Determination of structural and functional overlap/divergence of five proto-type galectins by analysis of the growth-regulatory interaction with ganglioside GM 1 in silico and in vitro on human neuroblastoma cells Sabine Andre ´ 1 , Herbert Kaltner 1 , Martin Lensch 1 , Roland Russwurm 1 , Hans-Christian Siebert 1 , Christine Fallsehr 2 , Emad Tajkhorshid 3 , Albert J.R. Heck 4 , Magnus von Knebel Doeberitz 2 , Hans-Joachim Gabius 1 and Juergen Kopitz 2 * 1 Institut fu ¨r Physiologische Chemie, Tiera ¨rztliche Fakulta ¨t, Ludwig-Maximilians-Universita ¨t, Mu ¨nchen, Germany 2 Institut fu ¨r Molekulare Pathologie, Klinikum der Ruprecht-Karls-Universita ¨t, Heidelberg, Germany 3 Theoretical and Computational Biophysics Group, Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA 4 Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands The growth-regulatory interplay between ganglioside GM 1 on human SK-N-MC neuroblastoma cells and an endogenous lectin provides a telling example for glycan (polysaccharide) functional- ity. Galectin-1 is the essential link between the sugar signal and the intracellular response. The emerging intrafamily complexity of galectins raises the question on defining extent of their structural and functional overlap/divergence. We address this problem for proto-type galectins in this system: ganglioside GM 1 as ligand, neuroblastoma cells as target. Using the way human galectin-1 interacts with this complex natural ligand as template, we first defined equivalent positioning for distinct substitutions in the other tested proto-type galectins, e.g., Lys63 vs. Leu60/Gln72 in galectins-2 and -5. As predicted from our in silico work, the tested proto-type galectins have affinity for the pentasaccharide of gan- glioside GM 1 . 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 negative growth control by competitively blocking access to the shared ligand without acting as effector. Thus, the quaternary structure of 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 exploited therapeutically by producing galectin variants tailored to interfere with galectin activities associated with the malignant phenotype. Moreover, the given strategy for comparative computational anal- ysis of extended binding sites has implications for the rational design 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 growth regulators in a clinically relevant tumor model. For the orientation of the reader, we start with a brief primer of the concept. The cell surface 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 essential means toward efficient information transfer. All three prerequisites are readily fulfilled by carbohydrate epitopes of cellular glycans (polysaccharides). In fact, their theoretical capacity for coding surpasses that of oligonucleotides and oligopeptides by orders of magnitude, 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 of glycan 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 to acquire a functional dimension. 3,9 –12 As a general theme directing our studies, we thus aim to provide evidence for the concept to link distinct characteristics of tumor cell glycosylation with aspects of the malignant phenotype. When interpreting oligosaccharides of glycan chains as code words, their message is expected to be biochemically decoded and then translated into cellular responses such as modulation of ad- hesion/migration or proliferation. 13,14 Laboratory applications of plant 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 that their expression matches that of enzymes involved in glycan assembly and remodeling in complexity strongly argue in favor of an 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 of endogenous lectins has evolved with specificity to this molecular category of targets, and this family is termed galectins. 17,21 In- triguingly, model studies with oligosaccharides representing branch-end epitopes of cell surface glycoconjugates and N-glycans harboring natural substitutions have already validated the pre- dicted impact of structural and conformational features of the sugar ligand on affinity to galectins. 22–24 Fitting the elaborate mechanisms to modify carbohydrate properties as ligands, two main factors on the side of galectins render effective fine-tuning and regulation likely: (i) the galectins’ diversification in up to 14 different family members in mammals with subdivision into three groups (proto-, chimera-, and tandem-repeat types) and (ii) the observations from RT-PCR and immunohistochemical analyses (galectin fingerprinting) that a tumor (or nonmalignant) cell can often 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. This issue 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 to the resolution of this problem. Due to the fact that neuroblastoma is a frequent extracranial solid tumor type in childhood, accounting for about 15% of pediatric cancer deaths, our project on endoge- nous growth regulators could spawn a clinical perspective. In detail, we have first shown that a distinct change in the glycomic profile, i.e., shift in the ganglioside population from higher sialy- lated forms to ganglioside GM 1 due to upregulation of a cell surface ganglioside sialidase (neuraminidase), is the crucial control element 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 f¨ ur Molekulare Pathologie, Klinikum der Ruprecht-Karls-Universit¨ at, 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 2004 DOI 10.1002/ijc.20699 Published 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