Terminal Sugar Residues Research Articles

IgG sialylation occurs in B cells pre antibody secretion.

Sialic acids as terminal sugar residues on cell surface or secreted proteins have many functional roles. In particular, the presence or absence of α2,6-linked sialic acid residues at the immunoglobulin G (IgG) Fc fragment can switch IgG effector functions from pro- to anti-inflammatory activity. IgG glycosylation is considered to take place inside the plasma blast/plasma cell while the molecule travels through the endoplasmic reticulum and Golgi apparatus before being secreted. However, more recent studies have suggested that IgG sialylation may occur predominantly post-antibody secretion. To what extent this extracellular IgG sialylation process contributes to overall IgG sialylation remains unclear, however. By generating bone marrow chimeric mice with a B cell-specific deletion of ST6Gal1, the key enzyme required for IgG sialylation, we now show that sialylation of the IgG Fc fragment exclusively occurs within B cells pre-IgG secretion. We further demonstrate that B cells expressing ST6Gal1 have a developmental advantage over B cells lacking ST6Gal1 expression and thus dominate the plasma cell pool and the resulting serum IgG population in mouse models in which both ST6Gal1-sufficient and -deficient B cells are present.

Abstract SY34-01: Plasticity, dormancy, and glycosylation of circulating tumor cell clusters in response to therapy

Abstract Cellular plasticity and stemness properties enable dynamic changes of circulating tumor cells (CTCs) during cancer dissemination, such as aggregation or cohesion of single CTCs into multicellular CTC clusters with 20-100 times higher metastatic propensity than the singles. Our work demonstrated that stemness glycoproteins on breast cancer stem cells, such as CD44, CD81, and ICAM1 drive CTC aggregation in metastatic breast cancer, such as triple negative breast cancer (TNBC). To determine the glycosylation patterns in CTCs and its association with clinical outcomes, we established multiple CTC analysis approaches including CellSearch, flow cytometry, and immunohistochemistry using patient blood and tissue sections collected at the pre-treatment baseline and after-therapy timepoints. We found that chemotherapy-evasive CTC clusters are relatively quiescent with a specific loss of terminal sugar residues α2,6-sialic acids in glycoproteins. CTCs showed dynamic hypo-sialylation in the blood with loss of the sialyl-transferase ST6GAL1, promoting CTC cluster formation with cellular quiescence (proliferative dormancy) and evading chemotherapy in breast cancer. Seeded tumor cells regained ST6GAL1 to enable metastatic colonization. Many adhesion proteins and stemness drivers as glycoprotein substrates of ST6GAL1 drive CTC clustering and metastatic seeding. Neutralizing antibodies against these clustering drivers inhibit CTC cluster formation and improve therapy response, thereby blocking lung metastasis in TNBC. Citation Format: Huiping Liu. Plasticity, dormancy, and glycosylation of circulating tumor cell clusters in response to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY34-01.

Terminal glycosylation pattern as a determinant of human iPSC-derived cardiomyocytes transduction with AAV vectors

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Young scientists grant MNS9/2021 Polish National Science Centre PRELUDIUM grant 2019/33/N/NZ1/03066 Introduction Although gene therapy has become a feasible alternative to regular treatment in numerous disorders, efficient targeting of human heart still seems out of reach. So far, adeno-associated viral vectors (AAVs) have been the most promising transgene carriers in in vivo applications. Transduction with AAVs relies on serotype-specific binding to the glycan residues on the cell surface. While AAV serotype 9 (AAV9) - binding galactose - was successfully used for delivery of therapeutic genes to cardiac muscle in murine models, its effect was underwhelming in large animals. Switching the serotype to AAV1 or AAV6 – binding sialic acids – enabled transduction of porcine hearts, and cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) in 2D in vitro culture. But, as evidenced in clinical trials where less than 1% of heart cells contained delivered transgene, such strategy was not suitable for in vivo application in humans. Purpose Taking into account differences in glycosylation patterns between humans and other mammals, we aimed to investigate the role of surface glycans in AAV6 and AAV9 transduction mechanism in 2D and 3D cultures of human cardiomyocytes. Methods We generated hiPSC- derived cardiomyocytes (hiPSC-CMs) and epicardial fibroblasts (hiPSC-CFs). Cells were cultured in regular 2D conditions or 3D co-cultures (as spheroids), since direct cells interaction may influence the availability of terminal sugar residues. Surface glycans were stained with lectins and imaged using confocal microscope. Results While in 2D conditions AAV6 transduces hiPSC-CMs very efficiently, it is significantly less potent in 3D culture. Conversely, AAV9 maintains most of its functionality in 3D model. Our data collected from spheroids, indicate that cells in such 3D cultures undergo significant changes in terminal glycosylation pattern. While the level of galactose (AAV9 receptor) increased from day 1 to day 7, the signal from sialic acids attached to galactose (AAV6 receptor) decreased. This effect was accompanied by the upregulation of sialidase-3 expression, that regulates availability of galactose residues on the cell surface. As a result, transduction efficiency was improved in spheroids exposed to AAV9 on day 7 of 3D culture, in comparison to those transduced on day 1. Inhibition of sialidases activity with NADNA reduced the level of terminal galactose in both, hiPSC-CMs and hiPSC-CFs, and was associated with more efficient transduction of cardiomyocytes with AAV6, and less efficient transduction with AAV9. Additionally, we observed that galactosylation of collagen can be another factor that influences cell-vector interaction in 2D and 3D culture. Conclusions Our results demonstrate that the availability of terminal sugar residues is a key factor regulating transduction of hiPSC-CMs with AAV vectors, and underline the need for development of appropriate models for testing of AAV functionality in preclinical studies.

The NKT cell TCR repertoire can accommodate structural modifications to the lipid and orientation of the terminal carbohydrate of iGb3

Isoglobotrihexosylceramide (iGb3) is a known NKT cell agonist, however the specific interactions required to trigger NKT cell TCR activation in response to this mammalian glycolipid are not fully understood. Here we report the synthesis of 1,3-β-Gal-LacCer (βG-iGb3) that displays a β-linked terminal sugar. βG-iGb3 activated NKT cells to a similar extent as iGb3 with a terminal α-linkage, indicating that the conformation of the terminal sugar residue of iGb3 is not essential to facilitate NKT cell TCR recognition. In addition, the immunological activity of four recently described iGb3 analogues with modifications to their terminal sugar or lipid backbone were also investigated. These iGb3 analogues all induced NKT cell proliferation, with IL-13 the predominate cytokine detected. This highlights the ability of the NKT cell TCR to accommodate variations in iGb3-based glycolipids and suggests that undiscovered NKT cell ligands may exist within the lacto-series of mammalian glycosphingolipids.

Combining oligomer build-up with alanine scanning to determine the flocculation protein mutants for enhancing oligosaccharide binding

ABSTRACT Oligosaccharide binding on protein plays an important role in cellular signalling and recognition. Protein mutation can alter the binding strength and thus regulate the relevant processes. In this work, we investigated the interactions between the mannose oligosaccharides and the wild type and mutant forms of yeast flocculation protein Flo1p from Saccharomyces cerevisiae. The binding strength of an oligosaccharide chain was estimated using molecular dynamics simulation and MM/GBSA binding free energy calculation, via the build-up of oligosaccharide from monosaccharide to disaccharide, trisaccharide and tetrasaccharide. At the protein-disaccharide stage, several mutation positions were identified via alanine scanning mutagenesis, leading to nine mutants, from which two mutants Q117N and Q117R were selected with significantly stronger binding free energies relative to the wild type protein. Furthermore, the two mutants also show enhanced binding at the trisaccharide and tetrasaccharide stages. Conformational stability and shape change were analysed to help understand the protein-oligosaccharide interactions. This study also presented a computational example to estimate the oligosaccharide binding strength using its terminal sugar residues.

Triterpene Glycosides from the Far Eastern Sea Cucumber Psolus chitonoides: Chemical Structures and Cytotoxicities of Chitonoidosides E1, F, G, and H.

Four new triterpene disulfated glycosides, chitonoidosides E1 (1), F (2), G (3), and H (4), were isolated from the Far-Eastern sea cucumber Psolus chitonoides and collected near Bering Island (Commander Islands) at depths of 100–150 m. Among them there are two hexaosides (1 and 3), differing from each other by the terminal (sixth) sugar residue, one pentaoside (4) and one tetraoside (2), characterized by a glycoside architecture of oligosaccharide chains with shortened bottom semi-chains, which is uncommon for sea cucumbers. Some additional distinctive structural features inherent in 1–4 were also found: the aglycone of a recently discovered new type, with 18(20)-ether bond and lacking a lactone in chitonoidoside G (3), glycoside 3-O-methylxylose residue in chitonoidoside E1 (1), which is rarely detected in sea cucumbers, and sulfated by uncommon position 4 terminal 3-O-methylglucose in chitonoidosides F (2) and H (4). The hemolytic activities of compounds 1–4 and chitonoidoside E against human erythrocytes and their cytotoxic action against the human cancer cell lines, adenocarcinoma HeLa, colorectal adenocarcinoma DLD-1, and monocytes THP-1, were studied. The glycoside with hexasaccharide chains (1, 3 and chitonoidoside E) were the most active against erythrocytes. A similar tendency was observed for the cytotoxicity against adenocarcinoma HeLa cells, but the demonstrated effects were moderate. The monocyte THP-1 cell line and erythrocytes were comparably sensitive to the action of the glycosides, but the activity of chitonoidosides E and E1 (1) significantly differed from that of 3 in relation to THP-1 cells. A tetraoside with a shortened bottom semi-chain, chitonoidoside F (2), displayed the weakest membranolytic effect in the series.

Structural characterization and immunomodulatory activities of two polysaccharides from Rehmanniae Radix Praeparata

The structures and immunomodulatory activities of two polysaccharides (SDH-WA and SDH-0.2A) from Rehmanniae Radix Praeparata (RRP) were investigated. RRP crude polysaccharide was obtained by water extraction and purified. Ion chromatography, high-performance gel permeation chromatography, Fourier-transform infrared spectroscopy, methylation analysis, gas chromatography–mass spectrometry, and nuclear magnetic resonance were used to characterize the polysaccharides. The main chain of SDH-WA was →6)-α-D-Galp-(1→6)-α-D-Galp-(1→5)-α-L-Araf-(1→3,5)-α-L-Araf-(1→, terminal sugar residue α-L-Araf-(1→ linked to residue →3,5)-α-L-Araf-(1→ on the main chain by an O-3 bond. The other two terminal sugar residues α-D-Galp-(1→ and →6)-β-D-Galp were linked to the end of the main chain. The main chain of SDH-0.2A was →2,4)-α-L-Rhap-(1→4)-α-D-GalpA-(1→. Three branched chains α-D-Galp-(1→6)-α-D-Galp-(1→5)-α-L-Araf-(1→3,5)-α-L-Araf-(1→, →3,6)-β-D-Galp-(1→5)-α-L-Araf-(1→, and →4)-β-D-Galp-(1→5)-α-L-Araf-(1→ were linked to the main chain residue →2,4)-α-L-Rhap-(1→ by an O-2 bond. Three terminal sugar residues α-D-Galp-(1→, α-L-Araf-(1→, and →6)-β-D-Galp were linked to the end of the chain. Both polysaccharides showed no cytotoxic effects on and significantly promoted the phagocytic activity of RAW264.7 cells. They dose-dependently improved lysozyme activity and stimulated the production of TNF-α and IL-6 by RAW264.7 cells, but attenuated the secretion of lysozymes, TNF-α, IL-6, IL-1β, and nitric oxide by lipopolysaccharide-induced RAW264.7 cells. The present studies suggest that PRR polysaccharide is a valuable source with immunomodulating.

Lewis A Glycans Are Present on Proteins Involved in Cell Wall Biosynthesis and Appear Evolutionarily Conserved Among Natural Arabidopsis thaliana Accessions

N-glycosylation is a highly abundant protein modification present in all domains of life. Terminal sugar residues on complex-type N-glycans mediate various crucial biological processes in mammals such as cell-cell recognition or protein-ligand interactions. In plants, the Lewis A trisaccharide constitutes the only known outer-chain elongation of complex N-glycans. Lewis A containing complex N-glycans appear evolutionary conserved, having been identified in all plant species analyzed so far. Despite their ubiquitous occurrence, the biological function of this complex N-glycan modification is currently unknown. Here, we report the identification of Lewis A bearing glycoproteins from three different plant species: Arabidopsis thaliana, Nicotiana benthamiana, and Oryza sativa. Affinity purification via the JIM84 antibody, directed against Lewis A structures on complex plant N-glycans, was used to enrich Lewis A bearing glycoproteins, which were subsequently identified via nano-LC-MS. Selected identified proteins were recombinantly expressed and the presence of Lewis A confirmed via immunoblotting and site-specific N-glycan analysis. While the proteins identified in O. sativa are associated with diverse functions, proteins from A. thaliana and N. benthamiana are mainly involved in cell wall biosynthesis. However, a Lewis A-deficient mutant line of A. thaliana showed no change in abundance of cell wall constituents such as cellulose or lignin. Furthermore, we investigated the presence of Lewis A structures in selected accessions from the 1001 genome database containing amino acid variations in the enzymes required for Lewis A biosynthesis. Besides one relict line showing no detectable levels of Lewis A, the modification was present in all other tested accessions. The data provided here comprises the so far first attempt at identifying Lewis A bearing glycoproteins across different species and will help to shed more light on the role of Lewis A structures in plants.

Characterization of O-glycan binding lectin from the red alga Hydropuntia eucheumatoides

The red alga, Hydropuntia eucheumatoides is one of the algal genera from which agar is commercially extracted, and is the main source of agar in the world. The lectin HEL from the red alga H. eucheumatoides was isolated by a combination of aqueous ethanol extraction, ethanol precipitation, ion exchange and filtration chromatography. Lectin gave a single band with molecular mass of 17,000 Da in both non-reducing and reducing SDS-PAGE conditions, therefore lectin exists in monomeric form. The hemagglutination activities of HEL were stable over a wide range of pH from 3 to 10, temperature up 60 oC and not affected by either the presence of EDTA or addition of divalent cations, indicating that lectin requires no metal for biological activity. The hemagglutination activities of HEL were not inhibited by monosaccharides and glycoproteins, D-glucose, D-mannose, D-galactose, D-xylose, N-acety-D-mannosamine, transferin, fetuin and yeast mannan, but strongly inhibited by monosaccharides containing acetamido groups at equatorial C2 position, such as N-acetyl-galactosamine, N-acetyl-glucosamine, N-acetyl-neuraminic acid and glycoprotein porcine stomach mucin bearing O-glycans. Thus, lectin is specific for O-glycans and may recognize the sequences GalNAcαSer/Thr, GalNAc(α1-3)[Fuc(α1-2)]Gal(β1-4)GlcNAc(β1-3)GalNAc- and GluNAc(α1-4)Gal- under interacting with the acetamido groups at equatorial C2 position of the terminal sugar residues in oligosaccharide structures of O-glycans. The red alga H. eucheumatoides could promise to be a source of valuable lectins for application in biochemistry and biomedicine.

Control of Innate Immunity by Sialic Acids in the Nervous Tissue.

Sialic acids (Sias) are the most abundant terminal sugar residues of glycoproteins and glycolipids on the surface of mammalian cells. The nervous tissue is the organ with the highest expression level of Sias. The ‘sialylation’ of glycoconjugates is performed via sialyltransferases, whereas ‘desialylation’ is done by sialidases or is a possible consequence of oxidative damage. Sialic acid residues on the neural cell surfaces inhibit complement and microglial activation, as well as phagocytosis of the underlying structures, via binding to (i) complement factor H (CFH) or (ii) sialic acid-binding immunoglobulin-like lectin (SIGLEC) receptors. In contrast, activated microglial cells show sialidase activity that desialylates both microglia and neurons, and further stimulates innate immunity via microglia and complement activation. The desialylation conveys neurons to become susceptible to phagocytosis, as well as triggers a microglial phagocytosis-associated oxidative burst and inflammation. Dysfunctions of the ‘Sia–SIGLEC’ and/or ‘Sia–complement’ axes often lead to neurological diseases. Thus, Sias on glycoconjugates of the intact glycocalyx and its desialylation are major regulators of neuroinflammation.

Principles of N-Linked Glycosylation Variations of IgG-Based Therapeutics: Pharmacokinetic and Functional Considerations.

The development of recombinant therapeutic proteins has been a major revolution in modern medicine. Therapeutic-based monoclonal antibodies (mAbs) are growing rapidly, providing a potential class of human pharmaceuticals that can improve the management of cancer, autoimmune diseases, and other conditions. Most mAbs are typically of the immunoglobulin G (IgG) subclass, and they are glycosylated at the conserved asparagine position 297 (Asn-297) in the CH2 domain of the Fc region. Post-translational modifications here account for the observed high heterogeneity of glycoforms that may or not impact the stability, pharmacokinetics (PK), efficacy, and immunogenicity of mAbs. These modifications are also critical for the Fc receptor binding, and consequently, key antibody effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Moreover, mAbs produced in non-human cells express oligosaccharides that are not normally found in serum IgGs might lead to immunogenicity issues when administered to patients. This review summarizes our understanding of the terminal sugar residues, such as mannose, sialic acids, fucose, or galactose, which influence therapeutic mAbs either positively or negatively in this regard. This review also discusses mannosylation, which has significant undesirable effects on the PK of glycoproteins, causing a decreased mAbs’ half-life. Moreover, terminal galactose residues can enhance CDC activities and Fc–C1q interactions, and core fucose can decrease ADCC and Fc–FcγRs binding. To optimize the therapeutic use of mAbs, glycoengineering strategies are used to reduce glyco-heterogeneity of mAbs, increase their safety profile, and improve the therapeutic efficacy of these important reagents.

Sialylation and Galectin-3 in Microglia-Mediated Neuroinflammation and Neurodegeneration.

Microglia are brain macrophages that mediate neuroinflammation and contribute to and protect against neurodegeneration. The terminal sugar residue of all glycoproteins and glycolipids on the surface of mammalian cells is normally sialic acid, and addition of this negatively charged residue is known as “sialylation,” whereas removal by sialidases is known as “desialylation.” High sialylation of the neuronal cell surface inhibits microglial phagocytosis of such neurons, via: (i) activating sialic acid receptors (Siglecs) on microglia that inhibit phagocytosis and (ii) inhibiting binding of opsonins C1q, C3, and galectin-3. Microglial sialylation inhibits inflammatory activation of microglia via: (i) activating Siglec receptors CD22 and CD33 on microglia that inhibit phagocytosis and (ii) inhibiting Toll-like receptor 4 (TLR4), complement receptor 3 (CR3), and other microglial receptors. When activated, microglia release a sialidase activity that desialylates both microglia and neurons, activating the microglia and rendering the neurons susceptible to phagocytosis. Activated microglia also release galectin-3 (Gal-3), which: (i) further activates microglia via binding to TLR4 and TREM2, (ii) binds to desialylated neurons opsonizing them for phagocytosis via Mer tyrosine kinase, and (iii) promotes Aβ aggregation and toxicity in vivo. Gal-3 and desialylation may increase in a variety of brain pathologies. Thus, Gal-3 and sialidases are potential treatment targets to prevent neuroinflammation and neurodegeneration.

A51 UTILIZING MUCIN-DERIVED SUGARS CONFERS A FITNESS ADVANTAGE TO THE ENTERIC PATHOGEN CITROBACTER RODENTIUM THAT PROMOTES INTESTINAL COLONIZATION

Abstract Background Citrobacter rodentium is an enteric murine pathogen used to model the human diarrheal pathogens. Following inoculation, C. rodentium colonizes the mouse cecum where it expands and ultimately spreads to the distal colon. During this process, C. rodentium has to compete with commensal microbes for available nutrients. Moreover, to spread throughout the gut, and infect the intestinal epithelium, C. rodentium has to cross through, and or dwell within the intestinal mucus layer which is composed of the heavily glycosylated protein Muc2. Muc-2 is glycosylated and coated by 5 distinct terminal sugar residues: galactose, N-acetylgalactosamine, N-acetylglucosamine, fucose, and sialic acid. Many commensal microbes have the ability to cleave and free these sugars from the Muc2 protein, releasing them for their own consumption, however pathogens appear to exploit this process. While studies have indicated that C. rodentium uses these terminal sugar residues as a nutritional source, their relative importance in the pathogenic strategy of C. rodentium (and other gut pathogens) remains unclear Aims Investigate the role played by mucin sugar residues in controlling C. rodentium pathogenesis Methods Deletions of agaW, nagE, mglB, galP, fucK, and nanT were generated on the chromosome of C. rodentium (Strepr) by overlap extension PCR. Growth assays were performed to examine the growth kinetics of mutants C. rodentium in minimal (M9) media supplemented with one of the 5 mucin sugars or M9 with whole mucin as control. Specific pathogen free (SPF) C57BL/6 mice, or germfree C57BL/6 mice were orally gavaged with wildtype C. rodentium (Strepr) or one of ΔagaW, ΔnagE, ΔmglB, ΔgalP, ΔfucK, or ΔnanT strains. Mice were euthanized at 6 days post-infection, and the cecum, colon, and spleen were collected and histologically scored for pathology and intestinal and systemic bacterial burden. Stool samples were collected throughout the 6 days to quantify C. rodentium burdens Results Growth assays confirmed that the specific sugar transporter/kinase mutant C. rodentium strains grew normally when placed in media supplemented with whole mucin, or with most sugars, only showing overt defects in growth when solely supplemented with the sugar for which they were impaired. Several of the C. rodentium mutants including ΔnanT showed overt defects in colonization/infection of SPF C57BL/6 mice, but their pathogenesis was normalized in germfree mice, or in mice treated with the antibiotic streptomycin at each day post-infection. These findings indicate that the impact of mucin sugar utilization on C. rodentium virulence is microbiota-dependent Conclusions C. rodentium uses mucin sugars as nutrient source in the mouse gut, and an inability to use these sugars impairs their ability to infect their hosts in a microbiota dependent manner Funding Agencies CAG, CCC, CIHR, NRC

The terminal sialic acid of stage-specific embryonic antigen-4 has a crucial role in binding to a cancer-targeting antibody

The terminal sialic acid of stage-specific embryonic antigen-4 has a crucial role in binding to a cancer-targeting antibody

CD23 is a glycan-binding receptor in some mammalian species

CD23, the low-affinity IgE receptor found on B lymphocytes and other cells, contains a C-terminal lectin-like domain that resembles C-type carbohydrate-recognition domains (CRDs) found in many glycan-binding receptors. In most mammalian species, the CD23 residues required to form a sugar-binding site are present, although binding of CD23 to IgE does not involve sugars. Solid-phase binding competition assays, glycoprotein blotting experiments, and glycan array analysis employing the lectin-like domains of cow and mouse CD23 demonstrate that they bind to mannose, GlcNAc, glucose, and fucose and to glycoproteins that bear these sugars in nonreducing terminal positions. Crystal structures of the cow CRD in the presence of α-methyl mannoside and GlcNAcβ1–2Man reveal that a range of oligosaccharide ligands can be accommodated in an open binding site in which most interactions are with a single terminal sugar residue. Although mouse CD23 shows a pattern of monosaccharide and glycoprotein binding similar to cow CD23, the binding is weaker. In contrast, no sugar binding was observed in similar experiments with human CD23. The absence of sugar-binding activity correlates with accumulation of mutations in the gene for CD23 in the primate lineage leading to humans, resulting in loss of key sugar-binding residues. These results are consistent with a role for CD23 in many species as a receptor for potentially pathogenic microorganisms as well as IgE. However, the ability of CD23 to bind several different ligands varies between species, suggesting that it has distinct functions in different organisms.

Lectin Treatment Affects Malignant Characteristics of TPC-1 Papillary Thyroid Cancer Cells

Objective: Abnormal glycosylation is a universal aspect of cancer cells. The altered glycosylation pattern has been originated from changings in expression of glycosylation enzymes which are up-regulating in reply to some oncoproteins in the biosynthetic pathway of glycans. In this study, it was aimed to show the presence of terminal α-2,3, α-2,6 sialic acid and α-1,6/α-1,2 fucose motifs in TPC-1 papillary thyroid cancer cells. Also it was aimed to examine the changes in viability and mobility of the cells after exogenously specific lectin treatment. Materials and Methods: In this study, the presence of terminal sugar residues in glycan chains on the cell surface was demonstrated using lectin histochemistry and lectin blotting techniques in TPC-1 cells. The changes in the cell viability and proliferation after lectin treatment were assessed using the WST-1 test. The Changes in the cell mobility after lectin treatment, however, were assessed using the wound healing test. Results: α-2,3, α-2,6 sialic acid and α-1,6/α-1,2 fucose motifs were widespread in the surface of TPC-1 cells. MAL-II (Maackia amurensis Lectin II) treatment increased the cell proliferation and mobility of TPC-1 cells. Although SNA (Sambucus nigra Aglutinin) and AAL (Aleuria aurantia Lectin) treatment did not significantly affect the cell proliferation, SNA and AAL treatment supported the mobility of TPC-1 cells. Conclusion: Lectin treatment affect cancerous properties differently depending on the cell type. Also lectin treatment can support the malignant behaviour of cancer. For this reason, it is necessary to understand the mechanisms of the lectin effect on the cancer cells.

Lectin Histochemistry Showed a Heterogeneous Population of Cells Among Human Mesenchymal Stem Cells Isolated From Adipose Tissue

Objectives: Adipose tissue as an appropriate source of Mesenchymal Stem Cells (MSCs) has the potential to differentiate into multiple lineages. Glycoconjugates content of the MSCs can be considered as biomarkers in self-renewal, pluripotency and differentiation processes. In this study, the lectin profile of MSCs isolated from adipose tissue was detected and according to that, a subpopulation was determined. Materials & Methods: MSCs were isolated from adipose tissue by explanting of the tissue pieces. The FITC-conjugated lectins, WGA, UEA, PNA, BSA and PWM were used to detect the terminal sugar residues. The cells were then counterstained with DAPI. The intensity of the reaction was evaluated by ImageJ software. The cells were also stained with PAS method. Results: MSCs were reacted with all lectins with different intensity of the reactions. The cells reacted with WGA, UEA, and BSA “strongly” and with PWM “moderately” and with PNA with “weak” intensity. The morphological analysis of the isolated MSCs revealed the existence of the two different cell types in the cultures. Two types of cells were detected according to nucleus size and lectin reactivity. The cells with large nuclei constitute 20.62% of the total cells and stained significant more intensity by UEA and less intense with PWM (both P=0.014) and PNA (P=0.044). Flow cytometry with CD34 shows that these large cells were not endothelial cells. Conclusion: The MSCs derived from adipose tissue seem to be a heterogeneous populations and lectin profile of the cells showed that they are different in the expression of the glycoconjugates.

Complex Carbohydrate Marker to Distinguish Mucins from Low Grade Neoplasms versus Adenocarcinomas in the Colon Causing Pseudomyxoma Peritonei

Introduction: Normal colonic epithelial cells produce mucins which are more than 50% carbohydrate and have characteristic terminal sugars on the oligosaccharide chains that can be recognized by lectins. A goblet cell in the upper portion of the colonic crypt makes mucin with a terminal sugar which binds the lectins Dolichos biflorus agglutinin (DBA) and soybean agglutinin (SBA). The lower portion of the crypt has mucin with a terminal sugar residue which binds to Ricinus communis agglutinin-1 (RCA-1). Colon cancer produces mucin that binds peanut agglutinin (PNA). The goal of this study was to use lectin binding patterns in order to differentiate mucins secreted by low grade neoplasms from mucins produced by adenocarcinomas in the colon causing pseudomyxoma peritonei. Methods: Normal and diseased colon specimens were obtained from 31 patients with mucinous lesions and prepared for fluorescent lectin microscopy using fluorescein isothicyanate (FITC)-conjugated lectins: DBA, SBA, RCA-1 and PNA. The FITC-lectin labeling and pathological interpretations were performed independently. Results: 23/23 of the adenocarcinoma cases were positive for FITC PNA labeling versus 3/7 of the low grade neoplasm cases, a difference that was statistically significant (p=0.001 by chi-square). Conclusion: PMP is a disease characterized by a mucinous fluid collection in the abdomen and pelvis caused by mucus-producing adenomas or adenocarcinomas. The disease can behave very aggressively clinically. In this study, PNA binds to the mucins in all tumor sections that are adenocarcinomas. It was expected that SBA and DBA would bind similarly to the same tumor section but that was not always the case. This might be due to subtle differences in what these two GalNAc-binding lectins recognize and has an undetermined significance. It is reasonable to theorize that a tumor section with strong PNA binding and weaker SBA, DBA, or RCA-1 binding could represent increased dysplasia within the tumor and it will be interesting to compare this data with clinical outcomes. Further work will include more cases of low grade neoplasms as well as long-term clinical outcomes in order to strengthen the hypothesis that the absence of PNA binding predicts improved outcomes. This information could be valuable in determining the best treatment strategy for different patients.

Identification of a gene involved in the biosynthesis pathway of the terminal sugar of the archaellin N-linked tetrasaccharide in Methanococcus maripaludis.

In Methanococcus maripaludis, the three archaellins which comprise the archaellum are modified at multiple sites with an N-linked tetrasaccharide with the structure of Sug-4-β-ManNAc3NAmA6Thr-4-β-GlcNAc3NAcA-3-β-GalNAc, where Sug is a unique sugar (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-L-erythro-hexos-5-ulo-1,5-pyranose, so far found exclusively in this species. In this study, a six-gene cluster mmp1089-1094, neighboring one of the genomic regions already known to contain genes involved with the archaellin N-glycosylation pathway, was examined for its potential involvement in the archaellin N-glycosylation or sugar biosynthesis pathway. The co-transcription of these six genes was demonstrated by RT-PCR. Mutants carrying an in-frame deletion in mmp1090, mmp1091 or mmp1092 were successfully generated. The Δmmp1090 deletion mutant was archaellated when examined by electron microscopy and mass spectrometry analysis of purified archaella showed that the archaellins were modified with a truncated N-glycan in which the terminal sugar residue and the threonine linked to the third sugar residue were missing. Both gene annotation and bioinformatic analyses indicate that MMP1090 is a UDP-glucose 4-epimerase, suggesting that the unique terminal sugar of the archaellin N-glycan might be synthesised from UDP-glucose or UDP-N-acetylglucosamine with an essential early step in synthesis catalysed by MMP1090. In contrast, no detectable phenotype related to archaellin glycosylation was observed in mutants deleted for either mmp1091 or mmp1092 while attempts to delete mmp1089, mmp1093 and mmp1094 were unsuccessful. Based on its demonstrated involvement in the archaellin N-glycosylation pathway, we designated mmp1090 as aglW.

A Panel of Recombinant Mucins Carrying a Repertoire of Sialylated O-Glycans Based on Different Core Chains for Studies of Glycan Binding Proteins.

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 (Galβ3GlcNAc) or type 2 (Galβ4GlcNAc) 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.