Tumor suppressor p16INK4a ) modulator of glycomic profile and galectin-1 expression to increase susceptibility to carbohydrate-dependent induction of anoikis in pancreatic carcinoma cells ´ Sabine Andre1, Hugo Sanchez-Ruderisch2, Hiroaki Nakagawa3, Malte Buchholz4, Jurgen Kopitz5, ă Pia Forberich6, Wolfgang Kemmner6, Corina Bock1, Kisaburo Deguchi3, Katharia M Detjen2, ă Bertram Wiedenmann2, Magnus von Knebel Doeberitz5, Thomas M Gress7, Shin-Ichiro Nishimura3, Stefan Rosewicz2 and Hans-Joachim Gabius1 Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University Munich, Germany ´ Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Charite-Universitatsmedizin Berlin, Germany ă Graduate School of Advanced Life Science, Frontier Research Center for Post-Genome Science and Technology, Hokkaido University, Sapporo, Japan Abteilung Innere Medizin I, Universitat Ulm, Germany ă Institut fur Angewandte Tumorbiologie, Klinikum der Ruprecht-Karls-Universitat, Heidelberg, Germany ă ă Clinic of Surgery and Surgical Oncology, Robert Roessle Hospital, Charite Campus Buch, Berlin, Germany Department of Gastroenterology, Endocrinology and Metabolism, University Hospital Giessen and Marburg, Germany Keywords anoikis; fibronectin receptor; galectin; glycosyltransferases; pancreas tumor Correspondence ´ S Andre, Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University Munich, Veterinarstr 13, 80539 Munich, Germany ă Fax: +49 89 21802508 Tel: +49 89 21803279 E-mail: Sabine.Andre@lmu.de (Received 21 February 2007, revised 23 March 2007, accepted 27 April 2007) doi:10.1111/j.1742-4658.2007.05851.x Expression of the tumor suppressor p16INK4a after stable transfection can restore the susceptibility of epithelial tumor cells to anoikis This property is linked to increases in the expression and cell-surface presence of the fibronectin receptor Considering its glycan chains as pivotal signals, we assumed an effect of p16INK4a on glycosylation To test this hypothesis for human Capan-1 pancreatic carcinoma cells, we combined microarray for selected glycosyltransferase genes with 2D chromatographic glycan profiling and plant lectin binding Major differences between p16-positive and control cells were detected They concerned expression of b1,4-galactosyltransferases (down-regulation of b1,4-galactosyltransferases-I ⁄ V and up-regulation of b1,4-galactosyltransferase-IV) as well as decreased a2,3-sialylation of O-glycans and a2,6-sialylation of N-glycans The changes are compatible with increased b1-integrin maturation, subunit assembly and binding activity of the a5b1-integrin Of further functional relevance in line with our hypothesis, we revealed differential reactivity towards endogenous lectins, especially galectin-1 As a result of reduced sialylation, the cells’ capacity to bind galectin-1 was enhanced In parallel, the level of transcription of the galectin-1 gene increased conspicuously in p16INK4a-positive cells, and even figured prominently in a microarray on 1996 tumor-associated genes and in proteomic analysis The cells therefore gain optimal responsiveness The correlation between genetically modulated galectin-1 levels and anoikis rates in engineered transfectants inferred functional significance To connect these findings to the fibronectin receptor, galectin-1 was shown to be co-immunoprecipitated We conclude that p16INK4a Abbreviations b4GalT, b1,4-galactosyltransferase; GalNAc, N-acetylgalactosamine; GalNAcT, N-acetylgalactosaminyltransferase; GnT-V, N-acetylglucosaminyltransferase V; LacNAc, N-acetyllactosamine; MAA, Maackia amurensis; ODS, octadecyl silane; PA, 2-aminopyridine; pI, isoelectric point; pRb, retinoblastoma tumor-suppressor gene FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS 3233 New function of p16INK4a ´ S Andre et al orchestrates distinct aspects of glycosylation that are relevant for integrin maturation and reactivity to an endogenous effector as well as the effector’s expression This mechanism establishes a new aspect of p16INK4a functionality Protein glycosylation has functional potential far beyond the structural roles exerted on the protein backbone [1–3] Endowed with a unique high-density coding capacity, oligosaccharides are versatile biochemical signals in glycoprotein maturation and routing intracellularly, as well as in diverse cell-surface activities such as adhesion or trigger mechanisms for signaling to regulate apoptosis ⁄ proliferation [4–7] This paradigm implies far-reaching functional consequences for the carefully mapped aberrations of glycosylation upon malignant transformation [8,9] In global terms, structural studies on N-glycans from virally transformed cells (polyoma, Rous sarcoma or hamster sarcoma virus carrying the v-ras oncogene) have traced distinct, nonrandom changes in the glycomic profile (i.e an increased degree of branching and chain extension) [10–12] Even more important, but still mostly descriptive, oncogene presence has been shown to affect particular components of the glycosylation machinery, as documented for H- and N-ras as well as the tyrosine kinase oncogenes src and her-2 ⁄ neu [13–16] Signaling for the measured transcriptional regulation of N-acetylglucosaminyltransferase V (GnT-V) and a2,6-sialyltransferase I (ST6Gal-I) is routed through Ras–Raf– Ets or Ral guanine exchange factors, respectively [15,16] However, the conclusion to invariably link the emergence of respective features, especially the increased b1,6-branching of N-glycans, with the malignant phenotype and therefore with an unfavorable prognosis in tumor patients, is not justified The opposite correlation was reported in tumor material from nonsmall cell lung cancer, neuroblastoma and bladder cancer [17–19] If it were known which glycoproteins are key targets, it could become possible to attribute altered glycosylation to a distinct functionality In this respect, studies with 3T3 fibroblasts transfected with the SV40 large T antigen gene, HD3 colon epithelial cells expressing oncogenic ras and human HT1080 fibrosarcoma cells overexpressing GnT-V (mentioned above) have illustrated target selection and, of special note, prominent appearance of the b1-integrin or the fibronectin receptor (a5b1-integrin) within this group [20–22] N-Glycosylation of the fibronectin receptor can be distributed over 14 potential sites in the a5-subunit and over 12 sites in the b1-chain, covering at least 35 types of oligosaccharides when analyzed for the protein from human placenta 3234 [23] The processing of these glycan chains is an important part of integrin maturation, and the glycosylation was shown to affect integrin association and clustering, the capacity for fibronectin or lectin binding and the interaction with the regulatory gangliosides GT1b and GD3 [21,24–29] These collective insights shape the hypothesis that remodeling the glycosylation of the fibronectin receptor can act as a molecular switch If we could select a cell system in which this integrin plays a major role for the fate of the cells, then it would be feasible to put our hypothesis to the experimental test The recent finding that the tumor suppressor p16INK4a restores susceptibility to anoikis induction in human Capan-1 pancreatic carcinoma cells by increasing a5b1-integrin expression and surface presentation offers such a suitable test system [30] We thus assumed that the presence of the tumor suppressor – beyond the transcriptional up-regulation of the a5-integrin gene [30] – may engender biologically significant influences on glycan synthesis and processing Three lines of evidence support the decision to test our hypothesis in this system First, constitutive p16INK4a expression in human A549 lung adenocarcinoma cells reduced global b1,4galactosyltransferase (b4GalT) activity on the cell surface by 25%, mainly as a result of reduced expression of the enzyme b4GalT-I [31] Analysis of transforming growth factor b1-induced rapid senescence of this cell type by northern blots revealed two- to fivefold increases in transcription for the b4GalT-II, -III, -V and -VI genes and abolishment of transcription of the b4GalT-IV gene [32] Second, comparison of DNA microarray-based expression profiles between specimens of pancreatic cancer and normal tissue revealed differential gene activities, especially up-regulation for b4GalT-V (factor 9.91) and b4GalT-I (factor 2.54) and an inverse regulation for GnT-IVa and b (factors 3.23 versus )20.27) [33] Third, the glycosyltransferases b4GalT-V and GnT-V were shown to be strongly expressed in a panel of eight human cancer lines [34] Transcriptional regulation of this b4GalT is under the control of the transcription factor Sp1 (which can activate genes for proteins with pro-growth ⁄ survival properties) and probably Ets-1; this property is shared with GnT-V [15,35,36] Gene FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS New function of p16INK4a ´ S Andre et al expression of b4GalT-V – and also of b4GalT-I [37] – can be enhanced by epidermal growth factor and dominant active ras [38] Intriguingly, both enzymes have a bearing on a5b1-integrin features b1-Integrin maturation, and transcription of the a5-integrin gene, were enhanced in human SHG44 glioma cells upon downregulating b4GalT-V expression [39], and the absence of GnT-V in murine embryonic fibroblasts led to a protein kinase C-dependent stimulation of transcription for the two integrin genes and of clustered cell-surface presentation of the fibronectin receptor [40] In contrast, human H7721 hepatocarcinoma cells responded to treatment with GnT-V-specific antisense cDNA with attenuation of gene expression for both integrins [41] Obviously, GnT-V-dependent effects in tumors and cells thus appear to be more difficult to predict than the consequences of b4GalT-V activity To delineate any p16INK4a effect on glycosylation at different levels, we devised a so-far unique, three-step strategy, starting with cDNA microarray analysis for glycosyltransferases Because the levels of mRNAs for these enzymes may or may not directly translate into the generation of respective oligosaccharides, we performed global product analysis using 2D chromatographic profiling The documented evidence on N-glycan alterations was the reason to focus on this type of glycosylation In order to find out the abundance of accessible surface glycans, we mapped the glycomic pattern of native cells using 24 plant lectins, reactive with N- and ⁄ or O-glycans, and then with human lectins The p16INK4a-positive cells were much more reactive with galectin-1 than control cells Picking up this trail, galectin-1 production was found to be significantly up-regulated when analyzed by two separate microarrays, proteomic profiling and flow cytofluorometry, its association with a5b1-integrin was shown and a positive correlation to anoikis rates was established The results presented thus reveal a connection between a tumor suppressor and glycosylation, at the molecular level probably between a5b1-integrin and galectin-1 This interplay is, at least in part, responsible for restoring susceptibility to anoikis in this cell system Results Profiling of gene expression for glycosyltransferases In the first set of experiments, we addressed the question of whether p16INK4a expression in Capan-1 pancreatic carcinoma cells will have an influence on the expression of glycosyltransferase genes The sensitivity of detection was refined to pick up minute signals from this class of often rather low-abundant mRNA species To avoid missing important clues, we monitored enzymes involved in N- and O-glycan as well as ganglioside biosynthesis Material from mock-treated and p16INK4a positive cells was processed under identical conditions, and the ratio of measured signal intensities for cDNA preparations from both types of clones was calculated for each enzyme In addition, the average signal intensity for the p16INK4a-positive cells served as a relative measure of the expression level When setting a threshold for a difference in ratio of ± 0.33, a total of 17 cases could be compiled; these are detailed in Table The overall mRNA supply for enzymatic capacity to attach N-acetylgalactosamine (GalNAc) to serine ⁄ threonine residues of a target protein by a UDPGalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAcT) of the two cell populations was measured for the initiation step and ensuing cluster building (here especially GalNAcTs-4 ⁄ -7) On average, it appeared to be rather similar The same applied to three tested cases for mucin-type O-glycan extension and its a2,6-sialylation at the proximal GalNAc moiety by ST6GalNAc-IV However, an increased expression level was measured in the cases of mRNA specific for two sialyltransferases involved in the synthesis of a-series gangliosides (Table 1) A high capacity for the synthesis of a2,3 ⁄ a2,6-disialyl Lea ⁄ Lec epitopes is an attribute of nonmalignant epithelial cells, reducing the presence of its sialyl Lea precursor Although this route of ganglioside synthesis could favor renormalization, the tested gene expression profile for core mucintype O-glycosylation did not reveal any impact of p16INK4a presence In view of the current literature on epithelial cancer or integrin glycosylation, it is reassuring to add that a marked influence on GalNAcT activity could not really be counted upon This situation is different for the branching of N-glycans and elaboration of their chain termini Turning therefore next to enzymes working on complex-type N-glycan structures, no major alteration was seen in the transcription of GnT-I, -III, -IVB and -V genes Because of its importance, the result on GnT-V was deliberately ascertained by independent PCR analysis The same picture emerged in three other groups of glycosyltransferases: b1,3-galactosyltransferases, b1,3-N-acetylglucosaminyltransferases, except for the type II/V protein (Table 1), and most a-fucosyltransferases except for a decrease to a ratio of 0.41 (signal intensity: 1249) for enzyme VIII introducing the core-fucose unit, as independently confirmed by real-time PCR (data not shown) As outlined in the Introduction, a different situation is anticipated for b4GalTs, and, indeed, the constitutive presence of FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS 3235 New function of p16INK4a ´ S Andre et al Table Microarray data of mRNA expression for glycosyltransferases (± 0.33 from ratio of 1) Accession Symbol Ratio p16 ⁄ mock NM_003774-0 NM_017423-0 NM_024642-0 NM_030965-0 NM_013443-0 GALNT4 GALNT7 GALNT12 ST6GALNAC5 ST6GALNAC6 0.63 1.50 0.55 1.67 1.77 670 2076 750 1058 2122 NM_006577 NM_032047 B3GNT2 B3GNT5 0.53 1.65 908 654 NM_001497 B4GALT1 0.33 1642 NM_003780 NM_003778 B4GALT2 B4GALT4 1.49 4.21 506 922 NM_004776 B4GALT5 0.29 3680 NM_006927 NM_006279 NM_003896 NM_006100 ST3GAL2 ST3GAL3 ST3GAL5 ST3GAL6 0.59 0.34 0.14 1.42 848 1240 1841 890 Signal p16 Enzyme functionality UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAcT-4) UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAcT-7) UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 12 (GalNAc-T-12) GD1a synthase (ST6GalNAc-V); a-series gangliosides GD1a synthase (ST6GalNAc-VI); a-series gangliosides, a3,6-disialyl Lec and Lea in a-series gangliosides b3-N-acetylglucosaminyltransferase (b3GnT-II); initiation and elongation of poly LacNAc b3-N-acetylglucosaminyltransferase (b3GnT-V); initiation and elongation of poly LacNAc, O-linked core 3, keratan sulfate, lactotriose b4-galactosyltransferase (b4GalT-I); branch extension of N- (b1,2-branch) and O-glycans (mucin, especially core 4, O-fuc, O-man), lactosylceramide b4-galactosyltransferase (b4GalT-II); branch extension of N- and O-glycans (see b4Gal-T-I) b4-galactosyltransferase (b4GalT-IV); poly LacNAc extension on N-glycans (b1,6-branch) and core O-glycans, neolacto series glycolipids, 6¢-O-sulfated LacNAc b4-galactosyltransferase (b4GalT-V); branch extension of N- (b1,6-branch) and O-glycans (mucin, O-fuc, O-man), lactosylceramide CMP-sialic acid:Galb1,3GalNAc a3-sialyltransferase (ST3Gal-II) CMP-sialic acid:Galb1,3 ⁄ 4GlcNAcb a3-sialyltransferase (ST3Gal-III) CMP-sialic acid:Galb1,4Glc-Cer a3-sialyltransferase (ST3Gal-V); GM3 synthase CMP-sialic acid:Galb1,4GlcNAcb a3-sialyltransferase (ST3Gal-VI); synthesis of 6¢-O-sulfated sLex p16INK4a made its mark on this group Overall, the most conspicuous changes were detected in the expression levels of three b4GalT proteins, explicitly downregulation of gene expression for proteins I and V and up-regulation of protein IV Of note, transcriptional regulation of b4GalTs-I ⁄ -V exhibits similarities noted in the Introduction In terms of signal intensity, b4GalTs-I ⁄ -V were the dominant species Set into relation with the rather low signal intensities for b1,3-galactosyltransferases, a preference for type-II termini of N-glycans is inferred Because the activity of b4GalTIV was increased, no drastic decrease in b1,4-galactosylation of glycan chains should occur Owing to potent galactosylation of the O-glycan core structures by b4GalT-IV, this synthetic route may be favored A similar trend with up- and down-regulation within one family was observed for N-glycan-specific a2,3-sialyltransferases (ST3Gal-III versus -VI) While the opposite direction of expression levels of these two a2,3-sialyltransferase genes for enzymes of similar substrate specificity for N-glycans may act in a compensatory manner, the reduction of gene expression of GM3 synthase (ST3Gal-V) may bear upon the capacity for ganglioside synthesis (Table 1) Looking at O-glycan a2,3-sialylation, gene expression for ST3Gal-II is reduced in the p16INK4a-positive cells In contrast, no modulation is seen for a2,6-sialyltransferase at the level of mRNA 3236 As with this case, it is essential to note, in general terms, that the measured extent of gene expression should not directly be extrapolated to enzyme and then product presence in a linear manner Of course, what matters for the cellular fate is the manifestation of a detected difference at the level of glycan production Naturally, post-transcriptional regulation and availability of the activated substrates at the appropriate site might also have their share in shifting glycan profiles Thus, we proceeded to the analysis of actual glycan profiles via different approaches Based on the presented results on significant differences in the display of mRNA levels of enzymes acting on N-glycans and the documented relevance of N-glycans for integrin maturation, we first focused on N-glycans to spot any major differences in their profile in situ For this purpose, we performed 2D chromatographic mapping of neutral N-glycans after labelling with 2-aminopyridine A major difference in the branching pattern, especially affecting b1,6-branching, will hereby be readily detectable Also, isomers can be separated and molar ratios determined Chromatographic profiling of N-glycans The total population of N-glycans was obtained from cellular extracts, and the reproducibility of results from seven individual cell batches of the stably transfected clones was ensured in the first set of experiments Each FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS New function of p16INK4a ´ S Andre et al Fig Quantitative profiling of N-glycans Complete representation of the N-glycan profiles of mock- and p16INK4a-transfected pancreatic carcinoma cells and their molar ratios determined by the 2D mapping technique The N-glycan structure is given for each case, and an inset is added for explanation individual peak from the first column was collected separately, and all oligosaccharides with a molar ratio above 1% were further analyzed, constituting a total of 22 different types of neutral N-glycans The elution properties of 18 N-glycan species are documented to underlie defined structures As follows, we present the effect of p16INK4a presence on the molar ratio and the structures of the major N-glycans In global terms, the presence of p16INK4a appeared to shift the balance from complex-type to oligomannosyl N-glycans, such as M6.1, M7.1 and M9.1 (Fig 1) Bi- and tetra-antennary N-glycans surpassed the 5% threshold of molar ratio in the mock-treated cells Invariably, the presence of b1,6-branching was reduced by p16INK4a expression (Fig 1) Only the biantennary complex-type N-glycan, with both a bisecting GlcNAc residue and core fucosylation, and the two type-Ibranched triantennary N-glycans without core fucosylation were slightly more abundant in the p16INK4a-expressing cells than in the control Of note, two N-glycans of unknown structure either in the region of mannose-rich compounds or between the trimannosyl core and the biantennary structure were only found for the p16INK4a-expressing cells, and no evidence for the emergence of an abundant N-glycan with poly N-acetyllactosamine extensions could be provided At this stage, it should be noted that work with whole cell extracts allows us to reach a statement on total glycan presence, at the level of sensitivity of this method Cytoplasmic N-glycans before and after maturation, as well as the cell-surface profile, irrespective of accessibility, will be simultaneously evaluated How the pattern of glycans presented on the cell surface and accessible for binding partners looks will have to be clarified by a different method Binding studies with glycan epitope-specific probes conducted on intact cells are suited to address this question For this purpose, forming a panel of plant lectins is a validated approach Although they will not monitor any glycosylation-dependent change of conformation or functional status of a glycoprotein, the glycan(s) in these cases directly acting on its (their) protein backbone, systematic application of these sensors for glycan structures is a step to define potential in situ effectors on the glycan side In order to cover the main classes of glycan constituents we selected 24 plant agglutinins The list of proteins and their sugar specificities are presented in Table Profiling of cell-surface glycans by plant lectins In the first step of these experiments, we established the concentration and sugar dependence of lectin binding, as illustrated in the supplementary material (Fig S1) These experiments were also instrumental in determining a common concentration to detect relative differences by comparative mapping and to avoid any toxic effects of lectins The experimental series was systematically performed in parallel under identical conditions for the two cell populations Hereby, any parameter change by prolonged or differential culture periods was avoided For convenient comparison, we measured the percentage of positive cells and mean fluorescence intensity in each panel, as listed in Fig In full accordance with the results on the abundance of mRNA for the GalNAcTs, the presence of GalNAc residues, measured with five different lectins, was rather similar except for VVA (Fig 2) This lectin may react preferentially with globo- and isoglobotetraosylceramides and not mucin-type O-glycans An apparent FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS 3237 New function of p16INK4a ´ S Andre et al Table Lectin panel for glycan profiling of cell surfaces listed in alphabetic order Latin name (common name) Acronym Monosaccharide specificity Artocarpus integrifolia (jack fruit) Arachis hypogaea (peanut) Canavalia ensiformis (jack bean) Datura stramonium (thorn apple) Dolichos biflorus (horse gram) Erythrina cristagalli (coral tree) Galanthus nivalis (snowdrop) Glycine max (soybean) Griffonia simplicifolia I Griffonia simplicifolia II Jacalin (JAC) PNA Con A DSA DBA ECA GNA SBA GSA I GSA II Gal ⁄ GalNAc Gal Man ⁄ Glc GlcNAc GalNAc Gal Man GalNAc GalNAc GlcNAc Lens culinaris (lentil) Lycopersicon esculentum (tomato) LCA LEA Man ⁄ Glc Maackia amurensis I (leukoagglutinin) Maackia amurensis II (haemagglutinin) Phaseolus vulgaris erythroagglutinin (kidney bean) Phaseolus vulgaris leukoagglutinin (kidney bean) Pisum sativum (garden pea) Sambucus nigra (elderberry) Solanum tuberosum (potato) Sophora japonica (pagoda tree) Triticum vulgare (wheat germ) Ulex europaeus I (gorse) Vicia villosa (hairy vetch) Viscum album (mistletoe) MAA I MAA II PHA-E b PHA-L b PSA SNA STA SJA WGA UEA I VVA VAA Man ⁄ Glc Gal ⁄ GalNAc a Potent oligosaccharidea Galb3GalNAca Galb3GalNAca GlcNAcb2Mana6(GlcNAcb2Mana3)Manb4GlcNAc (GlcNAc)n, Galb4GlcNAcb6(Galb4GlcNAcb2)Man (Galb4GlcNAc)3 GalNAca3GalNAca3Galb4Galb4Glc Galb4GlcNAcb6(Galb4GlcNAcb2)Man Mana6(Mana3)ManaR GalNAca3Galb6Glc GalNAca3Gal, GalNAca3GalNAcb3Gala4Galb4Glc GlcNAcb4GlcNAc, N-glycans with terminal, nonreducing-end GlcNAc N-glycan binding enhanced by core-fucosylation core and stem regions of high-mannose-type N-glycans, (GlcNAcb3Galb4GlcNAcb3Gal)n of complex-type N-glycans Neu5Aca3Galb4GlcNAc ⁄ Glc Neu5Aca3Galb3(a6Neu5Ac)GalNAc Bisected complex-type N-glycans: Galb4GlcNAcb2Mana6 (GlcNAcb2-Mana3)(GlcNAcb4)Manb4GlcNAc Tetra- and triantennary N-glycans with b6-branching b b b N-glycan binding enhanced by core-fucosylation Neu5Aca6Gal ⁄ GalNAc (GlcNAc)n with preference for high-mannose-type N-glycans GalNAcb6Gal, Galb3GalNAc (GlcNAc)n, Galb4GlcNAcb6Gal Fuca2Galb4GlcNAcb6R GalNAca3(6)Gal, GalNAcb3Gal Galb2(3)Gal, Gala3(4)Gal, Galb3(4)GlcNAc without ⁄ with a2,6-sialylation, Fuca2Gal b GalNAc GlcNAc ⁄ Neu5Ac Fuc GalNAc Gal Based on previously compiled information [121], extended and modified; preference for the Thomsen-Friedenreich epitope antigen was detected for p16INK4a-positive cells by PNA and jacalin This result may reflect either elevated core synthesis or efficient core masking by a2,3-sialylation in the mock control, as suggested by the microarray data It was therefore essential to study this issue in greater detail (please see the paragraph below) Using the standard concentration Maackia amurensis-II (MAA-II), the percentage of positive cells was enhanced for mock-treated clones, possibly attributable to different gene expression levels for the enzymes responsible for the two sialylation steps b no monosaccharide known as ligand N-Glycosylation, especially with core fucosylation, appeared to be accessible on the cell surface to a higher extent in the p16INK4a- versus mock-transfected cell populations Glycan profiling had revealed an increase for biantennary glycans of this type containing the additional bisecting GlcNAc unit, in contrast to an otherwise decreased level of core fucosylation (Fig 1), in full accord with array data of a-fucosyltransferase VIII Testing two probes with similar specificities (i.e LCA ⁄ PSA) served as an internal control Apparently, the chromatographic profiling, on average, detected more substituted N-glycan in mock controls Fig Profiling of cell-surface glycans by plant lectins Semilogarithmic representation of fluorescent surface staining by biotinylated plant lectins (for an explanation of the acronyms and listing of oligosaccharide specificity, please see Table 2) of mock-transfected (gray line) and p16INK4a-transfected (black line) Capan-1 pancreatic carcinoma cells determined in parallel assays Quantitative data on the percentage of positive cells and fluorescence intensity are given in each panel (first line: mock-treated cells; second line: p16INK4a-transfected cells) The concentration of the biotinylated lectins was 0.5 lgỈmL)1 except for SNA and DBA (1 lgỈmL)1), SJA and WGA (2 lgỈmL)1) and MAA-I (5 lgỈmL)1) 3238 FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS ´ S Andre et al FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS New function of p16INK4a 3239 New function of p16INK4a ´ S Andre et al when compared with the relatively increased lectin reactivity on the cell surface for p16INK4a-expressing cells, indicating disparities in the levels of accessibility and ligand preferences The chromatographic profiling and cell-surface detection of N-glycans with bisecting GlcNAc by PHA-E could easily be reconciled, product formation substantiating efficiency of only minute quantities of detectable mRNA for GnT-III, whereas no major accessibility difference could be discerned regarding lectin binding of the b1,6-branch by PHA-L (Fig 2) Due to the potential interference by a2,6-sialylation, this result, as noted for the Thomsen-Friedenreich antigen epitope and a2,3-sialylation above, had to be further scrutinized (please see below for the effect of sialidase treatment) A clear difference in lectin binding concerned the presence of accessible GlcNAc moieties measured by applying DSA ⁄ WGA but not seen to this extent with GSA-II and STA Expression changes in the b4GalT family may underlie this staining property LEA, a marker for extensions of N-glycan branches by N-acetyllactosamine (LacNAc) units, failed to provide clear evidence for marked cell-surface differences, arguing in favour of the concept of compensatory change within the tested b1,3-N-acetylglucosaminyltransferases and b4GalTs, as also seen in chromatographic profiling Because similar cell staining was measured with GNA, the contribution of the dual reactivity of LEA to high-mannose-type N-glycans will probably have no influence on this result Similarly, the abundance of accessible poly N-acetyllactosamine chains was rather similar, prompting a final check of chain-end galactosylation The two respective probes (VAA and ECA) revealed more intense staining of the p16INK4a-reconstituted clones than of the mock control (Fig 2; supplementary Fig S1) This result does not simply reflect the microarray data when adding up signal intensities for b4GalT-specific cDNAs As depicted above, sialylation will make its presence felt in this approach Because it can mask terminal galactose residues for lectins, measurement of its status was essential a2,3-Sialylation of N-glycans monitored comparatively with MAA-I at a fairly high concentration showed a slight preference for the p16INK4a-positive clone, indicating a compensatory balance for ST3Gal-III ⁄ -IV ⁄ -VI gene expression levels By contrast, cell positivity expressed as cell percentage was lowered in these cells when measuring lectin-reactive N-glycan-specific a2,6-sialylation, despite similar extents of ST6Gal-I gene expression (Fig 2) Ectopic ST6Gal-I expression in the p16INK4a-positive cells did not change this parameter (data not shown) As mentioned above, this observation on the nonidentical degree of sialylation makes it mandatory to deter3240 mine comparatively the impact of sialylation on the binding of plant lectins sensitive to its presence In addition to the inhibition control with haptenic sugar, the pre-exposure of cells to neuraminidase is, at the same time, a second control for ensuring carbohydrate-dependent binding of SNA Standard conditions for enzymatic treatment were established, minimizing the influence on the cell phenotype In line with the inhibition studies, enzymatic pretreatment significantly reduced (mock control) or almost completely abolished (p16INK4a-positive cells) SNA binding (Fig 3) The same effect was observed for MAA-II used at a nearly saturating concentration of lgỈmL)1 (Fig 3) In addition to its control character, these results support the evidence for a quantitative difference in the a2,6-sialylation status between the two cell populations Should the level of mucin-type O-glycan a2,3-sialylation also be lowered in the p16INK4a-positive cells, as suggested by the microarray data, then neuraminidase activity should enhance PNA staining of the control cells, with a minor influence on the p16INK4a-expressing cells and on DBA staining The importance of this aspect has been pointed out above Fittingly, PNA, but not DBA, positivity was markedly improved by the enzymatic removal of sialic acid residues from the cell surface for mock-transfected cells but not for the p16INK4a-positive cells (Fig 3) The minor effect on the p16INK4a-positive cells probably indicates the presence of mucin-type core tetrasaccharides, favored by an increased b4GalT-IV presence and reduced O-glycan a2,3-sialylation Thus, lectinaccessible mucin-type O-glycosylation appeared to be rather equally abundant, but the levels of its a2,3-sialylation were definitely different The apparent preference for mock-treated cells to carry O-glycan core a2,3-sialylation can be accounted for – at least in part – by the microarray data Because the presence of a2,6-linked sialic acids can impede PHA-L binding, the same procedure was also carried out in this case Using a lectin concentration of lgỈmL)1, increased staining was seen in both cell populations, and accessibility was still at an increased level for the p16INK4a-positive cells (Fig 3) To avoid underestimation of the presence of b1,6-branched N-glycans in the mock control, the remarkably different levels of SNA binding after standard neuraminidase treatment must be recalled In essence, data from chromatographic mapping square well with the lectin profiling A major result emerging from these experiments is that the extents of sialylation of N- and mucin-type O-glycans in the two cell populations (a2,6-substitution for N-glycans, a2,3-modification for O-glycans) are different In functional terms, these FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS New function of p16INK4a ´ S Andre et al Fig Effect of sialidase treatment on the cell-surface binding of plant lectins Semilogarithmic representation of fluorescent surface staining of mock-transfected (Mock) and p16INK4a-transfected (p16) Capan-1 pancreatic carcinoma cells without the incubation step using the biotinylated lectin (shaded) and after incubation with the labeled probe (0.5 lgỈmL)1 of PNA, lgỈmL)1 of PHA-L, lgỈmL)1 of SNA and MAA-II, as well as 10 lgỈmL)1 of DBA), without (gray line) or after (black line) sialidase treatment Quantitative data are presented as defined in the legend to Fig processes may generate or mask sites for contact in situ with endogenous lectins As the results with the b-galactoside-specific lectins VAA ⁄ ECA revealed, the levels of accessible galactose residues were remarkably different between the two cell populations Monitoring of cell-surface binding by plant lectins thus pinpointed disparities in accessible glycans These observations directed our interest to the detection of endogenous lectins They might turn these newly defined properties on the level of cell-surface glycosylation into effects, in this case on the level of susceptibility to anoikis With focus on sialylation ⁄ galactosylation, the main groups of human lectins that can read and translate such differences are the C-type lectins, siglecs and galectins [42] The ensuing microarray monitoring of expression of 42 C-type lectins and siglecs-2, -3, -5, -6, -7 and -9 failed to provide positive data or, if positive, a difference in signal intensities When testing the third mentioned lectin family, indications for transcription of galectin genes were collected As further ascertained by systematic RT-PCR analysis within this lectin family, transcription of genes for galectins-1, -3, -7 and -9, respectively, was detected, the most pronounced signal (i.e 5369) seen in the case of galectin-1 in p16INK4apositive cells (data not shown) Having herewith provided evidence for gene expression of members of this galactoside-specific lectin family, we next probed whether human adhesion ⁄ growth-regulatory galectins can bind to the cells, as shown for the galactoside- specific lectins ECA ⁄ VAA By testing more proteins than just galectin-1, the individual binding properties of the structurally closely related members of this family can also be profiled in one cell system, a comparison so far not reported For this purpose, we purified the lectins and then biotinylated them under activitypreserving conditions, ascertained a lack of harmful effects on sugar binding by activity assays and determined the labeling efficiency with 2–8 modified residues per carbohydrate-recognition domain As in the binding studies with plant lectins, we routinely performed experiments to assess concentration dependence and inhibition of galectin binding by haptenic sugar Profiling of galectin binding The measurements with the two galactoside-specific plant lectins and also with MAA-I (galectins tolerate a2,3- but not terminal a2,6-sialylation) led to the expectation that these human lectins may preferentially bind to p16INK4a-expressing cells with their increased presence of these epitopes Indeed, the respective studies confirmed this notion already in the first set of experiments with galectin-1, when documenting the dependence of cell staining on lectin concentration and on glycan binding (supplementary Fig S2, first and second panels) This homodimeric family member is a potent cross-linker for glycans on the cell surface FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS 3241 New function of p16INK4a ´ S Andre et al Besides using haptenic sugar to relate lectin activity to binding, we tested two mutants of human galectin-1 Their carbohydrate-binding activity was impaired by a crucial substitution (W68L, E71Q) in the carbohydrate-recognition domain The loss of binding to the p16INK4a-transfected cells compared with the His-tagged wild-type control protein served as an independent validation of the inhibition control (supplementary Fig S2, third panel) The concentrationand carbohydrate-dependent binding is also illustrated for the chimera-type galectin-3 In this case, it is obvious that the cells of the mock control are also rather reactive, when considering cell-percentage positivity (supplementary Fig S2) Given this indication for intergalectin differences, we systematically assayed a series of human galectins to define staining properties with these human effector proteins As shown in the supplementary material (Fig S3), there is a clear trend for galectin reactivity correlating with tumor suppressor presence The tandem-repeat-type galectin-8 reacted similarly to galectin-9 (data not shown) The most prominent change for the combination of both quantitative cell staining parameters was seen in the case of galectin-1 Glycan-dependent galectin binding to these cells is thus detectable, it is not a uniform characteristic, and, finally and even more importantly, the conspicuous difference of galectin-1 binding to the two cell populations gives further study a clear direction As a result of the blocking effect of terminal a2,6-sialylation on galectin-1 binding, we assumed that this type of sialylation will mask galectin-1-reactive sites on the cells of the mock control If therefore exposed to a sialidase, these cells should become reactive, as shown for SNA or PNA binding in Fig Indeed, reduction of sialylation under standard conditions increased cell binding markedly for the mock control, whereas the p16INK4a-transfected cells showed only slightly improved binding properties (Fig 4) Galectin-1 specificity renders it very likely that removing the blocking a2,6-sialylation underlies this parameter change That the same reactivity pattern was seen for galectin-3 constitutes not only an inherent control As galectin-3 tolerates a2,6-sialylation in poly N-acetyllactosamine chains already at the level of the dimer, in stark contrast to galectin-1 [43], these results signified no notable difference for the presence of such chain extensions between the cell types, in full agreement with LEA and microassay data In view of an effector function, the differential status of sialylation thus appeared to influence the accessibility to ligand sites effectively This result might become functionally relevant if a functional relationship between galectin-1 3242 Fig Effect of sialidase treatment on the cell-surface binding of human galectins Semilogarithmic representation of fluorescent staining of mock-transfected (Mock) and p16INK4a-transfected (p16) Capan-1 pancreatic carcinoma cells without the incubation step using the biotinylated lectin (shaded) and after incubation with labeled galectins-1 and -3 (gal-1 and gal-3 used at 10 lgỈmL)1) without (gray line) or after (black line) sialidase treatment Quantitative data are presented as defined in the legend to Fig and the expression of the p16INK4a protein could be delineated In this sense, the p16INK4a-dependent increase of the presentation of binding sites by reduced a2,6-sialylation might even be associated with enhanced galectin-1 expression, this regulatory event accomplishing optimal sensitivity We put this reasoning to the test in a stepwise manner by a gene array, by northern blotting ⁄ nuclear run-off experiments, by proteomic profiling and by flow cytofluorometry Identification of up-regulation of galectin-1 expression Quantitative determination of the presence of galectin1-specific mRNA indicated a p16INK4a-associated increase In detail, we comparatively probed 1996 cDNAs in an array designed for pancreas tissue and its cancer development and applied stringent criteria for defining up-regulation The threshold of 50% increase was surpassed by 16 signals Galectin-1 gene transcription was the most prominent defined case at a p16INK4a ⁄ mock ratio of 3.01 (supplementary FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS New function of p16INK4a ´ S Andre et al Table S1) Northern blotting and nuclear run-off experiments confirmed the array data and substantiated an increase in de novo transcription (data not shown) Extending our work from the mRNA level, we next performed a proteomic analysis with the intention of establishing whether the galectin-1 protein is produced at an amount reflecting gene expression With a total of 600–670 spots per gel and a reproducibility of spot assignment between different gels of 89.4–99%, we detected one spot among the 48 signals that showed a consistent increase in staining by 50% in p16INK4a-positive cells, relative to control cells, with mass ⁄ isoelectric point (pI) characteristics compatible with galectin-1 As shown in Fig 5, we confirmed this hypothesis by western blotting and mass-spectrometric fingerprinting On the basis of staining of protein by a dye or the western blotting procedure, galectin-1 presence as protein was found to be significantly up-regulated (P ¼ 0.0154, P ¼ 0.0027) The advantage of the proteomic profiling compared with western blotting after 1D electrophoresis, shown separately in Fig 6A as a control, is the exclusion of formation of any galectin-1 variants based on different pI values After synthesis, the protein underwent secretion, because we detected its presence in the medium (data not shown) As a consequence it may then associate with the cell surface, prompting flow-cytofluorometric analysis Applying the antigalectin-1-specific immunoglobulin for cell-surface detection, the difference in protein production translated into increased surface presence in the p16INK4a-positive cells (Fig 5) We deliberately tested several cell batches and consistently measured an enhanced cell-surface presentation in the p16INK4a-positive cells under standard culture conditions When determining the level of inhibition of galectin-1 binding by the haptenic sugar, lactose, a notable difference became apparent It was comparatively lower in this cell type than in the control cells, a measure for strong affinity of the endogenous lectin to a set of particular surface glycans In fact, endogenous galectin-1 could hardly be stripped off the cell surface, even in the presence of 200 mm lactose In comparison, lectin binding from the medium as source was much more sensitive to inhibitor presence, as observed from loading the cells with galectin-1 up to saturation (supplementary Fig S2), intimating visualization of the gradient of decreasing affinity for binding multivalent ligands seen in a recent model study [44] To exclude that the p16INK4a-dependent up-regulation of galectin-1 is a singular event confined to Capan-1 cells only, we tested Dan-G pancreatic cancer cells without and with p16INK4a expression Of note, these two cell lines give insight into the specificity of the effect as a result of their differences in the status of the retinoblastoma tumor-suppressor gene, pRb Increased galectin-1 expression was determined by western blotting in both clones of engineered transfectants with p16INK4a expression, despite maintained pRb status (data not shown) It is thus tempting to propose a functional correlation between the detected galectin-1 up-regulation, the increased presentation of galectin-1-binding sites in p16INK4a-expressing cells and acquisition of anoikis susceptibility associated with the fibronectin receptor Induction of anoikis by galectin-1 In order to test the hypothesis described above, we pursued two independent approaches First, we established stable clones with reduced galectin-1 production by transfection with a vector harboring full-length cDNA for galectin-1 in the antisense orientation After confirming the reduction of galectin-1 presence, to a level characteristic of wild-type cells, by western blotting, the cells of such a clone were subjected to monitoring levels of anoikis In line with our hypothesis, the extent of anoikis was correlated to the level of galectin-1 present (Fig 6A) Second, we forced an increase of galectin-1 production on wild-type cells weakly positive for galectin-1 binding, using proliferating cell nuclear antigen as an internal control Anoikis induction was enhanced, even showing a trend for dose dependence among the tested clones (Fig 6B) Corroborating this biological effect on wild-type cells artificially overexpressing galectin-1 we could, in parallel, elicit anoikis in regular wild-type cells by adding the lectin to medium at a concentration of 125 lgỈmL)1 (data not shown) The presence of haptenic sugar interfered with this process, revealing carbohydrate dependence, as did the presence of the predominantly monomeric galectin3, revealing a requirement for cross-linking (data not shown) Finally, to connect galectin-1 with the p16INK4a-associated increase of cell-surface presence of the fibronectin receptor, we reasoned that preparations of the integrin, when immunoprecipitated from p16INK4a-positive cells, should contain galectin-1 Using cells grown adherent or in suspension, we tested this assumption Western blotting revealed that galectin-1 was indeed co-immunoprecipitated with this glycoprotein, the levels of galectin-1 presence being consistently higher in p16INK4a-positive cells than in control cells (Fig 6C) Independently, the antibody against the a5-subunit was effective at markedly reducing the extent of binding of labeled galectin-1 to the cell surface (data not shown) These results imply that the a5b1-integrin is a binding partner of anoikis-inducing galectin-1 FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS 3243 New function of p16INK4a ´ S Andre et al Fig Enhanced galectin-1 expression in p16INK4a-reconstituted cells Aliquots of total protein (200 lg) from mock-transfected (Mock) and p16INK4a-transfected (p16) Capan-1 pancreatic carcinoma cells were subjected to 2D gel electrophoretic separation and silver staining The kDa-section where galectin-1 presence can be expected was marked (A, B) and the putative position of galectin-1 was labeled Western blot (WB) analysis with equal quantities of protein and lgỈmL)1 of galectin-1 antibody as probe revealed spots at this position after antigen visualization by enhanced chemiluminescence (top panel) Mass-spectrometric fingerprinting after digestion of the protein of this spot by trypsin ascertained identity to galectin-1, and quantification of the staining intensity in gel electrophoretic (2-DE) and WB analyses revealed statistically significant up-regulation (middle panel) Cell-surface detection of galectin-1 in flow-cytofluorometric analysis was performed using 20 lgỈmL)1 of polyclonal galectin-1 antibody as probe and fluorescent goat anti-rabbit IgG as the second-step reagent (bottom panel) 3244 FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS New function of p16INK4a ´ S Andre et al A B C Fig Role of galectin-1 in p16INK4a-mediated anoikis induction Quantification of p16INK4a and galectin-1 presence in wild-type (wt), p16INK4a-positive (p16) and p16INK4a ⁄ antisense galectin-1 (gal-1AS) double-transfected cells by western blot analysis and determination of anoikis rates of cells after 24 h in suspension in at least three independent experiments (***, P < 0.01 for p16INK4a cells versus wt; #, P < 0.05 for double transfectants versus p16INK4a cells; the study panel includes a mock control for second transfection) (A) Quantification of galectin-1 presence in wt, mock-treated and cells transfected with vector carrying galectin-1-specific cDNA showing different levels of galectin-1 positivity and determination of anoikis rates as given in panel A (***, P < 0.01, *, P < 0.05 for galectin-1 transfectants versus wt cells; sensitivity of detection was less than in panel A to avoid overexposure of the lane for clone gal-1 ⁄ 1) (B) Western blot detection of galectin-1 in preparations of immunoprecipitated fibronectin receptor obtained from cells grown while adherent (ad) or on polyhydroxyethylmethacrylate (PH) to keep them in suspension (C) Discussion Protein glycosylation affords a broad platform for highly versatile modulation of diverse functional aspects The presence of distinct glycan determinants in glycoproteins can underlie quantitative aspects of intracellular routing and transport to the cell surface as well as the regulation of activities of both protein and glycan parts at the final destination Deduced from the fundamental concept of the sugar code, cells derive much of their remarkable communication skills from presenting an array of glycan signals [3,5,6, 45,46] In this sense, glycan remodeling is gaining a functional dimension in our understanding, and even the introduction of at first sight minor substitutions, such as a bisecting GlcNAc or core fucose residues, has remarkable consequences for the shape and ligand activity of the N-glycan [47–49] It is thus intuitively attractive to assume that changes in the glycomic profile are nonrandom reprogramming events, acting on the protein (e.g conformation, protection from proteolysis, aggregate formation or ligand binding) and on the glycan (e.g conformation and affinity to lectins) Using the tumor suppressor, p16INK4a, the fibronectin receptor and the Capan-1 cell line as study objects, we herein have delineated a new route towards re-establishing susceptibility for anoikis The presented results lend credit to a scenario of finetuned and co-ordinated events translating into alterations of protein–protein and protein–carbohydrate recognition Towards this aim, we had designed a combined approach using a cDNA microarray for glycosyltransferases, 2D chromatographic profiling and binding studies with lectins on the cell surface The array data pinpoint several changes in expression of glycosyltransferase genes and hereby afforded first evidence for functional links To start with there is an increased potential for the synthesis of a2,3 ⁄ a2,6disialylated Lea ⁄ Lec-epitopes on gangliosides, an attribute of nonmalignant epithelial cells and ligand availability for siglec-7 [50,51] Automatically, the presence of a synthetic precursor (i.e the sialyl Lea epitope, which is present in wild-type Capan-1 cells and can act as mediator of tumor angiogenesis and metastasis) will be diminished [51,52] MAA-II staining at a probe concentration of lgỈmL)1 can be interpreted to reflect the differential presence of the respective disialylated core Next, the previously reported influence on b4GalT activity [31] was confirmed at the level of transcription and extended to divergent regulation between b4GalTs-I ⁄ -V and b4GalT-IV Of interest, the noted down-regulation of FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS 3245 New function of p16INK4a ´ S Andre et al b4GalT-V can be linked to integrin maturation and increased surface expression of a5b1-integrin To avoid severe effects caused by hypogalactosylation, which would otherwise even lead to symptoms of congenital disorder of glycosylation type IId [53,54], compensation within this family is essential The negative impact on core mucin-type O-glycan galactosylation can in principle be attenuated by b4GalTs-IV ⁄ -V [55,56], and it is b4GalT-IV whose gene expression is substantially elevated Its activity produces short core extensions, unless the synthetic precursor core is a2,3-sialylated [55–57] Fittingly, chromatographic profiling and LEA staining yielded no evidence for a major change in poly N-acetyllactosamine presence, and gene expression of a respective sialyltransferase decreased As a consequence, conspicuous diminution was substantiated on the level of the cell surface for a2,3-sialylation of mucin-type O-glycans, especially when using PNA staining without ⁄ with neuraminidase treatment Because exposure of human HT-29 colon cancer cells to GalNAc-a-Obenzyl resulted in reduced a2,3-sialylation, which engendered an impact on the apical delivery of glycoproteins [58], this parameter change may also have a bearing on integrin routing As noted for a2,3-sialylation of mucin-type O-glycosylation, cell-surface lectin staining likewise also unraveled a decrease in a2,6-sialylation of N-glycans, this one not predictable from our array data Any specific intermolecular mechanisms notwithstanding, these combined events already bring about a major parameter alteration At the cell surface, the degree of sialylation contributes markedly to charge distribution Its change can in itself elicit charge-sensitive processes, if, for example, electrostatic repulsion is lessened More specifically, a2,6-sialylation of the a5b1-integrin is known to have a negative impact on fibronectin binding to the extent of physiological relevance that induction of myeloid differentiation by phorbol ester targets ST6Gal-I expression [59] Further model studies on ST6Gal-I confirmed a more general role of this sialylation mode in cell adhesion and invasiveness [60–62] In our experimental series with plant lectins, it was essential to pay attention to alterations in the degree of sialylation, also The presence of a2,6-sialylation reduces the affinity of otherwise suitable binding partners for DSA and PHA-L so that this aspect needed to be further studied with sialidase-treated cells [63–66] Of note, the three-bond system of the a2,6-linkage generates an unusually high degree of intramolecular flexibility [67] Flanked by the controls with neuraminidase treatment, the different levels of sialylation were ascertained, whereas a 3246 major influence of p16INK4a on b1,6-branching and chain length was excluded Results from all three applied methods were in full agreement The essential prerequisite of a change in electrophoretic mobility of a5b1-integrin from the two cell populations as an indicator of altered glycosylation was also ascertained (data not shown) As an excellent measure of the sensitivity of the chromatographic profiling, a recent report documented the ability of the technique to spot quantitative differences reliably in the extent of b1,6branching between samples of normal bladder and superficial cancer [19] The array data are in full accordance with this interpretation and, furthermore, render any influence of the responsible enzyme GnT-V on the malignant phenotype by a nonenzymatic mechanism as unlikely (i.e its angiogenic property assigned to the basic region between amino acids 254–269 and responsible for mobilization of fibroblast growth factor-2 in situ) [68] At this stage, and with focus on the a5b1-integrin, we have thus detected at least two changes with proven impact on its routing and binding activity The binding of b-galactoside-specific plant lectins intimated a new role, attributing to the integrin’s glycans the potential to become ligands for endogenous lectins This finding prompted studies with human lectins, guided by array data with prominent positioning of galectin-1 among 1996 cancer-related genes Indeed, this endogenous lectin appears to exploit the increased level of a5b1-integrin expression with a2,6-hyposialylation of N-glycans Regarding GnT-V activity, it is fitting that galectin-1 does not discriminate between type-I- and type-II-branched triantennary N-glycans [69] In addition to the N-glycans, mucin-type core O-glycans, whose establishment and galactosylation is strongly favored by the up-regulation of b4GalT-IV [55], and the reduced core a2,3-sialylation can contribute to increased galectin-1 binding The differential degree of PNA positivity between p16INK4a-positive and mock control cells after sialidase treatment argues in favor of increased core presentation associated with the presence of p16INK4a The transit from core to core structures can prove advantageous, because model studies on asialofetuin with its three core disaccharides have shown no binding of galectins to these epitopes [44,70] Moreover, local cluster formation to accomplish high-density presentation of galactose residues will be beneficial for avidity, because short core structures appear to require chain extensions to acquire the capacity of high-affinity ligands [71] As galectin-1 binding to activated T cells attests [72], N- and O-glycans can both contribute to establish such a suitable topology The ensuing high-affinity FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS New function of p16INK4a ´ S Andre et al binding will then hardly be competed for by haptenic sugar, which was, in fact, noted experimentally The high-affinity binding therefore can be considered to be a cellular manifestation for results obtained in a recent model study on galectin binding to multivalent ligands, discovering negative co-operativity [44] In other words, the reduced level of a2,6-sialylation of N-glycans in concert with the reduced a2,3-sialylation of O-glycans, itself a factor promoting galectin-1 binding to Lec2 CHO mutant cells defective in transport of activated sialic acid into the Golgi [73], and the presence of short-length core structures presenting clustered galactose residues, enhance the binding avidity for an endogenous lectin That topology of ligand presentation is a salient factor for galectin binding is highlighted by systematic interaction analyses using natural glycoproteins and differential ligand selection on the level of glycoproteins between galectins despite their close sequence similarity [74–78] That the lectin is an endogenous effector is documented by its increased production in p16INK4a-reconstituted cells and the functional assays In this combination, our data epitomize an elegant orchestration of gene expression for glycosyltransferases and galectin-1 with functional consequence The general tissue-specific manner of expression of glycosyltransferase genes in murine organs [79,80], as well as the way that either tumor necrosis factor-a alters this parameter in endothelial cells [81] or the transcription factors Stat4 and T-bet prepare T cells for selectin binding [82,83], are quoted at this point to sensitize the reader to appraise the assumed wide range of this fundamental concept At the molecular level, it is, in our case, not yet clear whether the tumor suppressor acts on the expression of the quoted genes directly or indirectly With respect to tumor cells, the case of the differentiation-dependent activation of a cell-surface ganglioside sialidase, which engenders the inhibition of neuroblastoma proliferation in vitro by interaction between the product of its activity (i.e ganglioside GM1, and galectin-1 in neuroblastoma cells), even points to a therapeutic perspective [84–88] Scouring the records on galectin-1 teaches the lesson that it is not only a negative growth regulator in p16INK4a-positive cells and the mentioned SK-N-MC neuroblastoma model It is reactive, too, with other carcinoma cells [28] and was related to differentiation and apoptosis after its expression was induced by butyrate treatment in human LNCaP prostate cancer cells [89] Interestingly, its appearance followed p16INK4a induction by 5-azacytidine exposure of BL36 Burkitt lymphoma cells [90] Because the presence of p16 INK4a did not activate transcription nonspecifically (please see gene array and proteomics data as well as the illustrated proliferating cell nuclear antigen control) and Dan-G pancreatic carcinoma cells, which retain high expression of the pRb in contrast to Capan-1 cells [91], respond to the presence of p16INK4a with the same regulation of galectin-1, these data, albeit not allowing full exclusion of a common effect by tumor suppression, at least indicate an association to p16INK4a presence irrespective of the activity of pRb Based on the illustrated western blotting, an occurrence of galectin-1 variants, as described to be produced under the influence of fosB gene products [92], could definitely be ruled out When proceeding to look at tumors in situ, it might be expected that galectin-1 is not an abundant gene product in pancreas cancer Somewhat surprisingly, galectin-1 was found to be up-regulated in cancer tissue by expression profiling, an at-first puzzling result, but its localization was confined to fibroblasts and fibrotic tissue in and around tumors [33,93,94] By the way, the mode of localization of a galectin is a key issue also for tumor cells, as galectin-1 co-operates closely with oncogenic H-ras intracellularly [95] In summary, we have designed a combined strategy to discover changes in glycomic profile and lectin expression with general applicability The predictive value of individual data sets from the arrays was remarkable The presented results provide evidence for a co-ordinated regulation of glycosylation to increase galectin-1 binding and of expression of this endogenous lectin in p16INK4a-positive Capan-1 pancreatic carcinoma cells towards a common aim The coregulation of lectin ⁄ lectin ligand display is a biochemical means to acquire susceptibility to anoikis at the level of cell physiology It is evocative of mechanisms of cellular response to inflammation [6] This co-ordinated regulation of lectin ⁄ lectin ligand presentation is assumed to be of more general relevance in tumor biology To give an inspiring example, independent studies on glioma invasiveness have made a strong case for either a2,6-sialylation or galectin-1 as key factors without so far drawing a connection and envisioning a therapeutic perspective [96–99] When considering the next steps, the results give our research a clear direction to (a) pinpoint the molecular cause for reduced sialylation, which may for example reside in CMP-sialic acid transporter deficiency, as seen for two such protein types in the congenital disorders of glycosylation IIc and IIf [100,101], (b) monitor other cell and suppressor types to define the range and specificity of the detected effect, (c) delineate the intranetwork functionality of galectins, because galectin-3, a preferentially monomeric protein [102] produced and secreted from Capan-1 wild-type FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS 3247 New function of p16INK4a ´ S Andre et al cells and inactive to elicit anoikis, served as an endogenous inhibitor of galectin-1-dependent effects in neuroblastoma cells [86] and potent activator of K-ras [103], as well as (d) dissect the same aspect of intranetwork functionality in the cases of the b4GalTs and ST3Gals Experimental procedures Reagents and cells A panel of 22 biotinylated plant lectins, reactive with distinct building blocks of human glycans, was purchased from Vector Laboratories (distributed by Alexis Germany, Grună berg, Germany), supplemented by concanavalin A and Viscum album agglutinin and rigorously checked for activity by solid-phase and histochemical assays as positive controls, as described previously [104,105] The two mentioned plant lectins and the panel of human galectins obtained by recombinant production were purified by affinity chromatography on lactose- or mannose-bearing Sepharose 4B resins, prepared with divinyl sulfone (Fluka, Munich, Germany) for activation, and protein quality was routinely controlled by 1- and 2D gel electrophoresis, gel filtration and mass spectrometry as well as hemagglutination, solid-phase and cell assays [44,84,106–108] The megaprimer PCR technique was used to generate W68L ⁄ E71Q mutants of human galectin-1 as His-tagged proteins, which were purified by affinity chromatography on a Ni-CAMTM HC resin (Sigma, Munich, Germany) Biotinylation of the natural and mutant proteins was carried out with the N-hydroxysuccinimide ester derivative (Sigma) under activity-preserving conditions, product quality was ascertained by solid-phase assays and the extent of biotinylation quantitatively determined by measuring the pI alterations in 2D gel electrophoresis and setting them in relation to stepwise loss of free amino groups, as described previously [109,110] Cells of the human pancreatic carcinoma line Capan-1 (HTB 79; American Type Culture Collection, Rockville, MD, USA) stably transfected with full-length cDNA for human p16INK4a inserted into a pRC ⁄ CMV vector or with control vector (mock treatment) were routinely grown as monolayers in RPMI 1640 medium supplemented with 15% fetal bovine serum (Biochrom, Berlin, Germany), mm l-glutamine, 100 mL)1 of penicillin and 100 lgỈmL)1 of streptomycin, as described previously [30] Cell clones from wild-type cells overexpressing galectin1, or from p16INK4a-positive cells harboring galectin-1-specific cDNA in an antisense orientation, were generated using the pcDNA3.1 system and hygromycin at a concentration of 100 lgỈmL)1 for selection, as described previously [104] For determination of anoikis, · 105 cells per assay were cultured in suspension on a surface coated with polyhydroxyethylmethacrylate, and the cell cycle distribution, including percentage of cells in the pre-G1 fraction, was assessed using the cellquestTM program [30] 3248 Microarray and quantitative real-time PCR analyses RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions The quantity and quality of the products were determined by measurements in the NanoDrop ND-1000 UV ⁄ VIS spectrophotometer (Peqlab, Erlangen, Germany) and the Bioanalyzer (Agilent, Waldbronn, Germany), respectively, routinely resulting in RNA integrity number values between 9.5 and 10 The SuperScript Plus Direct cDNA Labeling System with Alexa Fluor aha-dUTPs (Invitrogen ⁄ Life Technologies, Karlsruhe, Germany) was used for the production of cDNA harboring fluorescent tagging Its efficiency was routinely monitored by adsorption measurements using a Nanodrop ND100 UV ⁄ VIS spectrophotometer Aliquots containing the labeled cDNA were diluted to a volume of 200 lL in Microarray Hybridization Solution, Version (Amersham Biosciences, Braunschweig, Germany) and hybridized onto the GlycoProfiler microarray (Scienion, Berlin, Germany) using an a-Hyb hybridization station (Miltenyi, Cologne, Germany) for 16 h at 42 °C Subsequent washing steps were then performed using · NaCl ⁄ Cit, 0.2% SDS as first, · NaCl ⁄ Cit as second and 0.05 · NaCl ⁄ Cit as third wash buffer The glass slides were dried in the centrifuge and scanned using the Agilent DNA Microarray Scanner, and the obtained data were subjected to analysis using the feature extraction 8.0 software (Agilent) The GlycoProfiler microarray comprising 60-mer gene sequences for selected human glycosyltransferases and lectins, in addition to internal housekeeping controls, is based on the quadruplicate spotting of each gene sequence onto glass slides Differential gene expression was examined by competitive array hybridization of fluorescently labeled probes (Alexa 555 versus Alexa 647) prepared from p16INK4a- and mock-transfected cells, respectively Dye biases were removed by using the feature extraction 8.1 software (Agilent) with Rank Consistent Probes as the dye-normalization probe selection method and Linear and Lowess as the normalization correction method, hereby accounting for signal intensitydependent dye biases The Linear and Lowess method applies a linear normalization to the entire range of data, then executes a nonlinear normalization to the linearized data set Errors were routinely computed using the setting ‘Most Conservative’, thereby considering a propagated error model, which is advantageous for low-intensity features, and a universal error model, instrumental for highintensity data Two independent series with four individual measurements in each series were carried out with standard deviations not surpassing the 10–12% range Quantitative real-time PCR controls used M-MLV reverse-transcriptasedriven cDNA synthesis Expression levels were determined in triplicate by assays-on-demand, delivered by Applied FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS New function of p16INK4a ´ S Andre et al Biosystems (Darmstadt, Germany), running the predeveloped b-actin assay 4310881E as a routine control After addition of TaqMan Universal ⁄ SYBR-Green PCR Master Mix, the standard experiment was run in a 7000 Sequence Detection System (Applied Biosystems) Thermal cycle conditions were as follows: 95 °C for 10 followed by 40 cycles of 95 °C for 15 s and then 60 °C for Data analysis was performed according to the DD threshold cycle method, using threshold cycle values of a cDNA stock prepared from cells of the colon carcinoma line LS174T as a calibrator [111] For an independent expression profiling of 1966 genes with a known or assumed role in pancreas cancer development [for a complete listing, please see http://www.uni-ulm.de/klinik/medklinik/innere1/ forschung/ag-gress/index.html (username: ag-gress, password genom)], a custom-made array was designed on Hybond N+ nylon membranes using a MicroGrid II arrayer (Bio Robotics Ltd, Cambridge, UK) Hybridization probes were generated from 30 lg of total RNA by oligodT-primed reverse transcription using the Superscript II enzyme (Gibco BRL, Karlsruhe, Germany) and [32P]dATP[aP] Probes were competed with 0.25 lgỈmL)1 of sonified cDNA from human placenta (Sigma) and COT-1 DNA (Gibco) for h Hybridizations were routinely performed in triplicate, the processed nylon membranes were scanned with a Storm 8600 Phosphoimager (Molecular Dynamics GmbH, Krefeld, Germany) and image analysis was performed with the arrayvision software (Imaging Research Inc., St Catharines, Canada), including correction for local background and normalization to mean signal intensities Glycan profiling by HPLC analysis N-Glycans from the cells were obtained and labeled by pyridylamination, as described previously [112,113] In detail, seven individual preparations of each cell type of  10 mg of total protein were carefully washed, heated to 90 °C for 15 and then lyophilized Then, each sample was dissolved in 200 lL of 50 mm Tris ⁄ HCl buffer (pH 8.0), containing 10 mm CaCl2, and digested with 100 lg each of trypsin (Sigma) and chymotrypsin (Calbiochem, Darmstadt, Germany) at 37 °C for 16 h After again heating at 90 °C for 10 to inactivate the proteases, samples were treated with 10 U of N-glycosidase F (recombinant; Roche Molecular Biochemicals, Mannheim, Germany) at 37 °C for 16 h to release N-glycans from glycoproteins The remaining peptides were digested with 100 lg of pronase (Calbiochem) before glycan purification by gel filtration chromatography The resulting N-glycans were fractionated using a Bio-Gel P-4 column (200–400 mesh; Bio-Rad, Munich, Germany) with H2O as the eluant and thoroughly 2-aminopyridylated via reductive amination with excess 2-aminopyridine (PA; Wako Pure Chemical, Osaka, Japan) ⁄ HCl (pH 6.8) and sodium cyanoborohydride (Sigma) at 90 °C for h [114] Derivatized N-glycans were separated from reagents on a Sephadex G-15 column (Amersham Biosciences) with 10 mm ammonium bicarbonate solution as eluant The fraction containing the glycans was acid-hydrolyzed at 90 °C for h with 0.01 m HCl (pH 2.0) to remove sialic acids from the oligosaccharides and subjected to the 2D mapping Structural assignment was based on the elution positions in the two modes of HPLC analysis by comparing these results with a database consisting of a total of 480 different types of N-glycans, as described previously [115], and publicly accessible with detailed structures and nomenclature (http://www glycoanalysis.info/index.html) An in-house control with PA-derivatized N-glycans from natural sources (e.g M5.1, M6.1 and M9.1 from ribonuclease B) was included In technical detail, the mixtures of neutral PA-derivatized N-glycans were first separated on an octadecyl silane (ODS) reverse-phase column (ShimPack HRC-ODS, · 150 mm; Shimadzu, Kyoto, Japan) by HPLC (D-7000 HPLC system equipped with an L-7485 fluorescence detector from Hitachi High-Technologies Co., Tokyo, Japan) Elution was performed at a flow rate of 1.0 mLỈmin)1 at 55 °C using a linear gradient elution system Solvent A was a 10 mm sodium phosphate buffer (pH 3.8) and solvent B a 10 mm sodium phosphate buffer (pH 3.8) containing 0.5% (v ⁄ v) 1-butanol The column was equilibrated with a mixture of solvents A and B (80 : 20, v ⁄ v) After sample injection, elution was performed using a linear gradient to a solvent ratio of 40 : 60 (A ⁄ B, v ⁄ v) in 80 Seven samples of each cell type revealed very similar patterns, enabling pooling for further analysis Material of each peak separated on the ODS column was collected and then applied to the amide-adsorption column (TSKgel Amide80, 4.6 · 250 mm) Elution from that column was performed with a flow rate of 1.0 mLỈmin)1 at 40 °C using a gradient elution system Solvents C and D were composed of 3% acetic acid-triethylamine buffer (pH 7.3) and acetonitrile at 35 : 65 and 50 : 50 (v ⁄ v), respectively The column was first equilibrated with solvent C, and elution was performed using a linear gradient from a solvent ratio of 100 : to 40 : 60 (v ⁄ v) of solvents C and D in 30 In both HPLC systems, the positions of elution of PA-derivatized N-glycans were spotted by a fluorescence detector using excitation and emission wavelengths of 320 and 400 nm, respectively The molar ratio of PA oligosaccharides was calculated based on peak areas of fluorescence intensity Glycan profiling by cell-surface lectin staining Quantitative determination of carbohydrate-dependent lectin binding using streptavidin ⁄ R-phycoerythrin (1 : 40) as a fluorescent marker (Sigma) was performed by flow cytofluorometry in a FACScan instrument (Becton-Dickinson, Heidelberg, Germany) Solutions with · 105 cells per FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS 3249 New function of p16INK4a ´ S Andre et al sample were carefully washed to remove any interfering serum compounds, the extent of protein binding by nonspecific interaction was reduced by an incubation step in Dulbecco’s phosphate-buffered saline containing 100 lgỈmL)1 of carbohydrate-free bovine serum albumin, and the biotinylated lectins were incubated with the cells for 30 at °C to minimize endocytic uptake, as described in detail previously [116–119] Control experiments without the incubation step with labeled lectin, or in the presence of haptenic sugar to block carbohydrate-dependent binding, as well as systematic titrations to define optimal concentrations, were run for each cell batch in parallel Sialidase treatment was performed for 60 at 37 °C with 0.2 mL)1 of sialidase (from Clostridium perfringens; Roche) Proteomic analysis and galectin-1 identification Samples of 200 lg of extract protein were prepared by lysis of frozen cells in 40 mm Tris ⁄ HCl containing m urea, 4% w ⁄ v 3-[[3-cholamidopropyl]-dimethylammonio]-1-propanesulfonate, 0.5% IPG buffer (pH 3–10; GE Healthcare Europe GmbH, Freiburg, Germany) and 20 mm dithiothreitol After incubation for h at room temperature, the solution was cleared by centrifugation for at 10 000 g with a 5415D centrifuge with F45-24-11 rotor (Eppendorf, Hamburg, Germany) 2D gel electrophoresis was carried out in a pI range of 3–10 in the IPGphorTM unit at  42 kVh, then the Hoefer SE 600 System was used for separation for h at 100 mA and h at 200 mA per four-gel set, as described previously [110] Silver staining and processing with the imagescanner (Labscan version 3.0; GE Healthcare Europe GmbH), using the ImageMasterÒ 2d elite software 3.1 package, yielded quantitative data for comparison of protein expression from six to seven different gels per cell population, further subjected to statistical analysis with the graphpad prism 3.0 package Galectin-1 identification was performed by western blotting using a specific polyclonal IgG fraction (1 : 1000) rigorously checked for the absence of cross-reactivity against other human galectins and for sensitive detection by enhanced chemiluminescence as well as by flow cytofluorometry on native cells using the commercial fluorescein thiocarbamyllabeled conjugate of goat anti-rabbit IgG (Sigma), as described previously [104] This procedure was also used for galectin-1 detection in preparations of the fibronectin receptor immunoprecipitated from 800 lg of total extract protein using a commercial antibody against the a5-integrin (Cymbus Biotechnology, Chandlers Ford, UK), as described previously [120] Mass-spectrometric fingerprinting of tryptic peptides was carried out after gel electrophoretic separation in two dimensions and Coomassie blue staining Following excision of the gel piece with the spot and its washing first with deionized water, then with acetonitrile in water (1 : 1, v ⁄ v) and finally with acetonitrile, protein was digested in situ using 100 ng of sequencing-grade trypsin (Promega, 3250 Mannheim, Germany) Sample preparation for mass-spectrometric analysis by cocrystallization with the matrix (a-cyano-4-hydroycinnamic acid) followed and the mass profile of the tryptic peptides was then recorded in the positiveion-reflector mode on a time-of-flight instrument (BrukerDaltronik, Bremen, Germany) Ion acceleration voltage was set to 26 kV and reflector voltage to 30 kV The spectrum was obtained by averaging 300 individual laser shots Database search for assignment was performed against the swissprot archive using the mascot search algorithm Acknowledgements We are indebted to Dr R Brossmer, Dr B Friday, Dr R Hase, Professor S Namirha and Dr A Villalobo for insightful discussions and critical reading of the manuscript, to the reviewers of our manuscript for their valuable input, to B Hofer, S Kudo and L Mantel for excellent technical assistance, as well as to the Deutsche Krebshilfe, Mizutani Foundation for Glycoscience, National Project on Functional Glycoconjugate Research Aimed at Developing for New Industry from the Ministry of Education, Science, Sports and Culture of Japan, Verein zur Forderung des biologisch-technolă ogischen Fortschritts in der Medizin e V and the Wilhelm-Sander Stiftung for generous financial support References Sharon N & Lis H (1997) Glycoproteins: structure and function In Glycosciences: Status and Perspectives (Gabius H-J & Gabius S, eds), pp 133–162 Chapman & Hall, Weinheim, Germany Reuter G & Gabius H-J (1999) Eukaryotic glycosylation: whim of nature or multipurpose tool? Cell Mol Life Sci 55, 368–422 Lowe JB & Marth JD (2003) A genetic approach to mammalian glycan function Annu Rev Biochem 72, 643–691 Laine R (1997) The information-storing potential of the sugar code In Glycosciences: Status and Perspectives (Gabius H-J & Gabius S, eds), pp 1–14 Chapman & Hall, Weinheim, Germany Hebert DN, Garman SC & Molinari M (2005) The glycan code of the endoplasmic reticulum: asparagine-linked carbohydrates as protein maturation and quality-control tags Trends Cell Biol 15, 364–370 Gabius H-J (2006) Cell surface glycans: the why and how of their functionality as biochemical signals in lectin-mediated information transfer Crit Rev Immunol 26, 43–80 ´ Villalobo A, Nogales-Gonzales A & Gabius H-J (2006) A guide to signalling pathways connecting protein– FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS New function of p16INK4a ´ S Andre et al 10 11 12 13 14 15 16 17 18 glycan interaction with the emerging versatile effector functionality of mammalian lectins Trends Glycosci Glycotechnol 18, 1–37 Alhadeff JA (1989) Malignant cell glycoproteins and glycolipids Crit Rev Oncol Hematol 9, 37–107 Hakomori S-i (1989) Aberrant glycosylation in tumors and tumor-associated carbohydrate antigens Adv Cancer Res 52, 257–331 Yamashita K, Ohkura T, TachibanaY, Takasaki S & Kobata A (1984) Comparative study of the oligosaccharides released from baby hamster kidney cells and their polyoma transformant by hydrazinolysis J Biol Chem 259, 10834–10840 Pierce M & Arango J (1986) Rous sarcoma virustransformed baby hamster kidney cells express higher levels of asparagine-linked tri- and tetraantennary glycopeptides containing [GlcNAc-b1,6Man-a1,6Man] and poly-N-acetyllactosamine sequences than baby hamster kidney cells J Biol Chem 261, 10772– 10777 Hubbard SC (1987) Differential effects of oncogenic transformation on N-linked oligosaccharide processing at individual glycosylation sites of viral glycoproteins J Biol Chem 262, 16403–16411 Easton EW, Bolscher JGM & van den Eijnden DH (1991) Enzymatic amplification involving glycosyltransferases forms the basis for the increased size of asparagine-linked glycans at the surface of NIH 3T3 cells expressing the N-ras proto-oncogene J Biol Chem 266, 21674–21680 Buckhaults P, Chen L, Fregien N & Pierce M (1997) Transcriptional regulation of N-acetylglucosaminyltransferase V by the src oncogene J Biol Chem 272, 19575–19581 Chen L, Zhang W, Fregien N & Pierce M (1998) The her-2 ⁄ neu oncogene stimulates the transcription of N-acetylglucosaminyltransferase V and expression of its cell surface oligosaccharide products Oncogene 17, 2087–2093 Dalziel M, Dall’Olio F, Mungul A, Piller V & Piller F (2004) Ras oncogene induces b-galactoside a2,6-sialyltransferase (ST6Gal I) via a RalGEF-mediated signal to its housekeeping promoter Eur J Biochem 271, 3623–3634 Dosaka-Akita H, Miyoshi E, Suzuki O, Itoh T, Katoh H & Taniguchi N (2004) Expression of N-acetylglucosaminyltransferase V is associated with prognosis and histology in non-small cell lung cancers Clin Cancer Res 10, 1773–1779 Inamori K-i, Gu J, Ohira M, Kawasaki A, Nakamura Y, Nakagawa T, Kondo A, Miyoshi E, Nakagawara A & Taniguchi N (2006) High expression of N-acetylglucosaminyltransferase V in favorable neuroblastomas: involvement of its effect on apoptosis FEBS Lett 580, 627–632 19 Ishimura H, Takahashi T, Nakagawa H, Nishimura S-I, Arai Y, Horikawa Y, Habuchi T, Miyoshi E, Kyan A, Hagisawa S et al (2006) N-Acetylglucosaminyltransferase V and b1–6 branching of N-linked oligosaccharides are associated with good prognosis of patients with bladder cancer Clin Cancer Res 12, 2506–2511 20 Asada M, Furukawa K, Segawa K, Endo T & Kobata A (1997) Increased expression of highly branched N-glycans at cell surface is correlated with the malignant phenotypes of mouse tumor cells Cancer Res 57, 1073–1080 21 Guo HB, Lee I, Kamar M, Akiyama SK & Pierce M (2002) Aberrant N-glycosylation of b1 integrin causes reduced a5b1 integrin clustering and stimulates cell migration Cancer Res 62, 6837–6845 22 Seales EC, Jurado GA, Singhal A & Bellis SL (2003) Ras oncogene directs expression of a differentially sialylated, functionally altered b1 integrin Oncogene 22, 7137–7145 23 Nakagawa H, Zheng M, Hakomori S-i, Tsukamoto Y, Kawamura Y & Takahashi N (1996) Detailed oligosaccharide structures of human integrin a5b1 analyzed by a three-dimensional mapping technique Eur J Biochem 237, 76–85 24 Akiyama SK, Yamada SS & Yamada KM (1989) Analysis of the role of glycosylation of the human fibronectin receptor J Biol Chem 264, 18011–18018 25 Zheng M, Fang H & Hakomori S-i (1994) Functional role of N-glycosylation in a5b1 integrin receptor De-N-glycosylation induces dissociation or altered association of a5 and b1 subunits and concomitant loss of fibronectin binding activity J Biol Chem 269, 12325–12331 26 Nadanaka S, Sato C, Kitajima K, Katagiri K, Irie S & Yamagata T (2001) Occurrence of oligosialic acids on integrin a5 subunit and their involvement in cell adhesion to fibronectin J Biol Chem 276, 33657– 33664 27 Wang X, Sun P, Al-Qamari A, Tai T, Kawashima I & Paller AS (2001) Carbohydrate-carbohydrate binding of ganglioside to integrin a5 modulates a5b1 function J Biol Chem 276, 8436–8444 28 Fischer C, Sanchez-Ruderisch H, Welzel M, Wieden´ mann B, Sakai T, Andre S, Gabius H-J, Khachigian L, Detjen KM & Rosewicz S (2005) Galectin-1 interacts with the a5b1 fibronectin receptor to restrict carcinoma cell growth via induction of p21 and p27 J Biol Chem 280, 37266–37277 ´ 29 Carcamo C, Pardo E, Oyanadel C, Bravo-Zehnder M, ´ Bull P, Caceres M, Martı´ nez J, Massardo L, Jacobelli ´ S, Gonzalez A et al (2006) Galectin-8 binds specific b1 integrins and induces polarized spreading highlighted by asymmetric lamellipodia in Jurkat T cells Exp Cell Res 312, 374–386 FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS 3251 New function of p16INK4a ´ S Andre et al 30 Plath T, Detjen K, Welzel M, von Marschall Z, Murphy D, Schirner M, Wiedenmann B & Rosewicz S (2000) A novel function for the tumor suppressor p16 (INK4a): induction of anoikis via upregulation of the a5b1 fibronectin receptor J Cell Biol 150, 1467–1478 31 Zhang SW, Fu XY, Cao SL, Shen ZH & Gu JX (1999) Down-regulation of b1,4-galactosyltransferase gene expression by cell-cycle suppressor gene p16 Biochim Biophys Acta 1444, 49–54 32 Kawado T, Hayashi O, Sato T, Ito H, Hayakawa S, Takayama E & Furukawa K (2004) Rapid cell senescence-associated changes in galactosylation of N-linked oligosaccharides in human lung adenocarcinoma A549 cells Arch Biochem Biophys 426, 306–313 33 Ide Y, Miyoshi E, Nakagawa T, Gu J, Tanemura M, Nishida T, Ito T, Yamamoto H, Kozutsumi Y & Taniguchi N (2006) Aberrant expression of N-acetylglucosaminyltransferase-IVa and IVb (GnT-IVa and b) in pancreatic cancer Biochem Biophys Res Commun 341, 478–482 34 Sato T, Shirane K, Kido M & Furukawa K (2000) Correlated gene expression between b-1,4-galactosyltransferase V and N-acetylglucosaminyltransferase V in human cancer cell lines Biochem Biophys Res Commun 276, 1019–1023 35 Ko JH, Miyoshi E, Noda K, Ekuni A, Kang R, Ikeda Y & Taniguchi N (1999) Regulation of the GnT-V promoter by transcription factor Ets-1 in various cancer cell lines J Biol Chem 274, 22941–22948 36 Sato T & Furukawa K (2004) Transcriptional regulation of the human b1,4-galactosyltransferase V gene in cancer cells: essential role of transcription factor Sp1 J Biol Chem 279, 39574–39583 37 Zhu X, Jiang J, Shen H, Wang H, Zong H, Li Z, Yang Y, Niu Z, Liu W, Chen X et al (2005) Elevated b1,4-galactosyltransferase I in highly metastatic human lung cancer cells Identification of E1AF as important transcription activator J Biol Chem 280, 12503–12516 38 Jiang J, Chen X, Shen J, Wei Y, Wu T, Yang Y, Wang H, Zong H, Yang J, Zhang S et al (2006) b1,4Galactosyltransferase V functions as a positive growth regulator in glioma J Biol Chem 281, 9482–9489 39 Chen X, Jiang J, Yang J, Chen C, Sun M, Wei Y, Guang X & Gu J (2006) Down-regulation of the expression of b1,4-galactosyltransferase V promotes integrin b1 maturation Biochem Biophys Res Commun 343, 910–916 40 Guo H-B, Lee I, Bryan BT & Pierce M (2005) Deletion of mouse embryo fibroblast N-acetylglucosaminyltransferase V stimulates a5b1 integrin expression mediated by the protein kinase C signaling pathway J Biol Chem 280, 8332–8342 3252 41 Guo P, Zhang Y, Zhao J-H, Wang L-y, Guo HB, Zhang X-y, Chen H & -1 (2003) Regulation on the expression and N-glycosylation of integrins by N-acetylglucosaminyltransferase V Biochem Biophys Res Commun 310, 619–626 42 Gabius H-J (1997) Animal lectins Eur J Biochem 243, 543–576 ´ 43 Ahmad N, Gabius H-J, Kaltner H, Andre S, Kuwabara I, Liu F-T, Oscarson S, Norberg T & Brewer CF (2002) Thermodynamic binding studies of cell surface carbohydrate epitopes to galectins-1, -3 and -7: evidence for differential binding specificities Can J Chem 80, 1096–1104 ´ 44 Dam TK, Gabius H-J, Andre S, Kaltner H, Lensch M & Brewer CF (2005) Galectins bind to the multivalent glycoprotein asialofetuin with enhanced affinities and a gradient of decreasing binding constants Biochemistry 44, 12564–12571 45 Gabius H-J (2001) Glycohistochemistry: the why and how of detection and localization of endogenous lectins Anat Histol Embryol 30, 3–31 ´ ´ 46 Gabius H-J, Siebert H-C, Andre S, Jimenez-Barbero J & Rudiger H (2004) Chemical biology of the sugar ă code ChemBioChem 5, 740764 47 Andre S, Unverzagt C, Kojima S, Frank M, Seifert J, Fink C, Kayser K, von der Lieth C-W & Gabius H-J (2004) Determination of modulation of ligand properties of synthetic complex-type biantennary N-glycans by introduction of bisecting GlcNAc in silico, in vitro and in vivo Eur J Biochem 271, 118–134 ´ 48 Andre S, Kojima S, Prahl I, Lensch M, Unverzagt C & Gabius H-J (2005) Introduction of extended LEC14type branching into core-fucosylated biantennary N-glycan FEBS J 272, 1986–1998 49 Park EI, Mi Y, Unverzagt C, Gabius H-J & Baenziger JU (2005) The asialoglycoprotein receptor clears glycoconjugates terminating with sialic acida2,6 GalNAc Proc Natl Acad Sci USA 102, 17125–17129 50 Itai S, Arii S, Tobe R, Kitahara A, Kim YC, Yamabe H, Ohtsuki H, Kirihara Y, Shigeta K & Kannagi R (1988) Significance of 2–3 and 2–6 sialylation of Lewisa antigen in pancreas cancer Cancer 61, 775–787 51 Miyazaki K, Ohmori K, Izawa M, Koike T, Kumamoto K, Furukawa K, Ando T, Kiso M, Yamaji T, Hashimoto Y et al (2004) Loss of disialyl Lewisa, the ligand for lymphocyte inhibitory receptor sialic acidbinding immunoglobulin-like lectin-7 (siglec-7), associated with increased sialyl Lewisa expression on human colon cancers Cancer Res 64, 4498–4505 ´ ´ 52 Peracaula R, Tabares G, Lopez-Ferrer A, Brossmer R, ´ de Bolos C & de Llorens R (2005) Role of sialyltransferases involved in the biosynthesis of Lewis antigens in human pancreatic tumour cells Glycoconjugate J 22, 135–144 FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS New function of p16INK4a ´ S Andre et al 53 Hanßke B, Thiel C, Lubke T, Hasilik M, Honing S, ă ă Peters V, Heidemann PH, Hoffmann GF, Berger EG, von Figura K et al (2002) Deficiency of UDP-galactose: N-acetylglucosamine b1,4-galactosyltransferase I causes the congenital disorder of glycosylation type IId J Clin Invest 109, 725–733 54 Kotani N, Asano M, Inoue N, Iwakura Y & Takasaki S (2004) Polylactosamine synthesis and branch formation of N-glycans in b1,4-galactosyltransferase-1-deficient mice Arch Biochem Biophys 426, 258–265 55 Ujita M, McAuliffe J, Schwientek T, Almeida R, Hindsgaul O, Clausen H & Fukuda M (1998) Synthesis of poly-N-acetyllactosamine in core branched O-glycans The requirement of novel b1,4-galactosyltransferase IV and b1,3-N-acetylglucosaminyltransferase J Biol Chem 273, 34843–34849 56 van Die I, van Tetering A, Schiphorst WECM, Sato T, Furukawa K & van den Eijnden DH (1999) The acceptor substrate specificity of human b4-galactosyltransferase V indicates its potential function in O-glycosylation FEBS Lett 450, 52–56 57 Brockhausen I, Matta KL, Orr J, Schachter H, Koenderman AH & van den Eijnden DH (1986) Mucin synthesis Conversion of R1-b1–3Gal-R2 to R1-b1–3 (GlcNAc b1–6) Gal-R2 and of R1-b1–3GalNAc-R2 to R1-b1–3 (GlcNAc b1–6) GalNAc-R2 by a b6-N-acetylglucosaminyltransferase in pig gastric mucosa Eur J Biochem 157, 463–474 58 Huet G, Hennebicq-Reig S, de Bolos C, Ulloa F, Lesuffleur T, Barbat A, Carriere V, Kim I, Real FX, Delannoy P et al (1998) GalNAc-a-O-benzyl inhibits NeuAca2–3 glycosylation and blocks the intracellular transport of apical glycoproteins and mucus in differentiated HT-29 cells J Cell Biol 141, 1311–1322 59 Semel AC, Seales EC, Singhal A, Eklund EA, Colley KJ & Bellis SL (2002) Hyposialylation of integrins stimulates the activity of myeloid fibronectin receptors J Biol Chem 277, 32830–32836 60 Zhu Y, Srivatana U, Ullah A, Gagneja H, Berenson CS & Lance P (2001) Suppression of a sialyltransferase by antisense DNA reduces invasiveness of human colon cancer cells in vitro Biochim Biophys Acta 1536, 148–160 61 Lin S, Kemmner W, Grigull S & Schlag PM (2002) Cell surface a2,6 sialylation affects adhesion of breast carcinoma cells Exp Cell Res 276, 101–110 62 Seales EC, Jurado GA, Brunson BA, Wakefield JK, Frost AR & Bellis SL (2005) Hypersialylation of b1 integrins, observed in colon adenocarcinoma, may contribute to cancer progression by up-regulating cell motility Cancer Res 65, 4645–4652 63 Hammarstrom S, Hammarstrom M-L, Sundblad G, ă ă Arnarp J & Lonngren J (1982) Mitogenic leukoă agglutinin from Phaseolus vulgaris binds to a pentasaccharide unit in N-acetyllactosamine-type 64 65 66 67 68 69 70 71 72 73 glycoprotein glycans Proc Natl Acad Sci USA 79, 1611–1615 Green ED & Baenziger JU (1987) Oligosaccharide specificities of Phaseolus vulgaris leukoagglutinating and erythroagglutinating phytohemagglutinins Interactions with N-glycanase-released oligosaccharides J Biol Chem 262, 12018–12029 Yamashita K, Totani K, Ohkura T, Takasaki S, Goldstein IJ & Kobata A (1987) Carbohydrate binding properties of complex-type oligosaccharides on immobilized Datura stramonium lectin J Biol Chem 262, 1602–1607 Bierhuizen MFA, Edzes HT, Schiphorst WECM, van den Eijnden DH & van DijkW (1987) Effect of a2– 6-linked sialic acid and a1–3-linked fucose on the interaction of N-linked glycopeptides and related oligosaccharides with immobilized Phaseolus vulgaris leukoagglutinating lectin (L-PHA) Glycoconjugate J 5, 85–97 ´ Siebert H-C, Rosen J, Seyrek K, Kaltner H, Andre S, Bovin NV, Nyholm PG, Sinowatz F & Gabius H-J (2006) a2,3 ⁄ a2,6-Sialylation of N-glycans: non-synonymous signals with marked developmental regulation in bovine reproductive tracts Biochimie 88, 399– 410 Saito T, Miyoshi E, Sasai K, Nakano N, Eguchi H, Honke K & Taniguchi N (2002) A secreted type of b1,6-N-acetylglucosaminyltransferase V (GnT-V) induces tumor angiogenesis without mediation of glycosylation: a novel function of GnT-V distinct from the original glycosyltransferase activity J Biol Chem 277, 17002–17008 ´ Andre S, Kojima S, Gundel G, Russwurm R, Schratt X, Unverzagt C & Gabius H-J (2006) Branching mode in complex-type triantennary N-glycans as regulatory element of their ligand properties Biochim Biophys Acta 1760, 768–782 Gupta D, Kaltner H, Dong X, Gabius H-J & Brewer CF (1996) Comparative cross-linking activities of lactose-specific plant and animal lectins and a natural lactose-binding immunoglobulin G fraction from human serum with asialofetuin Glycobiology 6, 843–849 Leppanen A, Stowell S, Blixt O & Cummings RD ă (2005) Dimeric galectin-1 binds with high affinity to a2,3-sialylated and non-sialylated terminal N-acetyllactosamine units on surface-bound extended glycans J Biol Chem 280, 5549–5562 Amano M, Galvan M, He J & Baum LG (2003) The ST6Gal I sialyltransferase selectively modifies N-glycans on CD45 to negatively regulate galectin-1-induced CD45 clustering, phosphatase modulation, and T cell death J Biol Chem 278, 7469–7475 Patnaik SK, Potvin B, Carlsson S, Sturm D, Leffler H & Stanley P (2006) Complex N-glycans are the major FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS 3253 New function of p16INK4a 74 75 76 77 78 79 80 81 82 83 84 ´ S Andre et al ligands for galectins-1, -3, and -8 on Chinese hamster ovary cells Glycobiology 16, 305–317 Wu AM, Wu JH, Tsai M-S, Kaltner H & Gabius H-J (2001) Carbohydrate specificity of a galectin from chicken liver (CG-16) Biochem J 358, 529–538 ´ Siebert H-C, Andre S, Lu S-Y, Frank M, Kaltner H, van Kuik JA, Korchagina EY, Bovin NV, Tajkhorshid E, Kaptein R et al (2003) Unique conformer selection of human growth-regulatory lectin galectin-1 for ganglioside GM1 versus bacterial toxins Biochemistry 42, 14762–14773 ´ ´ Lopez-Lucendo MF, Solı´ s D, Andre S, Hirabayashi J, Kasai K-i, Kaltner H, Gabius H-J & Romero A (2004) Growth-regulatory human galectin-1: crystallographic characterisation of the structural changes induced by single-site mutations and their impact on the thermodynamics of ligand binding J Mol Biol 343, 957–970 ´ Sturm A, Lensch M, Andre S, Kaltner H, Wiedenmann B, Rosewicz S, Dignass AU & Gabius H-J (2004) Human galectin-2: novel inducer of T cell apoptosis with distinct profile of caspase activation J Immunol 173, 3825–3837 ´ Wu AM, Singh T, Wu JH, Lensch M, Andre S & Gabius H-J (2006) Interaction profile of galectin-5 with free saccharides and mammalian glycoproteins: probing its fine specificity and the effect of naturally clustered ligand presentation Glycobiology 16, 524–537 Smith FI, Qu Q, Hong SJ, Kim KS, Gilmartin TJ & Head SR (2005) Gene expression profiling of mouse postnatal cerebellar development using oligonucleotide microarrays designed to detect differences in glycoconjugate expression Gene Expr Patterns 5, 740–749 Comelli EM, Head SR, Gilmartin T, Whisenant T, Haslam SM, North SJ, Wong N-K, Kudo T, Narimatsu H, Esko JD et al (2006) A focused microarray approach to functional glycomics: transcriptional regulation of the glycome Glycobiology 16, 117–131 Garcı´ a-Vallejo JJ, van Dijk W, van Het-Hof B, van Die I, Engelse MA, van Hinsbergh VWM & Gringhuis SI (2006) Activation of human endothelial cells by tumor necrosis factor-a results in profound changes in the expression of glycosylation-related genes J Cell Physiol 206, 203–210 White SJ, Underhill GH, Kaplan MH & Kansas GS (2001) Differential requirements for Stat4 in expression of glycosyltransferases responsible for selectin ligand formation in Th1 cells J Immunol 167, 628–631 Underhill GH, Zisoulis DG, Kolli KP, Ellies LG, Marth JD & Kansas GS (2005) A crucial role for T-bet in selectin ligand expression in T helper (Th1) cells Blood 106, 3867–3873 ´ Kopitz J, Andre S, von Reitzenstein C, Versluis K, Kaltner H, Pieters RJ, Wasano K, Kuwabara I, Liu 3254 85 86 87 88 89 90 91 92 93 F-T, Cantz M et al (2003) Homodimeric galectin-7 (p53-induced gene 1) is a negative growth regulator for human neuroblastoma cells Oncogene 22, 6277– 6288 Kopitz J, von Reitzenstein C, Burchert M, Cantz M & Gabius H-J (1998) Galectin-1 is a major receptor for ganglioside GM1, a product of the growth-controlling activity of a cell surface ganglioside sialidase, on human neuroblastoma cells in culture J Biol Chem 273, 11205–11211 ´ Kopitz J, von Reitzenstein C, Andre S, Kaltner H, Uhl J, Ehemann V, Cantz M & Gabius H-J (2001) Negative regulation of neuroblastoma cell growth by carbohydrate-dependent surface binding of galectin-1 and functional divergence from galectin-3 J Biol Chem 276, 35917–35923 ´ Andre S, Kaltner H, Lensch M, Russwurm R, Siebert H-C, Fallsehr C, Tajkhorshid E, Heck AJR, KnebelDoeberitz M, Gabius H-J et al (2005) Determination of structural and functional overlap ⁄ divergence of five proto-type galectins by analysis of the growth–regulatory interaction with ganglioside GM1 in silico and in vitro on human neuroblastoma cells Int J Cancer 114, 46–57 ´ Siebert H-C, Born K, Andre S, Frank M, Kaltner H, ´ von der Lieth C-W, Heck AJR, Jimenez-Barbero J, Kopitz J & Gabius H-J (2006) Carbohydrate chain of ganglioside GM1 as a ligand: identification of the binding strategies of three 15 mer peptides and their divergence from the binding modes of growth-regulatory galectin-1 and cholera toxin Chem Eur J 12, 388–402 Ellerhorst J, Nguyen T, Cooper DNW, Estrov Y, Lotan D & Lotan R (1999) Induction of differentiation and apoptosis in the prostate cancer cell line LNCaP by sodium butyrate and galectin-1 Int J Oncol 14, 225–232 Poirier F, Bourin P, Bladier D, Joubert-Caron R & Caron M (2001) Effect of 5-azacytidine and galectin-1 on growth and differentiation of the human B lymphoma line bl36 Cancer Cell Int 1, Plath T, Peters M, Detjen K, Welzel M, von Marschall Z, Radke C, Wiedenmann B & Rosewicz S (2002) Overexpression of pRB in human pancreatic carcinoma cells: function in chemotherapy-induced apoptosis J Natl Cancer Inst 94, 129–142 Nishioka T, Sakumi K, Miura T, Tahara K, Horie H, Kadoya T & Nakabeppu Y (2002) FosB gene products trigger cell proliferation and morphological alteration with an increased expression of a novel processed form of galectin-1 in the rat 3Y1 embryo cell line J Biochem (Tokyo) 131, 653–661 Berberat PO, Friess H, Wang L, Zhu Z, Bley T, Frigeri L, Zimmermann A & Buchler MW (2001) Comparative analysis of galectins in primary tumors and FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS New function of p16INK4a ´ S Andre et al 94 95 96 97 98 99 100 101 102 103 104 tumor metastasis in human pancreatic cancer J Histochem Cytochem 49, 539–549 Chen R, Pan S, Crispin DA & Brentnall TA (2006) Gene expression and proteomic analysis of pancreatic cancer: a recent update Cancer Genomics Proteomics 3, 1–10 ´ Rotblat B, Niv H, Andre S, Kaltner H, Gabius H-J & Kloog Y (2004) Galectin-1 (L11A) predicted from a computed galectin-1 farnesyl-binding pocket selectively inhibits Ras-GTP Cancer Res 64, 3112–3118 Rorive S, Belot N, Decaestecker C, Lefranc F, Gordower L, Micik S, Maurage C-A, Kaltner H, Ruchoux MM, Danguy A et al (2001) Galectin-1 is highly expressed in human gliomas with relevance for modulation of invasion of tumor astrocytes into the brain parenchyma Glia 33, 241–255 Yamamoto H, Oviedo A, Sweeley C, Saito T & Moskal JR (2001) a2,6-Sialylation of cell-surface N-glycans inhibits glioma formation in vivo Cancer Res 61, 6822–6829 Camby I, Belot N, Lefranc F, Sadeghi N, de Launoit Y, Kaltner H, Musette S, Darro F, Danguy A, Salmon I et al (2002) Galectin-1 modulates human glioblastoma cell migration into the brain through modifications to the actin cytoskeleton and levels of expression of small GTPases J Neuropathol Exp Neurol 61, 585–596 Dawson G, Moskal JR & Dawson SA (2004) Transfection of 2,6 and 2,3-sialyltransferase genes and GlcNActransferase genes into human glioma cell line U-373 MG affects glycoconjugate expression and enhances cell death J Neurochem 89, 1436–1444 Wild MK, Luhn K, Marquardt T & Vestweber D ă (2002) Leukocyte adhesion deciency II: therapy and genetic defect Cells Tissues Organs 172, 161–173 ´ Martinez-Duncker I, Dupre T, Piller V, Piller F, Candelier J-J, Trichet C, Tchernia G, Oriol R & Mollicone R (2005) Genetic complementation reveals a novel human congenital disorder of glycosylation of type II, due to inactivation of the Golgi CMP-sialic acid transporter Blood 105, 2671–2676 ´ Ahmad N, Gabius H-J, Andre S, Kaltner H, Sabesan S, Roy R, Liu B, Macaluso F & Brewer CF (2004) Galectin-3 precipitates as a pentamer with synthetic multivalent carbohydrates and forms heterogeneous cross-linked complexes J Biol Chem 279, 10841–10847 Elad-Sfadia G, Haklai R, Balan E & Kloog Y (2004) Galectin-3 augments K-Ras activation and triggers a Ras signal that attenuates ERK but not phosphoinositide 3-kinase activity J Biol Chem 279, 34922–34930 ´ Andre S, Kaltner H, Furuike T, Nishimura S-I & Gabius H-J (2004) Persubstituted cyclodextrin-based glycoclusters as inhibitors of protein-carbohydrate recognition using purified plant and mammalian lectins 105 106 107 108 109 110 111 112 113 114 115 and wild-type and lectin-gene-transfected tumor cells as targets Bioconjugate Chem 15, 87–98 ´ Manning JC, Seyrek K, Kaltner H, Andre S, Sinowatz F & Gabius H-J (2004) Glycomic profiling of developmental changes in bovine testis by lectin histochemistry and further analysis of the most prominent alteration on the level of the glycoproteome by lectin blotting and lectin affinity chromatography Histol Histopathol 19, 1043–1060 Gabius H-J (1990) Influence of type of linkage and spacer on the interaction of b-galactoside-binding proteins with immobilized affinity ligands Anal Biochem 189, 91–94 ´ Gabius H-J, Darro F, Remmelink M, Andre S, Kopitz J, Danguy A, Gabius S, Salmon I & Kiss R (2001) Evidence for stimulation of tumor proliferation in cell lines and histotypic cultures by clinically relevant low doses of the galactoside-binding mistletoe lectin, a component of proprietary extracts Cancer Invest 19, 114–126 Lensch M, Lohr M, Russwurm R, Vidal M, Kaltner ´ H, Andre S & Gabius H-J (2006) Unique sequence and expression profiles of rat galectins-5 and -9 as a result of species-specific gene divergence Int J Biochem Cell Biol 38, 1741–1758 Gabius H-J, Wosgien B, Hendrys M & Bardosi A (1991) Lectin localization in human nerve by biochemically defined lectin-binding glycoproteins, neoglycoprotein and lectin-specific antibody Histochemistry 95, 269–277 ´ ´ ´ ´ Purkrabkova T, Smetana K Jr, Dvorˇ ankova B, ´ ´ Holı´ kova Z, Bock C, Lensch M, Andre S, Pytl k R, ă Liu F-T, Klı´ ma J et al (2003) New aspects of galectin functionality in nuclei of cultured bone marrow stromal and epidermal cells: biotinylated galectins as tool to detect specific binding sites Biol Cell 95, 535–545 Livak KJ & Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the (-DDC(T)) method Methods 25, 402–408 Tomiya N, Awaya J, Kurono M, Endo S, Arata Y & Takahashi N (1988) Analyses of N-linked oligosaccharides using a two-dimensional mapping technique Anal Biochem 171, 73–90 Akama TO, Nakagawa H, Sugihara K, Narisawa S, Ohyama C, Nishimura S, O’Brien DA, Moremen KW, Millan JL & Fukuda MN (2002) Germ cell survival through carbohydrate-mediated interaction with Sertoli cells Science 295, 124–127 Yamamoto S, Hase S, Fukuda S, Sano O & Ikenaka T (1989) Structures of the sugar chains of interferon-c produced by human myelomonocyte cell line HBL-38 J Biochem (Tokyo) 105, 547–555 Takahashi N & Tomiya N (1992) Analysis of N-linked oligosaccharides: application of glycoamidase A In FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS 3255 New function of p16INK4a 116 117 118 119 120 ´ S Andre et al CRC Handbook of Endoglycosidases and Glycamidases (Muramatsu T & Takahashi N, eds), pp 199–332 CRC Press, Boca Raton ´ Andre S, Kojima S, Yamazaki N, Fink C, Kaltner H, Kayser K & Gabius H-J (1999) Galectins-1 and -3 and their ligands in tumor biology J Cancer Res Clin Oncol 125, 461–474 ´ Andre S, Liu B, Gabius H-J & Roy R (2003) First demonstration of differential inhibition of lectin binding by synthetic tri- and tetravalent glycoclusters from cross-coupling of rigidified 2-propynyl lactoside Org Biomol Chem 1, 3909–3916 ´ Andre S, Pei Z, Siebert H-C, Ramstrom O & Gabius ă H-J (2006) Glycodisuldes from dynamic combinatorial libraries as O-glycoside mimetics for plant and endogenous lectins: their reactivities in solid-phase and cell assays and conformational analysis by molecular dynamics simulations Bioorg Med Chem 14, 6314–6326 ´ Andre S, Maljaars CEP, Halkes KM, Gabius H-J & Kamerling JP (2007) Discovery of galectin ligands in fully randomized combinatorial one-bead-one-compound (glyco) peptide libraries Bioorg Med Chem Lett 17, 793–798 Detjen KM, Brembeck FH, Welzel M, Kaiser A, Haller H, Wiedenmann B & Rosewicz S (2000) Activation of protein kinase Ca inhibits growth of pancreatic 3256 cancer cells via p21 (cip)-mediated G1 arrest J Cell Sci 113, 3025–3035 121 Rudiger H & Gabius H-J (2001) Plant lectins: occură ence, biochemistry, functions and applications Glycoconjugate J 18, 589–613 Supplementary material The following supplementary material is available online: Fig S1 Controls for cell-surface staining with plant lectins Fig S2 Controls for cell-surface staining with human galectins Fig S3 Profiles of cell-surface binding of human galectins Table S1 Microarray data of mRNA expression for differentially regulated genes from a set of 1996 tumor-associated targets This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS ... for a co-ordinated regulation of glycosylation to increase galectin-1 binding and of expression of this endogenous lectin in p16INK4a- positive Capan-1 pancreatic carcinoma cells towards a common... p16INK4a- mediated anoikis induction Quantification of p16INK4a and galectin-1 presence in wild-type (wt), p16INK4a- positive (p1 6) and p16INK4a ⁄ antisense galectin-1 (gal-1AS) double-transfected cells by... detected galectin-1 up-regulation, the increased presentation of galectin-1- binding sites in p16INK4a- expressing cells and acquisition of anoikis susceptibility associated with the fibronectin receptor