT1 mutations in CMS patients do not appear to compromise global N-glycosylation in muscle cells. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
Open AccessArticle Two-Dimensional N-Glycan Distribution Mapping of Hepatocellular Carcinoma Tissues by MALDI-Imaging Mass Spectrometry
Biomolecules 2015, 5(4), 2554-2572; doi:10.3390/biom5042554
Received: 16 June 2015 / Revised: 18 September 2015 / Accepted: 28 September 2015 / Published: 15 October 2015
Cited by 7 | PDF Full-text (2315 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
A new mass spectrometry imaging approach to simultaneously map the two-dimensional distribution of N-glycans in tissues has been recently developed. The method uses Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS) to spatially profile the location and distribution of multiple N
[...] Read more.
A new mass spectrometry imaging approach to simultaneously map the two-dimensional distribution of N-glycans in tissues has been recently developed. The method uses Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS) to spatially profile the location and distribution of multiple N-linked glycan species released by peptide N-glycosidase F in frozen or formalin-fixed tissues. Multiple formalin-fixed human hepatocellular carcinoma tissues were evaluated with this method, resulting in a panel of over 30 N-glycans detected. An ethylation reaction of extracted N-glycans released from adjacent slides was done to stabilize sialic acid containing glycans, and these structures were compared to N-glycans detected directly from tissue profiling. In addition, the distribution of singly fucosylated N-glycans detected in tumor tissue microarray cores were compared to the histochemistry staining pattern of a core fucose binding lectin. As this MALDI-IMS workflow has the potential to be applied to any formalin-fixed tissue block or tissue microarray, the advantages and limitations of the technique in context with other glycomic methods are also summarized. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
Open AccessArticle A Panel of Recombinant Mucins Carrying a Repertoire of Sialylated O-Glycans Based on Different Core Chains for Studies of Glycan Binding Proteins
Biomolecules 2015, 5(3), 1810-1831; doi:10.3390/biom5031810
Received: 23 June 2015 / Revised: 3 August 2015 / Accepted: 4 August 2015 / Published: 12 August 2015
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Abstract
Sialylated glycans serve as key elements of receptors for many viruses, bacteria, and bacterial toxins. The microbial recognition and their binding specificity can be affected by the linkage of the terminal sugar residue, types of underlying sugar chains, and the nature of the
[...] Read more.
Sialylated glycans serve as key elements of receptors for many viruses, bacteria, and bacterial toxins. The microbial recognition and their binding specificity can be affected by the linkage of the terminal sugar residue, types of underlying sugar chains, and the nature of the entire glycoconjugate. Owing to the pathobiological significance of sialylated glycans, we have engineered Chinese hamster ovary (CHO) cells to secrete mucin-type immunoglobulin-fused proteins carrying terminal α2,3- or α2,6-linked sialic acid on defined O-glycan core saccharide chains. Besides stably expressing P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b cDNA (PSGL-1/mIgG2b), CHO cells were stably transfected with plasmids encoding glycosyltransferases to synthesize core 2 (GCNT1), core 3 (B3GNT6), core 4 (GCNT1 and B3GNT6), or extended core 1 (B3GNT3) chains with or without the type 1 chain-encoding enzyme B3GALT5 and ST6GAL1. Western blot and liquid chromatography-mass spectrometry analysis confirmed the presence of core 1, 2, 3, 4, and extended core 1 chains carrying either type 1 (Galb3GlcNAc) or type 2 (Galb4GlcNAc) outer chains with or without α2,6-linked sialic acids. This panel of recombinant mucins carrying a repertoire of sialylated O-glycans will be important tools in studies aiming at determining the fine O-glycan binding specificity of sialic acid-specific microbial adhesins and mammalian lectins. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
Open AccessArticle Complementary LC-MS/MS-Based N-Glycan, N-Glycopeptide, and Intact N-Glycoprotein Profiling Reveals Unconventional Asn71-Glycosylation of Human Neutrophil Cathepsin G
Biomolecules 2015, 5(3), 1832-1854; doi:10.3390/biom5031832
Received: 9 June 2015 / Revised: 20 July 2015 / Accepted: 6 August 2015 / Published: 12 August 2015
Cited by 8 | PDF Full-text (2604 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Neutrophil cathepsin G (nCG) is a central serine protease in the human innate immune system, but the importance of its N-glycosylation remains largely undescribed. To facilitate such investigations, we here use complementary LC-MS/MS-based N-glycan, N-glycopeptide, and intact glycoprotein profiling to
[...] Read more.
Neutrophil cathepsin G (nCG) is a central serine protease in the human innate immune system, but the importance of its N-glycosylation remains largely undescribed. To facilitate such investigations, we here use complementary LC-MS/MS-based N-glycan, N-glycopeptide, and intact glycoprotein profiling to accurately establish the micro- and macro-heterogeneity of nCG from healthy individuals. The fully occupied Asn71 carried unconventional N-glycosylation consisting of truncated chitobiose core (GlcNAcβ: 55.2%; Fucα1,6GlcNAcβ: 22.7%), paucimannosidic N-glycans (Manβ1,4GlcNAcβ1,4GlcNAcβ: 10.6%; Manβ1,4GlcNAcβ1,4(Fucα1,6)GlcNAcβ: 7.9%; Manα1,6Manβ1,4GlcNAcβ1,4GlcNAcβ: 3.7%, trace level of Manα1,6Manβ1,4GlcNAcβ1,4(Fucα1,6)GlcNAcβ), and trace levels of monoantennary α2,6- and α2,3-sialylated complex N-glycans. High-resolution/mass accuracy LC-MS profiling of intact nCG confirmed the Asn71-glycoprofile and identified two C-terminal truncation variants at Arg243 (57.8%) and Ser244 (42.2%), both displaying oxidation of solvent-accessible Met152. Asn71 appeared proximal (~19 Å) to the active site of nCG, but due to the truncated nature of Asn71-glycans (~5–17 Å) we questioned their direct modulation of the proteolytic activity of the protein. This work highlights the continued requirement of using complementary technologies to accurately profile even relatively simple glycoproteins and illustrates important challenges associated with the analysis of unconventional protein N-glycosylation. Importantly, this study now facilitates investigation of the functional role of nCG Asn71-glycosylation. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
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Open AccessArticle Extracellular Vesicles from Ovarian Carcinoma Cells Display Specific Glycosignatures
Biomolecules 2015, 5(3), 1741-1761; doi:10.3390/biom5031741
Received: 21 June 2015 / Revised: 22 June 2015 / Accepted: 28 July 2015 / Published: 4 August 2015
Cited by 2 | PDF Full-text (2635 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Cells release vesicles to the extracellular environment with characteristic nucleic acid, protein, lipid, and glycan composition. Here we have isolated and characterized extracellular vesicles (EVs) and total cell membranes (MBs) from ovarian carcinoma OVMz cells. EVs were enriched in specific markers, including Tsg101,
[...] Read more.
Cells release vesicles to the extracellular environment with characteristic nucleic acid, protein, lipid, and glycan composition. Here we have isolated and characterized extracellular vesicles (EVs) and total cell membranes (MBs) from ovarian carcinoma OVMz cells. EVs were enriched in specific markers, including Tsg101, CD63, CD9, annexin-I, and MBs contained markers of cellular membrane compartments, including calnexin, GRASP65, GS28, LAMP-1, and L1CAM. The glycoprotein galectin-3 binding protein (LGALS3BP) was strongly enriched in EVs and it contained sialylated complex N-glycans. Lectin blotting with a panel of lectins showed that EVs had specific glycosignatures relative to MBs. Furthermore, the presence of glycoproteins bearing complex N-glycans with α2,3-linked sialic acid, fucose, bisecting-GlcNAc and LacdiNAc structures, and O-glycans with the T-antigen were detected. The inhibition of N-glycosylation processing from high mannose to complex glycans using kifunensine caused changes in the composition of EVs and induced a decrease of several glycoproteins. In conclusion, the results showed that glycosignatures of EVs were specific and altered glycosylation within the cell affected the composition and/or dynamics of EVs release. Furthermore, the identified glycosignatures of EVs could provide novel biomarkers for ovarian cancer. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
Open AccessArticle Galectin Binding to Neo-Glycoproteins: LacDiNAc Conjugated BSA as Ligand for Human Galectin-3
Biomolecules 2015, 5(3), 1671-1696; doi:10.3390/biom5031671
Received: 28 May 2015 / Revised: 26 June 2015 / Accepted: 10 July 2015 / Published: 24 July 2015
Cited by 3 | PDF Full-text (2638 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Carbohydrate-lectin interactions are relatively weak. As they play an important role in biological recognition processes, multivalent glycan ligands are designed to enhance binding affinity and inhibitory potency. We here report on novel neo-glycoproteins based on bovine serum albumin as scaffold for multivalent presentation
[...] Read more.
Carbohydrate-lectin interactions are relatively weak. As they play an important role in biological recognition processes, multivalent glycan ligands are designed to enhance binding affinity and inhibitory potency. We here report on novel neo-glycoproteins based on bovine serum albumin as scaffold for multivalent presentation of ligands for galectins. We prepared two kinds of tetrasaccharides (N-acetyllactosamine and N,N-diacetyllactosamine terminated) by multi-step chemo-enzymatic synthesis utilizing recombinant glycosyltransferases. Subsequent conjugation of these glycans to lysine groups of bovine serum albumin via squaric acid diethyl ester yielded a set of 22 different neo-glycoproteins with tuned ligand density. The neo-glycoproteins were analyzed by biochemical and chromatographic methods proving various modification degrees. The neo-glycoproteins were used for binding and inhibition studies with human galectin-3 showing high affinity. Binding strength and inhibition potency are closely related to modification density and show binding enhancement by multivalent ligand presentation. At galectin-3 concentrations comparable to serum levels of cancer patients, we detect the highest avidities. Selectivity of N,N-diacetyllactosamine terminated structures towards galectin-3 in comparison to galectin-1 is demonstrated. Moreover, we also see strong inhibitory potency of our scaffolds towards galectin-3 binding. These novel neo-glycoproteins may therefore serve as selective and strong galectin-3 ligands in cancer related biomedical research. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
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Open AccessArticle Mammalian Cell Surface Display as a Novel Method for Developing Engineered Lectins with Novel Characteristics
Biomolecules 2015, 5(3), 1540-1562; doi:10.3390/biom5031540
Received: 17 May 2015 / Revised: 16 June 2015 / Accepted: 18 June 2015 / Published: 20 July 2015
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Abstract
Leguminous lectins have a conserved carbohydrate recognition site comprising four loops (A–D). Here, we randomly mutated the sequence and length of loops C and D of peanut agglutinin (PNA) and expressed the proteins on the surface of mouse green fluorescent protein (GFP)-reporter cells.
[...] Read more.
Leguminous lectins have a conserved carbohydrate recognition site comprising four loops (A–D). Here, we randomly mutated the sequence and length of loops C and D of peanut agglutinin (PNA) and expressed the proteins on the surface of mouse green fluorescent protein (GFP)-reporter cells. Flow cytometry, limiting dilution, and cDNA cloning were used to screen for several mutated PNAs with distinct properties. The mutated PNA clones obtained using NeuAcα2-6(Galβ1-3)GalNAc as a ligand showed preference for NeuAcα2-6(Galβ1-3)GalNAc rather than non-sialylated Galβ1-3GlcNAc, whereas wild-type PNA binds to Galβ1-3GlcNAc but not sialylated Galβ1-3GalNAc. Sequence analyses revealed that for all of the glycan-reactive mutated PNA clones, (i) loop C was eight amino acids in length, (ii) loop D was identical to that of wild-type PNA, (iii) residue 127 was asparagine, (iv) residue 125 was tryptophan, and (v) residue 130 was hydrophobic tyrosine, phenylalanine, or histidine. The sugar-binding ability of wild-type PNA was increased nine-fold when Tyr125 was mutated to tryptophan, and that of mutated clone C was increased more than 30-fold after His130 was changed to tyrosine. These results provide an insight into the relationship between the amino acid sequences of the carbohydrate recognition site and sugar-binding abilities of leguminous lectins. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
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Open AccessArticle Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform
Biomolecules 2015, 5(3), 1480-1498; doi:10.3390/biom5031480
Received: 26 May 2015 / Revised: 9 June 2015 / Accepted: 6 July 2015 / Published: 16 July 2015
Cited by 5 | PDF Full-text (1277 KB) | HTML Full-text | XML Full-text
Abstract
A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and
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A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10–100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
Open AccessArticle Co-Expression of NEU2 and GBA3 Causes a Drastic Reduction in Cytosolic Sialyl Free N-glycans in Human MKN45 Stomach Cancer Cells—Evidence for the Physical Interaction of NEU2 and GBA3
Biomolecules 2015, 5(3), 1499-1514; doi:10.3390/biom5031499
Received: 10 June 2015 / Revised: 6 July 2015 / Accepted: 7 July 2015 / Published: 16 July 2015
Cited by 2 | PDF Full-text (869 KB) | HTML Full-text | XML Full-text
Abstract
It is well known that the “free” form of glycans that are structurally related to asparagine (N)-linked glycans (“free N-glycans”) are found in a wide variety of organisms. The mechanisms responsible for the formation/degradation of high mannose-type free N-glycans have been
[...] Read more.
It is well known that the “free” form of glycans that are structurally related to asparagine (N)-linked glycans (“free N-glycans”) are found in a wide variety of organisms. The mechanisms responsible for the formation/degradation of high mannose-type free N-glycans have been extensively studied in mammalian cells. Recent evidence, however, also suggests that sialylated, complex-type free N-glycans are also present in the cytosol of various mammalian-derived cultured cells/tissues. We report herein on an investigation of the mechanism responsible for the degradation of such sialyl free N-glycans. The findings show that the amount of glycans is dramatically reduced upon the co-expression of cytosolic sialidase NEU2 with cytosolic β-glycosidase GBA3 in human stomach cancer-derived MKN45 cells. The physical interaction between NEU2 and GBA3 was confirmed by co-precipitation analyses as well as gel filtration assays. The NEU2 protein was found to be stabilized in the presence of GBA3 both in cellulo and in vitro. Our results thus indicate that cytosolic GBA3 is likely involved in the catabolism of cytosolic sialyl free N-glycans, possibly by stabilizing the activity of the NEU2 protein. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
Open AccessArticle Sulfatide-Hsp70 Interaction Promotes Hsp70 Clustering and Stabilizes Binding to Unfolded Protein
Biomolecules 2015, 5(2), 958-973; doi:10.3390/biom5020958
Received: 26 February 2015 / Revised: 24 April 2015 / Accepted: 7 May 2015 / Published: 15 May 2015
Cited by 3 | PDF Full-text (2035 KB) | HTML Full-text | XML Full-text
Abstract
The 70-kDa heat shock protein (Hsp70), one of the major stress-inducible molecular chaperones, is localized not only in the cytosol, but also in extracellular milieu in mammals. Hsp70 interacts with various cell surface glycolipids including sulfatide (3'-sulfogalactosphingolipid). However, the molecular mechanism, as well
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The 70-kDa heat shock protein (Hsp70), one of the major stress-inducible molecular chaperones, is localized not only in the cytosol, but also in extracellular milieu in mammals. Hsp70 interacts with various cell surface glycolipids including sulfatide (3'-sulfogalactosphingolipid). However, the molecular mechanism, as well as the biological relevance, underlying the glycolipid-Hsp70 interaction is unknown. Here we report that sulfatide promotes Hsp70 oligomerization through the N-terminal ATPase domain, which stabilizes the binding of Hsp70 to unfolded protein in vitro. We find that the Hsp70 oligomer has apparent molecular masses ranging from 440 kDa to greater than 669 kDa. The C-terminal peptide-binding domain is dispensable for the sulfatide-induced oligomer formation. The oligomer formation is impaired in the presence of ATP, while the Hsp70 oligomer, once formed, is unable to bind to ATP. These results suggest that sulfatide locks Hsp70 in a high-affinity state to unfolded proteins by clustering the peptide-binding domain and blocking the binding to ATP that induces the dissociation of Hsp70 from protein substrates. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
Open AccessArticle Overcoming Challenges and Opening New Opportunities in Glycoproteomics
Biomolecules 2013, 3(2), 270-286; doi:10.3390/biom3020270
Received: 20 February 2013 / Revised: 14 March 2013 / Accepted: 15 March 2013 / Published: 26 March 2013
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Abstract
Glycoproteomics has emerged as a prime area of interest within the field of proteomics because glycoproteins have been shown to function as biomarkers for disease and as promising therapeutic targets. A significant challenge in the study of glycoproteins is the fact that they
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Glycoproteomics has emerged as a prime area of interest within the field of proteomics because glycoproteins have been shown to function as biomarkers for disease and as promising therapeutic targets. A significant challenge in the study of glycoproteins is the fact that they are expressed in relatively low abundance in cells. In response, various enrichment methods have been developed to improve the detection of glycoproteins. One such method involves their capture via oxidation of their glycan chains and covalent attachment with hydrazide resins which, when catalyzed by PNGase F, release N-linked glycans and convert the glycosite Asn to Asp; this conversion is identifiable with LC/ESI-MS/MS as a corresponding increase of 0.984 Da in molecular weight. The present study builds on this body of work, providing evidence of three additional strategies that improve glycoprotein identification: (1) use of a high resolution mass spectrometer—the Q Exactive MS—which delivers 2–3 times more glycoprotein identifications than a low resolution MS; (2) optimization of instrument settings and database search parameters to reduce misidentification of N-linked glycopeptides to ~1 percent; and (3) labeling glycopeptides with 18O during PNGase F treatment to locate N-linked glycosites within peptides containing multiple N-linked sequons. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
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Open AccessArticle Binding of Sperm to the Zona Pellucida Mediated by Sperm Carbohydrate-Binding Proteins is not Species-Specific in Vitro between Pigs and Cattle
Biomolecules 2013, 3(1), 85-107; doi:10.3390/biom3010085
Received: 28 November 2012 / Revised: 7 January 2013 / Accepted: 16 January 2013 / Published: 25 January 2013
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Abstract
Carbohydrates are candidates for the basis of species-selective interaction of gametes during mammalian fertilization. In this study, we sought to clarify the roles of sugar residues in the species-selective, sperm–oocyte interaction in pigs and cattle. Acrosome-intact porcine and bovine sperm exhibited their strongest
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Carbohydrates are candidates for the basis of species-selective interaction of gametes during mammalian fertilization. In this study, we sought to clarify the roles of sugar residues in the species-selective, sperm–oocyte interaction in pigs and cattle. Acrosome-intact porcine and bovine sperm exhibited their strongest binding affinities for β-Gal and α-Man residues, respectively. Porcine-sperm specificity changed from β-Gal to α-Man after the acrosome reaction, while bovine-sperm specificity did not. Binding of acrosome-intact and acrosome-reacted sperm decreased after trypsinization, indicating that the carbohydrate-binding components are proteins. While immature oocytes bound homologous sperm preferentially to heterologous sperm, oocytes matured in vitro bound similar numbers of homologous and heterologous sperm. Lectin staining revealed the aggregation of α-Man residues on the outer surface of the porcine zona during maturation. In both species, zona-free, mature oocytes bound homologous sperm preferentially to heterologous sperm. The lectin-staining patterns of the zona pellucida and zona-free oocytes coincided with the carbohydrate-binding specificities of acrosome-intact and acrosome-reacted sperm, respectively, supporting the involvement of carbohydrates in gamete recognition in pigs and cattle. These results also indicate that sperm-zona pellucida and sperm–oolemma bindings are not strictly species-specific in pigs and cattle, and further suggest that sperm penetration into the zona and/or fusion with oolemma may be species-specific between pigs and cattle. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
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Open AccessArticle Application of Metabolic 13C Labeling in Conjunction with High-Field Nuclear Magnetic Resonance Spectroscopy for Comparative Conformational Analysis of High Mannose-Type Oligosaccharides
Biomolecules 2013, 3(1), 108-123; doi:10.3390/biom3010108
Received: 19 December 2012 / Revised: 10 January 2013 / Accepted: 15 January 2013 / Published: 25 January 2013
Cited by 13 | PDF Full-text (682 KB) | HTML Full-text | XML Full-text
Abstract
High mannose-type oligosaccharides are enzymatically trimmed in the endoplasmic reticulum, resulting in various processing intermediates with exposed glycotopes that are recognized by a series of lectins involved in glycoprotein fate determination in cells. Although recent crystallographic data have provided the structural basis for
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High mannose-type oligosaccharides are enzymatically trimmed in the endoplasmic reticulum, resulting in various processing intermediates with exposed glycotopes that are recognized by a series of lectins involved in glycoprotein fate determination in cells. Although recent crystallographic data have provided the structural basis for the carbohydrate recognition of intracellular lectins, atomic information of dynamic oligosaccharide conformations is essential for a quantitative understanding of the energetics of carbohydrate–lectin interactions. Carbohydrate NMR spectroscopy is useful for characterizing such conformational dynamics, but often hampered by poor spectral resolution and lack of recombinant techniques required to produce homogeneous glycoforms. To overcome these difficulties, we have recently developed a methodology for the preparation of a homogeneous high mannose-type oligosaccharide with 13C labeling using a genetically engineered yeast strain. We herein successfully extended this method to result in the overexpression of 13C-labeled Man9GlcNAc2 (M9) with a newly engineered yeast strain with the deletion of four genes involved in N-glycan processing. This enabled high-field NMR analyses of 13C-labeled M9 in comparison with its processing product lacking the terminal mannose residue ManD2. Long-range NOE data indicated that the outer branches interact with the core in both glycoforms, and such foldback conformations are enhanced upon the removal of ManD2. The observed conformational variabilities might be significantly associated with lectins and glycan-trimming enzymes. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
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Open AccessArticle Comparative Ultrastructure and Carbohydrate Composition of Gastroliths from Astacidae, Cambaridae and Parastacidae Freshwater Crayfish (Crustacea, Decapoda)
Biomolecules 2013, 3(1), 18-38; doi:10.3390/biom3010018
Received: 25 October 2012 / Revised: 27 November 2012 / Accepted: 28 November 2012 / Published: 21 December 2012
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Abstract
Crustaceans have to cyclically replace their rigid exoskeleton in order to grow. Most of them harden this skeleton by a calcification process. Some decapods (land crabs, lobsters and crayfish) elaborate calcium storage structures as a reservoir of calcium ions in their stomach wall,
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Crustaceans have to cyclically replace their rigid exoskeleton in order to grow. Most of them harden this skeleton by a calcification process. Some decapods (land crabs, lobsters and crayfish) elaborate calcium storage structures as a reservoir of calcium ions in their stomach wall, as so-called gastroliths. For a better understanding of the cyclic elaboration of these calcium deposits, we studied the ultrastructure of gastroliths from freshwater crayfish by using a combination of microscopic and physical techniques. Because sugars are also molecules putatively involved in the elaboration process of these biomineralizations, we also determined their carbohydrate composition. This study was performed in a comparative perspective on crayfish species belonging to the infra-order Astacidea (Decapoda, Malacostraca): three species from the Astacoidea superfamily and one species from the Parastacoidea superfamily. We observed that all the gastroliths exhibit a similar dense network of protein-chitin fibers, from macro- to nanoscale, within which calcium is precipitated as amorphous calcium carbonate. Nevertheless, they are not very similar at the molecular level, notably as regards their carbohydrate composition. Besides glucosamine, the basic carbohydrate component of chitin, we evidenced the presence of other sugars, some of which are species-specific like rhamnose and galacturonic acid whereas xylose and mannose could be linked to proteoglycan components. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
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Open AccessArticle Hyaluronidases Have Strong Hydrolytic Activity toward Chondroitin 4-Sulfate Comparable to that for Hyaluronan
Biomolecules 2012, 2(4), 549-563; doi:10.3390/biom2040549
Received: 13 October 2012 / Revised: 22 October 2012 / Accepted: 8 November 2012 / Published: 12 November 2012
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Abstract
Chondroitin sulfate (CS) chains are involved in the regulation of various biological processes. However, the mechanism underlying the catabolism of CS is not well understood. Hyaluronan (HA)-degrading enzymes, the hyaluronidases, are assumed to act at the initial stage of the degradation process, because
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Chondroitin sulfate (CS) chains are involved in the regulation of various biological processes. However, the mechanism underlying the catabolism of CS is not well understood. Hyaluronan (HA)-degrading enzymes, the hyaluronidases, are assumed to act at the initial stage of the degradation process, because HA is similar in structure to nonsulfated CS, chondroitin (Chn). Although human hyaluronidase-1 (HYAL1) and testicular hyaluronidase (SPAM1) can degrade not only HA but also CS, they are assumed to digest CS to only a limited extent. In this study, the hydrolytic activities of HYAL1 and SPAM1 toward CS-A, CS-C, Chn, and HA were compared. HYAL1 depolymerized CS-A and HA to a similar extent. SPAM1 degraded CS-A, Chn, and HA to a similar extent. CS is widely distributed from very primitive organisms to humans, whereas HA has been reported to be present only in vertebrates with the single exception of a mollusk. Therefore, a genuine substrate of hyaluronidases appears to be CS as well as HA. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
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Review

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Open AccessReview Notable Aspects of Glycan-Protein Interactions
Biomolecules 2015, 5(3), 2056-2072; doi:10.3390/biom5032056
Received: 5 August 2015 / Revised: 27 August 2015 / Accepted: 27 August 2015 / Published: 1 September 2015
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Abstract
This mini review highlights several interesting aspects of glycan-mediated interactions that are common between cells, bacteria, and viruses. Glycans are ubiquitously found on all living cells, and in the extracellular milieu of multicellular organisms. They are known to mediate initial binding and recognition
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This mini review highlights several interesting aspects of glycan-mediated interactions that are common between cells, bacteria, and viruses. Glycans are ubiquitously found on all living cells, and in the extracellular milieu of multicellular organisms. They are known to mediate initial binding and recognition events of both immune cells and pathogens with their target cells or tissues. The host target tissues are hidden under a layer of secreted glycosylated decoy targets. In addition, pathogens can utilize and display host glycans to prevent identification as foreign by the host’s immune system (molecular mimicry). Both the host and pathogens continually evolve. The host evolves to prevent infection and the pathogens evolve to evade host defenses. Many pathogens express both glycan-binding proteins and glycosidases. Interestingly, these proteins are often located at the tip of elongated protrusions in bacteria, or in the leading edge of the cell. Glycan-protein interactions have low affinity and, as a result, multivalent interactions are often required to achieve biologically relevant binding. These enable dynamic forms of adhesion mechanisms, reviewed here, and include rolling (cells), stick and roll (bacteria) or surfacing (viruses). Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
Open AccessReview Determinants of Glycosaminoglycan (GAG) Structure
Biomolecules 2015, 5(3), 2003-2022; doi:10.3390/biom5032003
Received: 29 May 2015 / Revised: 17 August 2015 / Accepted: 18 August 2015 / Published: 21 August 2015
Cited by 5 | PDF Full-text (156 KB) | HTML Full-text | XML Full-text
Abstract
Proteoglycans (PGs) are glycosylated proteins of biological importance at cell surfaces, in the extracellular matrix, and in the circulation. PGs are produced and modified by glycosaminoglycan (GAG) chains in the secretory pathway of animal cells. The most common GAG attachment site is a
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Proteoglycans (PGs) are glycosylated proteins of biological importance at cell surfaces, in the extracellular matrix, and in the circulation. PGs are produced and modified by glycosaminoglycan (GAG) chains in the secretory pathway of animal cells. The most common GAG attachment site is a serine residue followed by a glycine (-ser-gly-), from which a linker tetrasaccharide extends and may continue as a heparan sulfate, a heparin, a chondroitin sulfate, or a dermatan sulfate GAG chain. Which type of GAG chain becomes attached to the linker tetrasaccharide is influenced by the structure of the protein core, modifications occurring to the linker tetrasaccharide itself, and the biochemical environment of the Golgi apparatus, where GAG polymerization and modification by sulfation and epimerization take place. The same cell type may produce different GAG chains that vary, depending on the extent of epimerization and sulfation. However, it is not known to what extent these differences are caused by compartmental segregation of protein cores en route through the secretory pathway or by differential recruitment of modifying enzymes during synthesis of different PGs. The topic of this review is how different aspects of protein structure, cellular biochemistry, and compartmentalization may influence GAG synthesis. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
Open AccessReview Challenges in Antibody Development against Tn and Sialyl-Tn Antigens
Biomolecules 2015, 5(3), 1783-1809; doi:10.3390/biom5031783
Received: 9 June 2015 / Revised: 19 July 2015 / Accepted: 31 July 2015 / Published: 11 August 2015
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Abstract
The carbohydrate antigens Tn and sialyl-Tn (STn) are expressed in most carcinomas and usually absent in healthy tissues. These antigens have been correlated with cancer progression and poor prognosis, and associated with immunosuppressive microenvironment. Presently they are used in clinical trials as therapeutic
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The carbohydrate antigens Tn and sialyl-Tn (STn) are expressed in most carcinomas and usually absent in healthy tissues. These antigens have been correlated with cancer progression and poor prognosis, and associated with immunosuppressive microenvironment. Presently they are used in clinical trials as therapeutic vaccination, but with limited success due to their low immunogenicity. Alternatively, anti-Tn and/or STn antibodies may be used to harness the immune system against tumor cells. Whilst the development of antibodies against these antigens had a boost two decades ago for diagnostic use, so far no such antibody entered into clinical trials. Possible limitations are the low specificity and efficiency of existing antibodies and that novel antibodies are still necessary. The vast array of methodologies available today will allow rapid antibody development and novel formats. Following the advent of hybridoma technology, the immortalization of human B cells became a methodology to obtain human monoclonal antibodies with better specificity. Advances in molecular biology including phage display technology for high throughput screening, transgenic mice and more recently molecularly engineered antibodies enhanced the field of antibody production. The development of novel antibodies against Tn and STn taking advantage of innovative technologies and engineering techniques may result in innovative therapeutic antibodies for cancer treatment. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
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Open AccessReview Recent Advances in Cellular Glycomic Analyses
Biomolecules 2013, 3(1), 198-225; doi:10.3390/biom3010198
Received: 2 December 2012 / Revised: 28 January 2013 / Accepted: 14 February 2013 / Published: 21 February 2013
Cited by 8 | PDF Full-text (691 KB) | HTML Full-text | XML Full-text
Abstract
A large variety of glycans is intricately located on the cell surface, and the overall profile (the glycome, given the entire repertoire of glycoconjugate-associated sugars in cells and tissues) is believed to be crucial for the diverse roles of glycans, which are mediated
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A large variety of glycans is intricately located on the cell surface, and the overall profile (the glycome, given the entire repertoire of glycoconjugate-associated sugars in cells and tissues) is believed to be crucial for the diverse roles of glycans, which are mediated by specific interactions that control cell-cell adhesion, immune response, microbial pathogenesis and other cellular events. The glycomic profile also reflects cellular alterations, such as development, differentiation and cancerous change. A glycoconjugate-based approach would therefore be expected to streamline discovery of novel cellular biomarkers. Development of such an approach has proven challenging, due to the technical difficulties associated with the analysis of various types of cellular glycomes; however, recent progress in the development of analytical methodologies and strategies has begun to clarify the cellular glycomics of various classes of glycoconjugates. This review focuses on recent advances in the technical aspects of cellular glycomic analyses of major classes of glycoconjugates, including N- and O-linked glycans, derived from glycoproteins, proteoglycans and glycosphingolipids. Articles that unveil the glycomics of various biologically important cells, including embryonic and somatic stem cells, induced pluripotent stem (iPS) cells and cancer cells, are discussed. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
Open AccessReview Glycobiology Aspects of the Periodontal Pathogen Tannerella forsythia
Biomolecules 2012, 2(4), 467-482; doi:10.3390/biom2040467
Received: 2 September 2012 / Revised: 27 September 2012 / Accepted: 29 September 2012 / Published: 12 October 2012
Cited by 12 | PDF Full-text (502 KB) | HTML Full-text | XML Full-text
Abstract
Glycobiology is important for the periodontal pathogen Tannerella forsythia, affecting the bacterium’s cellular integrity, its life-style, and virulence potential. The bacterium possesses a unique Gram-negative cell envelope with a glycosylated surface (S-) layer as outermost decoration that is proposed to be
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Glycobiology is important for the periodontal pathogen Tannerella forsythia, affecting the bacterium’s cellular integrity, its life-style, and virulence potential. The bacterium possesses a unique Gram-negative cell envelope with a glycosylated surface (S-) layer as outermost decoration that is proposed to be anchored via a rough lipopolysaccharide. The S-layer glycan has the structure 4‑MeO-b-ManpNAcCONH2-(1→3)-[Pse5Am7Gc-(2→4)-]-b-ManpNAcA-(1→4)-[4-MeO-a-Galp-(1→2)-]-a-Fucp-(1→4)-[-a-Xylp-(1→3)-]-b-GlcpA-(1→3)-[-b-Digp-(1→2)-]-a-Galp and is linked to distinct serine and threonine residues within the D(S/T)(A/I/L/M/T/V) amino acid motif. Also several other Tannerella proteins are modified with the S‑layer oligosaccharide, indicating the presence of a general O‑glycosylation system. Protein O‑glycosylation impacts the life-style of T. forsythia since truncated S-layer glycans present in a defined mutant favor biofilm formation. While the S‑layer has also been shown to be a virulence factor and to delay the bacterium's recognition by the innate immune system of the host, the contribution of glycosylation to modulating host immunity is currently unraveling. Recently, it was shown that Tannerella surface glycosylation has a role in restraining the Th17-mediated neutrophil infiltration in the gingival tissues. Related to its asaccharolytic physiology, T. forsythia expresses a robust enzymatic repertoire, including several glycosidases, such as sialidases, which are linked to specific growth requirements and are involved in triggering host tissue destruction. This review compiles the current knowledge on the glycobiology of T. forsythia. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
Open AccessReview Sialyl-Tn in Cancer: (How) Did We Miss the Target?
Biomolecules 2012, 2(4), 435-466; doi:10.3390/biom2040435
Received: 29 August 2012 / Revised: 27 September 2012 / Accepted: 30 September 2012 / Published: 11 October 2012
Cited by 13 | PDF Full-text (853 KB) | HTML Full-text | XML Full-text
Abstract
Sialyl-Tn antigen (STn) is a short O-glycan containing a sialic acid residue a2,6-linked to GalNAca-O-Ser/Thr. The biosynthesis of STn is mediated by a specific sialyltransferase termed ST6GalNAc I, which competes with O-glycans elongating glycosyltransferases and prevents cancer cells from
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Sialyl-Tn antigen (STn) is a short O-glycan containing a sialic acid residue a2,6-linked to GalNAca-O-Ser/Thr. The biosynthesis of STn is mediated by a specific sialyltransferase termed ST6GalNAc I, which competes with O-glycans elongating glycosyltransferases and prevents cancer cells from exhibiting longer O-glycans. While weakly expressed by fetal and normal adult tissues, STn is expressed by more than 80% of human carcinomas and in all cases, STn detection is associated with adverse outcome and decreased overall survival for the patients. Because of its pan-carcinoma expression associated with an adverse outcome, an anti-cancer vaccine, named Theratope, has been designed towards the STn epitope. In spite of the great enthusiasm around this immunotherapy, Theratope failed on Phase III clinical trial. However, in lieu of missing this target, one should consider to revise the Theratope design and the actual facts. In this review, we highlight the many lessons that can be learned from this failure from the immunological standpoint, as well as from the drug design and formulation and patient selection. Moreover, an irrefutable knowledge is arising from novel immunotherapies targeting other carbohydrate antigens and STn carrier proteins, such as MUC1, that will warrantee the future development of more successful anti-STn immunotherapy strategies. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
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Open AccessReview Promiscuity of the Euonymus Carbohydrate-Binding Domain
Biomolecules 2012, 2(4), 415-434; doi:10.3390/biom2040415
Received: 23 August 2012 / Revised: 17 September 2012 / Accepted: 25 September 2012 / Published: 8 October 2012
Cited by 8 | PDF Full-text (642 KB) | HTML Full-text | XML Full-text
Abstract
Plants synthesize small amounts of carbohydrate-binding proteins on exposure to stress. For example, on exposure to drought, high salt, wounding and by treatment with some plant hormones or by pathogen attack. In contrast to the ‘classical’ plant lectins that are mostly located in
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Plants synthesize small amounts of carbohydrate-binding proteins on exposure to stress. For example, on exposure to drought, high salt, wounding and by treatment with some plant hormones or by pathogen attack. In contrast to the ‘classical’ plant lectins that are mostly located in the vacuolar compartment, this new class of inducible lectins is present in the cytoplasm and in the nucleus. Taking into account that any physiological role of plant lectins most likely relies on their specific carbohydrate-binding activity and specificity, the discovery of these stress-related lectins provides strong evidence for the importance of protein-carbohydrate-interactions in plant cells. Hitherto, six families of such nucleocytoplasmic lectins have been identified in plants. This review will focus on the nucleocytoplasmic lectins with one or more Euonymus lectin (EUL) domain(s). The carbohydrate-binding specificity of EUL proteins from a monocot, a dicot and a lower plant has been compared. Furthermore, modeling of the different EUL domains revealed a similar ß-trefoil fold consisting of three bundles of ß-sheet organized around a pseudo three-fold symmetry axis. Despite the sequence similarity and the conserved amino acids in the binding site, glycan array analyses showed that the EUL domain has a promiscuous carbohydrate-binding site capable of accommodating high mannose N-glycans, blood group B related structures and galactosylated epitopes. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
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Open AccessReview Comparative Studies of Vertebrate Platelet Glycoprotein 4 (CD36)
Biomolecules 2012, 2(3), 389-414; doi:10.3390/biom2030389
Received: 2 August 2012 / Revised: 6 September 2012 / Accepted: 18 September 2012 / Published: 24 September 2012
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Abstract
Platelet glycoprotein 4 (CD36) (or fatty acyl translocase [FAT], or scavenger receptor class B, member 3 [SCARB3]) is an essential cell surface and skeletal muscle outer mitochondrial membrane glycoprotein involved in multiple functions in the body. CD36 serves as a ligand receptor of
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Platelet glycoprotein 4 (CD36) (or fatty acyl translocase [FAT], or scavenger receptor class B, member 3 [SCARB3]) is an essential cell surface and skeletal muscle outer mitochondrial membrane glycoprotein involved in multiple functions in the body. CD36 serves as a ligand receptor of thrombospondin, long chain fatty acids, oxidized low density lipoproteins (LDLs) and malaria-infected erythrocytes. CD36 also influences various diseases, including angiogenesis, thrombosis, atherosclerosis, malaria, diabetes, steatosis, dementia and obesity. Genetic deficiency of this protein results in significant changes in fatty acid and oxidized lipid uptake. Comparative CD36 amino acid sequences and structures and CD36 gene locations were examined using data from several vertebrate genome projects. Vertebrate CD36 sequences shared 53–100% identity as compared with 29–32% sequence identities with other CD36-like superfamily members, SCARB1 and SCARB2. At least eight vertebrate CD36 N-glycosylation sites were conserved which are required for membrane integration. Sequence alignments, key amino acid residues and predicted secondary structures were also studied. Three CD36 domains were identified including cytoplasmic, transmembrane and exoplasmic sequences. Conserved sequences included N- and C-terminal transmembrane glycines; and exoplasmic cysteine disulphide residues; TSP-1 and PE binding sites, Thr92 and His242, respectively; 17 conserved proline and 14 glycine residues, which may participate in forming CD36 ‘short loops’; and basic amino acid residues, and may contribute to fatty acid and thrombospondin binding. Vertebrate CD36 genes usually contained 12 coding exons. The human CD36 gene contained transcription factor binding sites (including PPARG and PPARA) contributing to a high gene expression level (6.6 times average). Phylogenetic analyses examined the relationships and potential evolutionary origins of the vertebrate CD36 gene with vertebrate SCARB1 and SCARB2 genes. These suggested that CD36 originated in an ancestral genome and was subsequently duplicated to form three vertebrate CD36 gene family members, SCARB1, SCARB2 and CD36. Full article
(This article belongs to the Special Issue Challenges in Glycan, Glycoprotein and Proteoglycan Research)
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