Characterization of the interaction between galectin-1 and lymphocyte glycoproteins CD45 and Thy-1

Antony Symons1, Douglas N.Cooper3 and A.Neil Barclay2

Sir William Dunn School of Pathology, South Parks Road, University of Oxford, Oxford, OX1 3RE, UK and 3Department of Psychiatry, University of California San Francisco, 401 Parnassus Avenue, San Francisco, CA 94143–0984, USA

Received on July 16, 1999; revised on December 20, 1999; accepted on December 21, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Galectin-1 is a sugar binding protein specific for ß-galactosides and not requiring metal ions for binding activity. It exists as a soluble protein which forms a noncovalent homodimer and is expressed with a broad tissue distribution. Recently, galectin-1 has been shown to play a possible role in the immune system mediating apoptosis of activated T cells with indirect evidence suggesting that galectin-1 interacts with the heavily glycosylated, transmembrane, protein phosphotyrosine phosphatase CD45. The interaction of galectin-1 with purified lymphocyte cell surface proteins was studied using surface plasmon resonance in a BIAcoreTM. Galectin-1 was shown to bind CD45 and Thy-1 in a carbohydrate-dependent manner. Several galectin-1 molecules could bind each CD45 molecule. The dissociation constant of dimeric galectin-1 binding to CD45 was measured at ~5 µM, indicating the concentration at which cross-linking of cell surface glycoproteins by galectin-1 would occur. A possible role for galectin-1 in the organization of cell surface glycoproteins is discussed.

Key words: CD45/galectin-1/lectin/Thy-1


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Galectin-1 is a member of a family of sugar binding molecules specific for ß-galactosides, such as N-acetyllactosamine (Galß1–4GlcNAc [LacNAc]), and not requiring metal ions for binding activity (Barondes et al., 1994Go). It is a highly conserved protein found in the animal kingdom from vertebrates to nematodes (Hirabayashi et al., 1992Go). Examples of galectins have also been identified in marine sponges (Wagner-Hulsmann et al., 1996Go) and fungi (Cooper et al., 1997Go). In mammals, galectin-1 has a broad tissue distribution, expressed in muscle tissues, spleen, thymic epithelial cells, endothelial cells, lung, brain and the olfactory system (Baum et al., 1995aGo,b; Puche and Key, 1995Go; Ahmed et al., 1996Go; Chadli et al., 1997Go). The cellular localization is controversial. Galectin-1 has structural characteristics of a protein synthesized in the cytosol, however, it has been found in different intracellular compartments, at the cell surface and associated with the extracellular matrix (Cho and Cummings, 1995Go).

Galectin-1 forms a noncovalent homodimer (Barondes et al., 1994Go). The crystal structure in complex with a biantennary oligosaccharide (Bourne et al., 1994Go) revealed infinite chains of lectin dimers cross-linked through the oligosaccharides, suggesting that it could act to organize membrane-bound glycoproteins into a lattice (Sharon, 1994Go). Galectin-1 has been implicated in a variety of phenomena in multicellular organisms including development, mRNA splicing, differentiation, and cell adhesion (Kasai and Hirabayashi, 1996Go). Recently, it has been proposed that galectin-1 has a role in the immune system mediating apoptosis of activated T cells (Perillo et al., 1995Go, 1997). The apoptotic effect was shown to be dependent on expression of the transmembrane protein tyrosine phosphatase CD45 on the T cell (Perillo et al., 1995Go). Surprisingly, galectin-1 induced apoptosis was also blocked by an anti-CD45 monoclonal antibody (Perillo et al., 1995Go). Together these data suggest that galectin-1 induces apoptosis through binding to the CD45 glycoprotein. CD45 is a family of large, heavily glycosylated proteins expressed on all nucleated cells of hematopoietic origin (Thomas, 1989Go; Trowbridge and Thomas, 1994Go). Different isoforms of CD45 are generated by alternative splicing and expression of different CD45 isoforms is tightly regulated during development (Thomas, 1989Go). In lymphocytes, expression of CD45 is necessary for efficient activation through the antigen receptor (Trowbridge and Thomas, 1994Go).

In the following analysis, we have used purified proteins and surface plasmon resonance spectroscopy as implemented in the BIAcoreTM instrument, to directly show that galectin-1 binds to CD45 and also to the lymphocyte cell surface molecule Thy-1 in a carbohydrate-dependent manner. Measurement of the affinity of galectin-1 binding to CD45 also indicates at what concentration significant binding and cross-linking of cell surface glycoproteins will occur.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Galectin-1 interacts with cell-surface glycoproteins CD45 and Thy-1
Interactions between macromolecules can be followed in real time using the phenomenon of surface plasmon resonance. The protein of interest, galectin-1, was passed sequentially over four flow cells in the BIAcoreTM to which purified membrane proteins had been covalently bound. These included the ~200 kDa membrane glycoprotein CD45 purified from rat spleen, the ~25 kDa GPI-anchored Thy-1 glycoprotein purified from rat thymus and brain (Parekh et al., 1987Go), and a control protein lacking carbohydrate (GST-CD2). Thy-1 was chosen to compare to CD45 because it is expressed on thymocytes, and because the carbohydrate contents of the thymus and brain glycoprotein have been previously analyzed (Parekh et al., 1987Go). Figure 1 shows that galectin-1 binds to glycoproteins CD45 and Thy-1 but not to unglycosylated GST-CD2 control protein. The interaction was completely blocked by preincubation of galectin-1 with 20 mM lactose. These results demonstrate that binding of galectin-1 to CD45 is carbohydrate dependent and that galectin-1 can interact with other cell surface glycoproteins. Interestingly, the sensograms also show that the binding of galectin-1 to CD45 and Thy-1 is transient with a rapid dissociation phase.



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Fig. 1. Carbohydrate dependent binding of rat galectin-1 to CD45 and Thy-1. Proteins were immobilized on the sensor surface of a BIAcore chip by amine coupling. The amounts immobilized in each case in response units (RU) were: CD45 purified from rat spleen (6227 RU), Thy-1 purified from rat brain (8013 RU) and thymus (5517 RU), and recombinant GST-CD2 (5097 RU). Five microliters BSA (10 mg/ml) and galectin-1 (0.5 mg/ml) were injected sequentially through all four flow cells at 25°C and a flow rate of 1 µl/min. To remove any bound protein flow cells were then washed with 15 µl 0.1 M HCl for 3 min. 5 µl of BSA (10 mg/ml) and galectin-1 (0.5 mg/ml), preincubated with 20 mM lactose, were then injected at 1 µl/min. Injection of anti-rat CD45 (OX1) and anti-rat Thy-1 (OX7) mAb demonstrated active protein immobilized in each flow cell (35 µl at 5 µl/min). The arrows indicate the starting point of each injection.

 
The stoichiometry of the interaction between galectin-1 and the different glycoproteins on the sensor chip can be estimated at saturation. Binding of galectin-1 to the immobilized proteins had reached saturation at 35 µM as determined by injecting increasing concentrations of lectin (data not shown). The stoichiometry of each interaction was then calculated at 6.2 molecules of galectin-1 to 1 molecule of CD45, 0.16:1 galectin-1 to brain Thy-1 and 1.65:1 galectin-1 to thymic Thy-1. These figures indicate that there are several binding sites for galectin-1 on CD45. Rat CD45 is a highly glycosylated protein with 12–17 N-linked glycosylation sites and many more possible sites for O-glycosylation. In contrast, rat Thy-1 has only three N-linked glycosylation sites, which are fully occupied and the difference in binding to brain and thymic Thy-1 correlates with the different carbohydrate found on this protein in the different tissues (Parekh et al., 1987Go). Thy-1 has no O-linked sugars and hence the galectin-1 must be reacting with N-linked sugars at least in this protein.

Affinity measurements of the interaction between galectin-1 and CD45
It has been proposed that cross-linking CD45 at the lymphocyte cell surface by galectin-1 is involved in mediating apoptosis of T cells. It is important to determine the dissociation constant (Kd) of an interaction to establish at what concentration an effect will be seen. To measure the Kd of the interaction of galectin-1 with CD45, 2-fold serial dilutions of the lectin from 35 µM were injected over rat spleen CD45 immobilized on a BIAcoreTM sensor chip surface (Figure 3). Aggregates of galectin-1 that would give a disproportionately high affinity were removed by size-exclusion chromatography immediately prior to carrying out the BIAcoreTM analysis. Size-exclusion chromatography of galectin-1 at 10 or 1000 µg/ml (0.65 or 65 µM), identified a single peak at a retention volume equivalent to a protein with a molecular weight of 29 kDa (Figure 2). This indicates that the galectin-1 injected into the BIAcoreTM flow cell was dimeric across the concentrations used.



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Fig. 3. BIAcore analysis of the interaction between dimeric rat galectin-1 and rat spleen CD45. CD45 purified from rat spleen and GST-CD2 were immobilized on the sensor surface of a BIAcore chip by amine coupling giving 5938 RU of spleen CD45 and 3506 RU of GST-CD2 immobilizes. Galectin-1 (5 µl) was injected through both flow cells at 1 µl/min and 25°C. To remove any bound protein flow cells were then washed with 10 µl 0.1 M HCl for 2 min. The first two injections of galectin-1 are at the same concentration (35 µM) to show that regeneration with HCl does not affect binding. The subsequent series are 2-fold dilutions.

 



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Fig. 2. Rat galectin-1 is dimeric from analysis by gel filtration. (A) HPLC molecular sieve elution profiles, detected by absorption at 214 nm, are shown for separation of affinity-purified recombinant rat galectin-1 at 10 or 1000 µg/ml (PBS, 25mM lactose, 10 mM mercaptoethanol) and for size standards, cytochrome c (12.4 kDa) and serum albumin (66 kDa). Inset, the approximate molecular mass for the single galectin-1 peak in the HPLC elution profile was estimated to be 29 kDa by comparison with protein standards. (B) SDS–PAGE under reducing (R) and nonreducing (NR) conditions confirms the purity of monomeric rat galectin-1.

 
The response due to galectin-1 binding was corrected for the background signal due to a general increase in protein concentration as monitored in a flow cell with immobilized GST-CD2 and plotted in a Scatchard analysis. The Kd for the interaction of galectin-1 with CD45 calculated from the binding curve and Scatchard analysis was 5.01 µM (Figure 4). This clearly establishes that significant binding of dimeric galectin-1 to at least some of the abundant glycoproteins at the cell surface will occur in the concentration range 1–5 µM.



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Fig. 4. Analysis of the binding data for rat galectin-1 interacting with rat spleen CD45. (a) and (b) show the binding curve and Scatchard analysis, respectively, for the binding of rat galectin-1 interacting with splenic CD45 as measured using the BIAcoreTM. In (a) the response due to galectin-1 binding to CD45 (circles) is calculated by deducting the response in the GST-CD2 control flow cell (triangles) from the flow cell with CD45 immobilized (squares). The dissociation constant is then calculated from the best-fit binding curve (no symbols).

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
The results presented here demonstrate directly that galectin-1 can bind at least two major glycoproteins found on the lymphocyte cell surface, namely CD45 and Thy-1, in a carbohydrate dependent manner (Figure 1). Measurement of the dissociation constant for the interaction of dimeric rat galectin-1 with rat spleen CD45 (Figure 3) demonstrates that a significant amount of galectin-1 will be bound to the glycoprotein in the concentration range 1–5 µM. This represents a relatively weak interaction; however, yields of galectin-1 obtained from bovine spleen suggest an overall concentration of 2.5 µM in this tissue (Ahmed et al., 1996Go). Therefore, local concentrations of galectin-1 could be significantly higher and lead to binding.

The stoichiometry of the interaction between galectin-1 and the different glycoproteins emphasizes the carbohydrate dependence of galectin-1 binding. The lower level of galectin-1 binding to brain Thy-1 compared to thymic Thy-1 is consistent with the different amounts of N-acetyllactosamine (LacNAc) present on the different forms. Carbohydrate analysis has shown that 51% of the sugars on Thy-1 from rat thymus, and 23% on rat brain Thy-1, consist of complex structures which generally contain a single Galß1–4GlcNAc in each branch (Parekh et al., 1987Go). Furthermore, 20% of thymic Thy-1 was found to have repeating lactosamine units but no polylacto­samine was detected on brain Thy-1.

Very recent data have shown that galectin-1 can be used to immunopurify CD45 consistent with these studies (Pace et al., 1999Go; Walzel et al., 1999Go). However, the presence of multiple binding sites for galectin-1 on CD45 as shown above, raises questions as to how a mAb against a CD45 protein epitope can block the binding of galectin-1 to whole cells so effectively as indicated in (Perillo et al., 1995Go). Pace et al. (1999)Go extend the original observations and show that mAbs against CD3, CD4, CD8, CD43, CD45RA, CD45RB, and CD45RO all give around 50% inhibition of galectin-1 binding to T cells. How do all these mAbs specific for a variety of proteins give quite effective inhibition of a lectin binding carbohydrate? One possibility is that the binding of galectin-1 to cells in these circumstances is not through homogeneous dimeric galectin-1, but through traces of aggregates that bind in an anomalous manner or through some other intermediate. From the kinetic data one would expect the normal dimeric galectin-1 to dissociate from cells relatively quickly (Figure 1).

The presence of multiple binding sites for galectin-1 on CD45 also suggests how galectin-1 might organize specific glycoproteins at the cell surface. Glycoproteins with multiple LacNAc units will be the major ligands for galectin-1 because of an increased avidity. Dimeric binding would cross-link adjacent sugar sidechains on a glycoprotein or to another glyco­protein. Galectin-1 binding to carbohydrate will reach equilibrium very rapidly as demonstrated here by the real-time analysis of its interaction with CD45. Whilst the kinetic analysis of the on and off rates did not fit simple models, it is apparent that equilibrium binding is reached in <10 s and that dissociation of galectin-1 is equally rapid.

The dissociation constant for the interaction of dimeric galectin-1 with CD45 measured in this study (5 µM) is in close agreement with other measurements of galectin-1 binding to glycoproteins. Previously, the affinity of galectin-1 for laminin has been calculated to be ~1 µM (Zhou and Cummings, 1990Go). A recent study using iodinated galectin-1 binding to cells gave a similar affinity (Kopitz et al., 1998Go). In contrast, two studies have measured the monomeric affinity of galectin-1 for the disaccharide unit, N-acetyllactosamine at ~100 µM (Gupta et al., 1996Go).

In addition to the affinity of dimeric galectin-1 for carbohydrate, cross-linking of cell-surface glycoproteins will also be dependent on the equilibrium between monomeric and dimeric galectin-1. Our results (Figure 2) are consistent with the presence of stable dimers as gel filtration of the recombinant rat galectin-1 used in this study at low and high concentrations indicated all the material was dimeric. This was true even when preequilibrated at 10 µg/ml (0.7 µM subunit concentration) and regardless of buffer conditions (water or PBS, with or without lactose, with or without mercaptoethanol). This is contrary to an earlier study suggesting that the affinity of the dimerization was 7 µM and the dissociation of galectin-1 dimers was unusually slow (half-life of about of ~10 h; Cho and Cummings, 1995Go). However, it was in agreement with a more recent study (Giudicelli et al., 1997Go) and also studies on the thermal denaturation which indicated that the dimeric form was stable (Surolia et al., 1997Go; Schwarz et al., 1998Go). Together the available data suggest that significant cross-linking of cell surface glycoproteins will occur at concentrations of galectin-1 in the micromolar range. This fits well with the dose–response curve of galectin-1 induced apoptosis of T cells (Perillo et al., 1995Go), consistent with dimeric galectin-1 cross-linking of cell surface glycoproteins in this phenomenon.

Other important questions remain concerning the biological significance of the interaction between galectin-1 and lymphocyte cell surface glycoproteins. The cellular localization of galectin-1 remains controversial (Kasai and Hirabayashi, 1996Go). Galectins have many characteristics of intracellular proteins; they do not have a signal sequence, biosynthesis occurs on free ribosomes, the N-termini are acetylated, and all SH groups are in a free state (Barondes et al., 1994Go). Significantly, in the absence of carbohydrate some galectins lose the ability to bind sugars in an oxidizing environment, although the kinetics of this deactivation warrant further investigation. Galectin-1 has been identified in the cytosol but it has also been found at the cell surface and associated with the extracellular matrix; recombinant rat galectin-1 was secreted in yeast by a nonclassical mechanism (Cleves et al., 1996Go). Furthermore, a homolog of galectin-1 has been identified in fungi pointing to a more fundamental housekeeping function than immunity. However, it seems plausible to speculate that galectins function to organize glycoproteins at the cell surface by linking them through their carbohydrates. The subsequent loss of carbohydrate binding activity by exposure to the extracellular oxidizing environment would release the glycoprotein for display to other surface proteins.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Proteins and monoclonal antibodies
Native CD45 was purified from rat spleen by affinity chromatography with the mAb, OX1, as previously described (Sunderland et al., 1979Go). Rat thymus and brain Thy-1 were purified as described previously (Parekh et al., 1987Go). Sodium deoxycholate was removed by ethanol precipitation or extensive dialysis. Recombinant soluble GST-CD2 was expressed in E.coli and purified as described (Cyster et al., 1994Go). Recombinant rat galectin-1 was purified by lactose affinity chromatography (Cooper et al., 1991Go). Monoclonal antibodies (mAb) were produced as hybridoma culture supernatants. OX1 is specific for all isoforms of rat CD45 (Sunderland et al., 1979Go) and OX7 is specific for rat Thy-1 (Parekh et al., 1987Go).

Gel filtration chromatography
To estimate its approximate native molecular mass, affinity-purified recombinant rat galectin-1 was analyzed by gel filtration chromatography using a Superdex 75 HR 10/30 molecular sieve column (bed volume ~24 ml) (PharmaciaBiotech) and a Perkin-Elmer Series 4 HPLC system with detection at 214 nm. The protein was preequilibrated in a variety of buffers for at least 24 h at concentrations ranging from 10 to 1000 µg/ml. Sample was then injected into the HPLC, which had been equilibrated in the same buffer, and was chromatographed at a flow rate of 0.5 ml/min. Approximate molecular mass was calculated from peak elution volume by comparison with molecular weight standards (Sigma Chemical Co.).

BIAcore analysis of galectin-1 interactions with glycoproteins
All BIAcoreTM experiments were performed on a BIAcoreTM2000 biosensor (Pharmacia Biosensor, Uppsala) at 25°C in HBS running buffer (150 mM NaCl, 10 mM HEPES, pH 7.4, and 0.005% surfactant P20). Proteins were covalently coupled via amine groups onto the carboxymethylated dextran surface of CM5 (research-grade) sensor chips using the standard amine coupling kit (Pharmacia Biosensor) as recommended by manufacturer, with the following modifications. During coupling at a flow rate of 5 µl/min, splenic CD45 was injected for 7 min at 50 µg/ml in 10mM sodium acetate, pH 4.0. Thymic Thy-1 and brain Thy-1 were injected at 15 µg/ml in 10 mM sodium acetate, pH 5.0, 1% octyl glucoside and GST-CD2 was injected at 20–50 µg/ml in 10 mM sodium acetate, pH 5.0. All proteins were regenerated by injecting 100 mM HCl for 3 min. Binding experiments were performed at a flow rate of 1 µl/min. Rat galectin-1 was repurified immediately prior to binding analysis by size-exclusion chromatography on a Superdex 75 HR10/30 column, in 10 mM HEPES, pH 7.4, 150 mM NaCl. Serial 2-fold dilutions of galectin-1 from the dimeric peak were then made in HBS running buffer.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
This work was supported by the Medical Research Council, Arthritis Research Council, and the European Union Biotechnology program.


    Footnotes
 
1 Present address: The R.W. Johnson Pharmaceutical Research Institute, 3535 General Atomics Center, La Jolla, CA Back

2 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
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