Glycan Structures Of Glycoproteins Research Articles

Expression and Characterization of Human β-1, 4-Galactosyltransferase 1 (β4GalT1) Using Silkworm-Baculovirus Expression System.

Baculovirus expression vector system (BEVS) is widely known as a mass-production tool to produce functional recombinant glycoproteins except that it may not be always suitable for medical practice due to the differences in the structure of N-linked glycans between insects and mammalian. Currently, various approaches have been reported to alter N-linked glycan structures of glycoproteins derived from insects into terminally sialylated complex-type N-glycans. In the light of those studies, we also proposed in vitro maturation of N-glycan with mass-produced and purified glycosyltransferases by silkworm-BEVS. β-1,4-Galactosyltransferase 1 (β4GalT1) is known as one of type II transmembrane enzymes that transfer galactose in a β-1, 4 linkage to accepter sugars, and a key enzyme for further sialylation of N-glycans. In this study, we developed a large-scale production of recombinant human β4GalT1 (rhβ4GalT1) with N- or C-terminal tags in silkworm-BEVS. We demonstrated that rhβ4GalT1 is N-glycosylated and without mucin-type glycosylation. Interestingly, we found that purified rhβ4GalT1 from silkworm serum presented higher galactosyltransferase activity than that expressed from cultured mammalian cells. We also validated the UDP-galactose transferase activity of produced rhβ4GalT1 proteins by using protein subtracts from silkworm silk gland. Taken together, rhβ4GalT1 from silkworms can become a valuable tool for producing high-quality recombinant glycoproteins with mammalian-like N-glycans.

Abstract 1450: Tumor-associated glycans interact with macrophages through class-A scavenger receptor

Abstract Introduction. Tumor initiation and growth are associated with modifications to the glycan structure of glycoproteins, glycolipids and proteoglycans present on the cell surface. Suppression of the anti-tumor immune response is a putative mechanism linking tumor-associated-glycans to tumor progression. Within a tumor microenvironment, the conversion of macrophages from an activated, immunological type (referred to as M1) to an alternatively activated, IL-10 secreting type (referred to as M2) suppresses the immune response and potentiates tumor progression. We previously reported that the class A scavenger receptor (SR-A)-mediated macrophage activation enhances production of IL-10, indicating promotion of an immunosuppressive M2 phenotype. Therefore, tumor glycans may suppress immune responses by interacting with macrophages and promoting an M2 phenotype. Such interactions are most likely mediated by pattern recognition receptors such as SR-A. In the current study, we investigated whether tumor glycans interact with macrophages via binding to SRA. Methods. Binding of recombinant soluble SR-A to breast cancer cells and relevant glycan structures was assayed by flow cytometry and ELISA assays. N-glycosylation was inhibited by incubating cells with tunicamycin. Peritoneal macrophages were harvested from wild type (SR-A+/+) and knock-out (SR-A-/-) mice and used for ligand competition and functional assays. A peptide mimic of GlcNAc/GalNAc-containing tumor-associated carbohydrates was used for binding and competition assays. Results. To determine whether carbohydrates present on tumor cells are SR-A ligands, we examined the binding of a soluble SR-A protein to tumor cells. We found that soluble SR-A bound to breast cancer cells but not to normal epithelial cells, and that this tumor-specific binding of soluble SR-A was abolished by treating cells with tunicamycin. These results demonstrate that cell surface tumor-associated glycans are ligands for SR-A. Using purified glycans, we found that soluble SR-A bound to multiple breast cancer-associated glycans. To assess the potential consequence of glycan binding to SR-A on macrophage function, we adhered primary macrophages to plates coated with a carbohydrate-mimetic peptide and found that SR-A+/+ macrophages bound to CMP-coated plates showed increased IL-10 production relative to SR-A-/- macrophages. The CMP also competed for acetylated LDL binding to macrophages, indicating that it is a ligand for SR-A. These results suggest that SR-A binding to carbohydrate promotes macrophages to adopt an M2 phenotype. Conclusions. Together, our results indicate that tumor-associated glycans interact with macrophages via SR-A, and that this interaction leads to production of IL-10, which is a key characteristic of immunosuppressive (M2-like) macrophages. Citation Format: Behjatolah Monzavi Karbassi, Thomas Kieber-Emmons, Steven R. Post. Tumor-associated glycans interact with macrophages through class-A scavenger receptor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1450.

N-Propionylmannosamine: using biochemical glycoengineering to promote peripheral nerve regeneration.

N-Propionylmannosamine: using biochemical glycoengineering to promote peripheral nerve regeneration.

Lectin-probed western blot analysis.

Lectin-probed western blot analysis, the so-called lectin blot analysis, is a useful method to yield basic information on the glycan structures of glycoproteins, based on the carbohydrate-binding specificities of lectins. By lectin blot analysis, researchers can directly analyze the glycan structures without releasing the glycans from glycoproteins. Here, the author describes protocols for standard analysis, and applies analysis in combination with glycosidase digestion of blot.

UniCarbKB: building a knowledge platform for glycoproteomics

The UniCarb KnowledgeBase (UniCarbKB; http://unicarbkb.org) offers public access to a growing, curated database of information on the glycan structures of glycoproteins. UniCarbKB is an international effort that aims to further our understanding of structures, pathways and networks involved in glycosylation and glyco-mediated processes by integrating structural, experimental and functional glycoscience information. This initiative builds upon the success of the glycan structure database GlycoSuiteDB, together with the informatic standards introduced by EUROCarbDB, to provide a high-quality and updated resource to support glycomics and glycoproteomics research. UniCarbKB provides comprehensive information concerning glycan structures, and published glycoprotein information including global and site-specific attachment information. For the first release over 890 references, 3740 glycan structure entries and 400 glycoproteins have been curated. Further, 598 protein glycosylation sites have been annotated with experimentally confirmed glycan structures from the literature. Among these are 35 glycoproteins, 502 structures and 60 publications previously not included in GlycoSuiteDB. This article provides an update on the transformation of GlycoSuiteDB (featured in previous NAR Database issues and hosted by ExPASy since 2009) to UniCarbKB and its integration with UniProtKB and GlycoMod. Here, we introduce a refactored database, supported by substantial new curated data collections and intuitive user-interfaces that improve database searching.

Genomics of corneal wound healing: a review of the literature

Corneal wound healing is a complex process: its mechanisms and the underlying genetic control are not fully understood. It involves the integrated actions of multiple growth factors, cytokines and proteases produced by epithelial cells, stromal keratocytes, inflammatory cells and lacrimal gland cells. Following an epithelial insult, multiple cytokines are released triggering a cascade of events that leads to repair the epithelial defect and remodelling of the stroma to minimize the loss of transparency and function. In this review, we examine the literature surrounding the genomics of corneal wound healing with respect to the following topics: epithelial and stromal wound healing (including inhibition); corneal neovascularisation; the role of corneal nerves in wound healing; the endothelium; the role of aquaporins and aptamers. We also examine the effect of ectasia on corneal wound healing with regard to keratoconus and following corneal surgery. A better understanding of the cellular and molecular changes that occur during repair of corneal wounds will provide the opportunity to design treatments that selectively modulate key phases of the healing process resulting in scars that more closely resemble normal corneal architecture.

Improvement of mass spectrometry analysis of glycoproteins by MALDI-MS using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid

Protein glycosylation analysis is important for elucidating protein function and molecular mechanisms in various biological processes. We previously developed a glycan analysis method using a 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid liquid matrix (3-AQ/CHCA LM) and applied it to the quantitative glycan profiling of glycoproteins. However, information concerning glycosylation sites is lost; glycopeptide analysis is therefore required to identify the glycosylation sites in glycoproteins. Human epidermal growth factor receptor 2 (HER2) is a glycoprotein that plays a role in the regulation of cell proliferation, differentiation, and migration. Several reports have described the structure of HER2, but the structures of N-glycans attached to this protein remain to be fully elucidated. In this study, 3-AQ/CHCA LM was applied to tryptic digests of HER2 to reveal its N-glycosylation state and to evaluate the utility of this LM in characterizing glycopeptides. Peptide sequence coverage was considerably improved compared to analysis of HER2 using α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid. Most of the peaks observed using only this LM were localized at the inner or outer regions of sample spots. Furthermore, five of the peptide peaks that were enriched within the inner region were confirmed to be glycosylated by MS/MS analysis. Three glycosylation sites were identified and their glycan structures were elucidated. The reduction in sample complexity by on-target separation allowed for higher sequence coverage, resulting in effective detection and characterization of glycopeptides. In conclusion, these results demonstrate that MS-based glycoprotein analysis using 3-AQ/CHCA is an effective method to identify glycosylation sites in proteins and to elucidate the glycan structures of glycoproteins in complex samples.

Glycan‐Specific Metabolic Oligosaccharide Engineering of C7‐Substituted Sialic Acids

Intact and integral glycosylation of membrane-associated as well as secreted glycoproteins has been shown to be essential for many aspects of the proper function of biological systems. Recombinantly expressed glycoproteins, such as antibodies, growth factors, hormones, vaccines, and contrast agents are key elements in medical applications. The quality of these therapeutically administered glycoproteins can be efficiently improved by the incorporation of chemically functionalized monosaccharides into their glycan moieties, a process denoted as metabolic oligosaccharide engineering (MOE). In addition to these pharmaceutical applications, MOE has greatly advanced diagnostics by localizing and visualizing glycans even in living animals. To date, a multitude of chemically modified monosaccharides have been designed for MOE applications. Owing to their terminal position at glycan structures of glycoproteins and relevance for cellular recognition, sialic acids and their metabolic precursor N-acetylmannosamine (ManNAc), are the most prominent targets for MOE. Several ManNAc derivatives with N-acetyl side-chain modifications have been synthesized and metabolically incorporated by the sialic acid biosynthetic pathway into a corresponding sialic acid C5 analogue (Figure 1). This approach was beneficial to extending the understanding of the biological role of the N-acyl side chain of sialic acids, for example, in virus infection or neuronal differentiation. Alternatively, C9 modifications of sialosides have also been achieved by directly administering synthetic sialic acid analogues. Additionally, selective cleavage of the glycol moiety led to a truncated sialic acid equipped glycans with an aldehyde for labeling reactions (Figure 1). All of these modifications address sialylation of both, Nand O-glycosylation of glycoproteins, to almost the same extent. Herein we investigate whether the biosynthetic machinery for sialic acids also tolerates other ManNAc derivatives as substrates, which are modified directly at the six-membered carbohydrate ring. The modification of the C4 position appeared most attractive, because it is not enzymatically modified during cellular glycoprotein production and would deliver previously unknown C7-modified sialic acid containing glycoproteins (Figure 1). To probe the biosynthetic promiscuity, we targeted a C4-modified ManNAc derivative, N-acetyl-4-azido-4-deoxymannosamine (4-azido-ManNAc, 1), in our study to enable postglycosylational conjugation and visualization by bioorthogonal reactions. N-acetyl-(1,3,6-O-acetyl)-4-azido-4-deoxy-mannosamine (Ac3-4-azido-ManNAc) was generated by an optimized literature method (Figure S1 in the Supporting InformaFigure 1. Methods for the structural modification of glycan-bound sialic acids by application of chemically modified ManNAc or direct periodate oxidation of glycan-bound sialic acids (left). Specific modification of the C7 position of sialic acids was achieved by C4-modified ManNAc in this study (right; note that to date these methods were carried individually, resulting in only one modification of a single sialic acid molecule).

Strain-promoted alkyne-azide cycloadditions (SPAAC) reveal new features of glycoconjugate biosynthesis.

We have shown that 4-dibenzocyclooctynol (DIBO), which can easily be obtained by a streamlined synthesis approach, reacts exceptionally fast in the absence of a Cu(I) catalyst with azido-containing compounds to give stable triazoles. Chemical modifications of DIBO, such as oxidation of the alcohol to a ketone, increased the rate of strain promoted azide-alkyne cycloadditions (SPAAC). Installment of a ketone or oxime in the cyclooctyne ring resulted in fluorescent active compounds whereas this property was absent in the corresponding cycloaddition adducts; this provides the first example of a metal-free alkyne-azide fluoro-switch click reaction. The alcohol or ketone functions of the cyclooctynes offer a chemical handle to install a variety of different tags, and thereby facilitate biological studies. It was found that DIBO modified with biotin combined with metabolic labeling with an azido-containing monosaccharide can determine relative quantities of sialic acid of living cells that have defects in glycosylation (Lec CHO cells). A combined use of metabolic labeling/SPAAC and lectin staining of cells that have defects in the conserved oligomeric Golgi (COG) complex revealed that such defects have a greater impact on O-glycan sialylation than galactosylation, whereas sialylation and galactosylation of N-glycans was similarly impacted. These results highlight the fact that the fidelity of Golgi trafficking is a critical parameter for the types of oligosaccharides being biosynthesized by a cell. Furthermore, by modulating the quantity of biosynthesized sugar nucleotide, cells might have a means to selectively alter specific glycan structures of glycoproteins.

Glycobiomarkers by glycoproteomics and glycan profiling (glycomics): emergence of functionality

Glycans stand out from all classes of biomolecules because of their unsurpassed structural complexity. This is generated by variability in anomeric status of the glycosidic bond and its linkage points, ring size, potential for branching and introduction of diverse site-specific substitutions. What poses an enormous challenge for analytical processing is, at the same time, the basis for the fingerprint-like glycomic profiles of glycoconjugates and cells. What's more, the glycosylation machinery is sensitive to disease manifestations, earning glycan assembly a reputation as a promising candidate to identify new biomarkers. Backing this claim for a perspective in clinical practice are recent discoveries that even seemingly subtle changes in the glycan structure of glycoproteins, such as a N-glycan core substitution by a single sugar moiety, have far-reaching functional consequences. They are brought about by altering the interplay between the glycan and (i) its carrier protein and (ii) specific receptors (lectins). Glycan attachment thus endows the protein with a molecular switch and new recognition sites. Co-ordinated regulation of glycan display and presentation of the cognate lectin, e.g. in cancer growth regulation exerted by a tumour suppressor, further exemplifies the broad functional dimension inherent to the non-random shifts in glycosylation. Thus studies on glycobiomarkers converge with research on how distinct carbohydrate determinants are turned into bioactive signals.

Identification, affinity characterisation and biological interactions of lectin‐like peptide–carbohydrate complexes derived from human TNF‐α using high‐resolution mass spectrometry

A cyclic disulfide heptadecapeptide (TIP17ox; 2) derived from the lectin-like 17-amino acid domain of human tumor necrosis factor-alpha [TNF-alpha (100-116)] was synthesised and demonstrated to bind specifically to N,N-diacetylchitobiose, a disaccharide present in many glycan structures of glycoproteins. Although the TIP domain forms a loop structure in the native TNF-alpha protein, we show in this study by high-resolution ESI-FTICR mass spectrometry that a homologous linear heptadecapeptide (TIP17rd; 1) binds with comparable affinity to chitobiose, suggesting that cyclisation is not essential for carbohydrate binding. ESI-FTICR-MS was used as an efficient tool for the direct molecular characterisation of TIP peptide-carbohydrate complexes. The specific binding of the TNF-TIP domain to chitobiose and other carbohydrate motifs in glycoproteins may explain the high proteolytic stability of these peptides in biological fluids. A considerably higher proteolytic stability in human plasma was found by mass spectrometric analysis for the cyclic TIP peptide 2, compared to the linear peptide 1. Furthermore, affinity-proteomics studies using immobilised cyclic TIP peptide 2 provided the identification of specific interacting glycoproteins in plasma.

N-linked glycan structures of glycoproteins in suspension-cultured Arabidopsis thaliana MM2d cells

The oligosaccharide structures of total glycoproteins in suspension-cultured Arabidopsis thaliana MM2d cells are determined. The N-linked sugar chains released by hydrazinolysis were labeled with 2-aminopyridine, and analyzed by a combination of HPLC, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and exoglucosidase digestion. The glycan structures determined were GlcNAcMan3FucXylGlcNAc2 (8.5%), Man3FucXylGlcNAc2 (70.0%), Man3XylGlcNAc2 (13.7%), and GlcNAcMan3XylGlcNAc2 (7.8%). All the glycan structures of glycoproteins from Arabidopsis MM2d cells used in this study contain typical plant bisecting either b1,2-xylose or a1,3-fucose residues, showing that suspension-cultured MM2d cells have a glycosylation potential similar to that of tobacco BY2 suspension- cultured cells.

Printed covalent glycan array for ligand profiling of diverse glycan binding proteins.

Here we describe a glycan microarray constructed by using standard robotic microarray printing technology to couple amine functionalized glycans to an amino-reactive glass slide. The array comprises 200 synthetic and natural glycan sequences representing major glycan structures of glycoproteins and glycolipids. The array has remarkable utility for profiling the specificity of a diverse range of glycan binding proteins, including C-type lectins, siglecs, galectins, anticarbohydrate antibodies, lectins from plants and microbes, and intact viruses.

Glycoproteins secreted from suspension-cultured tobacco BY2 cells have distinct glycan structures from intracellular glycoproteins.

Glycan structures of glycoproteins secreted in the spent medium of tobacco BY2 suspension-cultured cells were analyzed. The N-glycans were liberated by hydrazinolysis and the resulting oligosaccharides were labeled with 2-aminopyridine. The pyridylaminated (PA) glycans were purified by reversed-phase and size-fractionation HPLC. The structures of the PA sugar chains were identified by a combination of the two-dimensional PA sugar chain mapping, MS analysis, and exoglycosidase digestion. The ratio (40:60) of the amount of glycans with high-mannose-type structure to that with plant-complex-type structure of extracellular glycoproteins is significantly different from that (ratio 10:90) previously found in intracellular glycoproteins [Palacpac et al., Biosci. Biotechnol. Biochem. 63 (1999) 35-39]. Extracellular glycoproteins have six distinct N-glycans (marked by *) from intracellular glycoproteins, and the high-mannose-type structures account for nearly 40% (Man5GlcNAc2, 28.8%; Man6GlcNAc2*, 6.4%; and Man7GlcNAc2*, 3.8%), while the plant-complex-type structures account for nearly 60% (GlcNAc2Man3Xyl1GlcNAc2*, 32.1%; GlcNAc1Man3Xyl1GlcNAc2 (containing two isomers)*, 6.2%; GlcNAc2Man3GlcNAc2*, 4.9%; Man3Xyl1Fuc1GlcNAc2, 8.3%; and Man3Xyl1GlcNAc2, 3.7%).

Protein glycosylation.

Glycan structures can modulate the biological properties and functions of glycoproteins. This has been shown by investigation of the biological activities and glycan structures of several recombinant glycoproteins. Glycan structures of glycoproteins differ according to the species and tissue producing them, and selection of an appropriate host-cell type can generate recombinant glycoproteins with new characteristics.