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 941430984, USA
Received on July 16, 1999; revised on December 20, 1999; accepted on December 21, 1999.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: CD45/galectin-1/lectin/Thy-1
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Galectin-1 forms a noncovalent homodimer (Barondes et al., 1994). The crystal structure in complex with a biantennary oligosaccharide (Bourne et al., 1994
) 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, 1994
). Galectin-1 has been implicated in a variety of phenomena in multicellular organisms including development, mRNA splicing, differentiation, and cell adhesion (Kasai and Hirabayashi, 1996
). Recently, it has been proposed that galectin-1 has a role in the immune system mediating apoptosis of activated T cells (Perillo et al., 1995
, 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., 1995
). Surprisingly, galectin-1 induced apoptosis was also blocked by an anti-CD45 monoclonal antibody (Perillo et al., 1995
). 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, 1989
; Trowbridge and Thomas, 1994
). Different isoforms of CD45 are generated by alternative splicing and expression of different CD45 isoforms is tightly regulated during development (Thomas, 1989
). In lymphocytes, expression of CD45 is necessary for efficient activation through the antigen receptor (Trowbridge and Thomas, 1994
).
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 Galß14GlcNAc in each branch (Parekh et al., 1987). 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., 1999; Walzel et al., 1999
). 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., 1995
). Pace et al. (1999)
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, 1990). A recent study using iodinated galectin-1 binding to cells gave a similar affinity (Kopitz et al., 1998
). In contrast, two studies have measured the monomeric affinity of galectin-1 for the disaccharide unit, N-acetyllactosamine at ~100 µM (Gupta et al., 1996
).
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, 1995). However, it was in agreement with a more recent study (Giudicelli et al., 1997
) and also studies on the thermal denaturation which indicated that the dimeric form was stable (Surolia et al., 1997
; Schwarz et al., 1998
). 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 doseresponse curve of galectin-1 induced apoptosis of T cells (Perillo et al., 1995
), 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, 1996). 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., 1994
). 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., 1996
). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 2050 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
2 To whom correspondence should be addressed
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barondes,S.H., Cooper,D.N., Gitt,M.A. and Leffler,H. (1994) Galectins. Structure and function of a large family of animal lectins. J. Biol. Chem., 269, 2080720810.
Baum,L.G., Pang,M., Perillo,N.L., Wu,T., Delegeane,A., Uittenbogaart,C.H., Fukuda,M. and Seilhamer,J.J. (1995a) Human thymic epithelial cells express an endogenous lectin, galectin-1, which binds to core 2 O-glycans on thymocytes and T lymphoblastoid cells. J. Exp. Med., 181, 877887.[Abstract]
Baum,L.G., Seilhamer,J.J., Pang,M., Levine,W.B., Beynon,D. and Berliner,J.A. (1995b) Synthesis of an endogeneous lectin, galectin-1, by human endothelial cells is up-regulated by endothelial cell activation. Glycoconj. J., 12, 6368.[ISI][Medline]
Bourne,Y., Bolgiano,B., Liao,D.I., Strecker,G., Cantau,P., Herzberg,O., Feizi,T. and Cambillau,C. (1994) Crosslinking of mammalian lectin (galectin-1) by complex biantennary saccharides. Nature Struct. Biol., 1, 863870.[ISI][Medline]
Chadli,A., LeCaer,J.P., Bladier,D., Joubert-Caron,R. and Caron,M. (1997) Purification and characterization of a human brain galectin-1 ligand. J. Neurochem., 68, 16401647.[ISI][Medline]
Cho,M. and Cummings,R.D. (1995) Galectin-1, a ß-galactoside-binding lectin in Chinese hamster ovary cells. II. Localization and biosynthesis. J. Biol. Chem., 270, 52075212.
Cleves,A.E., Cooper,D.N., Barondes,S.H. and Kelly,R.B. (1996) A new pathway for protein export in Saccharomyces cerevisiae. J. Cell Biol., 133, 10171026.[Abstract]
Cooper,D.N., Boulianne,R.P., Charlton,S., Farrell,E.M., Sucher,A. and Lu,B.C. (1997) Fungal galectins, sequence and specificity of two isolectins from Coprinus cinereus. J. Biol. Chem., 272, 15141521.
Cooper,D.N., Massa,S.M. and Barondes,S.H. (1991) Endogenous muscle lectin inhibits myoblast adhesion to laminin. J. Cell Biol., 115, 14371448.[Abstract]
Cyster,J.G., Fowell,D. and Barclay,A.N. (1994) Antigenic determinants encoded by alternatively spliced exons of CD45 are determined by the polypeptide but influenced by glycosylation. Int. Immunol., 6, 18751881.[Abstract]
Giudicelli,V., Lutomski,D., Levi-Strauss,M., Bladier,D., Joubert-Caron,R. and Caron,M. (1997) Is human galectin-1 activity modulated by monomer/dimer equilibrium? Glycobiology, 7(3), viiix.[Medline]
Gupta,D., Cho,M., Cummings,R.D. and Brewer,C.F. (1996) Thermodynamics of carbohydrate binding to galectin-1 from Chinese hamster ovary cells and two mutants. A comparison with four galactose-specific plant lectins. Biochemistry, 35, 1523615243.[ISI][Medline]
Hirabayashi,J., Satoh,M. and Kasai,K. (1992) Evidence that Caenorhabditis elegans 32-kDa ß-galactoside-binding protein is homologous to vertebrate ß-galactoside-binding lectins. cDNA cloning and deduced amino acid sequence. J. Biol. Chem., 267, 1548515490.
Kasai,K. and Hirabayashi,J. (1996) Galectins: a family of animal lectins that decipher glycocodes. J. Biochem. (Tokyo), 119, 18.[Abstract]
Kopitz,J., von Reitzenstein,C., Burchert,M., Cantz,M. and Gabius,H.J. (1998) Galectin-1 is a major receptor for ganglioside GM1, a product of the growth-controlling activity of a cell surface ganglioside sialidase, on human neuroblastoma cells in culture. J. Biol. Chem., 273, 1120511211.
Pace,K.E., Lee,C., Stewart,P.L. and Baum,L.G. (1999) Restricted receptor segregation into membrane microdomains occurs on human T cells during apoptosis induced by galectin-1. J. Immunol., 163, 38013811.
Parekh,R.B., Tse,A.G., Dwek,R.A., Williams,A.F. and Rademacher,T.W. (1987) Tissue-specific N-glycosylation, site-specific oligosaccharide patterns and lentil lectin recognition of rat Thy-1. EMBO J., 6, 12331244.[Abstract]
Perillo,N.L., Pace,K.E., Seilhamer,J.J. and Baum,L.G. (1995) Apoptosis of T cells mediated by galectin-1. Nature, 378, 736739.[ISI][Medline]
Perillo,N.L., Uittenbogaart,C.H., Nguyen,J.T. and Baum,L.G. (1997) Galectin-1, an endogenous lectin produced by thymic epithelial cells, induces apoptosis of human thymocytes. J. Exp. Med., 185, 18511858.
Puche,A.C. and Key,B. (1995) Identification of cells expressing galectin-1, a galactose-binding receptor, in the rat olfactory system. J. Comp. Neurol., 357, 513523.[ISI][Medline]
Schwarz,F.P., Ahmed,H., Bianchet,M.A., Amzel,L.M. and Vasta,G.R. (1998) Thermodynamics of bovine spleen galectin-1 binding to disaccharides: correlation with structure and its effect on oligomerization at the denaturation temperature. Biochemistry, 37, 58675877.[ISI][Medline]
Sharon,N. (1994) When lectin meets oligosaccharide. Nature Struct. Biol., 1, 843845.[ISI][Medline]
Sunderland,C.A., McMaster,W.R. and Williams,A.F. (1979) Purification with monoclonal antibody of a predominant leukocyte-common antigen and glycoprotein from rat thymocytes. Eur. J. Immunol., 9, 155159.[ISI][Medline]
Surolia,A., Swaminathan,C.P., Ramkumar,R. and Podder,S.K. (1997) Unusual structural stability and ligand induced alterations in oligomerization of a galectin FEBS Lett., 409, 417420.[ISI][Medline]
Thomas,M.L. (1989) The leukocyte common antigen family. Annu. Rev. Immunol., 7, 339369.[ISI][Medline]
Trowbridge,I.S. and Thomas,M.L. (1994) CD45: an emerging role as a protein tyrosine phosphatase required for lymphocyte activation and development. Annu. Rev. Immunol., 12, 85116.[ISI][Medline]
Wagner-Hulsmann,C., Bachinski,N., Diehl-Seifert,B., Blumbach,B., Steffen,R., Pancer,Z. and Muller,W.E. (1996) A galectin links the aggregation factor to cells in the sponge (Geodia cydonium) system. Glycobiology, 6, 785793.[Abstract]
Walzel,H., Schulz,U., Neels,P. and Brock,J. (1999) Galectin-1, a natural ligand for the receptor-type protein tyrosine phosphatase CD45. Immunol. Lett., 67, 193202.[ISI][Medline]
Zhou,Q. and Cummings,R.D. (1990) The S-type lectin from calf heart tissue binds selectively to the carbohydrate chains of laminin. Arch. Biochem. Biophys., 281, 2735.[ISI][Medline]