Regular ArticleCarbohydrate-Based Probes for Detection of Cellular Lectins☆
Abstract
Carbohydrate (spacered saccharide residue, Glyc) probes with various tags were synthesized as analytical tools for study of cellular lectins, i.e., Glyc–polyacrylamide–3H, Glyc–PAA–biotin, Glyc–PAA–fluorescein (flu), and Glyc–PAA–digoxigenin, where PAA is a soluble polyacrylamide carrier of ≈30 kDa. Binding of all types of probes, where Glyc is the sialyl Lewis X (SiaLeX) tetrasaccharide or a blank saccharide, was assessed using Chinese hamster ovary (CHO) cells either transfected with the E-selectin cDNA or mock-transfected. High binding of SiaLeX–PAA–3H to E-selectin-transfected cells and absence of binding to control cells (both native and permeabilized) allowed the conclusion that the polyacrylamide carrier and the spacer arm do not contribute significantly to the binding. The biotinylated probe showed a high level of nonspecific binding in cell enzyme-linked assays. A similarly built digoxigenin-labeled probe was significantly better. In flow cytometry assays, the fluorescein probe demonstrated a specific binding to E-selectin-transfected cells of a similar level to that given by an anti-E-selectin antibody. In addition, it could be inhibited by the anti-E-selectin antibody, further demonstrating specificity. Tumors were obtained from nude mice by injection of CHO E-selectin or mock-transfected cells. The fluorescent SiaLeX–PAA–flu probe could bind to tumor sections from E-selectin-positive CHO cells, but not from control CHO cells. These probes can thus be used to reveal specifically complex carbohydrate-binding sites on cells either in culture or on tissue sections.
References (17)
- D. Storm et al.
J. Immunol. Methods
(1996) - A. Adam et al.
J. Pharm. Biomed. Anal.
(1996) - A. Krzeslak et al.
Mech. Ageing Dev.
(1996) - T.M. Carlos et al.
Blood
(1994) - G. Weitz-Schmidt et al.
Anal. Biochem.
(1996) - S.M. Game et al.
Anal. Biochem.
(1998) - Q. Zhou et al.
Cell Surface Carbohydrates and Cell Development
(1992) - H.-J. Gabius
Eur. J. Biochem.
(1997)
Cited by (27)
Sulfated Lewis A trisaccharide on oviduct membrane glycoproteins binds bovine sperm and lengthens sperm lifespan
2019, Journal of Biological ChemistryCitation Excerpt :From 0.8 to 0.9 μmol of each glycan was coupled to 1 mg of polyacrylamide (54). Each fluorescein-conjugated glycan was added to the sperm suspension at 50 μg/ml (55) final concentration and incubated for 30 min at 39 °C. After the incubation, 10 μl of the sperm were transferred onto a microscope slide and covered.
A fraction of sperm deposited at mating or insemination reaches the oviduct isthmus, where sperm are retained and thereby form a reservoir. This reservoir delays capacitation, prevents polyspermy, selects a fertile population of sperm, and, foremost, increases sperm lifespan. The molecular interactions underlying the formation of a sperm reservoir are becoming clearer in mammals. Sperm lectins bind to oviductal glycans to form the reservoir. Herein, we found that the highest percentage of bovine sperm bound to the 3′-O-sulfated form of Lewis A (suLeA) trisaccharide and sialylated Lewis A and that fluoresceinated versions of each localized to receptors on the anterior head of the sperm. Following capacitation, binding to suLeA decreased significantly, a potential explanation for sperm release from the reservoir. MS and immunohistochemistry analyses indicated that suLeA motifs were present predominantly on O-linked glycans initiated by GalNAc residues, but no sialylated Lewis A was detected. To determine whether sperm binding to isolated suLeA in vitro could mimic in vivo sperm binding to oviduct cells and increase sperm longevity, we immobilized suLeA and incubated it with sperm. Using free-swimming sperm and sperm bound to immobilized laminin as controls, we observed that over 96 h, the viability of free-swimming sperm decreased to 10%, and that of sperm bound to immobilized laminin decreased to about 50%, whereas viability of sperm bound to immobilized suLeA was highest throughout the incubation and 60% at 96 h. These results indicate that bovine sperm binding to oviduct suLeA retains sperm for reservoir formation and extends sperm lifespan.
Glycoprobes as a tool for the study of lectins expressed on tumor cells
2010, Acta HistochemicaPolyacrylamide glycoconjugates, Glyc-PAA, having various tags or labels are convenient tools for analysis of cellular lectins. Adaptation of such glycoprobes for flow cytometry allows us to reveal lectins expressed on cell surface and analyze their carbohydrate specificity as well as functionality.
Localization of lectins is visualized by labeling of cells with fluorescein-tagged glycoprobes, Glyc-PAA-fluo, in combination with fluorescent microscopy techniques. Additionally, biotinylated glycoprobes can be immobilized on magnetic particles making it possible to separate a cell population according to its carbohydrate-binding profile. Here, we exemplify application of glycoprobes in the study of cellular siglecs and galectins, as well as lectin patterning of tumor cells. The specificity of sialic acid binding membrane-anchored lectins, siglecs-1, -5, -7, -8 and -9 was determined using this methodology. To study the carbohydrate-binding profile of soluble galactoside-binding lectins, galectins-1 or -3, these were loaded on (initially galectin free) Raji cells and probed using Glyc-PAA-fluo. Lessons learned from this model system allowed us to study the galectin distribution pattern of tumors: cells obtained from mice carrying mammary adenocarcinoma or lymphoma were probed with Glyc-PAA-fluo using flow cytometry. Disaccharide 6OSuLacdiNAc was shown to be the most potent probe for adenocarcinoma cells, demonstrating that 6OSuLacdiNAc-binding molecules accumulate on cell surface in a patch-wise distribution.
Affinity of low molecular weight fucoidan for P-selectin triggers its binding to activated human platelets
2009, Biochimica et Biophysica Acta - General SubjectsBackground: P-selectin is an adhesion receptor expressed on activated platelets and endothelial cells. Its natural ligand, P-selectin glycoprotein ligand-1, is expressed on leucocytes and the P-selectin/PSGL-1 interaction is involved in leukocyte rolling. We have compared the interaction of P-selectin with several low molecular weight polysaccharides: fucoidan, heparin and dextran sulfate.
Methods: Binding assays were obtained from the interaction of the polysaccharides with Sialyl Lewis X and PSGL-1 based constructs onto microtiter plates coated with P-selectin. SELDI TOF mass spectrometry was performed with anionic chips arrays coated with P-selectin in the absence or in the presence of polysaccharides. Kd were obtained from surface plasmon resonance experiments with immobilized P-selectin constructs, polysaccharides being injected in the mobile phase. Human whole blood flow cytometry experiments were performed with fluorescein isothiocyanate labelled polysaccharides with or without platelets activators.
Results: The fucoidan prevented P-selectin binding to Sialyl Lewis X with an IC50 of 20 nM as compared to 400 nM for heparin and < 25000 nM for dextran sulfate. It exhibited the highest affinity for immobilized P-selectin with a KD of 1.2 nM, two orders of magnitude greater than the KD of the other polysaccharides. Mass spectrometry evidenced the formation of a complex between P-selectin and fucoidan. The intensity of the fucoidan binding to platelets was dependent on the level of platelet activation. Competition between fucoidan and an anti P-selectin antibody demonstrated the specificity of the interaction.
General significance: Low molecular weight fucoidan is a promising therapeutic agent of natural origin for biomedical applications.
Real-time monitoring of the cell agglutination process with a quartz crystal microbalance
2008, Analytical BiochemistryThe real-time monitoring of the agglutination process of human hepatic normal cells (L-02) at the quartz crystal microbalance (QCM) gold (Au) electrode was performed. Two lectins, concanavalin A (Con A) and wheat germ agglutinin (WGA), induced the cell agglutination, resulting in the different Δf0 and ΔR1 responses from those caused by the normal cell attachment and growth. The cell–Con A–cell aggregates had higher affinity for the Au substrate due to the excellent adsorption ability of Con A, which was revealed by increased Δf0 and ΔR1 shifts and the obvious mass effect of QCM. In contrast, the lower adsorption ability of cell–WGA–cell aggregates was related to the same characteristic of WGA, presenting the decreased Δf0 and ΔR1 responses and the time-extended adhesion phase. Parallel microscopic observation experiments were also carried out and exhibited comparable results. The Δf0 responses during the processes of cell growth and cell agglutination were analyzed using the equations and , respectively. Furthermore, the current work proved that the QCM measurement technique based on cell agglutination was useful for discriminating hepatic normal cells (L-02) and hepatic cancer cells (Bel7402).
Probing Cell Surface Lectins with Neoglycoconjugates
2007, Lectins: Analytical TechnologiesKnowledge of carbohydrate-binding properties of cell surface lectins is important for understanding their functions. A particularity of carbohydrate—protein recognition of lectins is that both affinity and specificity are achieved due to polyvalent interaction, whereas affinity of single ligand to lectin can be very low or not registered at all. The use of polyvalent probes allows increasing of the binding due to multipoint interaction. Multivalency of binding requires a specific design of the probes; several copies of saccharide should be attached to a polymer carrier at an appropriate distance. The probe bears a direct label (e.g. fluorescein) or a tag for subsequent detection (e.g. biotin). The probes for cell studies should correspond to enormously strict requirements in respect of nonspecific interaction with cell components and matrix. Polyacrylamide-based glycoconjugates used for a long time for the study of specificity of soluble and membrane-associated lectins fit very well with requirements for probes of lectins. Biotinylated glycoprobes are convenient for lectin revealing based on CELISA (cell ELISA) technique, whereas fluorescein-labeled ones are appropriate for use in flow cytometry. Both biotinylated and fluorescein-labeled glycoprobes can be used in cytological and histological studies. Sometimes, more intense staining is observed when the probe is attached to a fluorescent particle. Glycoparticles prepared by immobilization of glycoconjugates on magnetic beads are convenient for isolation of a particular cell population, expressing carbohydrate-binding molecules.
Involvement of the Galβ1-3GalNAcβ structure in the recognition of apoptotic bodies by THP-1 cells
2003, European Journal of Cell BiologyA specific apoptotic glycosylation pattern may play an assistant or even a causative role in phagocytosis of apoptotic bodies. To elucidate the role of macrophages in lectin-mediated phagocytosis, an experimental system was used, where monocyte-derived THP-1 cells engulf the apoptotic bodies from the melanoma cell line MELJUSO. A flow cytometry assay was performed to reveal lectin expression and quantify the phagocytosis of apoptotic bodies. Taking into account that siglecs, a mannose receptor and galectins expressed on macrophages could be involved in engulfment of apoptotic bodies we studied their potential expression on THP-1 cells by means of polyacrylamide glycoconjugates. A strong binding of the cells to siglec ligands (3′SiaLac, 6′SiaLac, [Neu5Acα2 – 8]2) and galectin ligands (LacNAc, GalNAcβ1 – 4GlcNAc, Galβ1 – 3GalNAcβ and asialoGM1) was observed. To reveal the corresponding targets on apoptotic bodies, the carbohydrate pattern of MELJUSO cells was analyzed. The apoptotic membrane was characterized by a high level of glycans terminated by galactose or sialic acid. To study lectin-mediated phagocytosis of apoptotic bodies by THP-1 cells, an inhibitory phagocytosis assay was performed. Binding of Galβ1 – 3GalNAc- or LacNAc-specific reagents (lectins and antibodies) to apoptotic bodies abolished their engulfment by the THP-1 cells whereas blocking of Neu5Acα2 – 6 or Neu5Acα2 – 3 sites by the corresponding lectins was not effective. Furthermore, Galβ1 – 3GalNAcβ- or asialoGM1-PAA binding to the THP-1 cells decreased phagocytosis, whereas two other potent THP-1-binding probes, LacNAc-PAA and GalNAcβ1 – 4GlcNAc-PAA did not inhibit phagocytosis. Thus, Galβ1 – 3GalNAcβ-terminated chains represented on the apoptotic bodies but not the other tested galectin ligands appear to be a target for THP-1 cells.
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Fukuda, M.
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