Tumor suppressor p16INK4a − modulator of glycomic profile and galectin-1 expression to increase...

Tumor suppressor p16INK4a ) modulator of glycomic profileand galectin-1 expression to increase susceptibility tocarbohydrate-dependent induction of anoikis in pancreaticcarcinoma cellsSabine Andre1, Hugo Sanchez-Ruderisch2, Hiroaki Nakagawa3, Malte Buchholz4, Jurgen Kopitz5,Pia Forberich6, Wolfgang Kemmner6, Corina Bock1, Kisaburo Deguchi3, Katharia M. Detjen2,Bertram Wiedenmann2, Magnus von Knebel Doeberitz5, Thomas M. Gress7,Shin-Ichiro Nishimura3, Stefan Rosewicz2 and Hans-Joachim Gabius1

1 Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians-University Munich, Germany

2 Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Charite-Universitatsmedizin Berlin, Germany

3 Graduate School of Advanced Life Science, Frontier Research Center for Post-Genome Science and Technology, Hokkaido University,

Sapporo, Japan

4 Abteilung Innere Medizin I, Universitat Ulm, Germany

5 Institut fur Angewandte Tumorbiologie, Klinikum der Ruprecht-Karls-Universitat, Heidelberg, Germany

6 Clinic of Surgery and Surgical Oncology, Robert Roessle Hospital, Charite Campus Buch, Berlin, Germany

7 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

Fax: +49 89 21802508

Tel: +49 89 21803279

E-mail: [email protected]

(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-galacto-syltransferases (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 transcrip-

tion of the galectin-1 gene increased conspicuously in p16INK4a-positive

cells, and even figured prominently in a microarray on 1996 tumor-associ-

ated 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 signi-

ficance. 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

Protein glycosylation has functional potential far

beyond the structural roles exerted on the protein back-

bone [1–3]. Endowed with a unique high-density coding

capacity, oligosaccharides are versatile biochemical sig-

nals in glycoprotein maturation and routing intracellu-

larly, 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, struc-

tural 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 descrip-

tive, oncogene presence has been shown to affect par-

ticular 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-acetyl-

glucosaminyltransferase V (GnT-V) and a2,6-sialyl-transferase 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 malig-

nant phenotype and therefore with an unfavorable

prognosis in tumor patients, is not justified. The oppos-

ite 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 trans-

fected 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 poten-

tial 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

[23]. The processing of these glycan chains is an

important part of integrin maturation, and the glyco-

sylation 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 in-

tegrin 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 increas-

ing 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-inte-grin gene [30] – may engender biologically significant

influences on glycan synthesis and processing. Three

lines of evidence support the decision to test our hypo-

thesis in this system.

First, constitutive p16INK4a expression in human

A549 lung adenocarcinoma cells reduced global b1,4-galactosyltransferase (b4GalT) activity on the cell sur-

face 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 regula-

tion 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

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.

New function of p16INK4a S. Andre et al.

3234 FEBS Journal 274 (2007) 3233–3256 ª 2007 The Authors Journal compilation ª 2007 FEBS

expression of b4GalT-V – and also of b4GalT-I [37] –

can be enhanced by epidermal growth factor and dom-

inant active ras [38]. Intriguingly, both enzymes have a

bearing on a5b1-integrin features. b1-Integrin matur-

ation, and transcription of the a5-integrin gene, were

enhanced in human SHG44 glioma cells upon down-

regulating b4GalT-V expression [39], and the absence

of GnT-V in murine embryonic fibroblasts led to a pro-

tein kinase C-dependent stimulation of transcription for

the two integrin genes and of clustered cell-surface pres-

entation 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 abun-

dance 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 sig-

nificantly up-regulated when analyzed by two separate

microarrays, proteomic profiling and flow cytofluoro-

metry, 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 restor-

ing 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 inten-

sity for the p16INK4a-positive cells served as a relative

measure of the expression level. When setting a thresh-

old for a difference in ratio of ± 0.33, a total of 17

cases could be compiled; these are detailed in Table 1.

The overall mRNA supply for enzymatic capacity

to attach N-acetylgalactosamine (GalNAc) to serine ⁄threonine residues of a target protein by a UDP-

GalNAc: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 renormaliza-

tion, the tested gene expression profile for core mucin-

type O-glycosylation did not reveal any impact of

p16INK4a presence. In view of the current literature on

epithelial cancer or integrin glycosylation, it is reassur-

ing to add that a marked influence on GalNAcT activ-

ity could not really be counted upon. This situation is

different for the branching of N-glycans and elabor-

ation of their chain termini.

Turning therefore next to enzymes working on com-

plex-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-galactosyltrans-ferases, b1,3-N-acetylglucosaminyltransferases, except

for the type II/V protein (Table 1), and most a-fuco-syltransferases 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

S. Andre et al. New function of p16INK4a


p16INK4a made its mark on this group. Overall, the

most conspicuous changes were detected in the expres-

sion levels of three b4GalT proteins, explicitly down-

regulation 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 rela-

tion with the rather low signal intensities for b1,3-ga-lactosyltransferases, a preference for type-II termini of

N-glycans is inferred. Because the activity of b4GalT-

IV was increased, no drastic decrease in b1,4-galac-tosylation of glycan chains should occur. Owing to

potent galactosylation of the O-glycan core 2 struc-

tures by b4GalT-IV, this synthetic route may be fav-

ored. 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 sub-

strate specificity for N-glycans may act in a compensa-

tory 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.

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 availabil-

ity 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 documen-

ted 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 differ-

ence 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

Table 1. Microarray data of mRNA expression for glycosyltransferases (± 0.33 from ratio of 1).

Accession Symbol

Ratio

p16 ⁄mock

Signal

p16 Enzyme functionality

NM_003774-0 GALNT4 0.63 670 UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 4 (GalNAcT-4)

NM_017423-0 GALNT7 1.50 2076 UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 7 (GalNAcT-7)

NM_024642-0 GALNT12 0.55 750 UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 12 (GalNAc-T-12)

NM_030965-0 ST6GALNAC5 1.67 1058 GD1a synthase (ST6GalNAc-V); a-series gangliosides

NM_013443-0 ST6GALNAC6 1.77 2122 GD1a synthase (ST6GalNAc-VI); a-series gangliosides, a3,6-disialyl Lec and Lea in a-series

gangliosides

NM_006577 B3GNT2 0.53 908 b3-N-acetylglucosaminyltransferase 2 (b3GnT-II); initiation and elongation of poly LacNAc

NM_032047 B3GNT5 1.65 654 b3-N-acetylglucosaminyltransferase 5 (b3GnT-V); initiation and elongation of poly LacNAc,

O-linked core 3, keratan sulfate, lactotriose

NM_001497 B4GALT1 0.33 1642 b4-galactosyltransferase 1 (b4GalT-I); branch extension of N- (b1,2-branch) and O-glycans

(mucin, especially core 4, O-fuc, O-man), lactosylceramide

NM_003780 B4GALT2 1.49 506 b4-galactosyltransferase 2 (b4GalT-II); branch extension of N- and O-glycans (see b4Gal-T-I)

NM_003778 B4GALT4 4.21 922 b4-galactosyltransferase 4 (b4GalT-IV); poly LacNAc extension on N-glycans (b1,6-branch)

and core 2 O-glycans, neolacto series glycolipids, 6¢-O-sulfated LacNAc

NM_004776 B4GALT5 0.29 3680 b4-galactosyltransferase 5 (b4GalT-V); branch extension of N- (b1,6-branch) and O-glycans

(mucin, O-fuc, O-man), lactosylceramide

NM_006927 ST3GAL2 0.59 848 CMP-sialic acid:Galb1,3GalNAc a3-sialyltransferase (ST3Gal-II)

NM_006279 ST3GAL3 0.34 1240 CMP-sialic acid:Galb1,3 ⁄ 4GlcNAcb a3-sialyltransferase (ST3Gal-III)

NM_003896 ST3GAL5 0.14 1841 CMP-sialic acid:Galb1,4Glc-Cer a3-sialyltransferase (ST3Gal-V); GM3 synthase

NM_006100 ST3GAL6 1.42 890 CMP-sialic acid:Galb1,4GlcNAcb a3-sialyltransferase (ST3Gal-VI); synthesis of

6¢-O-sulfated sLex



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 oligomanno-

syl 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-I-

branched 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 tri-

mannosyl core and the biantennary structure were only

found for the p16INK4a-expressing cells, and no evi-

dence 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 mat-

uration, 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 glycosy-

lation-dependent change of conformation or functional

status of a glycoprotein, the glycan(s) in these cases

directly acting on its (their) protein backbone, system-

atic 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 con-

stituents we selected 24 plant agglutinins. The list of

proteins and their sugar specificities are presented in

Table 2.

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. 2.

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 isoglobotetraosyl-

ceramides and not mucin-type O-glycans. An apparent

Fig. 1. Quantitative profiling of N-glycans. Complete representation of the N-glycan profiles of mock- and p16INK4a-transfected pancreatic car-

cinoma 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.



preference for the Thomsen-Friedenreich epitope anti-

gen was detected for p16INK4a-positive cells by PNA

and jacalin. This result may reflect either elevated core 1

synthesis or efficient core 1 masking by a2,3-sialylationin 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.

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-fucosyltransferaseVIII. Testing two probes with similar specificities

(i.e. LCA ⁄PSA) served as an internal control. Appar-

ently, the chromatographic profiling, on average,

detected more substituted N-glycan in mock controls

Table 2. Lectin panel for glycan profiling of cell surfaces listed in alphabetic order.

Latin name (common name) Acronym

Monosaccharide

specificity Potent oligosaccharidea

Artocarpus integrifolia (jack fruit) Jacalin (JAC) Gal ⁄ GalNAc Galb3GalNAca

Arachis hypogaea (peanut) PNA Gal Galb3GalNAca

Canavalia ensiformis (jack bean) Con A Man ⁄ Glc GlcNAcb2Mana6(GlcNAcb2Mana3)Manb4GlcNAc

Datura stramonium (thorn apple) DSA GlcNAc (GlcNAc)n, Galb4GlcNAcb6(Galb4GlcNAcb2)Man (Galb4GlcNAc)3

Dolichos biflorus (horse gram) DBA GalNAc GalNAca3GalNAca3Galb4Galb4Glc

Erythrina cristagalli (coral tree) ECA Gal Galb4GlcNAcb6(Galb4GlcNAcb2)Man

Galanthus nivalis (snowdrop) GNA Man Mana6(Mana3)ManaR

Glycine max (soybean) SBA GalNAc GalNAca3Galb6Glc

Griffonia simplicifolia I GSA I GalNAc GalNAca3Gal, GalNAca3GalNAcb3Gala4Galb4Glc

Griffonia simplicifolia II GSA II GlcNAc GlcNAcb4GlcNAc, N-glycans with terminal, nonreducing-end

GlcNAc

Lens culinaris (lentil) LCA Man ⁄ Glc N-glycan binding enhanced by core-fucosylation

Lycopersicon esculentum (tomato) LEA b core and stem regions of high-mannose-type N-glycans,

(GlcNAcb3Galb4GlcNAcb3Gal)n of complex-type N-glycans

Maackia amurensis I (leukoagglutinin) MAA I b Neu5Aca3Galb4GlcNAc ⁄ Glc

Maackia amurensis II (haemagglutinin) MAA II b Neu5Aca3Galb3(a6Neu5Ac)GalNAc

Phaseolus vulgaris erythroagglutinin

(kidney bean)

PHA-E b Bisected complex-type N-glycans: Galb4GlcNAcb2Mana6

(GlcNAcb2-Mana3)(GlcNAcb4)Manb4GlcNAc

Phaseolus vulgaris leukoagglutinin

(kidney bean)

PHA-L b Tetra- and triantennary N-glycans with b6-branching

Pisum sativum (garden pea) PSA Man ⁄ Glc N-glycan binding enhanced by core-fucosylation

Sambucus nigra (elderberry) SNA Gal ⁄ GalNAc Neu5Aca6Gal ⁄ GalNAc

Solanum tuberosum (potato) STA b (GlcNAc)n with preference for high-mannose-type N-glycans

Sophora japonica (pagoda tree) SJA GalNAc GalNAcb6Gal, Galb3GalNAc

Triticum vulgare (wheat germ) WGA GlcNAc ⁄ Neu5Ac (GlcNAc)n, Galb4GlcNAcb6Gal

Ulex europaeus I (gorse) UEA I Fuc Fuca2Galb4GlcNAcb6R

Vicia villosa (hairy vetch) VVA GalNAc GalNAca3(6)Gal, GalNAcb3Gal

Viscum album (mistletoe) VAA Gal Galb2(3)Gal, Gala3(4)Gal, Galb3(4)GlcNAc without ⁄ with

a2,6-sialylation, Fuca2Gal

a Based on previously compiled information [121], extended and modified; b no monosaccharide known as ligand.

Fig. 2. 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 lgÆmL)1 except for SNA and DBA (1 lgÆmL)1), SJA and WGA (2 lgÆmL)1) and MAA-I

(5 lgÆmL)1).



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 regard-

ing 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-acetylglucosaminyl-

transferases and b4GalTs, as also seen in chromato-

graphic 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 micro-

array 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, meas-

urement 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 compensa-

tory 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, despitesimilar 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 noniden-

tical degree of sialylation makes it mandatory to deter-

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 carbohy-

drate-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 5 lgÆmL)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 popu-

lations. Should the level of mucin-type O-glycan

a2,3-sialylation also be lowered in the p16INK4a-posit-

ive 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 resi-

dues 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 2 tetrasaccha-

rides, favored by an increased b4GalT-IV presence

and reduced O-glycan a2,3-sialylation. Thus, lectin-

accessible 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

1 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

1 lgÆmL)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-branchedN-glycans in the mock control, the remarkably differ-

ent levels of SNA binding after standard neuramini-

dase treatment must be recalled. In essence, data from

chromatographic mapping square well with the lectin

profiling. A major result emerging from these experi-

ments 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



processes may generate or mask sites for contact in situ

with endogenous lectins. As the results with the b-gal-actoside-specific lectins VAA ⁄ECA revealed, the levels

of accessible galactose residues were remarkably differ-

ent 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 fam-

ily, 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 p16INK4a-

positive 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 fam-

ily can also be profiled in one cell system, a compar-

ison so far not reported. For this purpose, we purified

the lectins and then biotinylated them under activity-

preserving conditions, ascertained a lack of harmful

effects on sugar binding by activity assays and deter-

mined the labeling efficiency with 2–8 modified resi-

dues per carbohydrate-recognition domain. As in the

binding studies with plant lectins, we routinely per-

formed experiments to assess concentration depend-

ence 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.

Fig. 3. 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 biotinyl-

ated lectin (shaded) and after incubation with the labeled probe (0.5 lgÆmL)1 of PNA, 1 lgÆmL)1 of PHA-L, 5 lgÆmL)1 of SNA and MAA-II, as

well as 10 lgÆmL)1 of DBA), without (gray line) or after (black line) sialidase treatment. Quantitative data are presented as defined in the

legend to Fig. 2.



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 carbo-

hydrate-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 (sup-

plementary Fig. S2, third panel). The concentration-

and 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 posi-

tivity (supplementary Fig. S2). Given this indication

for intergalectin differences, we systematically assayed

a series of human galectins to define staining proper-

ties 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 react-

ive, as shown for SNA or PNA binding in Fig. 3.

Indeed, reduction of sialylation under standard condi-

tions 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 remov-

ing the blocking a2,6-sialylation underlies this param-

eter 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 agree-

ment 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

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 reason-

ing 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 galectin-

1-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

Fig. 4. 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 labe-

led galectins-1 and -3 (gal-1 and gal-3 used at 10 lgÆmL)1) without

(gray line) or after (black line) sialidase treatment. Quantitative data

are presented as defined in the legend to Fig. 2.



Table S1). Northern blotting and nuclear run-off

experiments confirmed the array data and substan-

tiated an increase in de novo transcription (data not

shown). Extending our work from the mRNA level,

we next performed a proteomic analysis with the inten-

tion 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 reproduci-

bility 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 pres-

ence as protein was found to be significantly up-regula-

ted (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 pro-

duction translated into increased surface presence in

the p16INK4a-positive cells (Fig. 5). We deliberately tes-

ted several cell batches and consistently measured an

enhanced cell-surface presentation in the p16INK4a-pos-

itive 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 pres-

ence 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 tes-

ted 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 expres-

sion, 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 sus-

ceptibility associated with the fibronectin receptor.

Induction of anoikis by galectin-1

In order to test the hypothesis described above, we pur-

sued 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 con-

firming 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 pre-

sent (Fig. 6A). Second, we forced an increase of galec-

tin-1 production on wild-type cells weakly positive for

galectin-1 binding, using proliferating cell nuclear anti-

gen 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 overex-

pressing galectin-1 we could, in parallel, elicit anoikis

in regular wild-type cells by adding the lectin to med-

ium at a concentration of 125 lgÆmL)1 (data not

shown). The presence of haptenic sugar interfered with

this process, revealing carbohydrate dependence, as did

the presence of the predominantly monomeric galectin-

3, 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 galec-

tin-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 redu-

cing 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-indu-

cing galectin-1.



Fig. 5. 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 1 lgÆmL)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 lgÆmL)1 of polyclonal galectin-1 antibody as probe and fluorescent goat anti-rabbit IgG as the second-step reagent (bottom panel).



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 fine-

tuned and co-ordinated events translating into altera-

tions of protein–protein and protein–carbohydrate

recognition.

Towards this aim, we had designed a combined

approach using a cDNA microarray for glycosyltrans-

ferases, 2D chromatographic profiling and binding

studies with lectins on the cell surface. The array data

pinpoint several changes in expression of glycosyl-

transferase genes and hereby afforded first evidence

for functional links. To start with there is an

increased potential for the synthesis of a2,3 ⁄a2,6-disialylated 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 stain-

ing at a probe concentration of 5 lgÆmL)1 can be

interpreted to reflect the differential presence of the

respective disialylated core. Next, the previously

reported influence on b4GalT activity [31] was con-

firmed at the level of transcription and extended to

divergent regulation between b4GalTs-I ⁄ -V and

b4GalT-IV. Of interest, the noted down-regulation of

A

B

C

Fig. 6. 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 ver-

sus 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 galec-

tin-1-specific cDNA showing different levels of galectin-1 positi-

vity 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 fi-

bronectin receptor obtained from cells grown while adherent

(ad) or on polyhydroxyethylmethacrylate (PH) to keep them in

suspension (C).



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 2 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 2 extensions, unless the synthetic

precursor core 1 is a2,3-sialylated [55–57]. Fittingly,

chromatographic profiling and LEA staining yielded

no evidence for a major change in poly N-acetyllacto-

samine presence, and gene expression of a respective

sialyltransferase decreased. As a consequence, con-

spicuous diminution was substantiated on the level of

the cell surface for a2,3-sialylation of mucin-type

O-glycans, especially when using PNA staining with-

out ⁄with neuraminidase treatment. Because exposure

of human HT-29 colon cancer cells to GalNAc-a-O-

benzyl resulted in reduced a2,3-sialylation, which

engendered an impact on the apical delivery of glyco-

proteins [58], this parameter change may also have a

bearing on integrin routing.

As noted for a2,3-sialylation of mucin-type O-gly-

cosylation, 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 distribu-

tion. 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 rele-

vance that induction of myeloid differentiation by

phorbol ester targets ST6Gal-I expression [59]. Fur-

ther 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 pres-

ence of a2,6-sialylation reduces the affinity of other-

wise 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 dif-

ferent levels of sialylation were ascertained, whereas a

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 indi-

cator of altered glycosylation was also ascertained

(data not shown). As an excellent measure of the sen-

sitivity of the chromatographic profiling, a recent

report documented the ability of the technique to spot

quantitative differences reliably in the extent of b1,6-branching 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 mech-

anism as unlikely (i.e. its angiogenic property assigned

to the basic region between amino acids 254–269 and

responsible for mobilization of fibroblast growth fac-

tor-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 bind-

ing activity.

The binding of b-galactoside-specific plant lectins

intimated a new role, attributing to the integrin’s gly-

cans the potential to become ligands for endogenous

lectins. This finding prompted studies with human lec-

tins, 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-hyposialyla-tion 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 2

O-glycans, whose establishment and galactosylation is

strongly favored by the up-regulation of b4GalT-IV

[55], and the reduced core 1 a2,3-sialylation can con-

tribute 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 2 presentation associated

with the presence of p16INK4a. The transit from core 1

to core 2 structures can prove advantageous, because

model studies on asialofetuin with its three core 1

disaccharides have shown no binding of galectins to

these epitopes [44,70]. Moreover, local cluster forma-

tion to accomplish high-density presentation of galac-

tose residues will be beneficial for avidity, because

short core 2 structures appear to require chain exten-

sions 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



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-gly-

cans 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 2 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 documen-

ted by its increased production in p16INK4a-reconstitu-

ted cells and the functional assays.

In this combination, our data epitomize an elegant

orchestration of gene expression for glycosyltrans-

ferases 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-aalters 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 activa-

tion of a cell-surface ganglioside sialidase, which

engenders the inhibition of neuroblastoma prolifer-

ation 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 per-

spective [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 buty-

rate 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 illus-

trated 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 suppres-

sion, at least indicate an association to p16INK4a pres-

ence 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 influ-

ence 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 surpris-

ingly, 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 clo-

sely 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 carci-

noma 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 regula-

tion 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) pin-

point 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



cells and inactive to elicit anoikis, served as an endog-

enous inhibitor of galectin-1-dependent effects in neur-

oblastoma 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 dis-

tinct building blocks of human glycans, was purchased from

Vector Laboratories (distributed by Alexis Germany, Grun-

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 recom-

binant production were purified by affinity chromatography

on lactose- or mannose-bearing Sepharose 4B resins, pre-

pared with divinyl sulfone (Fluka, Munich, Germany) for

activation, and protein quality was routinely controlled by

1- and 2D gel electrophoresis, gel filtration and mass spectr-

ometry 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 chro-

matography on a Ni-CAMTM HC resin (Sigma, Munich,

Germany). Biotinylation of the natural and mutant proteins

was carried out with the N-hydroxysuccinimide ester deriv-

ative (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 carci-

noma 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, Ber-

lin, Germany), 2 mm l-glutamine, 100 UÆmL)1 of penicillin

and 100 lgÆmL)1 of streptomycin, as described previously

[30]. Cell clones from wild-type cells overexpressing galectin-

1, or from p16INK4a-positive cells harboring galectin-1-speci-

fic cDNA in an antisense orientation, were generated using

the pcDNA3.1 system and hygromycin at a concentration of

100 lgÆmL)1 for selection, as described previously [104]. For

determination of anoikis, 2 · 105 cells per assay were cul-

tured in suspension on a surface coated with polyhydroxy-

ethylmethacrylate, and the cell cycle distribution, including

percentage of cells in the pre-G1 fraction, was assessed using

the cellquestTM program [30].

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 (Invi-

trogen ⁄Life Technologies, Karlsruhe, Germany) was used

for the production of cDNA harboring fluorescent tagging.

Its efficiency was routinely monitored by adsorption meas-

urements using a Nanodrop ND100 UV ⁄VIS spectro-

photometer. Aliquots containing the labeled cDNA were

diluted to a volume of 200 lL in Microarray Hybridization

Solution, Version 2 (Amersham Biosciences, Braunschweig,

Germany) and hybridized onto the GlycoProfiler microar-

ray (Scienion, Berlin, Germany) using an a-Hyb hybridiza-

tion station (Miltenyi, Cologne, Germany) for 16 h at

42 �C. Subsequent washing steps were then performed using

2 · NaCl ⁄ Cit, 0.2% SDS as first, 1 · 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 sub-

jected to analysis using the feature extraction 8.0 soft-

ware (Agilent). The GlycoProfiler microarray comprising

60-mer gene sequences for selected human glycosyltrans-

ferases 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 fea-

ture extraction 8.1 software (Agilent) with Rank Consis-

tent Probes as the dye-normalization probe selection

method and Linear and Lowess as the normalization cor-

rection method, hereby accounting for signal intensity-

dependent 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 fea-

tures, and a universal error model, instrumental for high-

intensity 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-transcriptase-

driven cDNA synthesis. Expression levels were determined

in triplicate by assays-on-demand, delivered by Applied



Biosystems (Darmstadt, Germany), running the predevel-

oped 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 min followed by

40 cycles of 95 �C for 15 s and then 60 �C for 1 min.

Data analysis was performed according to the DD thresh-

old 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 expres-

sion 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, pass-

word genom)], a custom-made array was designed on

Hybond N+ nylon membranes using a MicroGrid II arr-

ayer (Bio Robotics Ltd, Cambridge, UK). Hybridization

probes were generated from 30 lg of total RNA by oligo-

dT-primed reverse transcription using the Superscript II

enzyme (Gibco BRL, Karlsruhe, Germany) and [32P]dAT-

P[aP]. Probes were competed with 0.25 lgÆmL)1 of soni-

fied cDNA from human placenta (Sigma) and COT-1

DNA (Gibco) for 1 h. Hybridizations were routinely per-

formed 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 correc-

tion 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 min 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 (Calbio-

chem, Darmstadt, Germany) at 37 �C for 16 h. After again

heating at 90 �C for 10 min to inactivate the proteases,

samples were treated with 10 U of N-glycosidase F (recom-

binant; 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 1 h [114]. Derivatized N-glycans were

separated from reagents on a Sephadex G-15 column

(Amersham Biosciences) with 10 mm ammonium bicarbon-

ate solution as eluant. The fraction containing the glycans

was acid-hydrolyzed at 90 �C for 1 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,

6 · 150 mm; Shimadzu, Kyoto, Japan) by HPLC (D-7000

HPLC system equipped with an L-7485 fluorescence detec-

tor from Hitachi High-Technologies Co., Tokyo, Japan).

Elution was performed at a flow rate of 1.0 mLÆmin)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 mix-

ture of solvents A and B (80 : 20, v ⁄ v). After sample injec-

tion, elution was performed using a linear gradient to a

solvent ratio of 40 : 60 (A ⁄B, v ⁄ v) in 80 min. Seven sam-

ples 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 Amide-

80, 4.6 · 250 mm). Elution from that column was per-

formed with a flow rate of 1.0 mLÆmin)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 : 0 to 40 : 60 (v ⁄ v) of solvents C and D in 30 min.

In both HPLC systems, the positions of elution of PA-deri-

vatized N-glycans were spotted by a fluorescence detector

using excitation and emission wavelengths of 320 and

400 nm, respectively. The molar ratio of PA oligosaccha-

rides 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 cy-

tofluorometry in a FACScan instrument (Becton-Dickinson,

Heidelberg, Germany). Solutions with 4 · 105 cells per



sample were carefully washed to remove any interfering

serum compounds, the extent of protein binding by nonspe-

cific interaction was reduced by an incubation step in Dul-

becco’s phosphate-buffered saline containing 100 lgÆmL)1

of carbohydrate-free bovine serum albumin, and the biotin-

ylated lectins were incubated with the cells for 30 min at

4 �C to minimize endocytic uptake, as described in detail

previously [116–119]. Control experiments without the incu-

bation step with labeled lectin, or in the presence of hapten-

ic 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 min at 37 �C with 0.2 UÆmL)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 8 m urea, 4%

w ⁄ v 3-[[3-cholamidopropyl]-dimethylammonio]-1-propane-

sulfonate, 0.5% IPG buffer (pH 3–10; GE Healthcare

Europe GmbH, Freiburg, Germany) and 20 mm dithiothre-

itol. After incubation for 1 h at room temperature, the

solution was cleared by centrifugation for 5 min 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 1 h at 100 mA and 2 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 Image-

Master� 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 statis-

tical analysis with the graphpad prism 3.0 package. Galec-

tin-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 thiocarbamyl-

labeled conjugate of goat anti-rabbit IgG (Sigma), as des-

cribed previously [104]. This procedure was also used for

galectin-1 detection in preparations of the fibronectin recep-

tor immunoprecipitated from 800 lg of total extract protein

using a commercial antibody against the a5-integrin (Cym-

bus Biotechnology, Chandlers Ford, UK), as described pre-

viously [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,

Mannheim, Germany). Sample preparation for mass-spectr-

ometric analysis by cocrystallization with the matrix (a-cy-ano-4-hydroycinnamic acid) followed and the mass profile

of the tryptic peptides was then recorded in the positive-

ion-reflector mode on a time-of-flight instrument (Bruker-

Daltronik, 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. Data-

base 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 manu-

script, to the reviewers of our manuscript for their valu-

able 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-technol-

ogischen Fortschritts in der Medizin e. V and the Wil-

helm-Sander Stiftung for generous financial support.

References

1 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.

2 Reuter G & Gabius H-J (1999) Eukaryotic glycosyla-

tion: whim of nature or multipurpose tool? Cell Mol

Life Sci 55, 368–422.

3 Lowe JB & Marth JD (2003) A genetic approach to

mammalian glycan function. Annu Rev Biochem 72,

643–691.

4 Laine R (1997) The information-storing potential of

the sugar code. In Glycosciences: Status and Perspec-

tives (Gabius H-J & Gabius S, eds), pp. 1–14. Chap-

man & Hall, Weinheim, Germany.

5 Hebert DN, Garman SC & Molinari M (2005)

The glycan code of the endoplasmic reticulum:

asparagine-linked carbohydrates as protein matura-

tion and quality-control tags. Trends Cell Biol 15,

364–370.

6 Gabius H-J (2006) Cell surface glycans: the why and

how of their functionality as biochemical signals in lec-

tin-mediated information transfer. Crit Rev Immunol

26, 43–80.

7 Villalobo A, Nogales-Gonzales A & Gabius H-J (2006)

A guide to signalling pathways connecting protein–



glycan interaction with the emerging versatile effector

functionality of mammalian lectins. Trends Glycosci

Glycotechnol 18, 1–37.

8 Alhadeff JA (1989) Malignant cell glycoproteins and

glycolipids. Crit Rev Oncol Hematol 9, 37–107.

9 Hakomori S-i (1989) Aberrant glycosylation in tumors

and tumor-associated carbohydrate antigens. Adv

Cancer Res 52, 257–331.

10 Yamashita K, Ohkura T, TachibanaY, Takasaki S &

Kobata A (1984) Comparative study of the oligosac-

charides released from baby hamster kidney cells and

their polyoma transformant by hydrazinolysis. J Biol

Chem 259, 10834–10840.

11 Pierce M & Arango J (1986) Rous sarcoma virus-

transformed 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.

12 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.

13 Easton EW, Bolscher JGM & van den Eijnden DH

(1991) Enzymatic amplification involving glycosyltrans-

ferases forms the basis for the increased size of aspara-

gine-linked glycans at the surface of NIH 3T3 cells

expressing the N-ras proto-oncogene. J Biol Chem 266,

21674–21680.

14 Buckhaults P, Chen L, Fregien N & Pierce M (1997)

Transcriptional regulation of N-acetylglucosaminyl-

transferase V by the src oncogene. J Biol Chem 272,

19575–19581.

15 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.

16 Dalziel M, Dall’Olio F, Mungul A, Piller V & Piller F

(2004) Ras oncogene induces b-galactoside a2,6-sialyl-transferase (ST6Gal I) via a RalGEF-mediated signal

to its housekeeping promoter. Eur J Biochem 271,

3623–3634.

17 Dosaka-Akita H, Miyoshi E, Suzuki O, Itoh T, Katoh

H & Taniguchi N (2004) Expression of N-acetylgluco-

saminyltransferase V is associated with prognosis and

histology in non-small cell lung cancers. Clin Cancer

Res 10, 1773–1779.

18 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-acetylglu-

cosaminyltransferase 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-Acetylglucosami-

nyltransferase 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 malig-

nant 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 sia-

lylated, functionally altered b1 integrin. Oncogene 22,

7137–7145.

23 Nakagawa H, Zheng M, Hakomori S-i, Tsukamoto Y,

Kawamura Y & Takahashi N (1996) Detailed oligosac-

charide 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) Ana-

lysis of the role of glycosylation of the human fibro-

nectin 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, Martınez 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.



30 Plath T, Detjen K, Welzel M, von Marschall Z, Mur-

phy 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-galactosyltransferasegene 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 senes-

cence-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 & Tani-

guchi N (2006) Aberrant expression of N-acetylgluco-

saminyltransferase-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-galactosyl-transferase 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 can-

cer cell lines. J Biol Chem 274, 22941–22948.

36 Sato T & Furukawa K (2004) Transcriptional regula-

tion 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) b1,4-Galactosyltransferase 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-acetylglucosaminyltrans-

ferase V stimulates a5b1 integrin expression mediated

by the protein kinase C signaling pathway. J Biol

Chem 280, 8332–8342.

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, Kuwab-

ara 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 lec-

tins. 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

code. ChemBioChem 5, 740–764.

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 proper-

ties 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 LEC14-

type 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 acida2,6GalNAc. 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, Kumamo-

to 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 acid-

binding immunoglobulin-like lectin-7 (siglec-7), associ-

ated 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 sialyltrans-

ferases involved in the biosynthesis of Lewis antigens

in human pancreatic tumour cells. Glycoconjugate J 22,

135–144.



53 Hanßke B, Thiel C, Lubke T, Hasilik M, Honing S,

Peters V, Heidemann PH, Hoffmann GF, Berger EG,

von Figura K et al. (2002) Deficiency of UDP-galac-

tose: 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 forma-

tion of N-glycans in b1,4-galactosyltransferase-1-defici-ent 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 2 branched O-gly-

cans. The requirement of novel b1,4-galactosyltrans-ferase 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-galactosyl-transferase V indicates its potential function in

O-glycosylation. FEBS Lett 450, 52–56.

57 Brockhausen I, Matta KL, Orr J, Schachter H, Koen-

derman 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-acetyl-

glucosaminyltransferase 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 differ-

entiated 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 sti-

mulates 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,

Arnarp J & Lonngren J (1982) Mitogenic leuko-

agglutinin from Phaseolus vulgaris binds to a

pentasaccharide unit in N-acetyllactosamine-type

glycoprotein glycans. Proc Natl Acad Sci USA 79,

1611–1615.

64 Green ED & Baenziger JU (1987) Oligosaccharide spe-

cificities of Phaseolus vulgaris leukoagglutinating and

erythroagglutinating phytohemagglutinins. Interactions

with N-glycanase-released oligosaccharides. J Biol

Chem 262, 12018–12029.

65 Yamashita K, Totani K, Ohkura T, Takasaki S, Gold-

stein IJ & Kobata A (1987) Carbohydrate binding

properties of complex-type oligosaccharides on immo-

bilized Datura stramonium lectin. J Biol Chem 262,

1602–1607.

66 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 inter-

action of N-linked glycopeptides and related oligosac-

charides with immobilized Phaseolus vulgaris

leukoagglutinating lectin (L-PHA). Glycoconjugate J 5,

85–97.

67 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-syn-

onymous signals with marked developmental regula-

tion in bovine reproductive tracts. Biochimie 88, 399–

410.

68 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) indu-

ces tumor angiogenesis without mediation of glycosyla-

tion: a novel function of GnT-V distinct from the

original glycosyltransferase activity. J Biol Chem 277,

17002–17008.

69 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.

70 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.

71 Leppanen A, Stowell S, Blixt O & Cummings RD

(2005) Dimeric galectin-1 binds with high affinity to

a2,3-sialylated and non-sialylated terminal N-acetyl-

lactosamine units on surface-bound extended glycans.

J Biol Chem 280, 5549–5562.

72 Amano M, Galvan M, He J & Baum LG (2003) The

ST6Gal I sialyltransferase selectively modifies N-gly-

cans on CD45 to negatively regulate galectin-1-induced

CD45 clustering, phosphatase modulation, and T cell

death. J Biol Chem 278, 7469–7475.

73 Patnaik SK, Potvin B, Carlsson S, Sturm D, Leffler H

& Stanley P (2006) Complex N-glycans are the major



ligands for galectins-1, -3, and -8 on Chinese hamster

ovary cells. Glycobiology 16, 305–317.

74 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.

75 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 gan-

glioside GM1 versus bacterial toxins. Biochemistry 42,

14762–14773.

76 Lopez-Lucendo MF, Solıs 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 thermo-

dynamics of ligand binding. J Mol Biol 343, 957–970.

77 Sturm A, Lensch M, Andre S, Kaltner H, Wieden-

mann 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.

78 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.

79 Smith FI, Qu Q, Hong SJ, Kim KS, Gilmartin TJ &

Head SR (2005) Gene expression profiling of mouse

postnatal cerebellar development using oligonucleo-

tide microarrays designed to detect differences in

glycoconjugate expression. Gene Expr Patterns 5,

740–749.

80 Comelli EM, Head SR, Gilmartin T, Whisenant T,

Haslam SM, North SJ, Wong N-K, Kudo T, Narima-

tsu H, Esko JD et al. (2006) A focused microarray

approach to functional glycomics: transcriptional

regulation of the glycome. Glycobiology 16, 117–131.

81 Garcıa-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.

82 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.

83 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 1 (Th1)

cells. Blood 106, 3867–3873.

84 Kopitz J, Andre S, von Reitzenstein C, Versluis K,

Kaltner H, Pieters RJ, Wasano K, Kuwabara I, Liu

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.

85 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.

86 Kopitz J, von Reitzenstein C, Andre S, Kaltner H, Uhl

J, Ehemann V, Cantz M & Gabius H-J (2001) Nega-

tive regulation of neuroblastoma cell growth by carbo-

hydrate-dependent surface binding of galectin-1 and

functional divergence from galectin-3. J Biol Chem

276, 35917–35923.

87 Andre S, Kaltner H, Lensch M, Russwurm R, Siebert

H-C, Fallsehr C, Tajkhorshid E, Heck AJR, Knebel-

Doeberitz M, Gabius H-J et al. (2005) Determination

of structural and functional overlap ⁄ divergence of five

proto-type galectins by analysis of the growth–regula-

tory interaction with ganglioside GM1 in silico and in

vitro on human neuroblastoma cells. Int J Cancer 114,

46–57.

88 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-regula-

tory galectin-1 and cholera toxin. Chem Eur J 12,

388–402.

89 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.

90 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 lym-

phoma line bl36. Cancer Cell Int 1, 2.

91 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.

92 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.

93 Berberat PO, Friess H, Wang L, Zhu Z, Bley T, Fri-

geri L, Zimmermann A & Buchler MW (2001) Com-

parative analysis of galectins in primary tumors and



tumor metastasis in human pancreatic cancer. J Histo-

chem Cytochem 49, 539–549.

94 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.

95 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.

96 Rorive S, Belot N, Decaestecker C, Lefranc F, Gor-

dower 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 modu-

lation of invasion of tumor astrocytes into the brain

parenchyma. Glia 33, 241–255.

97 Yamamoto H, Oviedo A, Sweeley C, Saito T & Mos-

kal JR (2001) a2,6-Sialylation of cell-surface N-glycans

inhibits glioma formation in vivo. Cancer Res 61,

6822–6829.

98 Camby I, Belot N, Lefranc F, Sadeghi N, de Launoit

Y, Kaltner H, Musette S, Darro F, Danguy A, Sal-

mon 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.

99 Dawson G, Moskal JR & Dawson SA (2004) Transfec-

tion of 2,6 and 2,3-sialyltransferase genes and GlcNAc-

transferase genes into human glioma cell line U-373

MG affects glycoconjugate expression and enhances

cell death. J Neurochem 89, 1436–1444.

100 Wild MK, Luhn K, Marquardt T & Vestweber D

(2002) Leukocyte adhesion deficiency II: therapy and

genetic defect. Cells Tissues Organs 172, 161–173.

101 Martinez-Duncker I, Dupre T, Piller V, Piller F, Can-

delier 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 trans-

porter. Blood 105, 2671–2676.

102 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 hetero-

geneous cross-linked complexes. J Biol Chem 279,

10841–10847.

103 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 phosphoinosi-

tide 3-kinase activity. J Biol Chem 279, 34922–34930.

104 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

and wild-type and lectin-gene-transfected tumor cells

as targets. Bioconjugate Chem 15, 87–98.

105 Manning JC, Seyrek K, Kaltner H, Andre S, Sinowatz

F & Gabius H-J (2004) Glycomic profiling of develop-

mental 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.

106 Gabius H-J (1990) Influence of type of linkage and

spacer on the interaction of b-galactoside-binding pro-

teins with immobilized affinity ligands. Anal Biochem

189, 91–94.

107 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.

108 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.

109 Gabius H-J, Wosgien B, Hendrys M & Bardosi A

(1991) Lectin localization in human nerve by biochemi-

cally defined lectin-binding glycoproteins, neoglycopro-

tein and lectin-specific antibody. Histochemistry 95,

269–277.

110 Purkrabkova T, Smetana K Jr, Dvorankova B,

Holıkova Z, Bock C, Lensch M, Andre S, Pytlık R,

Liu F-T, Klıma J et al. (2003) New aspects of galec-

tin 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.

111 Livak KJ & Schmittgen TD (2001) Analysis of relative

gene expression data using real-time quantitative PCR

and the 2 (-DDC(T)) method. Methods 25, 402–408.

112 Tomiya N, Awaya J, Kurono M, Endo S, Arata Y &

Takahashi N (1988) Analyses of N-linked oligosacchar-

ides using a two-dimensional mapping technique. Anal

Biochem 171, 73–90.

113 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.

114 Yamamoto S, Hase S, Fukuda S, Sano O & Ikenaka T

(1989) Structures of the sugar chains of interferon-cproduced by human myelomonocyte cell line HBL-38.

J Biochem (Tokyo) 105, 547–555.

115 Takahashi N & Tomiya N (1992) Analysis of N-linked

oligosaccharides: application of glycoamidase A. In



CRC Handbook of Endoglycosidases and Glycamidases

(Muramatsu T & Takahashi N, eds), pp. 199–332.

CRC Press, Boca Raton.

116 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.

117 Andre S, Liu B, Gabius H-J & Roy R (2003) First

demonstration of differential inhibition of lectin bind-

ing by synthetic tri- and tetravalent glycoclusters from

cross-coupling of rigidified 2-propynyl lactoside. Org

Biomol Chem 1, 3909–3916.

118 Andre S, Pei Z, Siebert H-C, Ramstrom O & Gabius

H-J (2006) Glycodisulfides 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.

119 Andre S, Maljaars CEP, Halkes KM, Gabius H-J &

Kamerling JP (2007) Discovery of galectin ligands in

fully randomized combinatorial one-bead-one-com-

pound (glyco) peptide libraries. Bioorg Med Chem Lett

17, 793–798.

120 Detjen KM, Brembeck FH, Welzel M, Kaiser A, Hal-

ler H, Wiedenmann B & Rosewicz S (2000) Activation

of protein kinase Ca inhibits growth of pancreatic

cancer cells via p21 (cip)-mediated G1 arrest. J Cell Sci

113, 3025–3035.

121 Rudiger H & Gabius H-J (2001) Plant lectins: occur-

ence, biochemistry, functions and applications. Glyco-

conjugate 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 corres-

ponding author for the article.



Tumor suppressor p16INK4a − modulator of glycomic profile and galectin-1 expression to increase...

Documents

Transcript of Tumor suppressor p16INK4a − modulator of glycomic profile and galectin-1 expression to increase...