Determination of structural and functional overlap/divergence of five proto-typegalectins by analysis of the growth-regulatory interaction with ganglioside GM1 insilico and in vitro on human neuroblastoma cellsSabine Andre1, Herbert Kaltner1, Martin Lensch1, Roland Russwurm1, Hans-Christian Siebert1, Christine Fallsehr2,Emad Tajkhorshid3, Albert J.R. Heck4, Magnus von Knebel Doeberitz2, Hans-Joachim Gabius1 and Juergen Kopitz2*1Institut fur Physiologische Chemie, Tierarztliche Fakultat, Ludwig-Maximilians-Universitat, Munchen, Germany2Institut fur Molekulare Pathologie, Klinikum der Ruprecht-Karls-Universitat, Heidelberg, Germany3Theoretical and Computational Biophysics Group, Beckman Institute, University of Illinois at Urbana-Champaign,Urbana, IL, USA4Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute forPharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands

The growth-regulatory interplay between ganglioside GM1 onhuman SK-N-MC neuroblastoma cells and an endogenous lectinprovides a telling example for glycan (polysaccharide) functional-ity. Galectin-1 is the essential link between the sugar signal and theintracellular response. The emerging intrafamily complexity ofgalectins raises the question on defining extent of their structuraland functional overlap/divergence. We address this problem forproto-type galectins in this system: ganglioside GM1 as ligand,neuroblastoma cells as target. Using the way human galectin-1interacts with this complex natural ligand as template, we firstdefined equivalent positioning for distinct substitutions in theother tested proto-type galectins, e.g., Lys63 vs. Leu60/Gln72 ingalectins-2 and -5. As predicted from our in silico work, the testedproto-type galectins have affinity for the pentasaccharide of gan-glioside GM1. In contrast to solid-phase assays, cell surface pre-sentation of the ganglioside did not support binding of galectin-5,revealing the first level of regulation. Next, a monomeric proto-type galectin (CG-14) can impair galectin-1-dependent negativegrowth control by competitively blocking access to the sharedligand without acting as effector. Thus, the quaternary structureof proto-type galectins is an efficient means to give rise to func-tional divergence. The identification of this second level of regu-lation is relevant for diagnostic monitoring. It might be exploitedtherapeutically by producing galectin variants tailored to interferewith galectin activities associated with the malignant phenotype.Moreover, the given strategy for comparative computational anal-ysis of extended binding sites has implications for the rationaldesign of galectin-type-specific ligands.© 2004 Wiley-Liss, Inc.

Key words: apoptosis; galectin; ganglioside; lectin; neoglycoprotein;neuroblastoma

Our study focuses on an emerging class of endogenous growthregulators in a clinically relevant tumor model. For the orientationof the reader, we start with a brief primer of the concept. The cellsurface is the obvious site for presentation of sensors for the cells’communication with the environment. Spatial accessibility, bio-chemical hardware to enable high-density coding and the transla-tion of specific binding processes into signaling are essentialmeans toward efficient information transfer. All three prerequisitesare readily fulfilled by carbohydrate epitopes of cellular glycans(polysaccharides). In fact, their theoretical capacity for codingsurpasses that of oligonucleotides and oligopeptides by orders ofmagnitude, and a complex enzymatic machinery of glycosyltrans-ferases accounts for realization of an enormous structural diversi-ty.1–8 Spatially, the �-galactosides at antennae/branch ends ofglycan chains are especially well separated from the membrane.They can in principle be easily engaged in biomolecular recogni-tion. In this sense, the phenomenological mapping of disease-associated alterations in the glycomic profile is rather likely toacquire a functional dimension.3,9–12 As a general theme directingour studies, we thus aim to provide evidence for the concept to linkdistinct characteristics of tumor cell glycosylation with aspects ofthe malignant phenotype.

When interpreting oligosaccharides of glycan chains as codewords, their message is expected to be biochemically decoded andthen translated into cellular responses such as modulation of ad-hesion/migration or proliferation.13,14 Laboratory applications ofplant lectins extensively document the proof-of-principle versatil-ity of proteins with distinct carbohydrate specificity in this re-spect.14–16 The detection of endogenous lectins and the fact thattheir expression matches that of enzymes involved in glycanassembly and remodeling in complexity strongly argue in favor ofan elaborate in vivo system of protein(lectin)-glycan interac-tions.17–20 In full accord with the assumed active role of diverse�-galactosides in functional glycomics, one particular family ofendogenous lectins has evolved with specificity to this molecularcategory of targets, and this family is termed galectins.17,21 In-triguingly, model studies with oligosaccharides representingbranch-end epitopes of cell surface glycoconjugates and N-glycansharboring natural substitutions have already validated the pre-dicted impact of structural and conformational features of thesugar ligand on affinity to galectins.22–24 Fitting the elaboratemechanisms to modify carbohydrate properties as ligands, twomain factors on the side of galectins render effective fine-tuningand regulation likely: (i) the galectins’ diversification in up to 14different family members in mammals with subdivision into threegroups (proto-, chimera-, and tandem-repeat types) and (ii) theobservations from RT-PCR and immunohistochemical analyses(galectin fingerprinting) that a tumor (or nonmalignant) cell canoften express more than one galectin type.25–27 These recent in-sights raise the pertinent question of defining the extent of struc-tural and functional overlap/divergence among galectins. Thisissue characterizes the first main aim of our study.

Our previous work has defined a suitable tumor cell system, i.e.,human SK-N-MC neuroblastoma cells, for analysis to contribute tothe resolution of this problem. Due to the fact that neuroblastomais a frequent extracranial solid tumor type in childhood, accountingfor about 15% of pediatric cancer deaths, our project on endoge-nous growth regulators could spawn a clinical perspective. Indetail, we have first shown that a distinct change in the glycomicprofile, i.e., shift in the ganglioside population from higher sialy-lated forms to ganglioside GM1 due to upregulation of a cellsurface ganglioside sialidase (neuraminidase), is the crucial controlelement to switch cell behavior from proliferation to differentia-tion.28–30 Next, we pinpointed galectin-1 as major receptor for the

Grant sponsor: Mizutani Foundation for Glycoscience (Tokyo, Japan).*Correspondence to: Institut fur Molekulare Pathologie, Klinikum der

Ruprecht-Karls-Universitat, Im Neuenheimer Feld 220, 69120 Heidelberg,Germany. Fax: �49 6221 565981;E-mail: juergen.kopitz@med.uni-heidelberg.de

Received 2 June 2004; Accepted after revision 24 August 2004DOI 10.1002/ijc.20699Published online 2 November 2004 in Wiley InterScience (www.

interscience.wiley.com).

Int. J. Cancer: 114, 46–57 (2005)© 2004 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

ganglioside’s pentasaccharide chain.31 Sizable contributions of itsGalNAc/Neu5Ac moieties to the enthalpic gain of binding canexplain the notable selectivity of galectin-1 to pick this oligosac-charide from the wide panel of cell surface �-galactosides.32 Weproposed galectin-1 to be a regulator of neuroblastoma growth.Verifying our hypothesis, carbohydrate-dependent binding to gan-glioside GM1 by this homodimeric proto-type lectin impairedproliferation.33 Thus, we delineated a clear functional correlationbetween appearance of a distinct aspect of tumor cell glycosylationand its growth-regulatory functionality via an endogenous lectin.Interestingly, the p53-induced protein-1, another member of thegalectin family, referred to as galectin-7, proved to be a functionalhomolog in this aspect.34 In contrast, galectin-3, which shares thetarget specificity to ganglioside GM1, failed to affect cell growthand in consequence competitively inhibited the activity of galec-tins-1 and -7.33,34 This galectin is the only chimera-type galectin.It harbors a collagenase-sensitive stalk and a short N-terminalstretch with a phosphorylation site in addition to the carbohydraterecognition domain (CRD). In solution, it is predominantly mo-nomeric but can oligomerize to pentamers to a small extent withincreasing concentration.35–38 The cell biological result empha-sizes the importance of (i) ligand specificity, which in this caseengenders direct competition for the same glycan epitope, and (ii)topological mode of CRD presentation for eliciting growth regu-lation.

In order to systematically answer the question given above withfocus on proto-type proteins in a clinically relevant tumor system,we proceeded from the established basis in a stepwise manner.What follows in this paragraph is the rationale for our experimentroute: we started by selecting galectins-2 and -5 and, notably, thetwo chicken galectins CG-14 and �16 which are monomeric(CG-14) or dimeric (CG-16), as the test panel. We then examinedin silico how sequence variations among these galectins and alsogalectins-3, -4 and -7 affect ligand accommodation in this system.These computational data provide the first comparative mapping ofthe extended binding sites in complex with a physiological ligand.To set them in relation to experimental data on ligand affinity andgrowth regulation, we established recombinant production of theset of galectins and next performed binding studies in two settings:(i) a solid-phase assay using carrier-immobilized lysogangliosideGM1 and (ii) a cell-binding assay. The two assays naturally differin the topological aspect of the ganglioside’s presentation. Ligandselection on the cell surface was further probed by a gangliosideGM1-specific probe. Last but not least, functional assays gave theanswer on the extent of functional overlap/divergence. They wereflanked by competition assays focusing on cell growth. The majorlesson from our in silico and in vitro study is the detection offunctional divergence among proto-type galectins, a result relevantfor rationally optimizing or interfering with clinically relevantfunctions of galectins.

Material and methodsProcessing of sequence data

Amino acid sequences of human galectins-1 (accession no.P09382), -2 (P05162), -3 (P17931) and -7 (P47929), rat galec-tins-2 (Q9Z144), -4 (P38552) and -5 (P47967), mouse galectin-3(P16110), as well as chicken galectins CG-14 (P07583) andCG-16 (P23668) were available from the Swiss-Prot database(http://www.expasy.org/sprot) and edited using the default texteditor of Microsoft Windows (Redmond, WA, USA). Alignmentof the sequences was established using the program Multalin(http://prodes.toulouse.inra.fr/multalin/multalin.html; version 5.4.1).The ClustalW algorithm, available on the website of the EuropeanBioinformatics Institute (http://ebi.ac.uk/clustalw), was employedto calculate a tree cladogram illustrating putative phylogenic rela-tionships among exon sequences encoding the homologous regionof the CRD.

Molecular modeling of galectin-ligand complexesThe coordinates of the topological relationship between human

galectin-1 and the bound-state low-energy conformer of the pen-tasaccharide of ganglioside GM1 had been determined previous-ly.32 This structure was used as template for modeling the inter-action of the homologous CRDs with the ligand’s low-energyconformer, which is preferentially present in solution and selectedby galectin-1. CRD topologies were calculated by extensive ho-mology modeling. These structures were superimposed over thetemplate together with the carbohydrate ligand in its low-energyconformation, using especially the strictly conserved Trp residueand the neighboring Gly moiety as common point of reference, invisual molecular dynamics (VMD) with customized tcl scripts asdescribed.39 For internal control of the validity of the results of thehomology modeling procedure, we compared the datasets obtainedfrom our computations with those of available crystal structuresfor human galectin-2 (1HLC), galectin-3 (1A3K), galectin-7(1BKZ) and CG-16 (1QMJ). This comparison served to validatethe relevance of the results of calculations for cases in which nocrystal structure is listed in the databank. A color code is intro-duced to the illustration of the modeling results to allow the readerto spot substitutions in equivalent positions readily.

Cloning and recombinant production of the galectinscDNAs for human and rat galectin-2 and for rat galectin-5 were

cloned from total RNA of the human colon carcinoma line HT-29,rat duodenum and rat kidney, respectively, with primer sets de-signed on the basis of published cDNA sequences.40–42 BecauseCG-14 and CG-16 were both present in embryonic kidney,43

cDNAs for CG-14 and CG-16 were cloned from total kidney RNAeither with suitable primer sets by RT-PCR in the case of CG-1444

or by consecutive primer-directed RT-PCR and then 3�-RACE-PCR to complete the terminal sequence section for CG-16 (Swiss-Prot accession no. AY553270). Recombinant expression with thesystem combinations of pQE-60 (Qiagen, Hilden, Germany)/E.coli strain M15[pREP4] or pUC540 (KanR)/E. coli strain HB 101was performed in TB or 2YT media (Roth, Karlsruhe, Germany) at30°C or 37°C using final concentrations of isopropyl-�-D-thiogal-actoside of 25–500 �M. Galectins-1, -3 and -7 were produced asdescribed previously.33,34

Purification, labeling and activity controls of galectinsThe galectins were purified to homogeneity by affinity chroma-

tography on lactosylated Sepharose 4B, obtained by divinyl sul-fone activation, as a crucial step.45 Elution included stabilization ofthe lectin by iodoacetamide treatment to prevent loss of carbohy-drate-binding activity by oxidation.46 Purity controls were rou-tinely performed by 1- and 2-dimensional gel electrophoresis andgel filtration. Mass spectrometric analysis of the aggregation statusof rat galectin-5 was carried out with an LC-T nanoelectrosprayionization orthogonal time-of-flight mass spectrometer (Micro-mass, Manchester, UK) operating in the positive ion mode with 10pmol of protein sample in 1 �l using a solution of 50 mMammonium acetate at pH 6.8 to retain noncovalent interactions incontrast to a mixture of acetonitrile:water (1:1) with 0.1% formicacid to establish denaturing conditions.34 Analytical gel filtrationwith 100 �g aliquots was run on a prepacked Superose 12 HR10/30 column (24-ml bed volume) connected to a high perfor-mance liquid chromatography (HPLC) system (Hitachi-Merck,Darmstadt, Germany) with 50 mM PBS (pH 7.2) without/with 100mM lactose, its presence required to block galectin-matrix inter-actions and hereby preclude size-independent retardation, at a flowrate of 0.7 ml/min, as described.37 Hemagglutination assays withglutaraldehyde-fixed, trypsin-treated rabbit erythrocytes, biotiny-lation of galectins under conditions to maintain activity and as-sessment of extent of their labeling by a proteomics protocol wereperformed as described.47–49 Iodo beads (Pierce, Bonn, Germany)and carrier-free Na 125I (Amersham Biosciences, Freiburg, Ger-many) facilitated radioiodination of galectins in the presence of

47GALECTINS IN NEUROBLASTOMA GROWTH REGULATION

100 mM lactose to protect the carbohydrate-binding sites fromchemical modification.34

Solid-phase and cell-binding assaysPreparations of the sphingosine N-alkyl(sulfosuccinimidyl)ester

derivative of the lysoganglioside GM1 obtained from purifiedganglioside, its covalent coupling to carbohydrate-free BSA, ad-sorption of the resulting neoglycoprotein to the surface of micro-titer plate wells from solution (20 �g/ml in 20 mM PBS, pH 7.2)for 12 hr at 4°C and the solid-phase assays using biotinylatedgalectins in solution with parallel controls to determine the extentof carbohydrate-dependent binding using a mixture of 75 mMlactose and 0.5 mg asialofetuin/ml as glycoinhibitors followed thepreviously described protocols.34,50 For internal comparison ofgalectin activity toward N-glycans, the glycoproteins serum amy-loid P component with its single biantennary complex-type N-glycan at Asn32 per subunit in the pentamer and asialofetuin withits three predominantly (74%) triantennary N-glycans were alsoassayed as surface-immobilized ligands.48 Human neuroblastomacells (strain SK-N-MC) were routinely cultured in DMEM con-taining 10% FCS (PAA Laboratories, Colbe, Germany) and anti-biotics. Cells were seeded in 96-well plates and routinely grownfor 5 days to reach confluence with a density of about 105 cells/well in serum-supplemented medium. The medium was changed toserum-free DMEM with 25 mM HEPES (pH 7.4) and 0.01% BSA,cell culture continued for 16 hr and radioiodinated galectin wasadded in the absence of any inhibitor or in the presence of 25 �gcholera toxin B-subunit (Sigma, Munich, Germany)/ml, label-freegalectins or a mixture of 150 mM lactose and 0.5 mg asialofetuin/ml, as described.31,34 When the effects of galectin-1 binding onendocytosis were tested, the cultures were cooled to 12°C orpretreated (30 min) with 100 �M vinblastine prior to addingradiolabeled galectin-1.

Cell growth assaysCells were seeded at an initial density of 104 cells/well and

cultured for 16 hr to allow cell attachment. Then culture continued

for 48 hr in serum-supplemented medium containing galectins atthe standard concentration of 125 �g/ml, which has been deter-mined to be strongly inhibitory for cell growth in the cases ofgalectins-1 and -7, followed by quantitation of cell numbers byapplication of reagents of a commercial kit (CellTiter 96; Promega,Mannheim, Germany). Controls to ascertain inhibition of activityby presence of glycoinhibitors and assays to assess potency ofother galectins to block galectin-1 activity using a mixture, e.g.,galectin-1 and CG-14, followed routine procedures.33,34

Results and discussionSequence comparison as a measure to infer evolutionaryrelationship

Galectins share reactivity to �-galactosides and the jelly-roll-like folding pattern.17 Occurrence of a set of invariable aminoacids at sites crucial for ligand contact underlies the commonselectivity. The way the standard sequence signature can varywithin the group of proto-type galectins and relative to a tandem-repeat-type and the chimera-type galectin is illustrated by a de-tailed sequence alignment (Fig. 1). The basic requirement forpresence of the indolyl moiety (W68 in human galectin-1, placedat position 87 in the alignment of Fig. 1) engaged in stacking andC-H/�-interactions with the B-face of galactose is readily apparentby its strict conservation. It also becomes clear that considerablesequence diversification has taken place, an argument for func-tional divergence.

Further processing of these data was performed to infer thedegree of relationship based on sequence similarity. The obtainedresult indicates that galectins-1 and -7, which were both proven tobe growth regulators for neuroblastoma cells,33,34 are rather widelyseparated in the cladogram (Fig. 2). Remarkably, analyzing thesame galectin type from two mammalian species delineates effectsless pronounced than comparing different galectins in a species,and the chicken galectins have notable similarity to human galec-tin-1. Looking at these rather equal characteristics, functional

FIGURE 1 – Sequence comparison of mammalian and avian galectins. Complete amino acid sequences of proto-type human galectins-1, -2 and-7 (hGal-1, -2 and -7), rat galectins-2 and -5 (rGal-2 and -5) and chicken galectins CG-14 and -16, the homologous C-terminal part ofchimera-type human and mouse galectin-3 (hGal-3, mGal-3) as well as the N-terminal carbohydrate recognition domain of tandem-repeat-typerat galectin-4 (rGal-4N) were aligned using the program Multalin (http://prodes.toulouse.inra.fr/multalin/multalin.html; version 5.4.1). Identicalresidues invariably found in all sequences are indicated as white letters on black background, whereas residues that are identical or similarbetween at least five of the sequences are in black letters on gray background. A consensus (Cons) sequence calculated from the ten galectinsequences is added to the alignment; consensus symbols represent: !, I or V; $, L or M; %, F or Y; #, N, D, Q or E.

48 ANDRE ET AL.

divergence might then be solely based on differences in the qua-ternary structure. However, a serious caveat is to be raised: thegiven reasoning might lead to an error of judgement, becausesequence alignments are generally not weighted with respect to thestructural role of variable positions. Of course, it can be taken forgranted that the binding of the core part of a �-galactoside calls fora constant set of interactions, as chemically defined by thoroughmapping with deoxy- and fluoro- derivatives of lactose.51 But howa galectin accomplishes reaching its documented specificity to-ward particular complex �-galactoside-containing ligands on thecell surface or in the extracellular matrix, such as laminin, carci-noembryonic antigen, certain integrins or, as outlined above, gan-glioside GM1 in this cell model, is an open question.31,52 Mostlikely, an extension of the binding site and the recruitment offurther contact points on the complex glycan of the main cellulartarget beyond the disaccharide core should be operative.

The ensuing concept to explain the remarkable selectivity ofendogenous lectins to decode sugar-based information had re-cently been verified for P- and E-selectins. L-Fucose is the primarysite, with galactose, sialic acid (neuraminic acid) and suitablypositioned tyrosine sulfates serving as discriminatory auxiliarycontacts.53 P-Type lectins, too, engage more than the central sugarunit (the mannose-6-phosphate residue in this case) in the bindingprocess, the binding site encompassing a total of three mannosemoieties.54 Notably, the first study on a galectin dissecting itsinteraction with a complex ligand in structural terms in solution bynuclear magnetic resonance (NMR) spectroscopy and molecularmodeling was equally instrumental to highlight the importance ofamino acids in the vicinity of the primary contact site.32 Like thecore recognition structure which is bound in its low-energy syn-conformation,55,56 human galectin-1 selects a low-energy confor-mation of the complete pentasaccharide to bring the GalNAc/Neu5Ac residues in ganglioside GM1 in enthalpically favorablecontact with Arg48, His52, Lys63 and Glu71.32 Examining thesepositions in Figure 1 confronts us with sequence deviations. Thisresult teaches the lesson that the presented sequence alignment willnot be sufficient to answer questions on fine-specificity, when onesubstitution can matter. Owing to the availability of a detailed viewon how human galectin-1 accommodates the key-like low-energyconformation fitting of ganglioside GM1 like a lock, we were nowable to sort out common or disparate denominators in the galectins.To do this, we scrutinized the binding sites for this ligand by asophisticated computational approach, using the experimentallybased data on galectin-1 as template. In a broader context, testingthis procedure thoroughly will have merit to predict fine-specificityfeatures in other cases as well. Hereby, we are establishing a toolfor rational design of selective reagents.51

Comparative computational analysis of galectin-gangliosideGM1 interaction

Before we started the detailed monitoring, we performed ho-mology modeling of galectin structures. We set out from bovinegalectin-1 (1SLT) and compared the results obtained to coordi-nates of the crystal structures as far as available, i.e., for human

galectins-2, -3 and -7 and chicken liver galectin CG-16. Theremarkable ease of reconciling the datasets argues in favor of thevalidity of results from the computational process. Therefore, weproceeded to complete modeling also for those cases where nocrystal structure is known. The way the ganglioside’s low-energyconformation makes contacts to the individual binding sites willtranslate into predictions on ligand affinity. As shown in Figure 3,which uses a color code to allow rapid orientation for spottingstructurally equivalent positions, galectins-1 and -7 closely resem-ble each other, although they are widely separated in the cla-dogram (Fig. 2). A substitution from His52 to Thr56 (blue moietyin upper part) had no major bearing on total affinity in the bindingassays.34 The distance from His52 to Trp68 is 11.6 Å in humangalectin-1, whereas Thr56 is separated from Trp68 by 16.7 Å. Asa common denominator, the distance between Trp68 and Arg48varies only within the narrow range between 9.1 and 10.1 Å. Thisfeature, in combination with the depicted spatial display of theamino acids, will have to be kept in mind for the design ofgalectin-type-specific ligands (carbohydrates or glycomimetics)and of peptides substituting a galectin for therapeutic purpos-es.57–59

Substitutions with experimentally so far unknown impact in thebinding pocket concern Lys63, with Leu60 and Gln72 taking itsplace in human galectin-2 (and also the rat protein) and rat galec-tin-5, and His52, where these two lectins present Glu moieties.Looking at galectins-3 and -4 which bind GM1,34 Asn or Trp takethe place of His52 (not shown), without an impairment of affini-ty.34 Of special note, the two chicken galectins present no substi-tution relative to human galectin-1 in the spatial profile of relevantamino acids for the molecular rendezvous with the pentasaccha-ride. Consequently, their binding to the ganglioside is likely, anda difference in quaternary structure will make them attractive toolsto relate ability for cross-linking to biological activity. Whenpurified from the intestine or liver and subjected to gel filtration atphysiological ionic strength, CG-14 is monomeric; CG-16, incontrast, is dimeric and forms stoichiometric complexes with thetriantennary N-glycans of asialofetuin.60–62 To be independent ofnatural sources, we proceeded to facilitate recombinant productionof the galectins in the test panel. The availability of respectivevectors will also enable any mutational engineering to alter qua-ternary structure or to rationally optimize ligand properties in thefuture. Here, the purified proteins afforded to experimentally de-termine binding properties and relate the insights from computa-tional calculations to the proteins’ activities. In the first step, wedetermined the quaternary structures of the recombinant products,a process which also constitutes a purity control.

Solution structures of galectins in the test panelHemagglutination of glutaraldehyde-fixed and trypsin-treated

erythrocytes is a common assay to measure sugar-binding andcross-linking activities of lectins. When appropriate ligands arepresented on the cell surface, agglutination is an indicator foroligomer formation. Each reaction was completely abolished bylactose but not a nonspecific sugar, ascertaining carbohydrate

FIGURE 2 – Diagram of sequence-dependent relationship between galectins. The fact that one singular exon invariably codes for the core regionof the carbohydrate recognition domain of galectins intimated to focus the comparison of the galectins in the test panel on this sequence part.These translated sequences were used to calculate the galectins’ putative phylogenic relations, visualized in a tree cladogram, using the ClustalWalgorithm available on the website of the European Bioinformatics Institute (http://ebi.ac.uk/clustalw) with the given default parameter settings.

49GALECTINS IN NEUROBLASTOMA GROWTH REGULATION

FIGURE 3.

50 ANDRE ET AL.

dependence. The chicken galectins were widely separated in theiractivities by a factor of 60-fold for the minimal lectin quantity toyield a positive result, as already noted for these 2 lectins fromnatural sources.63 Human and rat galectins-2 were less reactivethan human galectin-1. Interestingly, their retention times and thatof human galectin-7 in gel filtration were also longer than forgalectin-1, albeit clearly not reaching the range for a monomer, sothat shape differences among dimeric proto-type galectins in so-lution become apparent. Controls in the presence of lactose ex-cluded retention by carbohydrate-dependent interaction to the ma-trix, as shown previously to be the case for galectin-3.37 Noevidence for dissociation or formation of higher aggregates couldbe obtained in gel filtration analysis, as was also previously seen ina study on human galectin-1 by small angle neutron scattering.64

Regarding rat galectin-5, we could confirm its weak but positivereaction as agglutinin and its behavior as monomer in gel filtration,which had been reported previously.41 To further analyze its ag-gregation status regardless of shape, we added the highly sensitivemethod of mass determination by nanoelectrospray ionizationmass spectrometry under conditions not harmful for noncovalentinteractions.34,65 In addition to recording spectra of samples ex-posed to acidic conditions, we applied a pseudophysiological mi-lieu as well, to look at the aggregation status. Previous experimen-tal series with galectins-1, -3 and -7 had ascertained the suitabilityof this approach to detect galectin dimerization.34

As illustrated in Figure 4, the galectin-5 preparation is pure.Analyzed in a denaturing (acidic) environment multiple ionizationoccurs (Fig. 4a), while this pattern is restricted under “native”conditions (Fig. 4b). The determined molecular masses of16,046.75 � 2.33 Da and 16,048.27 � 0.11 Da from the spectra inFigure 4a and b, respectively, are in accord with galectin-5 beingdevoid of the N-terminal methionine residue. In this form, thegalectin has a calculated mass of 16,048.23 Da. A minor peak at15,976.3 � 0.2 Da corresponds to a galectin-5 after the additionalloss of alanine from the N-terminus (calculated mass: 15,977.15Da). The spectrum under pseudophysiological conditions, whichmaintain dimerization of galectins-1 and -7 monitored previous-ly,34 raises no evidence for any aggregation of monomeric galec-tin-5 at a concentration of 10 �M (Fig. 4b). The concern thatsample processing will be harmful to an aggregate is furtherallayed by running an analysis in the presence of a carbohydrateligand. As similarly seen with galectin-7,34 occurrence of peaks oflectin-ligand complexes add to the evidence for stability of non-covalent complexes under these conditions (not shown). However,we cannot exclude the possibility that galectin-5, as galectin-3,36

might form an aggregate when exposed to a multivalent ligand ora cell surface. This process could explain the weak but significanthemagglutination activity. In solution, we thus deal with two (atleast preferentially) monomeric proto-type galectins in our testpanel, i.e., CG-14 and rat galectin-5. Although database mining forevidence for a human galectin-5 was negative (Lensch et al.,unpublished), rat galectin-5 together with CG-14 are valuable toolsfor the further experiments, i.e., binding and functional assays. To

exclude any variations of experimental conditions the followingbinding studies were performed in parallel for our test panel withthe same set of reagents or cell batches.

Solid-phase and cell binding assaysThe neoglycoprotein with the lysoganglioside as ligand part was

adsorbed to the surface of microtiter plate wells, hereby establish-ing a model for cell surface presentation. An advantage of thismodel is the uniform presence of only one class of ligand. But itshould also be mentioned that distinct features on the cell mem-brane such as local clustering can hereby not be mimicked. Asdocumented previously for galectins-1, -3, -7 and the N-terminaldomain of tandem-repeat-type galectin-4,34 binding of the biotin-ylated galectins was saturable and inhibitable by glycosubstances(0.5 mg asialofetuin/ml and 75 mM lactose). Examples of bindingcurves are shown in Figure 5. As further controls against theinfluence of carbohydrate-independent binding via protein/lipidparts of this neoglycoprotein, we performed parallel assays withthe glycoproteins’ serum amyloid P component and asialofetuinwith the same inhibition protocol. These series ascertained bindingactivity of the labeled lectins with one exception. CG-16 failed tobind to the lysoganglioside and reacted considerably weaker withthe glycoproteins than expected based on its cross-linking activitywith asialofetuin and the microcalorimetrically determined affini-ty.61,66 In this case, an impact of labeling (direct impairment orindirect effects, e.g., by further lowering its isoelectric point (pI)value, which already is the most acidic among the test panel)cannot be excluded. When the binding data were algebraicallytransformed, the obtained Scatchard plots were linear, as evidencefor presence of a single class of binding sites and absence ofcooperativity (Fig. 5). The calculated dissociation constants werein the range of those determined previously for galectins-1, -3, and-7 (and also galectin-4’s N-terminal domain with 1.41 � 0.34�M), CG-14 showing an increased affinity (Table I). Evidently,changes in the architecture of the extended binding sites, which areshown in Figure 3, will not cause dramatic changes in affinityunder these experimental conditions. These results should not yet

FIGURE 3 – Comparison of the architecture of the contact sites forthe pentasaccharide chain of ganglioside GM1. Calculations werebased on the experimentally and computationally derived structure ofthe complex between human galectin-1 and this pentasaccharide.32

The topological relationship between the bound-state pentasaccharideconformer and respective amino acids in the extended binding sites, asdefined previously in the case of human galectin-1,32 was assessedaccordingly by superimposition in VMD.39 Results of homology mod-eling were in complete agreement with available X-ray structures ofhuman galectins-1, -2 and -7 and of CG-16 (1GZW, 1HLC, 1BKZ,1QMJ). A color code based on the configuration in human galectin-1(hGal-1) is used to depict structurally equivalent substitutions in theother galectins, i.e., human galectin-7 (hGal-7), human galectin-2(hGal-2), rat galectin-5 (rGal-5) and the avian galectins CG-14 andCG-16. For complete sequence information and sites of substitutions,please see Figure 1.

FIGURE 4 – Determination of molecular mass and quaternary struc-ture of rat galectin-5. Nanoelectrospray ionization mass spectra of ratgalectin-5 were recorded under denaturing (a) and mild conditions, thelatter not being harmful for stability of noncovalent complexes (b).Denaturation leads to higher charge states (A9–A11), which were notdetectable during analysis of samples from processing in 50 mMNH4Ac (b).

51GALECTINS IN NEUROBLASTOMA GROWTH REGULATION

be automatically extrapolated to the physiological situationwith a lectin interacting with cell surface ligands. In fact, thefollowing caveats warrant to be acknowledged and should beaddressed: (i) presentation of the carbohydrate ligand in thismodel and on the cell surface can differ with implications foraffinity and (ii) any chemical modification can influence bind-ing properties so that further measurements, especially compe-tition and functional assays, are necessary. To address theseissues experimentally in a stepwise manner, we performed cellbinding studies.

Binding of the galectins was saturable and inhibitable by pres-ence of inhibitors of �-galactoside-dependent interaction (0.5 mgasialofetuin/ml and 150 mM lactose), as measured for galectins-1,-3 and -7 and as reported previously.33,34 The inherent control byusing the cholera toxin B-subunit as specific blocking reagentascertained that access to ganglioside GM1 is responsible for amajor contribution to cell surface binding of the tested galectins(Fig. 6). The calculation of the dissociation constants for cellsurface binding was based on binding curves, which are exemplar-ily shown in Figure 6. Scatchard plots yielded rather similar data,when compared to the previously obtained result set (Table I). Ininterspecies comparison, galectins-2 of rat and human origin be-haved indistinguishably. CG-16 proved active in this system andthe blocking effect of the cholera toxin B-subunit indicated inter-action with ganglioside GM1 (Fig. 6c). Also, the number of bind-ing sites at saturation was rather similar, implying comparableselection of binding sites. The only exception was galectin-5,

which failed to bind to the cell surface. To exclude that radio-iodination had impaired galectin-5’s binding activity towardganglioside GM1, which had been seen in the solid-phase assay,we performed an inhibition study with label-free galectin usingthe well-characterized binding of galectin-131,33,34 as standard.This assay was run for the complete panel beyond galectin-5 inorder to strengthen the evidence for common target specificityon the neuroblastoma cell surface. After all, analysis of bindingof sialylated �-galactosides with increasing chain length and of90K/MAC-2 BP had delineated distinct ligand preferences forgalectins-1 and -3.22,67 If a galectin—similar to the choleratoxin B-subunit—will home in on ganglioside GM1, it willreduce the extent of binding of galectin-1. The rather closerange of measured dissociation constant (KD)-values predictsefficient competition. Its extent was determined after strictcoincubation and also after coincubation preceded by a 1-hrpreincubation step with galectin-1.

Indeed, competition was effective for the galectins whichbound to the cell surface, as shown in Fig. 7a–c. When thegalectins used as competitors were included 1 hr later thangalectin-1, the binding of the latter was not completely abol-ished. In order to check the possibility that galectin-1 enhancesendocytosis, leaving less molecules of the ligand available, weconducted the same experiments at reduced temperature and inthe presence of vinblastine, an inhibitor of endocytosis. Sinceneither reduced temperature nor presence of the inhibitor af-fected residual galectin-1 binding after the addition of compet-

FIGURE 5 – Determination of theaffinity of galectins to the pen-tasaccharide chain of gangliosideGM1 in solid-phase assays. Resultsof quantitative assessment of car-bohydrate-inhibitable binding (ob-tained by subtracting the extent ofbinding not affected by presence ofglycoinhibitors from total extent ofbinding) of biotinylated human ga-lectin-2 (hGal-2) and chicken ga-lectin (CG-14) (insets) are pre-sented following the algebraictransformation of the Scatchardplot analysis. Representative plotsfrom a series of at least three indi-vidual measurements with dupli-cates in each assay are given.

TABLE I – DETERMINATION OF THE DISSOCIATION CONSTANT (KD) OF CARBOHYDRATE-DEPENDENT GALECTIN BINDING IN THE SOLID-PHASE ASSAYAND IN CELL ASSAYS TOGETHER WITH ASSESSMENT OF NUMBER OF BOUND PROBE MOLECULES AT SATURATION PER CELL (BMAX)

AND ACTIVITY AS GROWTH REGULATOR

Type of lectin Solid-phase assayKD (�M)

Cell assayActivity as

growth regulatorKD (�M) Bmax(106 molecules/cell)

Human galectin-1 1.62 � 0.551 0.76 � 0.181 2.23 � 0.291 ��3

Human galectin-2 1.09 � 0.88 0.63 � 0.22 2.07 � 0.41 ��Rat galectin-2 1.71 � 1.20 0.47 � 0.19 1.92 � 0.35 ��Murine galectin-3 2.85 � 0.641 0.94 � 0.251 2.70 � 0.372 –3

Rat galectin-5 1.45 � 1.10 n.d. n.d. –Human galectin-7 2.29 � 1.271 0.88 � 0.291 2.22 � 0.341 ��1

CG-14 0.19 � 0.09 0.51 � 0.27 2.22 � 0.39 –CG-16 n.d. 0.56 � 0.32 1.93 � 0.31 �

1From [34].–2From [31].–3From [33].–n.d., not detectable.

52 ANDRE ET AL.

itor, galectin-1 is unlikely to enhance endocytosis (data notshown). Galectin-5 failed to interfere with galectin-1 binding(Fig. 7d). Because galectin-5 was not chemically modified inthis assay, we can conclude that monomeric galectin-5—incontrast to monomeric CG-14 —shows no evidence for measur-able recognition of the ganglioside on the cell surface. Despitesimilar, albeit weak, activity in hemagglutination for both ga-lectins as measure for aggregation on a cell surface, the bindingcharacteristics in this system are markedly different. Of note,proto-type CG-14, with its close similarity to galectin-1, is asuitable tool to put the hypothesis to the test as to whether cellbinding without cross-linking is sufficient to elicit the biolog-ical response. Also, blocking of galectin-1 binding by CG-14could mean that presence of a monomeric proto-type galectin isa way to interfere with distinct aspects of functionality of

galectin-1. The monitoring of growth of cells exposed to ga-lectins will provide the respective answer.

Cell growth assays

In preliminary assays, we ascertained the reactivity of the cur-rently tested cell batches as control for constant responsivenessrelative to our previous studies. Having verified this essentialprerequisite, as documented exemplarily in Fig. 8a and b, we setforth to examine the growth-regulatory effects of cell binding forthe test panel. The following results were obtained: (i) galectin-2(human and rat) acted as functional homolog of human galectin-1,(ii) CG-16 was likewise an effector but to a reduced extent and (iii)the monomeric galectin-5 and CG-14 failed to affect growth (Figs.8 and 9; Table I). Because cell growth was unaffected by galectinsin the presence of glycoinhibitors to prevent carbohydrate-depen-

FIGURE 6 – Determination of theaffinity of galectins to surface li-gand(s) of neuroblastoma cells.Cells were cultured for five days toreach the final density of 105 cells/well in serum-supplemented me-dium and then for a further 16 hr inserum-free medium prior to a 2-hrincubation period with 125I-labeledgalectins, i.e., human galectin-2(a), CG-14 (b), CG-16 (c) and ratgalectin-5 (d). Parallel experimentswith glycoinhibitors determinedthe extent of carbohydrate-inhibit-able binding (inset) which wasused for calculations of Scatchardplot analysis. The low extent ofbinding of galectin-5 (d) precludedthis type of further analysis in thiscase. Binding studies were per-formed in the absence (●) and inthe presence (E) of cholera toxin asa measure of involvement of gan-glioside GM1 in cell surface bind-ing. Each given point representsthe means � standard deviation(SD) of at least three independentmeasurements with duplicates ineach series.

53GALECTINS IN NEUROBLASTOMA GROWTH REGULATION

dent cell binding (not shown), this process was essential for growthregulation. Presence of the glycoinhibitors did not affect cellgrowth (not shown). Apparently, cell binding and a dimeric struc-ture are necessary to lead to signaling for growth inhibition. Incontrast to functional differences among galectins-1 and -7 in invitro assays with blood and immune cells and in wound heal-ing68,69 galectins-1, -2, and -7 shared functionality in this cellmodel. To add further evidence to this conclusion, the results onCG-14 and galectin-5, shown in Figures 7–9, led us to the follow-ing experiment: to test both galectins as inhibitors of galectin-1’sactivity as negative growth regulator. The model we developedpredicts that CG-14 should interfere with galectin-1’s activity onthe level of ligand binding, hereby blocking galectin-1-dependentnegative growth regulation. In contrast, galectin-5 should be with-out functional effect. The microphotograph in Figure 8h and thedata in Figure 10 illustrate the validity of the concept. We haveadded a parallel series with CG-16 in Figure 10 to underscore itsactivity as effector in comparison to CG-14.

In summary, we thus detected divergence in the group of proto-type galectins on the structural and functional levels. The firstlesson emerging from this study is the requirement to monitorcomplex natural glycans with respect to their ligand properties. Forthis purpose, structurally well-characterized glycoproteins will besuited as well as glycolipids to unveil intrafamily regulation ofaffinity among galectins. These modifications can concern substi-

tutions in the chain (e.g., introduction of bisecting GlcNAc) or interminal �-galactosides (e.g., �1,2-fucosylation or �1,3-substitu-tions) as well as changes in the number of glycan antennae anddegree of clustering.23,24,70–73 Our studies follow the hypothesisthat distinct glycan modifications act as regulators of lectin activ-ity. Moreover, our current results underline the importance to takeresults from assays with a single target to the test by bindingstudies with cell surfaces. Whereas galectin-2, irrespective of itsorigin from rat or human tissue, and CG-16 are homologs in bothrespects, galectin-5 failed to bind ganglioside GM1 (and otherligand site(s)) on the cell surface. Once the major ligand(s) for acell-type-selective galectin effect have also been defined in othertumor cell systems, corresponding studies can follow the guidelineand strategy presented in this report. Hereby, it is then possible tofactor cell surface presentation of a glycan epitope into the assess-ment as ligand and to determine why distinct aspects of tumor cellglycosylation matter for establishing the malignant phenotype, ashas been shown in this system. With this knowledge acquired, theirexpression profiles can be manipulated on the level of the respon-sible glycosyltransferases or glycosidases, merging work withgalectins, as we are currently pursuing.

The second lesson enhances our understanding of the functionalityof the complex galectin network in tumor cells.74 Differences inquaternary structure, in this case the formation of monomeric anddimeric modules for cross-linking,75 translate into divergent effects on

FIGURE 7 – Competition studies of galectin-1-dependent cell binding using other proto-type galectins. The cells were seeded in 96-well platesat an initial density of 104 cells/well. After five days in culture, radioiodinated galectin-1 was added to the cells. The label-free galectin, i.e., eitherhuman galectin-2 (a), CG-14 (b), CG-16 (c) or rat galectin-5 (d), was added either simultaneously (●) or 1 hr after initial incubation with labeledhuman galectin-1 (E). The results are given as means � standard deviation (SD) of four independent measurements with duplicates in eachseries.

54 ANDRE ET AL.

the cellular level, i.e., triggering of growth regulation and its abroga-tion in this cell model. Thus, our data broaden the basis to refer togalectin-1 as a potent growth regulator in human neuroblastoma.Hereby, these data add dimeric galectins to the recently compiled

FIGURE 9 – Effect of proto-type galectins on cell proliferation. As-sessment of cell number in galectin-treated cultures after 48 hr forhuman and rat galectin-2, CG-16, CG-14, and rat galectin-5. Forcomparison, the arrow and open/filled arrowheads symbolize the ac-tivity of human galectins-1 (3), -3 (‹) and -7 (�) under identicalconditions. The results are given as means � standard deviation (SD)of 8 independent measurements with duplicates in each series.

FIGURE 10 – Effect of presence of proto-type galectins on galectin-1-dependent growth regulation. Standard assays for measuring growthregulation were run without any galectin as control and with humangalectin-1 to set the standard. Experiments using coincubation ofhuman galectin-1 with CG-14 and rat galectin-5, which are notgrowth-regulatory proteins, at a ratio of 1:1 or 1:10 (molar ratio ofabout 1:2 and 1:20) probed the influence of their presence on theactivity of human galectin-1. Presence of CG-16 was tested as furthercontrol for its activity (for competition studies on cell surface bindingof radiolabeled galectin-1 using these lectins, see Fig. 7b–d).

FIGURE 8 – Cell growth regulation by proto-type galectins. Photomicrographs of neuroblastoma cell preparations (magnification: 125)cultured in the absence (Control) or presence (b–h) of 125 �g lectins/ml, i.e., human galectin-1 as standard (hGal-1), human and rat galectin-2(hGal-2, rGal-2), dimeric CG-16, monomeric CG-14, monomeric rat galectin-5 (rGal-5) and human galectin-1 in the presence of a 10-fold excessof CG-14 (hGal-1 � CG-14), for an experimental period of 48 hr in serum-supplemented medium. For quantitative data, see Figure 9.

55GALECTINS IN NEUROBLASTOMA GROWTH REGULATION

panel of intracellular proteins relevant for cell cycle regulation andapoptosis in this tumor type, e.g., the Id2 protein and survivin.76 Theavailability of vector collection for galectins will be instrumental totaking the next step in our concept. Aside from ensuring a supply oftest material, it enables the design of variants, deliberately altering thefine-specificity and the quaternary structure. That efforts in this areaare promising has been recently demonstrated. Targeting the intracel-lular interaction of galectin-1 with oncogenic H-Ras, a custom-madegalectin-1 mutant (i.e., L11A) was designed, and it acted as a domi-nant negative effector for active H-Ras.77 This work and the perspec-tive raised by our report intimate that options for new treatmentmodalities might arise from rational exploitation of galectin function-ality.

Conclusions

Having defined cell surface �-galactosides as code words forcell communication and delineated the intrafamily diversity ofgalectins, the next issue is to take stock of the extent of structuraland functional overlap/divergence among galectins. Here, we fo-cused on proto-type galectins, including cross-species comparison.With the coordinates of the extended binding site of galectin-1 forganglioside GM1 and the potency of regulation of neuroblastoma

cell growth through the interaction at hand, we first defined distinctsubstitutions distinguishing combining sites for this glycan chain,e.g., between galectin-1 and galectins-2 and -5. This knowledge-based computational procedure will find further application todesign galectin-type-specific reagents and galectin-mimetic pep-tides. Regulation of galectin activity on cell growth was detectedon two levels, i.e., binding to the cell surface and triggering ofgrowth inhibition. Galectin-5, which showed affinity to the carrier-immobilized lysoganglioside in a solid-phase assay, failed to bindto ganglioside GM1 on the cell surface, and monomeric CG-14 didnot induce growth reduction. Because it blocked galectin-1’s ac-tivity competitively, the quaternary structure of proto-type galec-tins is a key factor for this type of functional divergence. Thisresult is proposed to have potential therapeutic relevance, i.e., incases where expression of a cross-linking galectin, for examplegalectin-1 in glioblastoma or colon cancer,74,78,79 is associatedwith the malignant phenotype.

Acknowledgements

We thank Drs. S. Namirha and S. Goldmann for helpful discus-sion and S. Himmelsbach, B. Hofer and L. Mantel for excellenttechnical assistance.

References

1. Brockhausen I, Schachter H. Glycosyltransferases involved in N- andO-glycan biosynthesis. In: Gabius HJ, Gabius S, eds. Glycosciences:status and perspectives. Weinheim-London: Chapman & Hall, 1997.79–113.

2. Laine RA. The information-storing potential of the sugar code. In:Gabius HJ, Gabius S, eds. Glycosciences: status and perspectives.Weinheim-London: Chapman & Hall, 1997. 1–14.

3. Reuter G, Gabius HJ. Eukaryotic glycosylation: whim of nature ormultipurpose tool? Cell Mol Life Sci 1999;55:368–422.

4. Harduin-Lepers A, Vallejo-Ruiz V, Krzewinski-Recchi MA, Samyn-Petit B, Julien S, Delannoy P. The human sialyltransferase family.Biochimie 2001;83:727–37.

5. Gabius HJ, Andre S, Kaltner H, Siebert HC. The sugar code: func-tional lectinomics. Biochim Biophys Acta 2002;1572:165–77.

6. Hennet T. The galactosyltransferase family. Cell Mol Life Sci 2002;59:1081–95.

7. Spiro RG. Protein glycosylation: nature, distribution, enzymatic for-mation, and disease implications of glycopeptide bonds. Glycobiology2002;12:43R–56R.

8. Ten Hagen KG, Fritz TA, Tabak LA. All in the family: the UDP-GalNAc: polypeptide N-acetylgalactosaminyl transferases. Glycobi-ology 2003;13:1R–16R.

9. Gabius HJ. Concepts of tumor lectinology. Cancer Invest 1997;15:454–64.

10. Brockhausen I, Schutzbach J, Kuhns W. Glycoproteins and theirrelationship to human disease. Acta Anat 1998;161:36–78.

11. Hakomori SI. Cancer-associated glycosphingolipid antigens: theirstructure, organization, and function. Acta Anat 1998;161:79–90.

12. Dall’Olio F, Chiricolo M. Sialyltransferases in cancer. Glycoconju-gate J 2001;18:841–50.

13. Solıs D, Jimenez-Barbero J, Kaltner H, Romero A, Siebert HC, vonder Lieth CW, Gabius HJ. Towards defining the role of glycans ashardware in information storage and transfer: basic principles, exper-imental approaches and recent progress. Cells Tissues Organs 2001;168:5–23.

14. Gabius HJ, Siebert HC, Andre S, Jimenez-Barbero J, Rudiger H.Chemical biology of the sugar code. ChemBioChem 2004;5:740–64.

15. Rudiger H, Gabius HJ. Plant lectins. Glycoconjugate J 2001;18:589–613.

16. Wu AM. Carbohydrate structural units in glycoproteins and polysac-charides as important ligands for Gal and GalNAc reactive lectins.J Biomed Sci 2003;10:676–88.

17. Gabius HJ. Animal lectins. Eur J Biochem 1997;243:543–76.18. Kaltner H, Stierstorfer B. Animal lectins as cell adhesion molecules.

Acta Anat 1998;161:162–79.19. Kilpatrick DC. Handbook of animal lectins. New York: J. Wiley &

Sons, Inc. 2000. 468 p.20. Gabius HJ, ed. Special issue: animal lectins. Biochim Biophys Acta

2002;1572:163–434.21. Leffler H, Carlsson S, Hedlund M, Quian Y, Poirier F. Introduction to

galectins. Glycoconjugate J 2002;19:433–40.22. Ahmad N, Gabius HJ, Kaltner H, Andre S, Kuwabara I, Liu FT, Oscar-

son S, Norberg T, Brewer CF. Thermodynamic binding studies of cellsurface carbohydrate epitopes to galectins-1, -3, and -7: evidence fordifferential binding specificities. Can J Chem 2002;80:1096–104.

23. Unverzagt C, Andre S, Seifert J, Kojima S, Fink C, Srikrishna G,Freeze HH, Kayser K, Gabius HJ. Structure-activity profiles of com-plex biantennary N-glycans with core fucosylation and with/withoutadditional �2,3/�2,6-sialylation: synthesis of neoglycoproteins andtheir properties in lectin assays, cell binding, and organ uptake. J MedChem 2002;45:478–91.

24. Andre S, Unverzagt C, Kojima S, Frank M, Seifert J, Fink C, KayserK, von der Lieth CW, Gabius HJ. Determination of modulation ofligand properties of synthetic complex-type biantennary N-glycans byintroduction of bisecting GlcNAc in silico, in vitro and in vivo. EurJ Biochem 2004;271:118–34.

25. Lahm H, Andre S, Hoflich A, Fischer JR, Sordat B, Kaltner H, WolfE, Gabius HJ. Comprehensive galectin fingerprinting in a panel of 61tumor cell lines by RT-PCR and its implications for diagnostic andtherapeutic procedures. J Cancer Res Clin Oncol 2001;127:375–86.

26. Cooper DNW. Galectinomics: finding themes in complexity. BiochimBiophys Acta 2002;1572:209–31.

27. Nagy N, Legendre H, Engels O, Andre S, Kaltner H, Wasano K, ZickY, Pector JC, Decaestecker C, Gabius HJ, Salmon I, Kiss R. Refinedprognostic evaluation in colon carcinoma using immunohistochemicalgalectin fingerprinting. Cancer 2003;97:1849–58.

28. Kopitz J, von Reitzenstein C, Muhl C, Cantz M. Role of plasmamembrane ganglioside sialidase of human neuroblastoma cells ingrowth control and differentiation. Biochem Biophys Res Commun1994;199:1188–93.

29. Kopitz J, von Reitzenstein C, Sinz K, Cantz M. Selective gangliosidedesialylation in the plasma membrane of human neuroblastoma cells.Glycobiology 1996;6:367–76.

30. Kopitz J, Muhl C, Ehemann V, Lehmann C, Cantz M. Effects of cellsurface ganglioside sialidase inhibition on growth control and differ-entiation of human neuroblastoma cells. Eur J Cell Biol 1997;73:1–9.

31. Kopitz J, von Reitzenstein C, Burchert M, Cantz M, Gabius HJ. Galec-tin-1 is a major receptor for ganglioside GM1, a product of the growth-controlling activity of a cell surface ganglioside sialidase, on humanneuroblastoma cells in culture. J Biol Chem 1998;273:11205–11.

32. Siebert HC, Andre S, Lu SY, Frank M, Kaltner H, van Kuik JA,Korchagina EY, Bovin NV, Tajkhorshid E, Kaptein R, VliegenthartJFG, von der Lieth CW, et al. Unique conformer selection of humangrowth-regulatory lectin galectin-1 for ganglioside GM1 versus bac-terial toxins. Biochemistry 2003;42:14762–73.

33. Kopitz J, von Reitzenstein C, Andre S, Kaltner H, Uhl J, Ehemann V,Cantz M, Gabius HJ. Negative regulation of neuroblastoma cell growthby carbohydrate-dependent surface binding of galectin-1 and functionaldivergence from galectin-3. J Biol Chem 2001;276:35917–23.

34. Kopitz J, Andre S, von Reitzenstein C, Versluis K, Kaltner H, Pieters RJ,Wasano K, Kuwabara I, Liu FT, Cantz M, Heck AJR, Gabius HJ.Homodimeric galectin-7 (p53-induced gene 1) is a negative growthregulator for human neuroblastoma cells. Oncogene 2003;22:6277–88.

35. Andre S, Liu B, Gabius HJ, Roy R. First demonstration of differential

56 ANDRE ET AL.

inhibition of lectin binding by synthetic tri- and tetravalent glycoclus-ters from cross-coupling of rigidified 2-propynyl lactoside. Org Bi-omol Chem 2003;1:3909–16.

36. Ahmad N, Gabius HJ, Andre S, Kaltner H, Sabesan S, Roy B, Liu B,Macaluso F, Brewer CF. Galectin-3 precipitates as a pentamer withsynthetic multivalent carbohydrates and forms heterogeneous cross-linked complexes. J Biol Chem 2004;279:10841–7.

37. Andre S, Kaltner H, Furuike T, Nishimura SI, Gabius HJ. Persubsti-tuted cyclodextrin-based glycoclusters as inhibitors of protein-carbo-hydrate recognition using purified plant and mammalian lectins andwild-type and lectin-gene-transfected tumor cells as targets. Biocon-jugate Chem 2004;15:87–96.

38. Morris S, Ahmad N, Andre S, Kaltner H, Gabius HJ, Brenowitz M,Brewer CF. Quaternary solution structures of galectins-1, -3, and -7.Glycobiology 2004;14:293–300.

39. Humphrey W, Dalke A, Schulten K. VMD-visual molecular dynam-ics. J Mol Graph 1996;14:33–8.

40. Gitt MA, Massa SM, Leffler H, Barondes SH. Isolation and expres-sion of a gene encoding L-14-II, a new human soluble lactose-bindinglectin. J Biol Chem 1992;267:10601–6.

41. Gitt MA, Wiser MF, Leffler H, Herrmann J, Xia YR, Massa SM,Cooper DNW, Lusis AJ, Barondes SH. Sequence and mapping ofgalectin-5, a �-galactoside-binding lectin, found in erythrocytes.J Biol Chem 1995;270:5032–8.

42. Oka T, Murakami S, Arata Y, Hirabayashi J, Kasai KI, Wada Y, FutaiM. Identification and cloning of rat galectin-2: expression is predom-inantly in epithelial cells of the stomach. Arch Biochem Biophys1999;361:195–201.

43. Stierstorfer B, Kaltner H, Neumuller C, Sinowatz F, Gabius HJ.Temporal and spatial regulation of expression of two galectins duringkidney development of the chicken. Histochem J 2000;32:325–36.

44. Ohyama Y, Hirabayashi J, Oda Y, Ohno S, Kawasaki H, Suzuki K,Kasai KI. Nucleotide sequence of chick 14K �-galactoside-bindinglectin from mRNA. Biochem Biophys Res Commun 1986;134:51–6.

45. Gabius HJ. Influence of type of linkage and spacer on the interactionof �-galactoside-binding proteins with immobilized affinity ligands.Anal Biochem 1990;189:91–4.

46. Powell JT, Whitney PL. Endogenous ligands of rat �-galactoside-binding protein (galaptin) isolated by affinity chromatography oncarboxymethylated-galectin-Sepharose. Biochem J 1984;223:769–74.

47. Gabius HJ, Engelhardt R, Rehm S, Cramer F. Biochemical charac-terization of endogenous carbohydrate-binding proteins from sponta-neous murine rhabdomyosarcoma, mammary adenocarcinoma, andovarian teratoma. J Natl Cancer Inst 1984;73:1349–57.

48. Andre S, Pieters RJ, Vrasidas I, Kaltner H, Kuwabara I, Liu FT, LiskampRMJ, Gabius HJ. Wedgelike glycodendrimers as inhibitors of binding ofmammalian galectins to glycoproteins, lactose maxiclusters, and cellsurface glycoconjugates. ChemBioChem 2001;2:822–30.

49. Purkrabkova T, Smetana K Jr, Dvorankova B, Holıkova Z, Bock C,Lensch M, Andre S, Pytlık R, Liu FT, Klıma J, Smetana K, Motlık J,et al. New aspects of galectin functionality in nuclei of cultured bonemarrow stromal and epidermal cells: biotinylated galectins as tool todetect specific binding sites. Biol Cell 2003;95:535–45.

50. Gabius S, Kayser K, Hellmann KP, Ciesiolka T, Trittin A, GabiusHJ. Carrier-immobilized derivatized lysoganglioside GM1 is a ligandfor specific binding sites in various human tumor cell types andperipheral blood lymphocytes and monocytes. Biochem Biophys ResCommun 1990;169:239–44.

51. Rudiger H, Siebert HC, Solıs D, Jimenez-Barbero J, Romero A, vonder Lieth CW, Dıaz-Maurino, Gabius HJ. Medicinal chemistry basedon the sugar code: fundamentals of lectinology and experimentalstrategies with lectins as targets. Curr Med Chem 2000;7:389–416.

52. Andre S, Kojima S, Yamazaki N, Fink H, Kaltner H, Kayser K,Gabius HJ. Galectins-1 and -3 and their ligands in tumor biology. JCancer Res Clin Oncol 1999;125:461–74.

53. Somers WS, Tang J, Shaw GD, Camphausen RT. Insights into themolecular basis of leukocyte tethering and rolling revealed by structuresof P- and E-selectin bound to sLex and PSGL-1. Cell 2000;103:467–79.

54. Dahms NM, Hancock MK. P-type lectins. Biochim Biophys Acta2002;1572:317–40.

55. Siebert HC, Gilleron M, Kaltner H, von der Lieth CW, Kozar T,Bovin NV, Korchagina EY, Vliegenthart JFG, Gabius HJ. NMR-based, molecular dynamics- and random walk molecular mechanics-supported study of conformational aspects of a carbohydrate ligand(Gal�1,2Gal�1,R) for an animal galectin in the free and in the boundstate. Biochem Biophys Res Commun 1996;219:205–12.

56. Asensio JL, Espinosa JF, Dietrich H, Canada FJ, Schmidt RR, Martın-Lomas M, Andre S, Gabius HJ, Jimenez-Barbero J. Bovine heartgalectin-1 selects a distinct (syn) conformation of C-lactose, a flexiblelactose analogue. J Am Chem Soc 1999;121:8995–9000.

57. Gabius HJ. Glycohistochemistry: the why and how of detection andlocalization of endogenous lectins. Anat Histol Embryol 2001;30:3–31.

58. Siebert HC, Lu SY, Frank M, Kramer J, Wechselberger R, Joosten J,Andre S, Rittenhouse-Olson K, Roy R, von der Lieth CW, Kaptein R,

Vliegenthart JFG, et al. Analysis of protein-carbohydrate interactionat the lower size limit of the protein part (15-mer peptide) by NMRspectroscopy, electrospray ionization mass spectrometry, and molec-ular modeling. Biochemistry 2002;41:9707–17.

59. Arnusch CJ, Andre S, Valentini P, Lensch M, Russwurm R, SiebertHC, Fischer MJE, Gabius HJ, Pieters RJ. Interference of the galac-tose-dependent binding of lectins by novel pentapeptide ligands.Bioorg Med Chem Lett 2004;14:1437–40.

60. Beyer EC, Zweig SE, Barondes SH. Two lactose-binding lectins fromchicken tissues. Purified lectin from intestine is different from those inliver and muscle. J Biol Chem 1980;255:4236–9.

61. Gupta D, Kaltner H, Dong X, Gabius HJ, Brewer CF. Comparativecross-linking activities of lactose-specific plant and animal lectins anda natural lactose-binding immunoglobulin G fraction from humanserum with asialofetuin. Glycobiology 1996;6:843–9.

62. Varela PF, Solıs D, Dıaz-Maurino T, Kaltner H, Gabius HJ, RomeroA. The 2.15 Å crystal structure of CG-16, the developmentally reg-ulated homodimeric chicken galectin. J Mol Biol 1999;294:537–49.

63. Schneller M, Andre S, Cihak J, Kaltner H, Merkle H, Rademaker GJ,Haverkamp J, Thomas-Oates JE, Losch U, Gabius HJ. Differentialbinding of two chicken �-galactoside-specific lectins to homologouslymphocyte subpopulations and evidence for inhibitor activity of thedimeric lectin on stimulated T cells. Cell Immunol 1995;166:35–43.

64. He L, Andre S, Siebert HC, Helmholz H, Niemeyer B, Gabius HJ.Detection of ligand- and solvent-induced shape alterations of cell-growth-regulatory human lectin galectin-1 in solution by small angleneutron and X-ray scattering. Biophys J 2003;85:511–24.

65. Heck AJR, van den Heuvel RHH. Investigation of intact protein com-plexes by mass spectrometry. Mass Spectrom Rev 2004;23:368–89.

66. Bharadwaj S, Kaltner H, Korchagina EY, Bovin NV, Gabius HJ,Surolia A. Microcalorimetric indications for ligand binding as afunction of the protein for galactoside-specific plant and avian lectins.Biochim Biophys Acta 1999;1472:191–6.

67. Tinari N, Kuwabara I, Huflejt ME, Shen PF, Iacobelli S, Liu FT.Glycoprotein 90K/MAC-2 BP interacts with galectin-1 and mediatesgalectin-1-induced cell aggregation. Int J Cancer 2001;91:167–72.

68. Cao Z, Said N, Amin S, Wu HK, Bruce A, Garate M, Hsu DK,Kuwabara I, Liu FT, Panjwani N. Galectins-3 and -7, but not galec-tin-1, play a role in re-epithelialization of wounds. J Biol Chem2003;277:42299–305.

69. Timoshenko AV, Gorudko IV, Maslakova OV, Andre S, Kuwabara I,Liu FT, Kaltner H, Gabius HJ. Analysis of selected blood and immunecell responses to carbohydrate-dependent surface binding of proto-and chimera-type galectins. Mol Cell Biochem 2003;250:139–49.

70. Andre S, Unverzagt C, Kojima S, Dong X, Fink C, Kayser K, GabiusHJ. Neoglycoproteins with the synthetic complex biantennary nona-saccharide or its �2,3/�2,6-sialylated derivatives: their preparation,assessment of their ligand properties for purified lectins, for tumorcells in vitro and in tissue sections, and their biodistribution intumor-bearing mice. Bioconjugate Chem 1997;8:845–55.

71. Wu AM, Wu JH, Tsai MS, Kaltner H, Gabius HJ. Carbohydratespecificity of a galectin from chicken liver (CG-16). Biochem J2001;358:529–38.

72. Wu AM, Wu JH, Tsai MS, Liu JH, Andre S, Wasano K, Kaltner H,Gabius HJ. Fine-specificity of domain-I of recombinant tandem-re-peat-type galectin-4 from rat gastrointestinal tract. Biochem J 2002;367:653–64.

73. Wu AM, Wu JH, Liu JH, Singh T, Andre S, Kaltner H, Gabius HJ.Effects of polyvalency of glycotopes and natural modifications ofhuman blood group ABH/Lewis sugars at the Gla�1-terminated coresaccharides on the binding of domain-I of recombinant tandem-repeat-type galectin-4 from rat gastrointestinal tract (G4-N). Bio-chimie 2004;86:317–26.

74. Lahm H, Andre S, Hoflich A, Kaltner H, Siebert HC, Sordat B, vonder Lieth CW, Wolf E, Gabius HJ. Tumor galectinology: insights intothe complex network of a family of endogenous lectins. Glycoconju-gate J 2004;20:227–38.

75. Brewer CF. Binding and cross-linking properties of galectins. Bio-chim Biophys Acta 2002;1572:255–62.

76. Borriello A, Roberto R, Ragione FD, Iolascon A. Proliferate andsurvive: cell division cycle and apoptosis in human neuroblastoma.Haematologica 2002;87:196–214.

77. Rotblat B, Niv H, Andre S, Kaltner H, Gabius HJ, Kloog Y. Galectin-1(L11A) predicted from a computed galectin-1 farnesyl-bindingpocket selectively inhibits ras-GTP. Cancer Res 2004;64:3112–8.

78. Camby I, Belot N, Lefranc F, Sadeghi N, de Launoit Y, Kaltner H,Musette S, Darro F, Danguy A, Salmon I, Gabius HJ, Kiss R. Galectin-1modulates human glioblastoma cell migration into the brain throughmodifications to the actin cytoskeleton and the levels of expression ofsmall GTPases. J Neuropathol Exp Neurol 2002;61:585–96.

79. Hittelet A, Legendre H, Nagy N, Bronckart Y, Pector JC, Salmon I,Yeaton P, Gabius HJ, Kiss R, Camby I. Upregulation of galectins-1and -3 in human colon cancer and their role in regulating cell migra-tion. Int J Cancer 2003;103:370–9.

57GALECTINS IN NEUROBLASTOMA GROWTH REGULATION