Homodimeric galectin-7 (p53-induced gene 1) is a negative ... - Nature

Oncogene (2003) 22, 6277–6288

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Homodimeric galectin-7 (p53-induced gene 1) is a negative growth regulator for human neuroblastoma cells Ju¨rgen Kopitz*,1, Sabine Andre´2, Carolina von Reitzenstein1, Kees Versluis3, Herbert Kaltner2, Roland J Pieters4, Kojiro Wasano5, Ichiro Kuwabara6, Fu-Tong Liu6, Michael Cantz1, Albert JR Heck3 and Hans-Joachim Gabius2 1

Institut fu¨r Molekulare Pathologie, Klinikum der Ruprecht-Karls-Universtia¨t, Im Neuenheimer Feld 220, 69120 Heidelberg, Germany; 2Institut fu¨r Physiologische Chemie, Tiera¨rztliche Fakulta¨t, Ludwig-Maximilians-universita¨t, Veterina¨rstr. 13, 80539 Mu¨nchen, Germany; 3Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands; 4Department of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, PO Box 80082, 3508 TB Utrecht, The Netherlands; 5Department of Anatomy and Cell Biology, Faculty of Medicine, Kyushu University, Fukuoka 812-0054, Japan; 6 Department of Dermatology, School of Medicine, University of California Davis, Sacramento, CA 95817, USA

The extracellular functions of galectin-7 (p53-induced gene 1) are largely unknown. On the surface of neuroblastoma cells (SK-N-MC), the increased GM1 density, a result of upregulated ganglioside sialidase activity, is a key factor for the switch from proliferation to differentiation. We show by solid-phase and cell assays that the sugar chain of this ganglioside is a ligand for galectin-7. In serum-supplemented proliferation assays, galectin-7 reduced neuroblastoma cell growth without the appearance of features characteristic for classical apoptosis. The presence of galectin-3 blocked this effect, which mechanistically resembles that of galectin-1. By virtue of carbohydrate binding, galectin-7 thus exerts neuroblastoma growth control similar to galectin-1 despite their structural differences. In addition to p53-linked proapoptotic activity intracellularly, galectin-7, acting as a lectin on the cell surface, appears to be capable of reducing cancer cell proliferation in susceptible systems. Oncogene (2003) 22, 6277–6288. doi:10.1038/sj.onc.1206631 Keywords: apoptosis; cell growth; galectin; ganglioside; mass spectrometry;neuroblastoma

Introduction The merit of molecular analysis of growth control is not limited to delineation of relevant pathways in basic science. In tumor biology, this knowledge will help to pinpoint physiological trigger mechanisms, which might later be exploited to restrict malignant cell proliferation therapeutically. Based on their strategic cell surface presentation and malignancy-associated structural variability, glycan chains of cellular glycoconjugates *Correspondence: J Kopitz; E-mail: [email protected] Dedicated to Professor Dr F Cramer on the occasion of his 80th birthday Received 16 January 2003; revised 4 April 2003; accepted 7 April 2003

deserve attention in this respect. In fact, in the interplay with endogenous lectins, such cell surface determinants participate in regulating cell adhesion, spreading, motility, and mitotic activity (Gabius, 1987, 1997; Raz and Lotan, 1987; Varki, 1993; Hakomori, 1998; Kaltner and Stierstorfer, 1998; Reuter and Gabius, 1999; Nangia-Makker et al., 2000; Solı´ s et al., 2001; Gabius et al., 2002; Kilpatrick, 2002). By often being positioned readily accessible at the tips of the branches of N- and mucin-type O-glycans, b-galactosides or derivatives thereof harbor ideal features to serve as docking sites for lectins (Gabius, 2000; Brewer, 2002). The occurrence of at least 13 b1,3(4)-galactosyltransferases gives reason to expect a fine-tuned regulation of synthesis for distinct determinants (Shaper et al., 2000), another argument for their role in biorecognition. Figuring prominently in this interplay, the members of the family of galectins (bgalactoside-specific lectins without Ca2 þ -requirement, which share structural homology based on the jelly-roll motif) home in on such epitopes, thereby completing an operative protein–carbohydrate recognition system (Barondes et al., 1994; Hirabayashi, 1997; Itzkowitz, 1997; Ohannesian and Lotan, 1997; Kaltner and Stierstorfer, 1998; Andre´ et al., 1999; Liu, 2000; Danguy et al., 2002; Rabinovich et al., 2002). Its consequences for cancer aspects are beginning to be detected clinically, for example, the galectin-1-induced apoptosis of CD7 þ T cells quite likely contributing to the accumulation of CD7 T cells during progression of the Se´zary syndrome (Rappl et al., 2002). With increasing insight into the intrafamily diversity of galectins (Cooper, 2002), the issue arises to assess the extent of their functional overlap or divergence. Indeed, human tumor cells express more than the genes of the commonly tested galectins-1 and -3, as monitored by recent RT-PCR (Lahm et al., 2001) and immunohistochemical profiling (Camby et al., 2001; Wollina et al., 2002). However, it is unclear whether and how the different galectins cooperate. To contribute to address the questions on cellular functions of the other family

Galectin - 7 inhibits neuroblastoma cell proliferation J Kopitz et al

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members and on their overlap to galectins-1 and -3, it is reasonable to test them in a system, in which the properties of galectins-1 and -3 have been wellcharacterized. For this purpose, we selected the human neuroblastoma cell line SK-N-MC. In these cells, the activity increase of a cell surface ganglioside sialidase, which shifts ganglioside presentation from higher sialylated forms to ganglioside GM1 and also the neutral glycolipid lactosylceramide, is the pivotal control element to switch cell behavior from proliferation to differentiation (Kopitz et al., 1994, 1996, 1997). In the next step of the investigations, homodimeric galectin-1 had been delineated as major receptor for this ganglioside, the ensuing protein–carbohydrate interaction triggering reduced cell growth without fitting the pattern of classical apoptosis (Kopitz et al., 1998, 2001). The chimera-type galectin-3 shared cell surface ligand(s), but failed to affect cell growth and could even block the galectin-1 activity competitively (Kopitz et al., 2001). Carbohydrate-dependent cell surface binding, a common characteristic of galectins following their secretion by a nonclassical route (Barondes et al., 1994; Hirabayashi, 1997; Hughes, 1999), and the specificity of the triggered response are thus two defined parameters to determine the properties of the test substances comparatively. Since analysed brain tumors lack the capacity for endogenous galectin-7 expression (Lahm et al., 2001), we set out to examine its activities in comparison to galectins-1 and -3 in this tumor model. Our interest in galectin-7 was spurred by previous reports relating its intracellular presence to the effector mechanisms of a tumor suppressor. The cDNA of galectin-7 was independently cloned in programs to identify markers associated with the normal keratinocyte phenotype (Madsen et al., 1995; Magnaldo et al., 1995). Its expression was abrogated in SV40transformed keratinocytes (K14) as well as TR146 and SCC13 squamous cell carcinoma cells, whereas sections of human basal and squamous cell carcinomas and chemically induced mouse papillomas still harbored residual immunopositivity (Madsen et al., 1995; Magnaldo et al., 1995, 1998). Using SCC13 cell transfection with a sense vector and UVB-irradiation as genotoxic stress, galectin-7 expression was linked to the p53related induction of epidermal apoptosis (Bernerd et al., 1999). These results fit in nicely with the preceding report that this marker of epithelial stratification belonged to the 14 out of 7202 identified transcripts in a screening by serial analysis of gene expression, which were upregulated before the onset of p53-induced apoptosis in the colorectal cancer line DLD-1 (Polyak et al., 1997). As alluded to above, this activity was honored by referring to galectin-7 as PIG1 (p53-induced gene 1). Its proapoptotic function in transfected HeLa cells was attributed to intracellular mechanisms upstream of JNK activation and cytochrome c release (Kuwabara et al., 2002). In developmental regulation, galectin-7 presence was assumed to accompany shifts in the balance between proliferation and differentiation (Magnaldo et al., 1998; Timmons et al., 1999). The fact that its cDNA belonged to a set of seven cloned Oncogene

transcripts upregulated in the course of rat mammary carcinogenesis by 1-methyl-1-nitrosourea and 7,12dimethylbenz[a]anthracene (but not azoxymethane-dependent colon tumor induction) cannot easily be reconciled with such a role (Lu et al., 1997). In addition to these reports on functional aspects, some information on its structure is also available. Compared to homodimeric and cross-linking galectin1, galectin-7 was considered as a monomeric protein (Madsen et al., 1995; Sato et al., 2002). It was crystallized as dimer with an unusual packing arrangement relative to galectins-1 and -2, not principally arguing against the assumed monomer status (Leonidas et al., 1998). The architecture of the carbohydrate recognition domain and the lengths and conformation of loop regions differed relative to the other two investigated prototype galectins (Leonidas et al., 1998). Thus, its fine-specificity for carbohydrates, but also its potential for protein–protein interactions might deviate from those of galectin-1. Concerning the latter aspect, oncogenic H-ras and Gemin4 are carbohydrate-independent galectin-1 binding partners in the cell (Park et al., 2001; Paz et al., 2001; Elad-Sfadia et al., 2002; Liu et al., 2002). Although no extracellular targets for protein–protein interactions are known, this second binding capacity should not be overlooked when dealing with cell surface interactions. Notably, the generation of a cholera toxin B subunit variant (His57Ala) retaining high-affinity GM1 binding with impaired postbinding signaling renders additional interactions of this lectin after initial sugar-dependent binding likely (Aman et al., 2001; Rodigherio et al., 2001). In detail, we addressed the following questions in this study: is galectin-7 monomeric or oligomeric in solution? Is the binding affinity to the glycan chain of ganglioside GM1 and to neuroblastoma cell surfaces similar or different to galectins-1 and -3? Do these galectins compete with galectin-7 for cell surface epitopes? Does exogenous addition of galectin-7 affect cell growth? Is any detected regulatory capacity dependent on lectin activity?

Results Mass spectrometric analysis Recombinant expression of human galectin-7 yielded a homogeneous protein with predicted electrophoretic mobility (not shown). The sensitivity of this standard quality control is not necessarily sufficient to exclude the presence of a low-molecular-weight post-translational modification of potential functional significance. To further investigate whether the actual molecular mass of the recombinant protein matches the theoretical sequence-based prediction, we employed the highly sensitive method of nano-electrospray ionization (ESI) mass spectrometry to look for the presence of any deviations not detectable in electrophoretic analysis. To record the spectra exclusively for the monomer, highly acidic conditions were established. In the obtained


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spectrum, the peaks originated from monomer ions with different levels of positive charges (Figure 1). No contamination was visible. Based on this experimental information, the molecular mass of human recombinant galectin-7 is 14 927.370.05 Da. At first sight, this value appeared to indicate an underestimation relative to the value given in the SWISS-PROT data bank (Accession number P47929) with 14 944 Da which would be difficult to account for biochemically. However, it should not be overlooked that cloning into the expression vector was facilitated by introducing a single-site mutation at the N-terminus. Due to the Ser2Ala substitution in the course of the generation of the NcoI cleavage site, the actual theoretical mass for the monomer is 14 926.88 Da. Thus, we can exclude that the recombinant protein is post-translationally modified. To answer the question on its quaternary structure, we then changed the conditions of the analysis markedly to reach a pseudophysiological milieu. As internal controls, we first performed the measurements with the related prototype galectin-1 and the chimera-type galectin-3. These proteins were selected based on the criteria of close relation and well-characterized behavior in solution to validate the experimental conditions. Human galectin-1 is exclusively a dimer even at low concentrations (2 mm) in native gel filtration (Giudicelli et al., 1997), and galectin-3, too, is at least primarily monomeric in size-exclusion chromatography (Roff and Wang, 1983; Frigeri et al., 1990; Ochieng et al., 1993). Focusing first on these two control proteins, we studied at pH 6.8 whether nano-ESI mass spectrometry will come up to these expectations from gel filtration, neither being detrimental to the dimer of galectin-1 nor initiating undue oligomerization of galectin-3. As shown in Figure 2a, b, the resulting spectra of galectins-1 and -3

Figure 2 Nano-ESI mass spectra of human galectin-1 (a), murine recombinant galectin-3 (b) and human recombinant galectin-7 (c) under pseudophysiological conditions to maintain noncovalent oligomer formation. Peak annotations designate the number of positive charges per monomer (M) and dimer (D)

Figure 1 Nano-ESI mass spectrum of human recombinant galectin-7 under acidic conditions favoring oligomer dissociation. Peak annotations designate the number of positive charges for the monomer ions

fulfilled the predictions. Galectin-3 was monomeric, while galectin-1 was predominantly dimeric. The charge distribution observed for galectin-3 is of interest due to a rather unusual occurrence of two maxima observed at approximately 24 (M24) and 11 (M11) charges, respectively (Figure 2b). Such a bimodal charge distribution is indicative of the presence of two distinct conformers of galectin-3 in solution. The more highly charged ions might then correspond to a more unfolded structure, whereas in this case the lower charge states could represent a more compact structure (Dobo and Kaltashov, 2001; Simmons and Konermann, 2002; van den Bremer et al., 2002). The relative abundance of these conformers may be influenced by low protein and salt concentration. For our line of reasoning, this essential quality control with galectins-1 and -3 thus ensured the suitability of the conditions for analysing the properties of galectin-7. The positions of galectin-7-derived protein Oncogene


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ions, especially for D10-D13, revealed a dimeric nature of galectin-7 (Figure 2c). Taking the relative distribution of signal intensities of the M/D-species with increasing charge differences into account, it is reasonable to assume that D12 (dimer with 12 charges) is the predominant form in the D12/M6 peak (Figure 2c). In comparison to the other two spectra, galectin-7’s ion peak pattern resembled that of galectin-1 with the ratio of dimer/(monomer þ dimer) ions reaching about 0.85 in both cases (Figure 2a, c). To further underscore that the conditions of nanoESI MS using an aqueous solution buffered with ammonium acetate at pH 6.8 indeed are gentle and suitable to maintain properties of the native protein, we added a set of experiments for galectin-7 in the presence of a carbohydrate ligand, that is, two wedge-like dendrimers with two or four isocyanate derivatives of lactose as the headgroup using 3,5-di(2-aminoethoxy)benzoic acid as a branching point. At equimolar lectin and ligand concentrations (10 mm) protein ions attributable to complexes of the ligand with the dimer were detected (Figure 3). Evidently, these conditions were not detrimental for ligand binding. Further increase of dendrimer concentration apparently entailed aggregation, a hallmark of receptors with at least bivalency, and no peaks could be recorded in the scanned molecular mass range (not shown). These results add to the weight of the conclusion that galectin-7 is – similar to galectin-1 – primarily a homodimer under these conditions. Although the galectins share binding properties to bgalactosides, their fine-specificities to natural ligands may or may not differ. Comparative binding studies with a biochemically purified glycan are one set of experiments to address this issue. Based on previous work with the neuroblastoma cells (Kopitz et al., 1994, 1996, 1997, 1998) the sugar chain of ganglioside GM1 is an adequate test model with relevance for further biological considerations. To assess whether and with what level of affinity galectin-7 will bind to the oligosaccharide chain of ganglioside GM1, we performed a solid-phase assay. To be able to correlate the data with the other galectins’ characteristics, we performed these measurements also with galectins-1 and -3. They are known to home in on this ligand when presented on the neuroblastoma cell surface (Kopitz et al., 1998). Solid-phase assay As ligand, we synthesized a neoganglioprotein covalently conjugating an average of two to three lysoganglioside GM1 units per carrier molecule. Relative to direct immobilization of the ganglioside to the plastic surface, use of the carrier is likely to improve ligand accessibility. Previous cyto- and histochemical measurements with this neoglycoprotein (Gabius et al., 1990; Kopitz et al., 1998; Kayser et al., 2001) and solid-phase assays with biantennary N-glycans (Andre´ et al., 1997) have documented that the protein-attached carbohydrate ligand retains its capacity to interact with lectins. Binding of galectins-1, -3 and -7 in this assay was Oncogene

Figure 3 Nano-ESI mass spectra of human recombinant galectin7 in the absence of a ligand (a) and in the presence of an equimolar concentration (10 mm) of dendrimer with either two (b) or four lactose headgroups (c). The symbols given in the figure symbolize the monomer () or dimer without bound ligand () and dimer with bound ligand where a square is added

saturable and inhibitable by glycosubstances (0.5 mg asialofetuin/ml and 75 mm lactose) blocking the lectin site, as shown in the binding curves (Figure 4). Obviously, the three galectins can accomodate the lysoganglioside’s sugar chain into their binding sites. Algebraic transformation of the data for sugar-inhibitable binding led to the Scatchard plots (Figure 4) and the calculation of the dissociation constant (KD). These data are compiled in Table 1. Despite galectin-typespecific characteristics in the sequence, the overall structural similarity of the three family members translated into rather similar affinities of about 1.5– 3 mm. To figure out whether binding of this ligand is also operative for a galectin from the third subgroup, these measurements were extended to a tandem-repeat-type galectin in the form of the N-terminal domain of


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Figure 4 Scatchard plot analysis of data of sugar-inhibitable binding (inset) of labeled galectin-1 (a), galectin-3 (b) and galectin-7 (c) to surface-immobilized neoganglioprotein presenting the oligosaccharide chain of lysoganglioside GM1 as ligand part (for calculated binding parameters, see Table 1). Total binding (&) was routinely reduced by the extent of nonspecific binding which was not inhibitable by presence of an excess of glycoligands (}) to yield carbohydrate-dependent binding (n). The figure shows a representative set of data from at least three independent experimental series

Table 1 Determination of the dissociation constant (KD) and the number of bound probe molecules at saturation (Bmax) for galectin binding in the solid-phase and cell assays Solid-phase assay

Cell assay

KD (mm)

KD (mm)

Bmax (molecules/cell)

Galectin-7 Galectin-1

2.2971.27 1.6270.55

Galectin-3

2.8570.64

0.8870.29 0.7670.18 (0.9870.21)a 0.94a70.25

2.22 10670.34 106 2.23 10670.29 106 (2.60 10670.39 106)a 2.70 106a70.37 106

Data were collected in at least three independent experimental series, exemplary binding curves and Scatchard plots are shown in Figures 4 and 5. a From Kopitz et al. (1998).

galectin-4. Actually, the two domains of members of this subfamily are nonidentical, requiring separate cloning for further biochemical analysis. Compared to the Nterminal domain, the C-terminal domain had less favorable solubility properties even as a fusion protein, thereby restricting the analysis to the N-terminal domain (Wu et al., 2002; see below). As observed for the prototype and chimeric galectins, the N-terminal carbohydrate recognition domain of this tandem-repeattype galectin accepted the neoganglioprotein ligand with a similar affinity (KD ¼ 1.4170.34 mm). Since complete galectin-4 is rather insoluble compared to galectins-1, -3, and -7 (e. g. isolation protocol from pig tongue included an extraction step in 4 m urea to remove other proteins and sonication in the presence of 9 m urea for the solubilization of galectin-4 (Chiu et al., 1992, 1994)), further cell binding and proliferation assays with

complete galectin-4 were precluded. In the course of these studies with galectin-7, we deliberately included parallel measurements with galectin-1, although tested thoroughly before (Kopitz et al., 1998, 2001), to be able to exclude any variation of cell surface properties unambiguously relative to the cell populations with which we had worked in our previous studies. Cell binding assay The solid-phase assays had shown that galectin-7 recognized the carbohydrate chain of the carrierimmobilized ganglioside presented on the plastic surface. However, this does not necessarily mean that galectin-7 will also be comparatively reactive with the tumor cell surface. Using radioiodinated probes retaining their binding activity this issue was resolved. Cell Oncogene


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binding studies controlled by inhibition to assess carbohydrate-independent cell association clearly revealed that galectin-7 – like galectin-1 – bound to the neuroblastoma cell surface (Figure 5). Binding parameters for galectin-7 and galectin-1 (determined in the course of this study with aliquots of the cell batches besides those which had initially been described in our former study (Kopitz et al., 1998)) were very similar to each other (Table 1). As an indication for ganglioside GM1 being involved in galectin-7 cell association, we demonstrated a conspicuous reduction of binding to cells when access to ganglioside GM1 was blocked by competitive inhibition with the GM1-specific cholera toxin B subunit (not shown). Systematic binding studies for the latter case resulted in the calculation of a KDvalue of cholera-toxin-B-subunit-insensitive binding of 480 nm with 0.93 106 galectin-7 molecules bound per cell (Figure 6). Ganglioside GM1 is thus likely to constitute a major cell surface ligand for galectin-7.

Figure 6 Influence of presence of the cholera toxin B subunit on extent of cell binding of 125I-labeled galectin-7. Scatchard plot analysis of data of carbohydrate-dependent cell surface binding (inset) of 125I-labeled galectin-7 in the absence of the ganglioside GM1-specific reagent () and in the presence of cholera toxin B subunit (J; 25 mg/well) was performed under experimental conditions described in the legend of Figure 5. Each point represents the mean7s.d. of three independent measurements with duplicates in each series

Figure 5 Scatchard plot analysis of data of carbohydratedependent cell surface binding (inset) of 125I-labeled galectin-7 () and galectin-1 (J). Cells were cultured for 5 days to reach a final density of 105 cells/well in the serum-supplemented medium and then for further 16 h in serum-free medium before the cells were exposed to galectin-containing solution for 2 h. Parallel experiments with galectin incubation in the presence of an inhibitor (150 mm lactose and 0.5 mg asialofetuin/ml) were performed to determine the extent of noninhibitable binding. The presented data are based on three independent measurements with duplicates for each point Oncogene

To add to the evidence of this conclusion implying very similar target selection of galectins-1, -3, and -7 in this cell system, we performed competitive binding studies with galectins-1 and -3. If they share ligand populations with galectin-7, then the level of noninhibitable binding will allow to estimate the extent of these differences. The differences, if detectable, might be attributed to different glycoligands or identical initial glycoligand selection with different galectin–protein (or lipid) interactions in the second step of a binding process. Despite fine-structural diversity in the same common folding pattern galectin-7 binding could be completely blocked by galectin-1 (Figure 7a). Displacement of bound galectin-7 proceeded to a level of only about 20% of residual binding (Figure 7a). The chimera-type galectin-3, too, served as an excellent inhibitor apparently homing in on the same set of ligands (or spatially closely associated glycans) (Figure 7b). No evidence for a distinct two-step binding


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Figure 8 Photomicrographs of neuroblastoma cell preparations cultured in the absence or presence of galectins. After seeding at an initial density of 104 cells/well and culture for 16 h to allow cell attachment, aliquots of individual cell batches were cultured in the following experimental period of 48 h in serum-supplemented medium (a) or this medium containing 125 mg galectin-1/ml (b) as controls. The effect of addition of 125 mg galectin-7/ml (c), and of galectin-7 in the presence of a 10-fold excess of galectin-3 (d) was determined in parallel under identical experimental conditions (magnification, 125)

Figure 7 Competitive studies of cell surface binding of 125I-labeled galectin-7 with unlabeled galectin-1 (a) and galectin-3 (b). The inhibitor was added in the standard binding assay (see legend to Figure 5) simultaneously with the probe () or 1 h after a preincubation step with labeled galectin-7 (J). The results are given as means7s.d. of four independent measurements with duplicates in each series

process of galectin-7 involving a protein or lipid could be delineated. These experiments demonstrated that cell surface binding of galectin-7 exhibited similar properties to that of galectins-1 and -3, prompting to address the ensuing issue as to whether galectin-7 binding will be bioactive concerning growth regulation in this system. Cell proliferation assays A culture of current cell batches in serum-supplemented medium fully reproduced the previously reported reduction of proliferation by galectin-1 (Kopitz et al., 2001), as shown in Figure 8a, b. This control rigorously excluded variability of this aspect of cell responsiveness. Under identical conditions, galectin-7 triggered the same effect (Figure 8c). As a quantitative measure, cell proliferation was assessed with a commercial kit. The result of these experiments was a significant reduction in

this parameter, comparable to galectin-1’s bioactivity (Figure 9a, b). Notably, the concentration dependence of this effect and the lack of a clear indication for triggering classical apoptosis were shared by these two galectins (not shown). Monitoring DNA cytograms for the presence of the typical sub-G0/G1 peak, searching for label incorporation into the DNA derived from internucleosomal fragmentation and testing caspase inhibitors did not come up with a signal as obtained in positive controls with staurosporine (not shown). That the carbohydrate targeting was essential to provoke the growth-inhibitory response was shown by blocking cell binding with a potent mixture of glycoinhibitors, which themselves behaved as bioinert additions to the medium on the cells (Figure 9c, d). Since galectin-3 could block galectin-7 binding to the cells, we finally examined whether galectin-3 presence will render the cells non-responsive to galectin-7. This was indeed the case, demonstrated by a photomicrograph of a representative part of a culture (Figure 8d) and by the results of the quantitative analysis (Figure 9e, f). Thus, the bioactivity of galectin-7 in this system depended on carbohydrate binding and was blocked by galectin-3. From these results, the concept of significant functional divergence within the galectin family even under conditions of at least very similar selection of cell surface ligands emerges. Oncogene


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Figure 9 Effect of galectin-7 on cell proliferation and the modulatory influence of galectin-3 on the galectin-7 activity. Details of the experimental design are given in the legend to Figure 8. Cell culture in the assay period was performed with serum-supplemented medium as control (a), with this medium containing 125 mg galectin-7/ml (b) or containing this galectin in the presence of inhibitor of sugar-dependent cell binding (c; 150 mm lactose and 0.5 mg asialofetuin/ml) and with this medium containing the inhibitor mixture without galectin-7 (d). The effect of galectin-3 on the galectin-7-mediated effect was tested by adding galectin-3 to the galectin-7-containing test medium in a 1:1 ratio (e) or a 10-fold excess (f). For comparison, the positions of the arrow and arrowhead on the y-axis depict the extent of influence of galectin-1 (arrow) and galectin-3 (arrowhead) at the same concentration (125 mg/ml) determined in parallel. These data sets for galectin-1 and -3 fully corroborate previously published results for these two galectins (Kopitz et al., 2001). The results are shown as means7s.d. of eight independent measurements

Discussion Using the highly sensitive method of nano-ESI mass spectrometry, the elucidation of galectin-7’s aggregation status was performed. Our analysis flanked by positive controls revealed an indistinguishable behavior of galectin-7 relative to homodimeric galectin-1. This result can completely be reconciled with the similar potency of both galectins in haemagglutination of trypsinized and glutaraldehyde-fixed rabbit erythrocytes, a hallmark of di- and oligovalent carbohydrate-binding proteins (Andre´ et al., 2001; Ahmad et al., 2002). Multivalency effects noted in inhibition assays with branched Nglycans (Andre´ et al., 2001) are in line with the detected consequences of aggregation processes in the presence of an excess of glycodendrimers in nano-ESI mass spectrometry. The fact that signals of lectin–ligand complexes were recorded at equimolar glycoligand concentration served as further control that the experimental condiOncogene

tions were very gentle. Association of galectin-7 to a homodimer in solution in contrast to previous reports is also reasonable when looking at the area size of the interface with 1484 A˚2 in the crystal, compared to 1093 and 1179 A˚2 for the homodimers galectin-1 and -2, respectively (Leonidas et al., 1998). One concern that could be raised against this conclusion might point out that the dimer was obtained by an intersubunit disulfide bridge involving the single cysteine residue at position 38. Precedents for none nzymatic oligomer formation involving free sulfhydryl groups had been reported in the literature for galectins-1 and -3 (Hirabayashi and Kasai, 1991; Woo et al., 1991). This possibility of a chemical dimer formation could definitely be excluded, because reductive conditions were maintained throughout purification. For use in cell biological assays, we had also treated the lectin with iodoacetamide during elution to stabilize its carbohydrate-binding activity, as first successfully carried out for this purpose with galectin-1 (Powell and Whitney, 1984). In that case, the carboxamidomethylation prevented any formation of internal disulfide bonds. Their generation had been related to the appearance of carbohydrate-independent transforming growth factor activity on BALB3T3 A31 cells and the function of oxidized galectin-1 as a regulatory repair factor in axonal regeneration (Yamaoka et al., 1993; Horie and Kadoya, 2000). Additionally, sulfhydryl modification in galectin-7 made any conversion to sulfenate or sulfinate impossible, seen in crystals of bovine galectin-1 and discussed to have potential impact for lectin activity (Liao et al., 1994; Varela et al., 1999). This property of galectin-7 was therefore deliberately protected, a key prerequisite for our experiments. In fact, the lectin property of the related galectin-1, acting as extracellular effector (Outenreath and Jones, 1992), was also relevant for inducing neurite outgrowth in vitro (Puche et al., 1996). Identification of the binding partner(s) in this context could bear medical relevance. The observation that upregulation of ganglioside GM1 levels by a cell surface ganglioside sialidase in hippocampal cells in culture apparently caused axon growth and regeneration (Rodriguez et al., 2001) added to the importance to analyse the binding properties of the galectins to the lysoganglioside GM1 conjugated to a histochemically inert carrier. Galectin-7 is known to harbor affinity to lactose (Lac) and N-acetyllactosamine (LacNAc), albeit to a lower degree than galectins-1 and -3, with whom it shares tolerance for a2,3-sialylation. In contrast to galectin-1 homing in on nonreducing terminal Lac/LacNAc units, galectin-7 (and also galectin-3) recognize both terminal and internal ligand sites (Ahmad et al., 2002). In the case of the GM1 pentasaccharide, the tested galectins bound to the matrix-immobilized ligand in a very similar manner. The results of the cell binding studies of sugar inhibitor (mixture of galectin-reactive glycoprotein and lactose), in the presence of the GM1-specific cholera toxin B subunit and of galectins-1 or -3 led to the conclusion that the carbohydrate chain of ganglioside GM1 is a major ligand for galectin-7 on the neuro-


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blastoma cell surface. Evidently, the three galectins compete for the same major docking site on the cell surface in this cell model. When using both galectins-1 and -3 as inhibitors of galectin-7 binding, the obtained results furthermore intimated to conclude that a differential second-step protein–protein interaction, inferred for cholera toxin B subunit by mutational analysis (Aman et al., 2001; Rodigherio et al., 2001), is not likely. That ligand selection by galectin-1 is, in principle, not confined to carbohydrate structures, is well-documented. As mentioned in the introduction, galectin-1 can be engaged intracellularly in recognition of distinct target protein epitopes (Park et al., 2001; Paz et al., 2001; Elad-Sfadia et al., 2002; Liu et al., 2002). On the surface of MS5.1 murine stromal cells galectin-1 appeared to participate in protein–carbohydrate and protein–protein interactions with Nalm6 pre-B cells, the latter involving the pre-B-cell receptor (Gauthier et al., 2002). These results and considerations left only two of the questions given at the end of the introduction open, that is, the question on a cellular response to galectin-7 binding to the cell surface and, if positive, the demonstration of its carbohydrate dependence. Previously, we had shown functional divergence between galectins-1 and -3 in this cell system (Kopitz et al., 2001). Current literature evidence has taught the lesson that such a divergence is also encountered between tandemrepeat-type galectins and members of the other two classes of this lectin family. Noting the differential activity profiles in nonsmall cell lung carcinoma cell (1299) adhesion of galectin-8 relative to galectins-1 and 3 or the lack of eosinophil chemoattractant activity of these two galectins relative to ecalectin/galectin-9 (Hadari et al., 2000; Matsushita et al., 2000), it becomes clear that bivalency of galectins will not necessarily afford identical functions. Our work revealed that galectin-7 is a negative growth regulator similar to galectin-1 in this cell system. The inherent pathway to net growth reduction did not present characteristics of classical apoptosis. In this respect, the galectin-mediated effect shared properties with the H-Ras-dependent nonapoptotic cell death without the involvement of the caspase cascade, which has been supposed to play a key role in spontaneous regression of neuroblastoma (Kitanaka et al., 2002). With these results, we complete answering the questions listed at the end of the introduction. For the first time, blocking of this carbohydrate-dependent activity of galectin-7 by galectin-3 was also reported. By virtue of its lectin activity, galectin-7 is thus an extracellular growth regulator, in addition to its intracellular functions. Consequently, external supply of galectin-7 or its secretion will be essential for this aspect of its functionality. So far, only few cell systems have been studied regarding this galectin. In culture, keratinocytes meet this requirement by secretion, documenting that biosynthesis of this galectin – as with other family members (Hirabayashi, 1997; Hughes, 1999; Gabius, 2001a, b) – can be linked to externalization despite the lack of a signal peptide (Madsen et al.,

1995). Ectopic gene expression in transiently transfected COS-1 and in HeLa cells yet failed to direct galectin-7 to the cell surface or the medium (Madsen et al., 1995; Kuwabara et al., 2002). These observations, corroborated by our own experience with galectin-1-overexpressing 293 cells (unpublished observations), give reason to cast doubt on the validity of a claim to be able to readily exploit the herein described function by transfection. In order to facilitate the necessary export as a step to cell surface binding vector, engineering including in-frame addition of a classical signal sequence could be a way to address this problem. Apart from this technical procedure, delineation of mechanisms of transfer of a galectin from cytoplasmic sites to the cell surface upon changes in differentiation status or cell density, seen for example in the case of galectin-1 in leukemia or these neuroblastoma cells (Lutomski et al., 1997; Kopitz et al., 2001), will also be helpful. In a general context, regarding the galectin network with implications on growth control, our results on galectin-7 in this system underscore the importance to extend respective studies beyond the commonly investigated galectins-1 and -3. The occurrence of galectin-4 in a gene cluster, which was found to be upregulated in association with peritoneal dissemination in gastric cancer lines and the suppressor-like galectin-8 expression profile in the course of malignancy development in colon cancer support this conclusion (Hippo et al., 2001; Nagy et al., 2002). As is the case with any activities of different galectins, simple extrapolation of galectin functionality from one cell type to other tumor classes also appears to be of questionable merit. Work on glioblastoma cells revealed a response to galectin-1 presence totally different from that of this neuroblastoma system. Galectin-1 stimulated tissue invasion, was preferentially expressed in invasive tumor parts in xenografts, and was clinically associated with shortterm survival of patients (Camby et al., 2001, 2002; Rorive et al., 2001). The search for proliferation and migration markers by serial analysis of gene expression in rat C6 gliosarcoma malignancy vs a primary astrocyte library provided corroborating evidence. Galectin-1 mRNA expression belonged to the set of upregulated transcripts in the tumor cells (Gunnersen et al., 2000). Fittingly, rat 9L glioma cells were subject to growth reduction by transfection with an antisense vector for galectin-1 (Yamaoka et al., 2000). In comparison to neuroblastoma cells, the biochemical nature of the ligand(s) in glioma cells and the impact of other galectins are to be clarified. In this sense, our study on neuroblastoma cells establishes a role model for the line of work to embark on. In neuroblastomas, N-myc, Id2 and survivin downregulation and abrogation of caspase-8 silencing have recently been suggested as potentially promising targets for therapeutic growth control (Borriello et al., 2002). Combined with the intracellular activities of a galectin, the growing knowledge about the modes of action of this effector family as a lectin can be instrumental, too, to devise strategies to rationally shift the balance from proliferation to differentiation/growth arrest. The way Oncogene


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in which regulation of ganglioside GM1 presentation is correlated to axon regeneration (Rodriguez et al., 2001) even points to a potential perspective of eventually utilizing a recombinant lectin such as galectin-7 in areas beyond cancer biology.

acetate was used. Mass spectra monitored by scanning the standard mass range of 700–6000 Th show the presence of differently charged monomer [M þ xH]x þ and dimer [2xM þ xH]x þ ions, referred to as Mx and Dx in the figures. To analyse ligand binding the same buffer system was used at a galectin-7 concentration of 10 mm. An equimolar concentration of the dendrimer with two or four lactose units was applied to monitor carbohydrate-dependent binding.

Materials and methods Reagents

Solid-phase assays

Galectin-1 was isolated from human lung, and murine galectin-3, the N-terminal section of rat galectin-4 and human galectin-7 were purified after recombinant expression (in detail, we used the plasmids prCBP35 s, kindly provided by JL Wang (East Lansing, MI, USA) (Agrwal et al., 1993), and Escherichia coli JA221 cells, pGEX-2 T containing a GST gene and the cDNA for the N-terminal part of rat tandem-repeat galectin-4 and E. coli BL21(DE3)pLysS cells (Wasano and Hirakawa, 1999), and pQE-60/hGal-7 (containing a Ser2Ala substitution to establish an NcoI cleavage site (Andre´ et al., 2001; Kuwabara et al., 2002)) and E. coli M15[ pREP14] cells, which were purchased from Qiagen (Hilden, Germany)) to electrophoretic homogeneity using affinity chromatography on lactosylated Sepharose 4B derived from ligand coupling to divinyl-sulfone-activated resin as a crucial step (Gabius, 1990; Andre´ et al., 1999, 2000). Homogeneity in one- and twodimensional gel electrophoretic analysis was ascertained as described (Kohnke-Godt and Gabius, 1989). The neoganglioprotein containing an average of two to three ganglioside GM1-derived oligosaccharide chains was prepared by covalently linking the sphingosine N-alkyl(sulfosuccinimidyl)ester derivative of lysoganglioside GM1, which had been activated by treatment with bis(sulfosuccinimidyl)suberate to carbohydrate-free bovine serum albumin as described (Gabius et al., 1990). Wedge-like glycodendrimers with 3,5-di(2-aminoethoxy)benzoic acid as a branching unit and two or four lactose head groups as galectin-reactive ligand were prepared and analysed for purity by NMR spectroscopy and mass spectrometry as described (Andre´ et al., 2001; Vrasidas et al., 2001). Cholera toxin B subunit was purchased from Sigma (Munich, Germany).

Carbohydrate-dependent binding of biotinylated galectins to surface-immobilized neoganglioprotein with the oligosaccharide chain of ganglioside GM1 as ligand was quantitated in a solid-phase assay in microtiter plates following an optimized protocol for this sugar receptor class (Andre´ et al., 1997, 2001). Briefly, coating of the neoganglioprotein was performed overnight at 41C with a solution containing 20 mg neoganglioprotein per ml of phosphate-buffered saline (20 mm, pH 7.2), and probing the concentration dependence of lectin binding up to saturation was performed for 1 h at 371C with galectin concentrations of 10–100 mg/ml. Subtraction of the extent of noninhibitable binding in the presence of galectin-blocking reagents (a mixture of 75 mm lactose and 0.5 mg/ml asialofetuin) from the total binding yielded the carbohydratedependent binding for the algebraic transformation of the data (duplicates for each concentration; at least three independent series) to calculate the dissociation constant and the number of bound probe molecules per well at saturation.

Mass spectrometric analysis Sample analysis was performed on an LC-T nanoelectrospray ionization orthogonal time-of-flight mass spectrometer (Micromass, Manchester, UK) operating in the positive ion mode. For spraying, needle voltage of 1450–1650 V, cone voltage of 45–100 V and source temperature of 851C were set. Borosilicate glass capillaries (Kwik-FilTM, World Precision Instruments Inc., Sarasota, FL, USA) were used on a P-97 puller (Sutter Instruments Co., Novato, CA, USA) to prepare the nanoelectrospray needles. They were subsequently coated with a thin gold layer (approx. 500 A˚) using a sputter coater (Edwards High Vacuum International, Crawley, UK). Typically, 10 pmol of galectin sample were introduced in 1 ml solution. For molecular mass determination of human recombinant galectin-7, the purified and lyophilized protein was dissolved in a (50:49:1) ammonium acetate:acetonitrile:formic acid solution and analysed. In order to monitor the quaternary structure of the galectins-1, -3, and -7, the conditions were changed to reduce a negative influence on the assembly of subunits, as previously described measuring dimers and tetramers of a plant lectin and their binding to carbohydrate ligands (Van Dongen and Heck, 2000). In detail, an aqueous solution buffered at pH 6.8 with 50 mm ammonium Oncogene

Cell binding and growth assays Human neuroblastoma cells (strain SK-N-MC) were routinely cultured in Eagle’s minimal essential medium containing 10% fetal calf serum (PAA Laboratories, Co¨lbe, Germany) and antibiotics. Cell binding studies with radioiodinated galectins, prepared with carrier-free Na125I (Amersham Biosciences Europe, Freiburg, Germany) and Iodo beads (Pierce, Bonn, Germany) in the presence of 100 mm lactose to protect the galectins’ carbohydrate-binding sites and reaching final specific radioactivites of 111 kBq/mg for galectin-1, 132 kBq/mg for galectin-3 and 144 kBq/mg for galectin-7, were performed in serum-free Eagle’s minimal essential medium supplemented with 25 mm HEPES (pH 7.4) and 0.01% (w/v) carbohydratefree bovine serum albumin as described (Kopitz et al., 1998). The cells seeded in 96-well tissue culture plates had been grown to confluency for 5 days (approximately 105 cells/well) and were further incubated for 16 h in serum-free medium. To determine the carbohydrate-dependent level of galectin binding the inhibitory mixture of 150 mm lactose and 0.5 mg asialofetuin/ml was used, and algebraic transformation of the binding data led to calculation of the characteristics of cell binding as described for the solid-phase assay above and also for monitioring cell binding previously (Kopitz et al., 1998). Inhibition studies of galectin-7 binding with galectins-1 and -3 were performed with unlabeled galectins at increasing concentrations in a heterologous system (Kopitz et al., 2001) and with cholera toxin B subunit using 25 mg per well added 1 h prior to radioiodinated galectin-7 as described (Kopitz et al., 1998). The effect of galectins on cell growth was determined under routine culture conditions 16 h after seeding in the presence of serum for an experimental period of 48 h using a commercial kit to quantitate cell proliferation (CellTiter 96; Promega, Mannheim, Germany) including tests with caspase inhibitors as described (Kopitz et al., 2001). Monitoring for apoptotic cells in the cell populations was performed with a commercial in situ cell death detection kit and by the


6287 fluorescence-activated cell sorting analysis of stained DNA as described (Kopitz et al., 2001). Acknowledgements We gratefully acknowledge Dr JL Wang for kindly providing the expression vector for murine galectin-3, Dr BB

Namirha for helpful advice and L Mantel for excellent technical assistance. Furthermore, we are indebted to the EURO-conference program and the DAAD/NSF cooperation program for travel grants, and to the Wilhelm Sander-Stiftung (Munich, Germany) for generous financial support.

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