Preferential Lectin Binding of Cancer Cells upon Sialic Acid Treatment Under Nutrient Deprivation

The terminal monosaccharide of glycoconjugates on a eukaryotic cell surface is typically a sialic acid (Neu5Ac). Increased sialylation usually indicates progression and poor prognosis of most carcinomas. Here, we utilize two human mammary epithelial cell lines, HB4A (breast normal cells) and T47D (breast cancer cells), as a model system to demonstrate differential surface glycans when treated with sialic acid under nutrient deprivation. Under a starved condition, sialic acid treatment of both cells resulted in increased activities of α2→3/6 sialyltransferases as demonstrated by solid phase assay using lectin binding. However, a very strong Maackia amurensis agglutinin I (MAL-I) staining on the membrane of sialic acid-treated T47D cells was observed, indicating an increase of Neu5Acα2→3Gal on the cell surface. To our knowledge, this is a first report showing the utility of lectins, particularly MAL-I, as a means to discriminate between normal and cancer cells after sialic acid treatment under nutrient deprivation. This method is sensitive and allows selective detection of glycan sialylation on a cancer cell surface.     

Determination and distribution of N-acetyl- and N-glycolylneuraminic acids in culture media and cell-associated glycoconjugates from human malignant mesothelioma and adenocarcinoma cells

Sialic acids containing glycoconjugates are very common in human neoplasias and their expression frequently correlates with malignant phenotype and the tumor grade. The majority of tumor markers containing sialic acids in man involve changes in the amount of total sialic acids and in the presence of the two main sialic acid types, Neu5Ac and Neu5Gc, and their derivatives. The aim of the present study was to examine whether malignant mesothelioma cell lines synthesize sialic acid containing glycoconjugates at both the extracellular and cell membrane levels and particularly whether the type and the content of Neu5Ac and Neu5Gc are of biological importance for mesothelioma cell differentiation and evaluation of its prognosis. The study was performed in three human malignant mesothelioma cell lines, two with a fibroblast like phenotype (STAV-FCS and Vester) and one of epithelial differentiation (STAV-AB), which developed from the pleural effusions of patients with malignant mesothelioma and in one human adenocarcinoma cell line (Wart). Neu5Ac and Neu5Gc were determined following a mild hydrolysis step and a sample clean-up procedure. The determination was performed by reversed-phase HPLC after the NeuAc and NeuGc had been converted to per-O-benzoylated derivatives. It was found that Neu5Gc is the major sialic acid in the culture media of all cell lines examined. Molar ratios of Neu5Ac to Neu5Gc showed that Neu5Gc is the predominant sialic acid in the culture medium of the fibroblast-like mesothelioma cells. Neu5Ac is almost undetectable in the cell membrane, whereas Neu5Gc is present in considerable amounts. The obtained results suggest that the type and the content of Neu5Ac and Neu5Gc in culture media are of biological importance for mesothelioma cell differentiation and may be of value in the evaluation of prognosis.     

Expression and distribution of N-acetyl and N-glycolylneuraminic acids in secreted and cell-associated glycoconjugates by two human osteosarcoma cell lines

N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the dominant sialic acids (Sia) in mammals usually found in the non-reducing terminal of oligosaccharide side chains in glycoproteins and glycolipids. Their expression and distribution pattern have been correlated both with the malignant phenotype and tumor grade of human cancers. The aim of the present study was to determine by reversed-phase HPLC method the amounts of Neu5Ac and Neu5Gc as well as their distribution among the culture media and cell surface of MG-63 and Saos-2 human osteosarcoma cell lines of high and low metastatic potential. It was determined that MG-63 cells produce up to 5-fold more total sialic acid as compared with the Saos 2 cells. Neu5Ac accounts for ca 60% of the total sialic acids secreted by MG-63 cells, whereas Neu5Gc is the predominant sialic acid present on the MG-63 cell membrane. Saos 2 cells secrete considerable amounts of Neu5Ac to culture media. The obtained data indicate that the human osteosarcoma cells express both forms of Sia-containing glycoconjugates; the differences in the amounts of each of the two major Sia types and their distribution may be related to their differences in morphology and/or metastatic potentials.     

Tumor Carbohydrate Associated Antigen Analogs as Potential Binders for Siglec-7

We investigated two recently synthesized and characterized sialyl derivatives, bearing the Neu5Ac-α-(2-6)-Gal epitope, as promising binders for Siglec-7, an inhibitory Siglec mainly found on natural killer cells. A variety of sialoglycan structures can be recognized by Siglec-7 with implications in the modulation of immune responses. Notably, overexpression of sialylated glycans recognized by Siglec-7 can be associated with the progression of several tumors, including melanoma and renal cell carcinoma. NOE-based NMR techniques, including Saturation Transfer Difference and transferred-NOESY NMR, together with molecular docking and dynamic simulations were combined to shed light on the molecular basis of Siglec-7 recognition of two conformationally constrained Sialyl-Tn antigen analogs. We, therefore, identify the ligands epitope mapping and their conformational features and propose 3D models accurately describing the protein-ligand complexes. We found that the binding site of Siglec-7 can accommodate both synthetic analogs, with the sialic acid mainly involved in the interaction. Moreover, the flexibility of Siglec-7 loops allows a preferred accommodation of the more rigid compound bearing a biphenyl moiety at position 9 of the sialic acid that contributed to the interaction to a large extent. Our findings provided insights for developing potential novel high affinity ligands for Siglec-7 to hinder tumor evasion.     

Sialyldisaccharide conformations: a molecular dynamics perspective

Sialyldisaccharides are significant terminal components of glycoconjugates and their negative charge and conformation are extensively utilized in molecular recognition processes. The conformation and flexibility of four biologically important sialyldisaccharides [Neu5Acα(2-3)Gal, Neu5Acα(2-6)Gal, Neu5Acα(2-8)Neu5Ac and Neu5Acα(2-9)Neu5Ac] are studied using Molecular Dynamics simulations of 20 ns duration to deduce the conformational preferences of the sialyldisaccharides and the interactions which stabilize the conformations. This study clearly describes the possible conformational models of sialyldisaccharides deduced from 20 ns Molecular Dynamics simulations and our results confirm the role of water in the structural stabilization of sialyldisaccharides. An extensive analysis on the sialyldisaccharide structures available in PDB also confirms the conformational regions found by experiments are detected in MD simulations of 20 ns duration. The three dimensional structural coordinates for all the MD derived sialyldisaccharide conformations are deposited in the 3DSDSCAR database and these conformational models will be useful for glycobiologists and biotechnologists to understand the biological functions of sialic acid containing glycoconjugates.     

Efficient Synthesis of the Disialylated Tetrasaccharide Motif in N‐Glycans through an Amide‐Protection Strategy

A disialylated tetrasaccharide, Neu5Ac(α2,3)Gal(β1,3)[Neu5Ac(α2,6)]GlcNAc ( 1 ), which is found at the termini of some N‐glycans, has been synthesized. Compound 1 was obtained through an α‐sialylation reaction between a sialic acid donor and a trisaccharide that was synthesized from the glycosylation of a sialylated disaccharide with a glucosaminyl donor. This synthetic route enabled the synthesis of the as‐described disialylated structure. A more‐convergent route based on the glycosylation of two sialylated disaccharides was also established to scale up the synthesis. Protection of the amide groups in the sialic acid residues significantly increased the yield of the glycosylation reaction between the two sialylated disaccharides, thus suggesting that the presence of hydrogen bonds on the sialic acid residues diminished their reactivity.     

Divalent Sialylated Precision Glycooligomers Binding to Polyomaviruses and the Effect of Different Linkers

Divalent precision glycooligomers terminating in N‐acetylneuraminic acid (Neu5Ac) or 3′‐sialyllactose (3′‐SL) with varying linkers between scaffold and the glycan portions are synthesized via solid phase synthesis for co‐crystallization studies with the sialic acid‐binding major capsid protein VP1 of human Trichodysplasia spinulosa‐associated Polyomavirus. High‐resolution crystal structures of complexes demonstrate that the compounds bind to VP1 depending on the favorable combination of carbohydrate ligand and linker. It is found that artificial linkers can replace portions of natural carbohydrate linkers as long as they meet certain requirements such as size or flexibility to optimize contact area between ligand and receptor binding sites. The obtained results will influence the design of future high affinity ligands based on the structures presented here, and they can serve as a blueprint to develop multivalent glycooligomers as inhibitors of viral adhesion.     

One‐Pot Synthesis of N‐Acetyl‐ and N‐Glycolylneuraminic Acid Capped Trisaccharides and Evaluation of Their Influenza A(H1 N1) Inhibition

Human lung epithelial cells natively offer terminal N‐acetylneuraminic acid (Neu5Ac) α(2→6)‐linked to galactose (Gal) as binding sites for influenza virus hemagglutinin. N‐Glycolylneuraminic acid (Neu5Gc) in place of Neu5Ac is known to affect hemagglutinin binding in other species. Not normally generated by humans, Neu5Gc may find its way to human cells from dietary sources. To compare their influence in influenza virus infection, six trisaccharides with Neu5Ac or Neu5Gc α(2→6) linked to Gal and with different reducing end sugar units were prepared using one‐pot assembly and divergent transformation. The sugar assembly made use of an N‐phthaloyl‐protected sialyl imidate for chemoselective activation and α‐stereoselective coupling with a thiogalactoside. Assessment of cytopathic effect showed that the Neu5Gc‐capped trisaccharides inhibited the viral infection better than their Neu5Ac counterparts.     

Theoretical investigation on the binding specificity of fluorinated sialyldisaccharides Neu5Acα(2–3)Gal and Neu5Acα(2–6)Gal with influenza hemagglutinin H1 – A Molecular Dynamics Study

In the present study, the hydroxyl groups at the C4 and C7 positions of sialic acid and C6 position of galactose in Neu5Acα(2–3)Gal (N23G) and the hydroxyl groups at the C8 position of sialic acid and C3 and C4 positions of galactose in Neu5Acα(2–6)Gal (N26G) were substituted with fluorine atoms, respectively. Molecular dynamics simulations of 100 ns duration were carried out to investigate the structural and dynamical behavior of H1 bound with the tri-fluorinated N23G and N26G (FN23G and FN26G). Based on energy analysis, it was concluded that FN26G should be a better binder for hemagglutinin (H1) than FN23G and it might act as an inhibitor for influenza.     

The Microbial Pathology of Neu5Ac and Gal Epitopes

Glycans play a vital role in modulating many physiological and pathological phenomena of microbes and humans, such as bacterial adhesion, colonization, host-microbial interactions, cancer recognition, and blood group determination. The aim of the current review is to provide an account of the functions of N-acetyl sialic acid (Neu5Ac) and galactose (Gal) residues in microbial pathology. Specifically, an overview of the biosynthesis and metabolism of Neu5Ac and Gal residues in different bacterial species will provide a better understanding of microbial pathogenesis in the human body.     

Monomeric inhibitors of influenza neuraminidase enhance the hemagglutination inhibition activities of polyacrylamides presenting multiple C-sialoside groups

Background: Influenza viruses use hemagglutinin (HA) arrays to bind to sialic acid moieties on the surface of cells; crosslinking of erythrocytes by this mechanism leads to hemagglutination. A number of synthetic polymers containing multiple sialic acid (Neu5Ac) groups as side chains are potent inhibitors of this process. Inhibition may be due to two mechanisms: polyvalent binding of the inhibitor's multiple Neu5Ac side chains to multiple HA sites on the viral surface, or steric stabilization of the viral particle by a layer of the adsorbed, water-swollen polymer, which prevents adhesion to the erythrocyte. The balance between these two effects is not yet known.Results: Polyacrylamides with multiple C-sialosides (PA(Neu5Ac)) were 2–20 fold more effective as inhibitors of virally mediated hemagglutination when assayed in the presence of Neu2en-NH₂, a potent monomeric inhibitor of influenza neuraminidase (NA). The ability of monomeric inhibitors of NA to enhance the inhibition of hemagglutination in this assay correlated with the affinity of the monomer for NA.Conclusions: We propose that inhibitors of NA act by competing with the C-sialosides of PA(Neu5Ac) for binding to the active sites of the NA. Competitive displacement of Neu5Ac causes an expansion of the layer of polymeric gel adsorbed to the virus, enhancing its inhibitory effect. This study provides an example of synergy between two ligands directed toward the active sites of two different proteins, and reinforces the conclusion that steric stabilization is important for the activity of polyvalent inhibitors.     

Molecular recognition of sialic acid end groups by phenylboronates

A multinuclear NMR study of the interaction between phenylboronic acid (PBA) and sialic acid (Neu5 Ac) has been performed. The latter compound is known to be overexpressed on the cell surface of tumor cells. The results of this investigation suggest that the binding of PBA to sialic acid is pH dependent. 17O NMR experiments with glycolic acid as the model compound prove that an interaction at the alpha-hydroxycarboxylate occurs at pH < 9, while a study with threonic and erythronic acids shows that the PBA group interacts selectively with the vicinal diol functions at higher pH. Similarly, Neu5 Ac binds PBA through its alpha-hydroxycarboxylate at low pH (< 9) and through its glycerol side chain at higher pH values. The conditional stability constant of the phenylboronate ester at pH 7.4 is 11.4. On cell surfaces, sialic acid is connected to the neighboring sugar unit through the 2-hydroxy group. To mimic this the 2-alpha-O-methyl derivative of Neu5 Ac was included in this study. The erythro configuration of the hydroxy substituents prevents stable-complex formation at positions C7 and C8 and, consequently, the strongest interaction is observed at positions C8 and C9, leading to a five-membered 2-boron-1,3-dioxalate. In addition, a relatively small amount of the C7-C9 six-membered complex was observed. Molecular modeling studies confirm that the C8-C9 boronate complex has the lowest energy.     

Bifunctional CD22 ligands use multimeric immunoglobulins as protein scaffolds in assembly of immune complexes on B cells

CD22 is a B cell-specific sialic acid-binding immunoglobulin-like lectin (Siglec) whose function as a regulator of B cell signaling is modulated by its interaction with glycan ligands bearing the sequence NeuAc alpha2-6Gal. To date, only highly multivalent polymeric ligands (n = 450) have achieved sufficient avidity to bind to CD22 on native B cells. Here we demonstrate that a synthetic bifunctional molecule comprising a ligand of CD22 linked to an antigen (nitrophenol; NP) can use a monoclonal anti-NP IgM as a decavalent protein scaffold to efficiently drive assembly of IgM-CD22 complexes on the surface of native B cells. Surprisingly, anti-NP antibodies of lower valency, IgA (n = 4) and IgG (n = 2), were also found to drive complex formation, though with lower avidity. Ligands bearing alternate linkers of variable length and structure were constructed to establish the importance of a minimal length requirement, and versatility in the structural requirement. We show that the ligand drives assembly of IgM complexes exclusively on the surface of B cells and not other classes of white blood cells that do not express CD22, which lends itself to the possibility of targeting B cells in certain hematopoietic malignancies.     

Chemical Remodeling of Cell‐Surface Sialic Acids through a Palladium‐Triggered Bioorthogonal Elimination Reaction

We herein report a chemical decaging strategy for the in situ generation of neuramic acid (Neu), a unique type of sialic acid, on live cells by the use of a palladium‐mediated bioorthogonal elimination reaction. Palladium nanoparticles (Pd NPs) were found to be a highly efficient and biocompatible depropargylation catalyst for the direct conversion of metabolically incorporated N‐(propargyloxycarbonyl)neuramic acid (Neu5Proc) into Neu on cell‐surface glycans. This conversion chemically mimics the enzymatic de‐N‐acetylation of N‐acetylneuramic acid (Neu5Ac), a proposed mechanism for the natural occurrence of Neu on cell‐surface glycans. The bioorthogonal elimination was also exploited for the manipulation of cell‐surface charge by unmasking the free amine at C5 to neutralize the negatively charged carboxyl group at C1 of sialic acids.     

Chemoenzymatic synthesis of sialylated glycopeptides derived from mucins and T-cell stimulating peptides.

The Tn, T, sialyl-Tn, and 2,3-sialyl-T antigens are tumor-associated carbohydrate antigens expressed on mucins in epithelial cancers, such as those affecting the breast, ovary, stomach, and colon. Glycopeptides carrying these antigens are of interest for development of cancer vaccines and a short, chemoenzymatic strategy for their synthesis is reported. Building blocks corresponding to the Tn (GalNAc alpha-Ser/Thr) and T [Gal beta(1-->3)GalNAc alpha-Ser/Thr] antigens, which are relatively easy to obtain by chemical synthesis, were prepared and then used in the synthesis of glycopeptides on the solid phase. Introduction of sialic acid to give the sialyl-Tn [Neu5Ac alpha(2-->6)GalNAc alpha-Ser/Thr] and 2,3-sialyl-T [Neu5Ac alpha(2-->3)Gal beta(1-->3)GalNAc alpha-Ser/Thr] antigens is difficult when performed chemically at the building block level. Sialylation was therefore carried out with recombinant sialyltransferases in solution after cleavage of the Tn and T glycopeptides from the solid phase. In the same manner, the core 2 trisaccharide [Gal beta 1-->3(GlcNAc beta 1-->6)GalNAc] was incorporated in glycopeptides containing the T antigen by using a recombinant N-acetylglucosaminyltransferase. The outlined chemoenzymatic approach was applied to glycopeptides from the tandem repeat domain of the mucin MUC1, as well as to neoglycosylated derivatives of a T cell stimulating viral peptide.     

Detection of carbohydrates using new labeling reagent 1-(2-naphthyl)-3-methyl-5-pyrazolone by capillary zone electrophoresis with absorbance (UV)

Surface plasmon resonance (SPR) was used to screen the interaction between a variety of affinity ligands and hemagglutinin (HA) from human influenza virus, with the aim of identifying low affinity ligands useful for the development of a rapid bioanalytical sensor. Three sialic acid-based structures and four lectins were evaluated as sensor ligands. The sialic acid-based ligands included a natural sialic acid-containing glycoprotein, human alpha1-acid glycoprotein (alpha1-AGP), and two synthetic 6'-sialyllactose-conjugates, with varying degree of substitution. The interaction of HA with the four lectin-based ligands, concanavalin A (Con A), wheat germ agglutinin (WGA), Maackia amurensis lectin (MAL), and Sambucus nigra agglutinin (SNA), showed a wide variation of affinity strengths. Affinity and kinetics data were estimated. Strong affinities were observed for Con A, WGA, alpha1-AGP, and a 6'-sialyllactose-conjugate with a high substitution degree, and low affinities were observed for MAL and a 6'-sialyllactose-conjugate with low substitution. The main objective, to identify a low affinity ligand which could be used for on-line monitoring and product quantification, was met by a 6'-sialyllactose-ovalbumin conjugate that had 0.6 mol ligand per mol carrier protein. The apparent affinity of this ligand was estimated to be 1.5+/-0.03 microM (K(D)) on the SPR surface. Vaccine process samples containing HA were analyzed in the range 10-100 microg HA mL(-1) and correlated with single-radial immunodiffusion. The coefficient of variation on the same chip was between 0.010 and 0.091.     

Neoglycolipid probes prepared via oxime ligation for microarray analysis of oligosaccharide-protein interactions

Neoglycolipid technology is the basis of a microarray platform for assigning oligosaccharide ligands for carbohydrate-binding proteins. The strategy for generating the neoglycolipid probes by reductive amination results in ring opening of the core monosaccharides. This often limits applicability to short-chain saccharides, although the majority of recognition motifs are satisfactorily presented with neoglycolipids of longer oligosaccharides. Here, we describe neoglycolipids prepared by oxime ligation. We provide evidence from NMR studies that a significant proportion of the oxime-linked core monosaccharide is in the ring-closed form, and this form selectively interacts with a carbohydrate-binding protein. By microarray analyses we demonstrate the effective presentation with oxime-linked neoglycolipids of (1) Lewis(x) trisaccharide to antibodies to Lewis(x), (2) sialyllactose analogs to the sialic acid-binding receptors, siglecs, and (3) N-glycans to a plant lectin that requires an intact N-acetylglucosamine core.     

Elucidating the Anomalous Binding Enhancement of Isoquinoline Boronic Acid for Sialic Acid Under Acidic Conditions: Expanding Biorecognition Beyond Vicinal Diols

A zwitterionic heterocyclic boronic acid based on 4-isoquinolineboronic acid (IQBA) exhibits the highest reported binding affinity for sialic acid or N-acetylneuraminic acid (Neu5Ac, K=5390±190 m ⁻¹) through the formation of a cyclic boronate ester complex under acidic conditions (pH 3). This anomalous pH-dependent binding enhancement does not occur with common neutral saccharides (e.g., glucose, fructose, sorbitiol), because it is mediated via selective complexation to a α-hydroxycarboxylate moiety forming a stable ion pair and ternary complex with Neu5Ac in phosphate buffer. IQBA expands biorecognition beyond classical vicinal diols under neutral or alkaline buffer conditions, which enables the direct analysis of Neu5Ac by native fluorescence with sub-micromolar detection limits.     

Steering Siglec–Sialic Acid Interactions on Living Cells using Bioorthogonal Chemistry

Sialic acid sugars that terminate cell‐surface glycans form the ligands for the sialic acid binding immunoglobulin‐like lectin (Siglec) family, which are immunomodulatory receptors expressed by immune cells. Interactions between sialic acid and Siglecs regulate the immune system, and aberrations contribute to pathologies like autoimmunity and cancer. Sialic acid/Siglec interactions between living cells are difficult to study owing to a lack of specific tools. Here, we report a glycoengineering approach to remodel the sialic acids of living cells and their binding to Siglecs. Using bioorthogonal chemistry, a library of cells with more than sixty different sialic acid modifications was generated that showed dramatically increased binding toward the different Siglec family members. Rational design reduced cross‐reactivity and led to the discovery of three selective Siglec‐5/14 ligands. Furthermore, glycoengineered cells carrying sialic acid ligands for Siglec‐3 dampened the activation of Siglec‐3⁺ monocytic cells through the NF‐κB and IRF pathways.