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Glycobiology Advance Access originally published online on February 6, 2008
Glycobiology 2008 18(4):315-324; doi:10.1093/glycob/cwn009
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© The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Galectin-loaded cells as a platform for the profiling of lectin specificity by fluorescent neoglycoconjugates: A case study on galectins-1 and -3 and the impact of assay setting

Eugenia M Rapoport1,2, Sabine André3, Olga V Kurmyshkina2, Tatiana V Pochechueva2, Vyacheslav V Severov2, Galina V Pazynina2, Hans-J Gabius3 and Nicolai V Bovin1,2

2 Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, 117997, ul. Miklukho-Maklaya 16/10, Moscow, Russia
3 Faculty of Veterinary Medicine, Institute of Physiological Chemistry, Ludwig-Maximilians-University, Veterinärstr, 13, D-80539 Munich, Germany

1 To whom correspondence should be addressed: Fax: + 7-495-3305592; e-mail: rapoport{at}carbohydrate.ru, bovin{at}carb.ibch.ru

Received on October 2, 2007; revised on January 29, 2008; accepted on February 1, 2008


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 Funding
 Conflict of interest statement
 References
 
The involvement of galectins as pleiotropic regulators of cell adhesion and growth in disease progression explains the interest to define their ligand-binding properties. Toward this end, it is desirable to approach in vivo conditions to attain medical relevance. In order to simulate physiological conditions with cell surface glycans as recognition sites and galectins as mediators of intercellular contacts we developed an assay using galectin-loaded Raji cells. The extent of surface binding of fluorescent neoglycoconjugates depended on the lectin presence and the type of lectin, the nature of the probes’ carbohydrate headgroup and the density of unsubstituted β-galactosides on the cell surface. Using the most frequently studied galectins-1 and -3, application of this assay led to rather equal binding levels for linear and branched oligomers of N-acetyllactosamine. A clear preference of galectin-3 for {alpha}1-3-linked galactosylated lactosamine was noted. In parallel, a panel of 24 neoglycoconjugates was tested as inhibitors of galectin binding from solution to N-glycans of surface-immobilized asialofetuin. These two assays differ in presentation of the galectin and ligand, facilitating identification of assay-dependent properties. Under the condition of the cell assay, selectivity among oligosaccharides for the lectins was higher, and extraordinary affinity of galectin-1 to 3'-O-sulfated probes in a solid-phase assay was lost in the cell assay. Having introduced and validated a cell assay, the comprehensive profiling of ligand binding to cell-surface-presented galectins is made possible.

Key words: Galectin / lactosamine / lectin / neoglycoconjugate / oligosaccharide O-sulfate


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 Funding
 Conflict of interest statement
 References
 
The cell surface presents a wide variety of sugar-encoded signals to the environment. They can be translated into responses such as adhesion or growth control by endogenous lectins (Gabius 2006Go). In this respect, the spatially accessible determinants at the tip of glycan antennae will likely be preferential contact sites. Homing primarily in on these often-substituted β-galactosides, the members of the galectin family are receiving special attention owing to their emerging involvement in various clinically relevant processes. For instance, galectins factor into immune regulation as well as tumor progression and spread (Leffler 2004Go; Liu 2005Go; Liu and Rabinovich 2005Go). The presently accrued insights into the functional spectrum of galectins in vivo predict an exquisite fine-tuning in the structure–function relation between individual aspects of the glycomic profile and these lectins, and work on model (neo)glycoproteins has even revealed the impact of topological factors (e.g., substitutions in the glycan core or altering local density) on affinity (Wu et al. 2006Go; André et al. 2007Go). It is thus an obvious aim to define ligand selection by different galectins, at best under in vitro/in vivo conditions.

A series of methods has so far been used to map the binding of glycan ligands to galectins, starting with classical inhibition of hemagglutination (Teichberg et al. 1975Go). As such, solid-phase assays with surface-immobilized (neo)glycoconjugates or glycans and labeled galectins or determination of direct ligand binding in solution using fluorescence polarization, frontal affinity chromatography, and isothermal titration calorimetry showed merit to detect differences in binding properties among galectins (Leffler and Barondes 1986Go; Lee et al. 1990Go; Ahmad et al. 2002Go; Hirabayashi et al. 2002Go; Sörme et al. 2004Go; Leppänen et al. 2005Go). In order to substitute the artificial matrix by a physiological surface, the design of the inhibition assay had been extended to using a human tumor or Chinese hamster ovary cells in vitro (André et al. 2006Go; Patnaik et al. 2006Go). This experimental setting with cells, the multivalency of galectins apparent from their activity as hemagglutinins and the versatility of neoglycoconjugates, serving as potent lectin ligands (Rapoport et al. 2007Go), prompted us to explore the following approach: to combine availability of a panel of labeled neoglycoconjugates, which present different carbohydrate headgroups, with cell surface binding of galectins. The aim was to develop a new cell-based type of specificity assay for galectins. Intuitively, effector functionality of the lectins implies full cis saturation of their binding sites by endogenous ligands. Of note in this respect, the natural presentation of galectin-1 on SK-N-MC neuroblastoma cells was shown to maintain stoichiometric accessibility of the homodimeric lectin for its ligand ganglioside GM1 (Kopitz et al. 1998Go). We therefore proposed that binding of bi- to multivalent lectins to a cell surface can entail to decorate cells with accessible binding sites for a labeled neoglycoconjugate when using the carrier-immobilized glycans at a noninhibitory concentration. The advantages of this assay will be (a) to avoid protein labeling; (b) to map binding properties of galectins when bound to naturally complex glycans, a factor which can affect global aspects of the lectin structure (He et al. 2003Go); and (c) to consider potential for secondary effects on a natural surface (Horan et al. 1999Go).

In what follows, we first report on the technical aspects of this new assay procedure. A cell line, which lacks endogenous production of galectins (Lahm et al. 2004Go), was selected as a tool to avoid confounding data interpretation. Having established the experimental protocol, we present results on initial application, testing human galectins-1 and -3 with priority. These two proteins stand out from the galectin family as being the most frequently studied. The measurements revealed characteristic features in the profiles of ligand binding and a so far not described marked intergalectin difference for 3'-O-sulfated ligands. These data were set into relation to results from solid-phase assays with the galectins in the solution and asialofetuin as a surface-immobilized ligand. This comparison delineated relative differences in binding properties. The reported validation of the assay and the first results give further research on profiling galectin specificity and on drug design a clear direction.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 Funding
 Conflict of interest statement
 References
 
Loading of Raji cells surface with galectins
The two human galectins and non-cross-reactive antibodies were used to test whether loading of cell surfaces with a reactive lectin could be accomplished. The incubation steps were carried out at 4°C to minimize lateral mobility in the membrane and cis clustering as well as endocytic uptake. The extent of galectin association with the cell surface critically depended on the protocol for removing the galectin-containing solution. Standard washing in three steps led to a drastic reduction of the fluorescence signal in the ensuing immunocytochemical detection. When a single round of cell pelleting by centrifugation was tested, strong signals comprising nearly the complete cell populations in immunofluorimetric monitoring were measured (Figure 1). Thus, cell surfaces can be furnished with galectins-1 and -3. To examine whether this system is a suitable platform for specificity assays, we next tested a panel of fluorescent neoglycoconjugates for binding. Should they associate with cell surfaces depending on the galectin presence, it would mean that the comparative profiling of neoglycoconjugate binding will be feasible. Thus, we set out to test this reasoning experimentally.


Figure 1
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  		Fig. 1 Loading of Raji cell surfaces with galectin-1 (A) and galectin-3 (B). Flow cytometric analysis of cells probed with the corresponding antibody preparations followed by application of secondary antibodies as described in Materials and methods. The logs of fluorescence intensity are plotted against cell number. The gray area defines background fluorescence of processed galectin-free cells (negative control). The number given for the black curve represents the percentage of cells reactive with the antibody, the standard for reactivity of the cell surface for galectins.

 
Flow cytofluorimetric analysis
The first lesson which emerged from Figures 2 and 3 is that fluorescent neoglycoconjugates could definitely interact with loaded cells. Binding depended on the presence of the galectins (please compare black curves with the gray area of control cells) and the nature of the carbohydrate headgroup (the panel of tested glycocompounds and the changes in fluorescence intensity are summarized in Table I). Strong signals were invariably recorded for linear and branched N-acetyllactosamine (LN) oligomers. Decreases of oligomer length by presenting only the disaccharide (Lec or LN) or branches lacking galactose reduced the extent of binding (Figures 2 and 3, Table I). The agalacto derivative of (GlcNAc)23',6' LN consequently had no ligand capacity, underscoring the inherent requirement for accessible galactose residues. The Galβ1-3GalNAc terminus was moderate reactive with galectin-1 when presented in the context of the asialoganglioside GM1 tetrasaccharide but not as a disaccharide. Of note, any sulfated disaccharide tested and {alpha}2,3-sialylated N-acetyllactosamine did not bind to galectin-1 presented on Raji cell surfaces.


Figure 2
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  		Fig. 2 Probing the galectin-1-loaded Raji cells with fluorescent neoglycoconjugates in flow cytometry. The logs of fluorescence intensity are plotted against the cell number. The gray area defines background fluorescence of the galectin-free cells incubated with the probe. The numbers given for each black curve obtained for the series of glycocompounds (see name in the top part of each panel) represent the percentage of cells reactive with the fluorescent neoglycoconjugates, the measure for comparing galectin reactivity.

 

Figure 3
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  		Fig. 3 Probing the galectin-3-loaded Raji cells with fluorescent neoglycoconjugates in flow cytometry. The logs of fluorescence intensity are plotted against the cell number. The gray area defines background fluorescence of galectin-free cells incubated with the probe. The numbers given for each black curve obtained for the series of glycocompounds (see name in the top part of each panel) represent the percentage of cells reactive with the fluorescent neoglycoconjugates, the measure for comparing galectin reactivity.

 

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  		Table I List of tested glycans and their reactivity to galectins-1 and -3

 
The binding profile to galectin-3-loaded cells showed not only similarities for LacNAc oligomers but also differences. The LNnT tetrasaccharide with reducing-end Glc terminus was rather reactive and, even more important, an {alpha}1,3-galactose extension at LacNAc, known as xenoantigen, proved effective as a docking site (Figure 3 and Table I). Weak binding was seen for LacdiNAc and the 3'-O-sulfated Lec derivative, and all other compounds listed in Table I were inactive. Evidently, the two galectins not only shared properties but also exhibited distinct characteristics, as summarized in Table I. They led us to examine whether spatial blocking with a plant lectin (peanut agglutinin, PNA) reactive with β-galactosides will delineate differences at the levels of loading and reactivity. We also examined enzymatic treatment of galectin-3 (proteolytic truncation) and cells, reducing the presence of unsubstituted β-galactosides.

Blocking of galectin-binding sites
PNA is reactive with the cell surface in a carbohydrate-dependent manner leading to 91% positive cells (not shown). Its presence reduced the reactivity of Raji cells for galectin-1 (as probed with LNnT) markedly (Figure 4A). In contrast, fluorescence intensity of galectin-3-loaded cells probed with Gal{alpha}3'LN after PNA treatment was not affected (Figure 4B). Epitopes for galectin binding to cell surfaces were thus, at least in part, disparate between the two human lectins. This conclusion is further supported by the enzymatic removal of β-galactosides controlled by PNA recognizing β-galactosides (Figure 5A). This process markedly reduced galectin-1 and galectin-3 loading (Figure 5B). Desialylation did not affect loading in both cases despite a conspicuous reduction in SNA reactivity, signaling efficient removal of {alpha}2,6-linked sialic acid (Figure 5A). Evidently, eliminating {alpha}2, 6-sialylation will not automatically turn the unmasked β-galactosides into galectin ligands (Figure 5B).


Figure 4
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  		Fig. 4 Probing the galectin-loaded Raji cells with fluorescent neoglycoconjugates after incubation of cells with the plant lectin PNA in flow cytometry. The logs of fluorescence intensity are plotted against cell number. Gray area defines fluorescence of cells without pretreatment, black curves represent the fluorescence distribution of cell populations pretreated with PNA. (A) Galectin-1 probed with LNnT-PAA-fluo; (B) galectin-3 probed with Gal{alpha}3'LN-PAA-fluo.

 

Figure 5
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  		Fig. 5 Flow cytometric analysis of binding of galectin-1- and galectin-3 to Raji cells after enzymatic treatment of cells. Cells were treated with sialidase followed by β-galactosidase, and stained with SNA, MAA-I, PNA (A) or with anti-galectin antibodies (B). Fluorescence increase was calculated as [(Fi/F0) x 100] – 100, where Fi is the fluorescence intensity of cells after incubation with digoxigenin-labeled lectins (A) or antibodies (B) and F0 is the fluorescence intensity of cells stained with secondary antibodies (B).

 
The tendency of the chimera-type galectin-3 to pentamerize when in contact to multivalent ligands (Ahmad et al. 2004Go) afforded the opportunity to address the issue whether oligomerization has a bearing on this assay setting. Indeed, proteolytic degradation of the N-terminus mainly involved in oligomerization but not sugar binding nearly abolished signal generation to LNnT as a ligand (Figure 6). Cell binding of the potent trisaccharide with {alpha}1-3 galactose extension was completely abolished (not shown). Accessibility of galectin-3 for ligands thus depended on the capacity of the chimera-type galectin to efficiently oligomerize.


Figure 6
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  		Fig. 6 Difference in flow cytofluorimetric profiles of cells loaded with full-length and proteolytically truncated galectin-3, respectively. The gray line defines the fluorescence profile for cells treated with the LNnT-PAA-fluo probe only, while the black lines represent respective profiles after incubation with full-length galectin-3 (thin lines; also see Figure 3) or its C-terminal CRD (bold lines).

 
Sensivity of probe binding to enzymatic treatment of galectin-loaded cells
Physiological ligands of galectins in general include unsubstituted and substituted β-galactosides. In order to infer whether dynamic events after cell surface loading depended on free β-galactoside termini we performed extensive β-galactosidase treatment controlled by the application of the plant lectins DSA and PNA probes to verify the reduction of accessible β-galactosides shown for DSA in Figure 7A. Evidently, we herewith decreased the density of β-galactosides and restricted sites for galectin binding to substituted β-galactosides not susceptible to this enzyme treatment. Immunofluorimetric controls revealed no dissociation of galectins from loaded cells, shown for galectin-3 in Figure 7B. As a consequence of enzyme treatment, the extent of fluorescence intensity for the rather weak ligand LN increased (Figure 7C, D). Apparently, unsubstituted cell surface β-galactosides, that is cis sites, can have an inhibitory effect on probe binding. The density of reactive sites on the cell surface and ligand properties of the headgroup of the fluorescent neoglycoconjugate appeared to factor into the overall results. This result also underscored the potential for dynamic processes on the cell surface not simulated by solid-phase assays. This aspect and the further parameters setting cell-based experiments apart from the solid-phase system prompted us to examine whether the nature of the assay system may influence the reactivity of glycocompounds for galectins. To address this issue we tested the same panel of neoglycoconjugates (Table I) as inhibitors of galectin binding from the solution to a matrix established by asialofetuin.


Figure 7
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  		Fig. 7 Probing galectin-1- and galectin-3-loaded Raji cells after enzymatic degalactosylation of the cells with β-galactosidase. Staining with DSA (A), anti-galectin-3 antibody (B), or probed with fluorescent LN-PAA (C, D). Fluorescence increase was calculated as [(Fi/F0) x 100] –100, where Fi is the fluorescence intensity of galectin-loaded cells after incubation with the secondary antibody (A, B) or with the probe and F0 is the fluorescence intensity of galectin-free cells exposed to secondary antibody (A, B) or to LN-PAA-fluo (C, D). (A) Data for galectin-3; similar results were obtained in the case of galectin-1.

 
Analysis by solid-phase assays
The matrix presented a single glycoprotein, and galectin binding from solution was inhibited in a competitive manner by a panel of neoglycoconjugates. The assay was performed at 4°C and 37°C, reaching very similar data (data not shown). With respect to neutral sugars, no major differences in reactivity profiles between galectins-1 and -3 were noted (Table I). The {alpha}1-3Gal extension did not improve inhibitory reactivity with respect to galectin-3. However, 3'-O-sulfation led to a strong preference for galectin-1 (Table I). When competing with a neutral ligand as a docking site, both 3'-O-sulfated disaccharides were potent inhibitors for galectin-1 but not for galectin-3. The comparison given in Table I revealed that the nature of the assay conditions (i.e., galectin loading of cells versus free galectin and cell surface ligands versus asialofetuin) affected the reactivity profile of neoglycoconjugates.


   Discussion
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 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 Funding
 Conflict of interest statement
 References
 
Emerging evidence reveals the strategic orchestration of cell glycosylation and lectin functionality. Elucidation of the ways the tumor suppressor p16INK4a can mediate anoikis induction via galectin-1 or the internalization of growth factor receptors bearing β1-6-branches in N-glycans appears to be regulated by galectin-3 is capturing further attention for the activities of the members of the galectin family (Lau et al. 2007Go). These clinical implications add interest to comprehensively define the ligand properties of human galectins in a setting as close as possible to the physiological context. It was the central aim of our report to establish a cell-based assay and validate its application for these two most frequently studied family members.

Toward this end we used the favorable properties of neoglycoconjugates to present carbohydrate ligands in a multivalent manner. This characteristic already facilitated detection of cell surface lectin activity (Rapoport et al. 2007Go). By selecting a suitable cell line as a platform for galectin presentation we intended to set up conditions which simulate establishment of intercellular contacts by galectins. By monitoring this new receptor property on the cell surface with a panel of fluorescent neoglycoconjugates a comparative analysis between galectins becomes possible. The presented results enabled us to draw the following conclusions:

  1. Cells, which lack the presence of endogenous lectins, were selected to simplify the interpretation of results. Loading was accomplished, and the multivalent neoglycoconjugates obviously formed stable contacts with the presented galectins. Binding of the probes depended on the presence of terminal galactose residues. Cells without the loading step were not reactive. The set-up thus simulated a cell system primed for intercellular contacts.
  2. The length of the carbohydrate chain and its degree of branching were important parameters for binding. The disaccharide LacNAc was not effective. This result is in full accord with previous reports of lectin reactivity to LacNAc oligomers when tested with surface-immobilized (neo)glycoproteins (Stowell et al. 2004Go; Leppänen et al. 2005Go). In contrast, oligomer presentation to the galectin in the solution has in general no or minor effects on affinity (Di Virgilio et al. 1999Go; Bachhawat-Sikder et al. 2001Go; Ahmad et al. 2002Go, 2004Go; Hirabayashi et al. 2002Go). Because the crystal structures of human galectins-1 and -3 provide no evidence for a series of LacNAc-specific sites (Seetharaman et al. 1998Go; López-Lucendo et al. 2004Go), kinetic factors are likely to account for the measured binding preference.
  3. To challenge the assay's quality we deliberately added the monitored xenoantigen determinant and LacdiNAc to our test panel. Both determinants had been reported to target galectin-3 (van den Berg et al. 2004Go; Jin et al. 2006Go). Our cell assay confirmed avid binding and revealed conspicuous selectivity. Although being structurally closely related and sharing affinity to certain natural ligands such as the pentasaccharide of ganglioside GM1 (Kopitz et al. 1998Go), galectins-1 and -3 also have divergent ligand preferences, as revealed by our analysis. The question immediately arises on properties of the other members of this lectin family. Evidently, this assay will become a valuable tool for systematic screening.
  4. The effect of sulfation strictly specific for its location on the galactose moiety is published before (Allen et al. 1998Go). In our solid-phase assay 3-O-Su-Galβ1-3(4)GlcNAc probes abnormally high affinity to galectin-1, order of magnitude more than neutral ligands. The situation is opposite in the cell assay where sulfated probes bind to cell-loaded galectin-1 at a background level. The reason for this difference remains unclear. At any rate, the comparison of results from the two assays types intimated a dependence of binding properties on assay conditions. This outcome is to be reckoned with in the quest to design selective inhibitors.
  5. If we take into consideration only neutral ligands, ranking of ligands in two assaying system is practically identical (Table I), but the cell assay discriminates "good" and "poor" ligands much better compared to a solid-phase assay. Therefore the cell assay seems to be more precise instrument to identify fine affects.
  6. Enzymatic tailoring of cell surface glycans, rigorously controlled by plant lectins, reveals a cis-effect of β-galactosides on neoglycoconjugate binding. Equally important, removal of {alpha}2,6-sialic acid, often considered as a block for galectin binding, will not necessarily turn the unmasked β-galactosides at the cell surface into operative ligands.

In summary, we have established and validated a cell-based specificity assay for human galectins. Controls with oligosaccharides lacking terminal galactose confirm the specificity of the assay. Differences between the two lectins were detected as probed with neoglycoconjugates. Based on this work the comparative profiling of the properties of human galectins can now be performed using the presented cell-based assay.


   Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
Funding
 Conflict of interest statement
 References
 
Reagents
The label-free and fluorescent neoglycoconjugates with a polyacrylamide backbone were obtained from Lectinity (Moscow, Russia). Carbohydrate-free bovine serum albumin (BSA) was from Merck (Darmstadt, Germany), asialofetuin, β-galactosidase of E. coli, digoxigenin-labeled lectins from Arachis hypogea (PNA), Datura stramonium (DSA), MAA-I (Maackia amurensis), SNA (Sambucus nigra), and FITC-labeled anti-digoxigenin antibodies were purchased from Roche (Mannheim, Germany); neuraminidase of E. coli were from Sigma (St. Louis, MO). Monoclonal mouse anti-rabbit IgG labeled with horseradish peroxidase (IgG-PO) from Sigma (Munich, Germany) as well as goat anti-rabbit IgG-FITC and rabbit anti-mouse IgG-FITC from the Gamaleya Research Institute of Experimental Microbiology (Moscow, Russia).

Galectins and their specific antibodies
The human galectins originated from recombinant production and were then purified by affinity chromatography on lactosylated Sepharose 4B, prepared by divinyl sulfone activation (Gabius 1990Go), as a crucial step, analyzed for purity by one- and two-dimensional gel electrophoresis, gel filtration, and nano-electrospray ionization mass spectrometry (Kopitz et al. 2001Go; André et al. 2004Go), and checked for binding activity prior to and after biotinylation by hemagglutination, solid-phase assays, and cell binding (André et al. 2006Go). Proteolytic truncation of full-length galectin-3 used extensive collagenase digestion to yield the C-terminal CRD (Kopitz et al. 2001). Polyclonal antibodies against galectins-1 and -3 were raised in rabbits under the constant control of the titer; the IgG fractions were isolated by affinity chromatography using protein-A Sepharose 4B (Pharmacia, Freiburg, Germany) and checked for the lack of cross-reactivity against other members of this lectin family by Western blotting and ELISAs (Kaltner et al. 2002Go). The hybridoma line producing the Mac-2 monoclonal antibody (no. TIB-166) was obtained from ATCC (Rockville, MD, USA).

Loading of Raji cell surfaces with galectins
Raji cells (human B-lymphocyte origin, ATCC no. CCL86) were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum and 2 mM glutamine at 37°C in an atmosphere of 5% CO2.

Cells were washed three times with phosphate-buffered saline containing 0.2% BSA (PBA) to deplete the solution of glycoproteins from the serum using centrifugation at 800 rpm/min and 4°C. Aliquots of the cell suspension (1 x 105 cells in 50 µL) were incubated with 50 µL of galectin-containing solution (0.4 mg/mL) for 30 min at 4°C under gentle agitation on a shaker. Cell pelleting then separated cells from the solution. Loading controls were performed with the galectin-type-specific antibodies and corresponding fluorescent second-step reagents used in incubation steps lasting 20 min at 4°C. Flow cytometry was performed using a FACScan instrument (Becton-Dickinson, Heidelberg, Germany) equipped with the software WiNMDI 2.8.

Probing the galectin-loaded cells with neoglycoconjugates
Cells loaded with a galectin were centrifuged at 800 rpm/min. The cells (1 x 105 per well in 50 µL) were carefully resuspended in PBA and incubated in 50 µL with the Glyc-PAA-fluo probes in PBA (final concentration: 100 µM) for 40 min at 4°C under gentle agitation. Thereafter, the cells were washed twice with PBA and analyzed by flow cytometry as given above. Mock-treated cells were used as negative control.

Masking of distinct β-galactosides
Raji cells were incubated with the plant lectin PNA in PBA (1 µg/mL) for 30 min at 4°C. After thorough washing with PBA cells were then loaded with galectins as above followed by probing with fluorescent neoglycoconjugate. After final washing fluorescence profiling was performed by flow cytofluorimetry.

To control glycan accessibility to galectins cells were incubated with neuraminidase (4 U/mL) followed by β-galactosidase, controls for effective hydrolysis were performed by using labeled plant lectins with affinity for {alpha}2,3/6-linked sialic acid (MAA-I, SNA) and β-galactosides (PNA). Galectins were loaded on enzyme treated cells, binding of galectins to cells was analyzed with the galectin-type-specific antibodies as described above.

Probing of galectin-loaded cells after their enzymatic treatment
The galectin-loaded cells were incubated with β-galactosidase from E. coli (4 U/mL) for 3 h at 37°C under gentle agitation followed by centrifugation, washing and probing with LN-PAA-fluo as described above.

Solid-phase assays for galectins
Surfaces of microtiter plate wells were coated with asialofetuin (10 µg/mL) at 4°C overnight. Residual non-specific sites for protein binding were saturated by incubation with 150 µL of a 3% (w/v) solution of BSA in PBS for 60 min at 37°C, and the wells were washed three times with PBS containing 0.05% Tween-20 (P-Tw buffer). Between all following steps the plates were carefully washed three times with P-Tw buffer. Neoglycoconjugates were coincubated with galectin-1 (10 µg/mL) or biotinylated galectin-3 (10 µg/mL) at concentrations ranging from 500 µg/mL to 100 ng/mL for 60 min at 37°C, followed either by immunodetection of bound galectin-1 with peroxidase-labeled antibody as a second-step reagent (1 µg/mL) or by biotin detection with a streptavidin-peroxidase conjugate (0.25 µg/mL) and signal generation with the chromogenic substrates 0.04% O-phenylenediamine and 0.03% H2O2. The percentage of inhibition was calculated as (ODA–OD1) x 100/ODA, where ODA is the mean value of the optical density in the absence of inhibitor and OD1 is the mean value of the optical density in the presence of the inhibitor.


   Funding
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Funding
Conflict of interest statement
 References
 
The Russian Foundation for Basic Research (07-04-00969), the National Institutes of Health (5 U54 GM062116-05), the Russian Academy of Sciences Program "Molecular and Cell Biology", the research initiative LMUexcellent and the EC Marie Curie Research Training Network Program (contract no. MCRTN-CT-2005-19561).


   Conflict of interest statement
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Funding
 Conflict of interest statement
References
 
None declared.


   Acknowledgements
 
We are indebted to Drs. B. Friday, G. Ippans, and S. Namirha for valuable comments.


   Abbreviations
 
Biot, biotin; BSA, bovine serum albumin; CRD, carbohydrate recognition domain; galectin-3C, N-terminally truncated (collagenase-treated) galectin-3; Glyc, carbohydrate residue; fluo, fluorescein; mAb, monoclonal antibody; OD, optical density; PAA, polyacrylamide; pAb, polyclonal antibody; PBS, phosphate-buffered saline, pH 7.2; PBA, PBS containing 0.2% BSA; P-Tw, PBS containing 0.05% Tween-20


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Funding
 Conflict of interest statement
 References
 
Ahmad N, Gabius H-J, Kaltner H, André S, Kuwabara I, Liu F-T, Oscarson S, Norberg T, Brewer CF. Thermodynamic binding studies of cell surface carbohydrate epitopes to galectins-1, -3, and -7: Evidence for differential binding specificities. Can J Chem (2002) 80:1096–1104.

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