Báo cáo khóa học: Determination of modulation of ligand properties of synthetic complex-type biantennary N-glycans by introduction of bisecting GlcNAc in silico, in vitro and in vivo pot

Determination of modulation of ligand properties of synthetic
complex-type biantennary N-glycans by introduction of bisecting
GlcNAc
in silico
,
in vitro
and
in vivo
Sabine Andre
´
1
, Carlo Unverzagt
2,
*, Shuji Kojima
3
, Martin Frank
4
, Joachim Seifert
2,
†, Christian Fink
5
,
Klaus Kayser
6
, Claus-Wilhelm von der Lieth
4
and Hans-Joachim Gabius
1
1
Institut fu
¨

r Physiologische Chemie, Tiera
¨
rztliche Fakulta
¨
t, Ludwig-Maximilians-Universita
¨
tMu
¨
nchen, Germany;
2
Institut fu
¨
r
Organische Chemie und Biochemie, Technische Universita
¨
tMu
¨
nchen, Garching, Germany;
3
Faculty of Pharmaceutical Sciences,
Tokyo University of Science, Japan;
4
Zentrale Spektroskopie, Deutsches Krebsforschungszentrum, Heidelberg, Germany;
5
Radiologische Diagnostik und Therapie, Deutsches Krebsforschungszentrum, Heidelberg, Germany;
6
Institut fu
¨
r Pathologie,
Charite

´
, Humboldt Universita
¨
t, Berlin, Germany
We have investigated the consequences of introducing
a bisecting GlcNAc moiety into biantennary N-glycans.
Computational analysis of glycan conformation with pro-
longed simulation periods in vacuo and in a solvent box
revealed two main effects: backfolding of the a1–6 arm and
stacking of the bisecting GlcNAc and the neighboring Man/
GlcNAc residues of both antennae. Chemoenzymatic syn-
thesis produced the bisecting biantennary decasaccharide
N-glycan and its a2–3(6)-sialylated variants. They were
conjugated to BSA to probe the ligand properties of
N-glycans with bisecting GlcNAc. To assess affinity altera-
tions in glycan binding to receptors, testing was performed
with purified lectins, cultured cells, tissue sections and ani-
mals. The panel of lectins, including an adhesion/growth-
regulatory galectin, revealed up to a sixfold difference in
affinity constants for these neoglycoproteins relative to data
on the unsubstituted glycans reported previously [Andre
´
,S.,
Unverzagt,C.,Kojima,S.,Dong,X.,Fink,C.,Kayser,K.&
Gabius, H J. (1997) Bioconjugate Chem. 8, 845–855]. The
enhanced affinity for galectin-1 is in accord with the
increased percentage of cell positivity in cytofluorimetric and
histochemical analysis of carbohydrate-dependent binding
of labeled neoglycoproteins to cultured tumor cells and
routinely processed lung cancer sections. Intravenous injec-

tion of iodinated neoglycoproteins carrying galactose-ter-
minated N-glycans into mice revealed the highest uptake in
liver and spleen for the bisecting compound compared with
the unsubstituted or core-fucosylated N-glycans. Thus, this
substitution modulates ligand properties in interactions with
lectins, a key finding of this report. Synthetic glycan tailoring
provides a versatile approach to the preparation of newly
substituted glycans with favorable ligand properties for
medical applications.
Keywords: bisecting GlcNAc; drug targeting; lectin; neo-
glycoprotein; tumor imaging.
A major challenge in the post-genomic era is the
functional analysis of post-translational protein modifica-
tions leading to medical applications. With about two
thirds of eukaryotic protein sequences reported to harbor
the N-glycosylation sequon, this type of modification is
typical of membrane and secretory proteins [1]. The
complex enzymatic machinery located in the endoplasmic
reticulum and Golgi network, representing a notable
investment in terms of genomic coding capacity, is known
to produce a large variety of N-glycans [2,3]. These two
aspects, i.e. frequent protein glycosylation and large panel
of glycosyltransferases, suggest a nonrandom, albeit not
template-driven, synthesis with a strong impact on cellular
activities [3,4]. The glycan chains produced, referred to as
the glycomic profile, reflect cellular parameters such as
differentiation and disease processes [3,5]. Current efforts
are directed to relating distinct glycan sequence motifs to
key effector mechanisms at the cellular level. In this
context our study focuses on defining the role of the

bisecting GlcNAc residue.
A key regulatory step in N-glycan processing is the
addition of a b1–4-linked GlcNAc residue to the central
b-mannose unit of the core pentasaccharide. This reaction
is catalyzed by N-acetylglucosaminyltransferase III
(EC 2.4.1.144; GnT-III) placing this GlcNAc residue
(Fig. 1, bottom panel) between the a1–3 and a1–6 arms,
Correspondence to S. Andre
´
, Institut fu
¨
r Physiologische Chemie,
Tiera
¨
rztliche Fakulta
¨
t, Ludwig-Maximilians-Universita
¨
tMu
¨
nchen,
Veterina
¨
rstr. 13, 80539 Mu
¨
nchen, Germany.
Fax: + 49 89 2180 2508, Tel.: + 49 89 2180 2290,
E-mail:
Abbreviations: E-PHA, erythroagglutinating phytohemagglutinin;
GnT-III, N-acetylglucosaminyltransferase III; MD, molecular

dynamics.
Enzymes: N-acetylglucosaminyltransferase III (EC 2.4.1.144;
GnT-III).
*Present address: Bioorganische Chemie, Geba
¨
ude NW1, Universita
¨
t
Bayreuth, 95440 Bayreuth, Germany.
Present address: 3 Hi-Tech Court, Brisbane Technology Park,
Eight Mile Plains, Brisbane, QLD 4113, Australia.
Dedication: dedicated to and in thankful commemoration of Professor
F. Cramer who died recently three months before his 80th birthday.
(Received 25 September 2003, revised 28 October 2003,
accepted 6 November 2003)
Eur. J. Biochem. 271, 118–134 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03910.x
which extend from the core pentasaccharide [2]. The
introduction of a bisecting GlcNAc residue will probably
perturb conformational aspects proximal and also distal to
its position in the glycan chain. Evidence has been provided
for a shift of the conformational equilibrium of the torsional
angle x of the C5–C6 bond of the a1–6 linkage between the
two positions around 180 ° and )60 ° to the 180 ° position
for the biantennary complex-type N-glycan and to the )60 °
position for the hybrid-type N-glycan [6–11]. Next, in the
presence of chain elongation beyond the core pentasaccha-
ride (but not in its absence), the a1–3 linkage becomes
considerably more restrained than in the N-glycan without a
bisecting GlcNAc unit [12–14]. In summary, these reports of
NMR experiments and molecular modeling intimate that

the bisecting GlcNAc acts like a wedge with implications for
the shape of the N-glycan. The occurrence of distinct
alterations in shape induced by this substitution prompted
us to examine the ligand properties of the substituted
N-glycans in detail. Undoubtedly, this work would gain
substantial relevance and importance, if biological effects
were known associated with cellular expression of this
substitution.
Indeed, the second premise for our study was provided
by respective reports. The applied methods included
up-regulation of GnT-III activity, studies on normal or
tumor cells with natural or increased GnT-III activity,
ectopic GnT-III expression, and generation of GnT-III-
deficient mice. In detail, the mutant phenotype of LEC10
CHO cells displaying resistance to ricin was attributed to
induction of GnT-III activity [15,16]. Transcriptional up-
regulation of GnT-III gene expression by forskolin and thus
increased production of N-glycans with bisecting GlcNAc
led to the decreased cell surface presence of lysosomal-
associated membrane glycoprotein-1 and c-glutamyltrans-
peptidase [17]. This exemplary result links the bisecting
motif to intracellular routing. Regarding tumors, GnT-III
was markedly increased in the preneoplastic stage of rat (but
not mouse) liver carcinogenesis, the blast crisis of chronic
myelogenous leukemia, and pediatric brain tumors [18]. The
bisecting GlcNAc then makes its presence felt in tumor
progression. This process can apparently be influenced
nonuniformly, as attested by analysis of different cell types.
GnT-III overexpression yielded suppression of lung meta-
stasis for mouse B16 melanoma cells, increased adhesivity

Fig. 1. Neoglycoproteins with BSA as carrier
for the test panel of six complex-type bianten-
nary N-glycans. The upper part shows the
structures of the biantennary nonasaccharide
(Bi9) and its a2–6(3)-sialylated undecasac-
charides (Bi1126, Bi1123). The bottom panel
illustrates the corresponding decasaccharides
and dodecasaccharides with bisecting Glc-
NAc. For expample, the abbreviation
BiB1226-BSA stands for biantennary bisecting
N-glycan 12-mer a2–6-sialylated BSA conju-
gate. The linker arm and the attachment point
to an e-amino group of lysine of the carrier
protein are also shown.
Ó FEBS 2003 N-glycans with bisecting GlcNAc as ligands (Eur. J. Biochem. 271) 119
and decreased migration activity was observed for human
U373 glioma cells bearing mutual overexpression of GnT-
III relative to GnT-V (shift from biantennary N-glycans
with bisecting GlcNAc to highly branched N-glycans), and
GnT-III-overexpressing human K562 erythroleukemic cells
rather effectively colonized the spleen in athymic mice with
acquisition of resistance to NK cell cytotoxicity [19–21].
Moreover, hemopoiesis supported by bone marrow stroma
requires, in part, integrity of N-glycans, and hemopoietic
dysfunction with lowered production of nonadherent cells in
transgenic mice with GnT-III overexpression demonstrates
that respective changes in the N-glycan profile account for
suppression of proliferation and differentiation of hemopoi-
etic cells [22,23]. Altered glycosylation appears to translate
into cellular responses via biochemical pathways. Apart

from having a bearing on the protein part’s folding or access
to binding partners, the capacity of protein-bound glycan
chains to Ôimpart a discrete recognitional role to the proteinÕ
[24] deserves attention in this respect. Explicitly, the bisecting
GlcNAc has potential either to be directly engaged in
intermolecular interactions with receptors, e.g. endogenous
lectins [4,25–30], or to modulate binding processes at other
sites in the chain through the shape alterations induced by its
presence. That bisecting GlcNAc constitutes a docking point
for protein–carbohydrate interactions is known from the
plant lectin erythroagglutinating phytohemagglutinin
(E-PHA), whereas the presence of a bisecting GlcNAc in a
core-fucosylated biantennary N-glycan did not affect bind-
ing to the
L
-fucose-specific lectin from Aleuria aurantia
[31–35]. Annexin V, a cell surface marker of apoptotic cells,
has recently been reported to share this property with the
plant lectin E-PHA [36].
To determine if and to what extent ligand properties are
conveyed and/or affected by this natural substitution, and
then to devise routes for potential medical application, we
combined synthetic, biochemical, cell biological, histopatho-
logical and in vivo procedures. The question of whether
introduction of the bisecting GlcNAc changes the ligand
properties (binding affinities) of biantennary N-glycans for
branch-end-specific lectins as well as for cells and organs has
so far not been tackled. To this end, it is desirable to obtain
pure test substances, i.e. N-glycans free of natural micro-
heterogeneity, by a convenient preparative route. After

developing a chemoenzymatic synthesis for biantennary
N-glycans with this distinct substitution, the preparation of
the corresponding glycan-bearing neoglycoproteins made
the required tools available. In a previous study, we prepared
neoglycoproteins carrying unsubstituted biantennary
N-glycans [37]. These compounds were examined for their
properties in enzyme-linked lectin and cell binding assays,
histopathological monitoring, and organ biodistribution
[37]. The utility of our combined approach and the
validity of the concept were then tested by assessing the
properties of N-glycans modified with a1–6 core fucose.
Changes in cell biological parameters could be attributed
to the N-glycan modification [38]. Pursuing this line of
research, we report results obtained with neoglycoproteins
carrying biantennary N-glycans with the bisecting Glc-
NAc motif. The chemoenzymatic synthesis of bisecting
N-glycans with terminal galactose or sialic acid residues is
presented followed by the generation of neoglycoproteins.
To take advantage of the enormous strides made over
recent years in acquiring, storing and handling increasing
quantities of data from molecular modeling calculations,
we devoted part of the study to scrutinizing the
conformational properties of the substituted and unsub-
stituted N-glycans. Their behavior patterns were compar-
atively analyzed by refined computational protocols
in vacuo and, compared with previous calculations, in
the presence of a defined solvent. The simulation periods
were significantly extended. Experimentally, the ligand
characteristics of the N-glycans as part of the neoglyco-
proteins (Fig. 1) were evaluated in biochemical, cell

biological and histopathological assays including deter-
mination of biodistribution in vivo relative to the control
compounds (complex-type biantennary N-glycans lacking
bisecting GlcNAc). It is shown that the bisecting substi-
tution triggers alteration of ligand properties, implying
functional significance of the bisecting GlcNAc as a
modulator of N-glycan biorecognition.
Materials and methods
Synthetic and analytical procedures
NMR spectra were recorded with a Bruker AMX 500
spectrometer. HPLC separations were performed on a
Pharmacia LKB gradient system 2249 equipped with a
Pharmacia LKB Detector VWM 2141 (Freiburg, Germany).
For size-exclusion chromatography, a Pharmacia Hi Load
Superdex 30 column (600 · 16 mm) was used. RP-HPLC
was performed on a Macherey-Nagel Nucleogel RP 100-10
column (300 · 25 mm). BSA, b1–4-galactosyltransferase,
a2–6-sialyltransferase and nucleotide sugars were purchased
from Sigma (Munich, Germany), and alkaline phosphatase
(calf intestine, molecular biology grade; EC 3.1.3.1) from
Roche Diagnostics (Heidelberg, Germany). We are grateful
to J. C. Paulson (Cytel Corp., San Diego, USA) for a sample
of recombinant a2–3-sialyltransferase. ESI mass spectra
were recorded on a Finnigan TSQ 700 in methanol/water.
MALDI-TOF mass spectra were collected by D. Renauer at
the Boehringer Mannheim research facility (Tutzing, Ger-
many) on a Voyager Biospectrometry workstation (Vestec/
Perseptive) MALDI-TOF mass spectrometer, using 2,5-
dihydroxybenzoic acid as a matrix. The structures of the
synthetic N-glycans were invariably confirmed by the

following 2D NMR experiments: TOCSY, NOESY,
HMQC (heteronuclear multiple-quantum coherence),
HMQC-COSY, HMQC-DEPT (distortionless enhance-
ment by polarization transfer), HMQC-TOCSY. Signals of
NMR spectra were assigned according to the following
convention including the spacer.
N
1
-(6-Benzyloxycarbonyl-6-aminohexanoylamido)-2-ace-
tamido-2-deoxy-b-
D
-glucopyranosyl)-(1 fi 2)-a-
D
-mann-
opyranosyl-(1 fi 3)-[2-acetamido-2-deoxy-b-
D
-glucopy-
ranosyl-(1 fi 4)]-[2-acetamido-2-deoxy-b-
D
-glucopyran-
120 S. Andre
´
et al.(Eur. J. Biochem. 271) Ó FEBS 2003
osyl-(1 fi 2)-a-
D
-mannopyranosyl-(1 fi 6)]-b-
D
-mann-
opyranosyl-(1 fi 4)-2-acetamido-3,6-di-O-benzyl-2-de-
oxy-b-

D
-glucopyranosyl-(1 fi 4)-2-acetamido-3,6-di-O-
benzyl-2-deoxy-b-
D
-glucopyranoside (3; Bzl3-BiB8AH-
Z). To a portion of 61.1 mg (32.1 lmol) glycosylazide (1)
dissolved in 2 mL methanol were added 65 lLtriethylam-
ine. The flask was flushed with argon followed by addition
of 200 lL propane-1,3-dithiol [39]. After completion of the
reaction (3.5 h; TLC: R
f
amine ¼ 0.22; propan-2-ol/1
M
ammonium acetate, 4 : 1, v/v), the solution was evaporated
and dried in high vacuo for 15 min. The remainder was
allowed to react with a solution of activated Z-aminohex-
anoic acid (2) prepared as follows: 236 mg (0.89 mmol,
20 eq.) Z-aminohexanoic acid were dissolved in 3.5 mL
N-methylpyrrolidone. Subsequently, 136.3 mg (0.89 mmol,
20 eq.) N-hydroxybenzotriazol (HOBT), 285.8 mg
(0.89 mmol, 20 eq.) TBTU [(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium tetrafluoroborate] and 233 lL
(1.32 mmol) di-isopropylethylamine were added, following
a protocol for standard peptide chemistry activation [40].
The mixture was stirred until a clear solution was obtained
and adjusted to pH ¼ 9 by adding 130 lL di-isopropyl-
ethylamine.
The dried glycosylamine was dissolved in 2.5 mL of the
solution prepared as above, and the pH was adjusted to
9.0 with di-isopropylethylamine. After 30 min at ambient

temperature (TLC: propan-2-ol/1
M
ammonium acetate,
4 : 1, v/v), 0.5 mL of the solution was added and stirring
was continued for 5 min. The reaction mixture was
evaporated and dried in high vacuo. Purification of the
remainder was performed by RP-HPLC [acetonitrile/water,
gradient of 35–45% acetonitrile run in 40 min; flow
rate ¼ 8mLÆmin
)1
, Macherey–Nagel Nucleogel RP 100-
10 (300 · 25 mm)]. The yield was 36.8 mg (54.0%), and
analytical data were as follows: R
f
(amine) ¼ 0.22 (propan-
2-ol/1
M
ammonium acetate, 2 : 1) and R
f
(3) ¼ 0.47
(propan-2-ol/1
M
ammonium acetate, 2 : 1); ½a
22
D
¼ ) 2.3°
(0.7, CH
3
CN/H
2

O, 1 : 1) and C
100
H
139
N
7
O
43
(M ¼ 2127.22). ESI-MS: M
calcd
¼ 2125.9, M
found
¼
1064.3 (M + 2 H)
2+
. (Complete set of
1
H/
13
C-NMR data
not shown.)
N
1
-(6-Aminohexanoylamido)-2-acetamido-2-deoxy-b-
D
-
glucopyranosyl)-(1 fi 2)-a-
D
-mannopyranosyl-(1 fi 3)-
[2-acetamido-2-deoxy-b-

D
-glucopyranosyl-(1 fi 4)]-[2-
acetamido-2-deoxy-b-
D
-glucopyranosyl-(1 fi 2)-a-
D
-
mannopyranosyl-(1 fi 6)]-b-
D
-mannopyranosyl-(1 fi 4)-
2-acetamido-2-deoxy-b-
D
-glucopyranosyl-(1 fi 4)-2-ace-
tamido-2-deoxy-b-
D
-glucopyranoside (4; BiB8AH). A
35.8 mg (16.8 lmol) portion of the protected octasaccha-
ride (3) was dissolved in a mixture of 6.7 mL methanol and
330 lL acetic acid. After addition of 61 mg palladium-(II)-
oxide hydrate (84.6%; Pd), the suspension was stirred under
hydrogen at atmospheric pressure for 24 h (TLC: propan-2-
ol/1
M
ammonium acetate, 2 : 1, v/v). To drive the reaction
to completion, 50 mg palladium-(II)-oxide hydrate and
300 mL acetic acid were added, and hydrogenation was
continued for 6 h. The catalyst was removed by centrifu-
gation and washed three times with 10% (v/v) acetic acid in
methanol. The combined supernatants were concentrated,
and the remainder (36.0 mg) was purified by gel filtration

(column: Pharmacia Hi Load Superdex 30, 600 · 16 mm;
mobile phase: 100 m
M
NH
4
HCO
3
; flow rate: 750 lLÆmin
)1
;
detection: 220 and 260 nm) and lyophilized. The yield was
26.1 mg (95.0%) and analytical data were as follows:
R
f
¼ 0.29 (propan-2-ol/1
M
ammonium acetate, 2 : 1,
v/v), ½a
22
D
¼ )1.0 ° (1.7, H
2
O) and C
64
H
109
N
7
O
41

(M ¼ 1632.59). ESI-MS: M
calcd
¼ 1631.68, M
found
¼
816.9 (M + 2H)
2+
. (Complete set of
1
H/
13
C-NMR data
not shown.)
N
1
-(6-Aminohexanoylamido)-b-
D
-galactopyranosyl-(1 fi 4)-
2-acetamido-2-deoxy-b-
D
-glucopyranosyl-(1 fi 2)-a-
D
-mannopyranosyl-(1 fi 3)-[2-acetamido-2-deoxy-b-
D
-
glucopyranosyl-(1 fi 4)]-[b-
D
-galactopyranosyl-(1 fi 4)-
2-acetamido-2-deoxy-b-
D

-glucopyranosyl-(1 fi 2)-a-
D
-
mannopyranosyl-(1 fi 6)]-b-
D
-mannopyranosyl-(1 fi 4)-
2-acetamido-2-deoxy-b-
D
-glucopyranosyl-(1 fi 4)-2-ace-
tamido-2-deoxy-b-
D
-glucopyranoside (5; BiB10AH). A
6.8 mg portion (4.17 lmol) octasaccharide (4) was dissolved
in 2.4 mL 20 m
M
sodium cacodylate buffer, pH 7.4,
containing 1.7 mg BSA, 4.34 lmol NaN
3
,2.43lmol
MnCl
2
,8.3 mg(12.5 lmol) UDP-galactose, 10.3 U alkaline
phosphatase and 206 mU GlcNAc-b1–4-galactosyltrans-
ferase (EC 2.4.1.22). The reaction mixture was incubated for
48 h at 37 °C. After complete reaction (TLC: propan-2-ol/
1
M
ammonium acetate, 2 : 1, v/v), the precipitate was
removed by centrifugation. The supernatant was concen-
trated to a volume of 450 lL, purified by gel filtration

(column: Pharmacia Hi Load Superdex 30, 600 · 16 mm;
mobile phase: 100 m
M
NH
4
HCO
3
; flow rate: 750 lLÆmin
)1
;
detection: 220 and 260 nm) and lyophilized. The yield was
7.3 mg (89.4%) and analytical data were as follows:
R
f
¼ 0.15 (propan-2-ol/1
M
ammonium acetate, 2 : 1, v/v),
½a
22
D
¼ +2.6° (0.46; H
2
O) and C
76
H
129
N
7
O
51

(M ¼ 1956.87). ESI-MS: M
calcd
¼ 1955.79, M
found
¼ 978.9
(M + 2H)
2+
. (Complete set of
1
H/
13
C-NMR data not
shown.)
N
1
-(6-Aminohexanoylamido)-(5-acetamido-3,5-dideoxy-
a-
D
-glycero-
D
-galacto-2-nonulopyranulosonic acid)-
(2 fi 6)-b-
D
-galactopyranosyl-(1 fi 4)-2-acetamido-2-
deoxy-b-
D
-glucopyranosyl-(1 fi 2)-a-
D
-mannopyranosyl-
(1 fi 3)-[2-acetamido-2-deoxy-b-

D
-glucopyranosyl-
(1 fi 4)]-[(5-acetamido-3,5-dideoxy-a-
D
-glycero-
D
-gal-
acto-2-nonulopyranulosonic acid)-(2 fi 6)b-
D
-galactopy-
ranosyl-(1 fi 4)-2-acetamido-2-deoxy-b-
D
-glucopyrano-
syl-(1 fi 2)-a-
D
-mannopyranosyl-(1 fi 6)]-b-
D
-man-
nopyranosyl-(1 fi 4)-2-acetamido-2-deoxy-b-
D
-glucopy-
ranosyl-(1 fi 4)-2-acetamido-2-deoxy-b-
D
-glucopyrano-
side (6; BiB1226AH). A 6.8 mg portion (4.17 lmol) of
octasaccharide (4) was dissolved in 2.4 mL 20 m
M
sodium
cacodylate buffer, pH 7.4, containing 1.7 mg BSA,
4.34 lmol NaN

3
,2.43lmol MnCl
2
, 8.3 mg (12.5 lmol)
UDP-galactose, 10.3 U alkaline phosphatase and 206 mU
GlcNAc-b1–4-galactosyltransferase. The reaction mixture
was incubated for 48 h at 37 °C. After complete reaction
(TLC: propan-2-ol/1
M
ammonium acetate, 2 : 1, v/v),
7.0 mg (9.1 lmol) CMP-N-acetylneuraminic acid and
75 mU b-galactoside-a2–6-sialyltransferase (EC 2.4.99.1)
were added. After the pH had been adjusted to 6.0,
incubation at 37 °C was continued for 24 h. A second
portion of 7.0 mg (9.1 lmol) CMP-N-acetylneuraminic
acid and 75 mU b-galactoside-a2–6-sialyltransferase was
Ó FEBS 2003 N-glycans with bisecting GlcNAc as ligands (Eur. J. Biochem. 271) 121
added (pH 6.0). Incubation at 37 °C for 24 h was followed
by removal of the precipitate by centrifugation. The
supernatant was concentrated to a volume of 450 lL,
purified by gel filtration (column: Pharmacia Hi Load
Superdex 30, 600 · 16 mm; mobile phase: 100 m
M
NH
4
HCO
3
; flow rate: 750 lLÆmin
)1
; detection: 220 and

260 nm) and lyophilized. The yield was 8.4 mg (79.5%),
and analytical data were as follows: R
f
¼ 0.11 (propan-2-ol/
1
M
ammonium acetate, 2 : 1, v/v), ½a
22
D
¼ +8.1° (0.28;
H
2
O) and C
98
H
163
N
9
O
67
(M ¼ 2539.39). ESI-MS:
M
calcd
¼ 2537.96, M
found
¼ 1270.3 (M + 2H)
2+
. (Com-
plete set of
1

H/
13
C-NMR data not shown.)
N
1
-(6-Aminohexanoylamido)-(5-acetamido-3,5-dideoxy-
a-
D
-glycero-
D
-galacto-2-nonulopyranulosonic acid)-
(2 fi 3)-b-
D
-galactopyranosyl-(1 fi 4)-2-acetamido-2-
deoxy-b-
D
-glucopyranosyl-(1 fi 2)-a-
D
-mannopyranosyl-
(1 fi 3)-[2-acetamido-2-deoxy-b-
D
-glucopyranosyl-
(1 fi 4)]-[(5-acetamido-3,5-dideoxy-a-
D
-glycero-
D
-gal-
acto-2-nonulopyranulosonic acid)-(2 fi 3)-b-
D
-galacto-

pyranosyl-(1 fi 4)-2-acetamido-2-deoxy-b-
D
-glucopyra-
nosyl-(1 fi 2)-a-
D
-mannopyranosyl-(1 fi ]-b-
D
-manno-
pyranosyl-(1 fi 4)-2-acetamido-2-deoxy-b-
D
-glucopyran-
osyl-(1 fi 4)-2-acetamido-2-deoxy-b-
D
-glucopyranoside
(7; BiB1223AH). A 6.8 mg portion (4.17 lmol) of
octasaccharide (4) was dissolved in 2.4 mL 20 m
M
sodium
cacodylate buffer, pH 7.4, containing 1.7 mg BSA,
4.34 lmol NaN
3
,2.43lmol MnCl
2
, 8.3 mg (12.5 lmol)
UDP-galactose, 10.3 U alkaline phosphatase and 206 mU
GlcNAc-b1–4-galactosyltransferase. The reaction mixture
was incubated for 48 h at 37 °C. After complete reaction
(TLC: propan-2-ol/1
M
ammonium acetate, 2 : 1, v/v),

7.0 mg (9.1 lmol) CMP-N-acetylneuraminic acid and
103 mU b-galactoside-a2–3-sialyltransferase (EC 2.4.99.6)
were added. After the pH had been adjusted to 6.0,
incubation at 37 °C was continued for 24 h. A second
portion of 7.0 mg (9.1 lmol) CMP-N-acetylneuraminic
acid and 103 mU b-galactoside-a2–3-sialyltransferase were
added (pH 6.0). Incubation at 37 °C for 24 h was followed
by removal of the precipitate by centrifugation. The
supernatant was concentrated to a volume of 450 lL,
purified by gel filtration (column: Pharmacia Hi Load
Superdex 30, 600 · 16 mm; mobile phase: 100 m
M
NH
4
HCO
3
; flow rate: 750 lLÆmin
)1
; detection: 220 and
260 nm) and lyophilized. The yield was 6.5 mg (61.4%) and
analytical data were as follows: R
f
¼ 0.14 (propan-2-ol/1
M
ammonium acetate, 2 : 1, v/v), ½a
22
D
¼ +11.2° (0.1;
H
2

O) and C
98
H
163
N
9
O
67
(M ¼ 2539.39). ESI-MS:
M
calcd
¼ 2537.96, M
found
¼ 1270.3 (M + 2H)
2+
. (Com-
plete set of
1
H/
13
C-NMR data not shown.)
To obtain the neoglycoproteins from the spacered N-gly-
cans, the terminal amino group was converted into a reactive
isothiocyanate [41,42]. In detail, a 0.34 lmol portion of each
6-aminohexanoyl-N-glycan (5–7) was dissolved in 200 lL
dilute NaHCO
3
(100 mg Na
2
CO

3
/10 mL H
2
O) in a 1.5 mL
plastic vessel. A solution of 1 lL(13.1 lmol) thiophosgene in
200 lL dichloromethane was added, and the biphasic
mixture was vigorously stirred. After the amine had been
consumed (1.5 h; TLC: 2-propanol/1
M
ammonium acetate
2 : 1, v/v; R
f
of the decasaccharide derivative ¼ 0.45; R
f
of
the a2–3-disialylated derivative ¼ 0.35; R
f
of the a2–6-
disialylated derivative ¼ 0.28), the phases were separated by
centrifugation, and the aqueous phase was collected. The
organic phase was extracted twice with 100 mL dilute
NaHCO
3
. To remove traces of thiophosgene, the combined
aqueous phases were extracted twice with dichloromethane.
A 2 mg portion of carbohydrate-free BSA was dissolved in
the aqueous solution containing the isothiocyanate deriv-
ative. The pH was adjusted to 9.0 by addition of 1
M
NaOH.

After 6 days at ambient temperature, the neoglycoprotein
was purified by gel filtration (column: Pharmacia Hi Load
Superdex 30, 600 · 16 mm; mobile phase: 100 m
M
NH
4
HCO
3
; flow rate: 750 lLÆmin
)1
; detection: 220 and
260 nm). The product-containing solution was lyophilized.
To calculate the extent of oligosaccharide incorporation into
the protein carrier, a colorimetric assay was employed [43].
Gel electrophoretic analysis under denaturing conditions
combined with silver staining of the neoglycoproteins was
performed as described [37,38]. In addition to these three
neoglycoproteins, lactosylated albumin was produced as
control by the diazonium and phenylisothiocyanate reac-
tions with p-aminophenyl b-lactoside [41,42].
Molecular modeling
The simulations were performed on an IBM-SP2 parallel
machine using the program
DISCOVER
on four or eight
processors in parallel. Typically, they took several days of
CPU time until completed and produced history files with a
size ranging from several hundred megabytes to about 2 GB.
In detail, molecular dynamics calculations of the N-glycans
using parametrization by the CFF91 force field and

automatic procedures of
INSIGHT II
(Molecular Simulations,
San Diego, CA, USA) were run at 1000 K in vacuum using a
dielectric constant of four and at 300 K, 400 K and 450 K in
a solvent box with explicit water molecules in the program
frame
DISCOVER
version 2.98 (Accelrys Inc., San Diego,
USA). An additional force was applied to the pyranose rings
at 1000 K to avoid ring inversions. After an equilibration
period of 100 ps, simulations proceeded for 50–100 ns in the
gas phase and for 1–25 ns in the solvent box. To be able to
describe the conformational space, which is occupied during
the simulation, several characteristic distances between
pseudo atoms were evaluated. The pseudo atom co-ordinates
of each sugar moiety are defined by the arithmetic mean
value of the co-ordinates of its heavy atoms. Conformational
analysis of the data obtained used suitable software tools,
as described previously [44,45].
Lectin-binding assay
Purification of galactoside-specific lectins from dried leaves
of mistletoe (Viscum album L) and bovine heart and of the
b-galactoside-binding IgG fraction from human serum, the
purity controls, biotinylation of the sugar receptors under
activity-preserving conditions, and quantitation of carbo-
hydrate-specific binding to surface-immobilized neoglyco-
proteins have been described in detail previously [37,38,46].
The experimental series with increasing concentrations of
labeled markers and duplicates at each concentration step

were performed at least four times up to saturation of
binding, and each data set was algebraically transformed to
obtain the K
d
value and the number of bound sugar
receptor molecules at saturation.
122 S. Andre
´
et al.(Eur. J. Biochem. 271) Ó FEBS 2003
Flow cytofluorimetry
Automated flow cytofluorimetric analysis of carbohydrate-
dependent binding of biotinylated marker to the surface of
a panel of human tumor cells of different histogenetic
origin [BLIN-1, pre-B cell line; Croco II, B-lymphoblastoid
cell line; CCRF-CEM, T-lymphoblastoid cell line; K-562,
erythroleukemia cell line; KG-1, acute myelogenous
leukemia cell line; HL-60, promyelocytic cell line;
DU4475, mammary carcinoma cell line; NIH:OVCAR-3,
ovarian carcinoma cell line; C205, SW480, and SW620,
colon adenocarcinoma cell lines; Hs-294T, melanoma cell
line; HS-24, nonsmall cell (epidermoid) lung carcinoma cell
line] using the streptavidin–R-phycoerythrin conjugate as
fluorescent indicator (1 : 40 dilution; Sigma, Munich,
Germany) was performed on a FACScan instrument
(Becton-Dickinson, Heidelberg, Germany) equipped with
the software
CONSORT
30, as described previously [37,38].
To reduce nonspecific binding by protein–protein interac-
tions, cells were incubated with 100 lg ligand-free carrier

protein (BSA) per mL for 30 min at 4 °C before incubation
with the biotinylated neoglycoprotein in Dulbecco’s
phosphate-buffered saline containing 0.1% (w/v) ligand-
free BSA. The extent of noncarbohydrate-dependent
fluorescence intensity was subtracted in each case from
the total binding.
Glycohistochemical processing
Following an optimized procedure for visualizing carbohy-
drate-ligand-dependent binding of the neoglycoproteins to
sections of bronchial tumour [small cell (18 cases) and the
three types of nonsmall cell lung cancer (total number of
cases 60; 20 for each type), mesothelioma (20 cases) and
carcinoid (20 cases)], the specimens were processed under
identical conditions with ABC kit reagents and the
substrates diaminobenzidine/H
2
O
2
for development of the
colored, water-insoluble product [42,47,48]. A case was
considered to be positive when at least clusters of tumor cells
were specifically stained and the controls excluded binding
of the labeled neoglycoprotein via the protein, the spacer or
the biotin moieties [42,47,48]. Also, control experiments
without the incubation step with the marker ruled out
staining by binding of kit reagents, i.e. the glycoprotein
avidin or horseradish peroxidase [49].
Biodistribution of radioiodinated neoglycoproteins
Incorporation of
125

I into the neoglycoproteins to a specific
activity of 11.5 MBqÆ(mg protein)
)1
was achieved by the
chloramine-T method using limiting amounts of reagents
[50]. The retention of radioactivity in organs of Ehrlich-
solid-tumor-bearing ddY mice (7 weeks old; Nihon Clea
Co., Tokyo, Japan) after injection of 28.75 kBq per animal
into the tail vein was determined by a c-counter (Aloka
ARC 300, Tokyo, Japan) and expressed as percentage of the
injected dose per gram of wet tissue or per milliliter of blood
for a group of four mice for each type of neoglycoprotein
and for each time point, as described [37,51,52]. The mice
were treated and/or handled according to the Guide
Principles for the Care and Use of Laboratory Animals of
the Japanese Pharmacological Society and with the
approval of Tokyo University of Science’s Institutional
Animal Care and Use Committee.
Results
Synthetic chemistry
A synthetic biantennary heptasaccharide N-glycan with a
single unprotected hydroxy group at the 4¢ position of the
b-linked mannose moiety was used to introduce the
bisecting GlcNAc residue in a yield of 56%. This reaction
pathway was planned as an extension of the basic chemo-
enzymatic strategy of N-glycan synthesis and required no
change in protecting group manipulations [53,54]. After
completion of the synthesis of the bisecting octasaccharide,
the removal of the base-labile protective groups was
straightforward and led to compound 1 (Fig. 2). Selective

reduction of the azido function at the anomeric center was
achieved by the propanedithiol method. After removal of
the volatile compounds in high vacuo, the intermediate
glycosylamine was coupled to the 6-aminohexanoic acid
derivative 2 using standard peptide chemistry activation
with TBTU/HOBt which generates the intermediate active
ester of 2 (Fig. 2). An excess of the activated spacer 2 was
required in the coupling step, suggesting that the remaining
traces of propane-1,3-dithiol were scavenging the active
ester. The purified octasaccharide spacer conjugate 3 was
obtained in 54% yield after preparative HPLC. In the final
deprotection step, catalytic hydrogenation was used to
simultaneously remove the four permanent benzyl groups
from the chitobiosyl core and liberate the primary amino
function in the spacer part. Compound 4 was easily purified
by gel filtration and processed further to the final carbo-
hydrate derivatives 5–7 by enzymatic elongation of the
carbohydrate chain using glycosyltransferases. The presence
of alkaline phosphatase ensured removal of inhibitors [55].
In the first enzymatic step, bovine milk b1–4-galactosyl-
transferase attached galactosyl moieties to each of the
terminal GlcNAc residues of the a1–3 and a1–6 arms. As
expected, the bisecting GlcNAc residue was not a substrate,
presumably because of sterically blocked access for the
enzyme exerted by the two antennae [56]. The resulting
digalactosylated dodecasaccharide 5 (Fig. 2) was purified
(89% yield after gel filtration), and portions were subse-
quently incubated in a one-pot reaction with CMP-sialic
acid and either a2–6 or a2–3-sialyltransferase. After puri-
fication, the desired sialylated dodecasaccharides 6 and 7

were obtained in a yield of 80% and 61%, respectively. To
ensure the structural identity of all reaction products shown
in Fig. 2, compounds 1–7 were routinely analyzed by
electrospray ionization MS (detailed in Materials and
methods) and by complete assignment of the ring carbons
and hydrogens by state-of-the-art 2D NMR techniques
(not shown). TOCSY, NOESY, HMQC, HMQC-COSY,
HMQC-DEPT and HMQC-TOCSY experiments were
employed. Conjugation of the three N-glycans 5–7 to
BSA as cytochemically and histochemically inert carrier was
acomplished by selective activation of the terminal amino
group, as illustrated in Fig. 3. The free amines were
converted into isothiocyanates by thiophosgene in a bipha-
sic reaction, which was followed conveniently by TLC. The
isothiocyanates [10 eq.Æ(mol BSA)
)1
] were added directly to
Ó FEBS 2003 N-glycans with bisecting GlcNAc as ligands (Eur. J. Biochem. 271) 123
carbohydrate-free BSA and allowed to react for 6 days at
pH 9.0. After purification of the neoglycoproteins, success-
ful conjugation was first visualized by the shift in electro-
phoretic mobility in standard SDS/PAGE (Fig. 4). The
mean N-glycan incorporation into the carriers (measured by
a colorimetric assay) for decasaccharide (5) was 3.6
N-glycan molecules per BSA molecule. The reactions with
the sialylated dodecasaccharides 6 and 7 resulted in 4.9 and
3.1 carrier-conjugated glycan chains, respectively. Two main
effects of the bisecting GlcNAc unit on the conformation of
the biantennary glycan were pinpointed graphically by
molecular modeling. In comparison with previous work

[6–11], we (a) extended the simulation periods considerably,
(b) added calculations in a solvent box, and (c) assessed
probabilities of population density in the conformational
space with improved statistical reliability and without the
strict dependence on time-averaged distance constraints.
Computational chemistry
Computational methods were used to analyze the dynamic
behavior of the a1–3/a1–6 branches in the absence and
presence of the bisecting GlcNAc residue. The calculated
xyz population densities of all monosaccharide building
blocks were translated into a strict free-energy grading using
the Boltzmann equation. Isocontour plots at a constant free
energy level of 1.5 kcalÆmol
)1
were drawn to visualize the
inherent flexibility at each point of the branches and the
relation of individual flexibility to structural changes
(Fig. 5). Interestingly, the molecular dynamics (MD) calcu-
lations set to vacuum or a solvent box with water mole-
cules gave very similar results. A prevailing influence of
van der Waals dispersive and repulsive forces on the
conformational properties of this system is consistent with
this outcome. Relative to the absence of solvent, the
simulated presence of water molecules had a dampening
effect on the extent of conformational fluctuations and
dynamics of intramolecular mobility. The way in which the
bisecting substituent shapes the population density of the
biantennary nonasaccharide and decasaccharide is shown in
Fig. 5B,C. As part of the sugar chain, the bisecting GlcNAc
induced separation of the accessed space of the a1–6 arm

into two sections (Fig. 5C). In comparison with the
behavior of the unsubstituted N-glycan, the vicinity of the
core trisaccharide becomes fairly accessible for terminal
residues of the a1–6 arm, a process referred to as backfold-
Fig. 2. Chemical and enzymatic steps to pro-
duce galactosylated and sialylated N-glycans
substituted with bisecting GlcNAc. (a) 1,pro-
panedithiol, Et
3
N, MeOH; 2, N-benzyloxy-
carbonyl-6-aminohexanoic acid, TBTU, 1-
hydroxybenzotriazole (HOBT) (1–2: 54%).
(b) Pd-H
2
, AcOH, MeOH (95%). (c) b1–4-
galactosyltransferase, UDP-Gal, alkaline
phosphatase (89%). (d) a2–6-sialyltransferase,
CMP-NeuNAc, alkaline phosphatase (c + d:
80%). (e) a2–3-sialyltransferase, CMP-Neu-
NAc, alkaline phosphatase (c + e: 61%).
124 S. Andre
´
et al.(Eur. J. Biochem. 271) Ó FEBS 2003
ing. The wedge-like central GlcNAc moiety accounts for
other changes presented in Fig. 5. The inherent flexibility of
the a1–3 arm is clearly restricted in the bisecting compound
(Fig. 5B,C). Backfolding and restrained fluctuations have
been confirmed by experimental NMR analysis with model
oligosaccharides [6–11]. The contribution of carbohydrate
stacking to chain flexibility becomes apparent when two

energetically favored conformations are scrutinized (Fig. 6).
Regions I and II in Fig. 6A,B comprise conformations from
the MD trajectories, with distances between pseudo atoms
characteristic of stacking. Examples of the topological
arrangements of the chain constituents from these regions
are illustrated in Fig. 6C,D. To compare major aspects of
the conformational ensembles of the N-glycans, including
the sialylated compounds without/with bisecting GlcNAc,
representative snapshots from the MD trajectories are
presented in Fig. 7. A topological constellation showing
how the pronounced flexibility of the a1–6 branch can lead
to a dramatic reduction in intramolecular contact with the
bisecting GlcNAc, a situation especially encountered in
region III, is depicted in Fig. 7C,D.
At this point, the frequent occurrence of N-glycosylation
should be recalled. It is still difficult to gauge the influence of
the protein backbone on glycan flexibility in individual
instances. In this context, it is reassuring to note that
oligosaccharides from N-glycoproteins, for example the
Man
5)8
N-glycans from RNase B [57,58], can exhibit
conformational behavior similar to that when attached to
the protein. Therefore, it can be assumed that constant
dynamics or only slightly changed level of mobility will also
be encountered in other cases, especially for neoglycopro-
teins with spacer-bound glycans, where spatial constraints
exerted by the protein are reduced by adding a linker.
However, emerging evidence for the varying influence of a
bisecting GlcNAc on the processing of branch ends for

several glycoproteins precludes general apriorideductions
being drawn [59]. Nonetheless, Figs 5–7, combined with the
experimental evidence reviewed in the Introduction, show
how the addition of the bisecting GlcNAc modifies the
conformational behavior of the N-glycan. To determine
whether binding of sugar receptors to the branches is
affected by the bisecting modification, we performed solid-
phase assays with the same set of lectins and antibodies, as
described previously [37]. With galectin-1 as a test substance,
we selected an endogenous lectin that mediates tumor cell
adhesion to extracellular matrix glycoproteins, a key step in
the metastatic process, invasion of the parenchyma, and
growth regulation after ligand cross-linking [27,28,60–67].
Solid-phase assay
The incorporation of 3.1–4.9 N-glycan chains per albumin
molecule was comparable to the yields in our previous
studies [37,38]. The neoglycoproteins obtained were thus
Fig. 3. Activation of the amino group of the
spacer at the reducing end of the synthetic
N-glycans represented by dodecasaccharide (6)
and the coupling to BSA. (a) Thiophosgene,
CH
2
Cl
2
/H
2
O, NaHCO
3
, pH 8.5 (quant.); (b)

BSA, H
2
O, NaHCO
3
,pH9.0.
Fig. 4. Visualization of the gel electrophoretic mobility under denaturing
conditions of carbohydrate-free BSA (A) and the neoglycoproteins car-
rying the decasaccharide with bisecting GlcNAc (5) (B) and the a2–3-
sialylated (C) and a2–6-sialylated (D) derivatives (substances 6 and 7 in
Fig. 2). Positions of marker proteins for molecular mass designation
are indicated by arrowheads.
Ó FEBS 2003 N-glycans with bisecting GlcNAc as ligands (Eur. J. Biochem. 271) 125
expected to have sufficient ligand density for interaction. In
contrast with natural glycoproteins, the problem of micro-
heterogeneity of the N-glycans is not encountered with these
synthetic products. To monitor the ligand properties
comparatively, we maintained the test profile with a dimeric
plant lectin, mammalian homodimeric galectin-1 and
b-galactoside-specific IgG fraction from human serum.
Similar to the interaction of a lectin with a cell surface and
following the protocol of our two previous studies, the
neoglycoproteins were first bound to the surface and the
glycan-binding proteins assayed in solution. Thereby, any
distortion of the lectin/antibody structures by adsorption to
plastic was avoided. In the solid-phase assay, saturable and
carbohydrate-dependent binding was measured, and the
resulting Scatchard plots gave straight lines indicating the
presence of a single class of binding sites in each case (not
shown). To allow convenient comparisons, we summarize
our data together with previous results on unsubstituted

Fig. 5. Illustration of the nomenclature system for the N-glycan constituents (A) and of their inherent flexibility by isocontour plots (derived from
analysis of xyz population densities of each monosaccharide unit) at a constant energy level of 1.5 kcalÆmol
-1
for the biantennary nonasaccharide (B; first
structure in Fig. 1) and the decasaccharide containing the bisecting GlcNAc (C; structure 4 in Fig. 1 and substance 5 in Fig. 2). For convenient
comparison, the conformations were positioned in space in the same way by superimposing the ring atoms of mannose units of the pentasaccharide
core. Access to the conformational space in the vicinity of the linear part of the core for the terminal galactose moiety of the a1–6 arm is emphasized
by introducing the term ÔbackfoldedÕ into the figure (C; also Fig. 7C,D).
Fig. 6. Analysis of the involvement of the bisecting GlcNAc unit in stacking interactions by measuring pseudo atom distances between this residue
(Fig. 5A, 9) and spatially neighbouring residues 4, 5 and 4¢,5¢ of the two branches, respectively, in MD simulations. The pseudo atom co-ordinates of
each sugar moiety are defined by the arithmetic mean of the co-ordinates of its heavy atoms. Two energetically favored conformational families with
glycosidic torsion angle sets of the GlcNAcb1–4Man linkage of F ¼ 50 °/Y ¼ 20 ° (A; global minimum) or F ¼ )30 °/Y ¼ )30 ° (B; side
minimum) were separately analyzed. The two illustrated conformations representing examples from regions I and II were taken from the MD
trajectories in explicit solvent (for clarity, water molecules are not shown). Distance values between residues 9 and 5 (5¢) located in region I (A) are
characteristic of occurrence of stacking indicated by arrows (C). Spatial proximity to the 4 (4¢) residues can still be maintained for the bisecting
GlcNAc in region II (A, B, D). In region III, populated by the flexible a1–6 branch, the intramolecular contact with the bisecting GlcNAc is clearly
diminished.
126 S. Andre
´
et al.(Eur. J. Biochem. 271) Ó FEBS 2003
N-glycans [37] in Table 1. The general conclusion is that the
presence of a bisecting GlcNAc affects lectin affinity.
In the case of mistletoe lectin, the introduction of a
bisecting GlcNAc into the glycan was unfavorable for
binding. The affinity of the galactose-dependent interaction
was about sixfold lower (Bi9-BSA vs. BiB10-BSA, Table 1).
A similar result was obtained for the a2–6-sialylated
derivative, which is a ligand with even greater binding
affinity than the sialic-acid-free N-glycan (Bi1126-BSA vs.
BiB1226-BSA, Table 1). a2–3-Sialylation produced an un-

favorable docking site. The introduction of the glycan
substituent at a distance from the actual contact site for the
lectin can thus indeed modulate ligand properties, without
being directly involved in binding. Remarkably, the plant
Fig. 7. Illustration of major aspects of the conformational ensembles of sialylated derivatives of the biantennary nonasaccharide Bi9 (A) and of the
decasaccharide BiB10 (for nomenclature, see Fig. 1) with the bisecting GlcNAc (B–D). The conformations presented were taken from the MD
trajectories in explicit solvent (for clarity, water molecules are not shown). The terminal sialic acid moieties of the branches are designated as N and
N¢ to allow easy visualization in the extended (A, B) and backfolded structures (C, D). A description of the populated conformational space has
been given in Fig. 5B,C and that the conformations shown in Fig. 6C,D complement the illustration of the extended structure of the biantennary
N-glycan with bisecting GlcNAc. Stacking interactions with this moiety (indicated by an arrow between residue 4 in the a1–3 branch as the main
contact point and the bisecting GlcNAc) are also possible in backfolded structures (D).
Table 1. Apparent dissociation constants (K
d
) for the interaction of (neo)glycoproteins with sugar receptors and the number of bound probe molecules at
saturation for Viscum album agglutinin (VAA), bovine galectin-1 and the human b-galactoside-binding IgG subfraction from human serum in a solid-
phase assay. K
d
isgiveninn
M
; B
max
is expressed as bound probe molecules per well. The assays with VAA and the neoglycoprotein BiB1226-BSA
were performed with 0.25 lg as matrix. For Lac-BSA (diazo) and Lac-BSA (thio) BSA was glycosylated by covalent attachment of either the
diazophenyl derivative (diazo) or the p-isothiocyanatophenyl derivative (thio) of p-aminophenyl b-
D
-lactoside. Each value is the mean ± SD from
at least four independent experimental series, the quantity of (neo)glycoprotein for coating in lg/well being given for each type of substance.
Matrix
VAA Galectin-1 IgG
K

d
B
max
K
d
B
max
K
d
B
max
BiB10-BSA (0.5 lg) 163.3 ± 79.4 1.9 ± 0.4 · 10
10
518.6 ± 118 2.1 ± 0.6 · 10
10
73.9 ± 31.6 2.3 ± 1.9 · 10
10
BiB1223-BSA (0.5 lg) 1063 ± 347 1.4 ± 0.6 · 10
10
817.9 ± 493 1.6 ± 0.6 · 10
10
36.1 ± 26.5 2.9 ± 1.1 · 10
10
BiB1226-BSA (0.5 lg) 49.8 ± 19.9 4.2 ± 2.3 · 10
10
829.6 ± 50.6 2.2 ± 0.6 · 10
10
71.8 ± 48.9 2.5 ± 1.1 · 10
10
Bi9-BSA (0.5 lg)

a
26.7 ± 11.6 4.6 ± 1.9 · 10
10
900.1 ± 176 42.8 ± 12.5 · 10
10
32.9 ± 19.6 0.35 ± 0.1 · 10
10
Bi1123-BSA (0.5 lg)
a
938.4 ± 661 8.2 ± 4.4 · 10
10
829.5 ± 501 42.0 ± 16.5 · 10
10
87.3 ± 62.7 0.38 ± 0.1 · 10
10
Bi1126-BSA (0.5 lg)
a
8.7 ± 4.5 6.1 ± 1.4 · 10
10
1025.5 ± 619 48.7 ± 18.4 · 10
10
33.9 ± 4.6 0.46 ± 0.1 · 10
10
Lac-BSA (diazo) (3 lg) 312.4 ± 190 4.7 ± 2.3 · 10
10
1127.2 ± 53.3 34.6 ± 17.6 · 10
10
139.0 ± 87.6 0.70 ± 0.1 · 10
10
Lac-BSA (thio) (0.5 lg) 13.4 ± 7.3 5.1 ± 0.2 · 10

10
516.0 ± 20.3 83.3 ± 6.5 · 10
10
7.6 ± 5.2 0.65 ± 0.1 · 10
10
Asialofetuin (1 lg) 7.4 ± 2.6 4.9 ± 0.5 · 10
10
819.0 ± 268 37.5 ± 10.7 · 10
10
69.2 ± 40.2 0.43 ± 0.1 · 10
10
a
From [37].
Ó FEBS 2003 N-glycans with bisecting GlcNAc as ligands (Eur. J. Biochem. 271) 127
lectin tested shares its high toxicity with the biohazard ricin
via rRNA N-glycosidase activity [68]. The specificity of ricin
for galactosides is also very similar to Viscum album
agglutinin, as tested with a series of monosaccharides and
disaccharides and derivatives of methyl b-lactoside [69,70].
This information is relevant for the interpretation of
molecular aspects of the increasing ricin resistance in
LEC10 mutant cells with induced GnT-III expression
[15,16]. After all, our model matrix of neoglycoproteins
with bisecting GlcNAc mimics a cell surface presentation.
Thus, our data strongly argue in favor of the reduced affinity
for toxin binding to the neoglycoprotein contributing to the
resistance linked to GnT-III expression. Moreover, our
measurements on a cell-surface-like support agree with
previous measurements using free N-glycans, supporting the
above conclusions. When the chromatographic retention of

free N-glycans on a ricin column was monitored, a shift to a
shorter retention time was seen for the bisecting N-glycan
relative to the unsubstituted biantennary N-glycan [71]. This
result supports the notion that the conformational behavior
of the BSA-bound N-glycans is similar to that of the
corresponding free N-glycans.
Having shown that the substitution induces changes in
bioaffinity, we next monitored a growth-regulatory mam-
malian lectin, galectin-1. Affinity modulation should have
obvious consequences, for example on removal of activated
T-cells by the pro-apoptotic activity of this lectin [27,29,65].
In this case, an increase in affinity with concomitant
decrease in probe binding (B
max
) was found (Table 1). A
similar tendency had been noted for the animal lectin in the
presence of the a1–6 core fucose unit [38]. Likewise,
presentation of lactose maxiclusters at high density (lactos-
ylated BSA) but not of triantennary N-glycans of asialo-
fetuin improved the affinity in this assay (Table 1).
Surprisingly, when presented on a surface under constant
conditions, biantennary N-glycans without/with bisecting
GlcNAc reacted differently with the two tested galactoside-
specific lectins. In principle, affinity regulation is shown. It
can be positive or negative depending on the nature of the
lectin. Our next result shows that it is also possible for no
apparent effect to be detectable, underscoring the import-
ance of receptor protein parameters. Under identical
experimental conditions, a b-galactoside-binding antibody
fraction from human serum was tested. It did not react

sensitively to the structural modification. Binding affinities
to the IgG fraction did not reflect the structural difference
between unsubstituted N-glycans and their bisecting deriv-
atives. To further examine the ligand properties of the
neoglycoproteins, we determined their binding to cells and
to tissue sections as well as their biodistribution in mice after
injection of radioiodinated material.
Binding to cells and organs
In these experiments, the carrier protein was labeled (by
biotinylation or iodination) without affecting the glycan
part. Relative to the solid-phase assays with purified lectins,
quantitation of cell binding comprises the complex display
of carbohydrate-binding activities including the occurrence
of E-PHA-like human lectins. We monitored the ligand
properties of the carrier-immobilized N-glycans by FAC-
Scan analysis, by staining tissue sections and quantitating
organ uptake. First, we used a panel of established and well-
characterized leukemia/lymphoma, carcinoma and melan-
oma lines to assess surface binding to native cells. Working
with human tumor cells can be considered a model for drug
targeting. To exclude any effect on lectin expression of
varying the duration of the cells in culture or different
numbers of passages, measurements were always performed
with aliquots of the same cell batch on the same day. Under
these controlled conditions, human tumor cells in culture
consistently bound probes in a carbohydrate-dependent
manner. Mixtures of lactose and (asialo)fetuin were expec-
ted to be potent inhibitors. Carbohydrate-independent
binding of the neoglycoprotein and biotin-independent
binding of the second-step reagent was subtracted from the

results to determine the level of specific (glycan-dependent)
binding. Having confirmed the validity of the experimental
Table 2. Flow cytofluorimetric analysis of binding of neoglycoproteins with the biantennary N-glycan (BiB10-BSA) or the a2–3/a2–6-sialylated
derivatives (BiB1223, BiB1226) to human tumor cell lines of diverse histogenetic origin. The median signal of binding of the fluorescent probe is given
as extent of carbohydrate-ligand-dependent binding by subtracting carbohydrate-independent binding from total binding for each cell line.
Cell line
BiB10-BSA BiB1223-BSA BiB1226-BSA
Median
fluorescence
%of
positive cells
Median
fluorescence
%of
positive cells
Median
fluorescence
%of
positive cells
BLIN-1 7.8 80.3 10.8 80.7 7.9 77.7
Croco II 30.2 48.3 26.7 27.2 43.8 34.5
CCRF-CEM 113.7 68.2 129.0 65.7 177.8 66.2
K-562 4.5 27.2 4.9 14.1 5.7 15.5
KG-1 7.1 45.3 17.7 70.6 22.6 75.1
HL-60 11.6 16.8 5.7 21.6 9.9 27.8
DU4475 17.9 21.5 34.0 48.2 48.7 49.3
NIH:OVCAR-3 7.5 17.5 6.0 13.4 11.7 18.5
C205 3.1 16.3 3.6 16.0 5.3 23.4
SW480 11.0 47.3 3.8 30.2 5.4 24.9
SW620 14.4 66.8 5.9 34.0 10.1 43.8

Hs-294T 14.9 31.2 3.5 10.4 3.4 16.5
HS-24 11.8 79.6 6.7 85.4 2.5 88.9
128 S. Andre
´
et al.(Eur. J. Biochem. 271) Ó FEBS 2003
design, we collected binding data to address two issues: (a)
to examine staining with respect to cellular parameters and
(b) to define any effect of the introduction of the substitu-
tion. The results, expressed as median fluorescence intensity
and percentage of positive cells, reflected (a) the histogenetic
origin of the cell type and the level of differentiation and
(b) the glycan structure.
The progression from a pre-B (BLIN-1) to a B-lympho-
blastoid (Croco II) status changed the extent of binding in
the cell populations markedly (Table 2). T-lymphoblastoid
CCRF-CEM cells were clearly distinguished from their
B-lymphoblastoid counterparts by dramatic differences in
median fluorescence intensity (Table 2). When the staining
profiles were examined using the two neoglycoproteins
with/without bisecting GlcNAc, staining intensity was
significantly higher for the pre-B cells when using the
unsubstituted N-glycan-bearing neoglycoprotein (Bi9; 53.4
[37,38]) instead of the bisecting congener BiB10-BSA (7.8;
for nomenclature, see Fig. 1). The nonuniform ligand
properties of BiB10/Bi9 were underscored by measuring
the percentage of positive cells under identical conditions
for leukemia/lymphoma lines (CCRF-CEM, K562 and
KG-1) but not the promyelocytic HL60 cells and especi-
ally for colon/lung carcinoma cells (Fig. 8). As an
additional control for the specificity of binding, the

sialylation status, an important factor in affinity for
endogenous lectins in the case of the siglecs [72], was
noted to affect binding properties. Compared with the
solid-phase assays, the studies of cell binding allowed
determination of the ligand properties of biantennary
N-glycans [37] with either bisecting GlcNAc (this study)
Fig. 8. Comparison of the percentage of positive cells in cytofluorimetric
analysis using the biotinylated neoglycoproteins presenting as cyto-
chemical ligand part the biantennary N-glycan without substitution
(nonasaccharide Bi9) and its derivatives with core fucosylation (deca-
saccharide BiF10) or with bisecting GlcNAc (decasaccharide BiB10) as
givenintheinset.Data for the Bi/BiF substances have previously been
published and are shown for comparison [37,38].
Fig. 9. Quantitative evaluation of the percentage of positive cases in
sections of small cell anaplastic lung carcinoma (SCLC; N = 18), non-
small cell lung carcinoma (N = 60) [i.e. adenocarcinoma (AC; N = 20),
epidermoid carcinoma (EC; N = 20) and large cell carcinoma (LC;
N = 20)], mesothelioma (N = 20) and carcinoid (N = 20), grouped for
the three tested neoglycoproteins as given in the inset (for glycan
nomenclature, see Fig. 1).
Fig. 10. Comparison of the positivity profiles in lung cancer sections
after processing with biotinylated neoglycoproteins presenting as histo-
chemical ligand part the biantennary N-glycans without substitution
(nonasaccharides and undecasaccharides Bi9, Bi1123, Bi1126) and their
derivatives with core fucosylation (decasaccharides and dodecasaccha-
rides BiF10, BiF1223, BiF1226) or with bisecting GlcNAc (decassccha-
rides and dodecasaccharides BiB10, BiB1223, BiB1226) as given in the
insets. Data for the Bi/BiF substances have previously been published
and are shown for comparison [37,38].
Ó FEBS 2003 N-glycans with bisecting GlcNAc as ligands (Eur. J. Biochem. 271) 129

or a1–6 core fucose units [38] for distinct lectins and also
the natural cell surface panel. In comparison with
published data for the core-fucosylated neoglycoprotein
BiF10-BSA [38], the properties of BiB10-BSA binding to
acute myelogenous leukemic KG-1 cells, breast carcinoma
DU4475 cells and colon carcinoma cells showed a distinct
profile (Table 2, Fig. 8). These data illustrate that the cell
surface (as well as the two model lectins in the solid-phase
assays) differentiates between the individual types of
N-glycan in a quantitative manner.
Having shown that the neoglycoproteins interact via
their N-glycans with cell surface receptors on native cells,
the probes were next used in glycohistochemistry [42,73–
75]. Routinely fixed sections of different types of lung
tumor were processed following an optimized protocol. The
results of staining showed that the carrier-immobilized
N-glycans find accessible carbohydrate-binding sites in
a substantial percentage of cases (Fig. 9). Whereas the
differences between the three probes were less pronounced
for the three nonsmall cell lung cancer types (AC, EC, LC),
small cell lung cancer sections (SCLC) showed reduced
staining by the a2–3-sialylated BiB1223-BSA relative to the
nonsialylated BiB10-BSA (Fig. 9). To compare the effect of
the bisecting GlcNAc residue or core fucosylation with
unsubstituted N-glycans in this assay, the three data sets
were combined in one figure, adding relevant information
from previously published reports [37,38]. As shown in
Fig. 10, the binding properties were clearly not uniform.
Because most chemical properties, i.e. the nature of the
carrier protein, the spacer for the glycan and the labeling,

were kept identical and the same set of tumor cases was
examined, the differences in staining can be attributed to
the structure of the N-glycan ligands. In line with the solid-
phase and cell surface binding studies, the profiles of tumor
cell positivity displayed probe-dependent alterations. In
agreement with the data from cytofluorimetric analysis
shown in Fig. 8, there is a general tendency for increased
reactivity to substituted N-glycans. These results add
further evidence to the impact of GnT-III activity on the
ligand characteristics of N-glycans. By measuring the
biodistribution of the iodinated neoglycoproteins after
intravenous injection into mice, a further test system was
applied to relate changes in N-glycan structure with cell
binding, using the observed organ retention as a model for
drug targeting [76,77].
The radioactivity, expressed as percentage of injected
dose per g of tissue (mL of blood), was determined 1 h
and 6 h after the injection. Blood clearance was obviously
retarded for the sialylated derivatives BiB1223-BSA
and BiB1226-BSA (Table 3). However, the extent of
retardation was smaller than for the unsubstituted sialyl-
ated derivatives, especially the a2–6-sialylated compound
Bi1126-BSA, with 13.95 ± 0.45% of the injected dose still
being in circulation after 6 h [37] compared with
5.56 ± 0.14% for BiB1226. When considering means of
prolonging the presence in the serum of a glycosylated
carrier (e.g. a pharmacologically active glycoprotein), a
biantennary N-glycan with a2–3 sialylation appears to be a
good candidate, with no improvement gained by further
addition of a bisecting GlcNAc or an a1–6 core Fuc unit

(Fig. 11). On the other hand, organ uptake may be
enhanced by these substitutions. Figure 11 presents a
comparison of the biodistribution data for the three series
of neoglycoproteins and major organ sites for uptake. In
the case of the neoglycoprotein with the bisecting
decasaccharide (BiB10-BSA), radioactivity counts in the
liver and spleen were highest when compared with the
unsubstituted and core-fucosylated forms. Adding a
bisecting GlcNAc substituent to a nonsialylated N-glycan
could therefore be exploited when targeting to the liver
and spleen is an important factor such as in imaging. By
administering the a2–6-sialylated derivative of the core-
fucosylated N-glycan (BiF1226), uptake in the liver and
spleen could be slightly enhanced [38]. Interestingly, this
derivative gave weaker signals for kidney than the related
dodecasaccharide with bisecting GlcNAc (BiB1226-BSA),
pointing to a combination of the effects of sialylation and
core substitution. Also, the low accumulation of the
neoglycoprotein with the N-glycan chain of BiF1226
(1.36 ± 0.09% after 6 h) in tumors compared with the
Table 3. Biodistribution of
125
I-neoglycoproteins (% injected dose per g of tissue or ml of blood) in Ehrlich solid-tumor-bearing mice after 1 h and 6 h.
Each value represents the mean ± SEM for four animals.
Tissue
1h 6h
BiB10-BSA BiB1223-BSA BiB1226-BSA BiB10-BSA BiB1223-BSA BiB1226-BSA
Blood 3.11 ± 0.14 21.23 ± 1.36 12.19 ± 0.35 1.39 ± 0.10 11.20 ± 0.83 5.56 ± 0.14
Liver 6.32 ± 0.37 3.91 ± 0.26 4.04 ± 0.22 2.32 ± 0.13 2.22 ± 0.20 1.68 ± 0.07
Kidneys 3.01 ± 0.32 4.04 ± 0.19 4.10 ± 0.41 1.33 ± 0.12 2.59 ± 0.45 2.39 ± 0.30

Spleen 3.26 ± 0.34 3.30 ± 0.20 2.26 ± 0.13 1.05 ± 0.07 1.49 ± 0.15 1.16 ± 0.08
Heart 1.01 ± 0.05 3.12 ± 0.20 2.00 ± 0.19 0.38 ± 0.04 1.78 ± 0.15 1.01 ± 0.04
Lung 1.82 ± 0.15 2.69 ± 0.14 2.26 ± 0.10 0.85 ± 0.10 1.92 ± 0.17 1.42 ± 0.09
Thymus 1.13 ± 0.11 1.63 ± 0.24 1.60 ± 0.05 0.74 ± 0.13 1.58 ± 0.14 1.16 ± 0.11
Pancreas 0.92 ± 0.06 1.14 ± 0.09 1.35 ± 0.08 0.53 ± 0.12 0.86 ± 0.11 0.81 ± 0.42
Lymph node 0.75 ± 0.03 2.00 ± 0.11 1.17 ± 0.22 0.33 ± 0.05 1.57 ± 0.15 0.95 ± 0.06
Muscle 0.31 ± 0.01 0.44 ± 0.05 0.46 ± 0.02 0.17 ± 0.02 0.33 ± 0.04 0.34 ± 0.02
Vertebrae 0.86 ± 0.06 1.51 ± 0.13 1.17 ± 0.07 0.37 ± 0.04 0.81 ± 0.05 0.70 ± 0.04
Brain 0.13 ± 0.01 0.33 ± 0.02 0.25 ± 0.01 0.04 ± 0.00 0.20 ± 0.02 0.11 ± 0.01
Tumor 1.47 ± 0.09 4.04 ± 0.31 2.92 ± 0.15 0.67 ± 0.09 3.44 ± 0.36 2.39 ± 0.16
130 S. Andre
´
et al.(Eur. J. Biochem. 271) Ó FEBS 2003
data for BiB1226 (2.39 ± 0.16%) and Bi1126
(3.46 ± 0.25%) underscores how the parameter variation,
i.e. glycan structure, is able to affect the biodistribution
and its kinetics. Uptake of the neoglycoproteins into
organs other than liver, kidney and spleen was rather low.
This is in accordance with our previous results and with
studies of the uptake of tyrosine-modified oligosaccharides
from bovine fetuin and porcine fibrinogen [78,79]. Overall,
these data show that manipulation of N-glycans can
endow them with medically favorable properties.
Discussion
The enzymology of N-glycan biosynthesis has been thor-
oughly analyzed and this knowledge has then been used to
pinpoint the mutated site(s) in glycosylation mutants (e.g. in
LEC cell lines) or to modulate distinct glycosyltransferase
activities by creating deficient or overexpressing cells and
mice (see the Introduction). Furthermore, sophisticated

protocols have been devised for glycoconjugate analysis to
detect the presence of N-glycan variants in the glycomic
profile [80,81]. The evidence accrued suggests that changes
in the glycomic profile should not be considered events that
occur solely by chance. Correlation of structural changes in
the glycan population with measurable effects is not a
straightforward process, especially at cell or organism level.
Preparing synthetic N-glycans with defined substitutions
has allowed us to produce neoglycoproteins lacking phy-
siological microheterogeneity. These synthetic probes can be
used to determine whether structural alterations affect the
properties of N-glycans in intermolecular recognition. Thus,
a combination of chemoenzymatic synthesis with biochemi-
cal, cell biological and histopathological assays can be used
to assess the biological relevance of particular glycan
modifications.
In continuation of our two previous studies on bianten-
nary N-glycans and their core-fucosylated derivatives
[37,38], we have devised a strategy for synthesizing spacer-
modified bisecting N-glycans followed by enzymatic chain
elongation and conjugation to BSA. State-of-the-art
molecular modeling, using the available experimental
evidence, indicated that, relative to the unmodified bianten-
nary N-glycan, the presence of the bisecting GlcNAc residue
alters the conformational space accessible for the a1–3 and
a1–6 arms by backfolding and stacking. These topological
changes are the most likely candidates to explain the change
in affinity for two galactoside-specific lectins, because they
exclusively bind to terminal galactose residues without
direct interaction with the bisecting GlcNAc. These results

thus provide strong initial evidence that the conformational
modulation triggered by adding a substituent to a carbo-
hydrate ligand can alter the affinity for distinct lectins,
notably including a mammalian lectin. This message
broadens our view on the levels of affinity modulation for
carbohydrate ligands. Besides alterations in sequence and
local density by clustering, the presence of a substitution is
to be reckoned with, making complex glycans study objects
of choice to define the binding profile of a lectin, e.g. a
galectin [82,83]. In comparison with the tested lectins,
GnT-V interacts with substrates (acceptors with/without
bisecting GlcNAc) with similar K
m
values, but a dramatic
impairment at the level of the catalytic step was reported
when the substitution was present [84].
Using the neoglycoproteins to probe binding in cells,
tissues and organs, the data produced support our
reasoning that naturally occurring N-glycan modifications
would translate into changes in biological properties. The
occurrence of ricin resistance in CHO cell mutants being
attributed to GnT-III activity [15,16] is an example in
which an ensuing decrease in affinity in plant lectin
binding can help to explain the new cell feature. In the
same way, it is reasonable to suggest galectin-1 and
galectin-3 as candidates to rationalize the increase in
Fig. 11. Comparison of the numerical values for the percentage of
injected dose per g of tissue (or ml of blood) of
125
I-neoglycoproteins

presenting as bioactive ligand part the biantennary N-glycans without
substitution (nonasaccharides and undecasaccharides Bi9, Bi1123,
Bi1126) and their derivatives with core fucosylation (decasacchrides and
dodecasaccharides BiF10, BiF1223, BiF1226) or with bisecting GlcNAc
(decasccharides and dodecasaccharides BiB10, BiB1223, BiB1226), as
given in the insets, present in blood, liver, kidneys, and spleen of mice 1 h
after injection. Data for the Bi/BiF substances have previously been
published and are shown for comparison [37,38]; note different scales
of the y-axis in the three graphs.
Ó FEBS 2003 N-glycans with bisecting GlcNAc as ligands (Eur. J. Biochem. 271) 131
percentage of positive cells, shown in Fig. 8, as their cell
surface presentation had been ascertained for the colon
cancer cell lines tested [85]. From the decreased cell
migration of stable GnT-III-overexpressing transfectants
of the human glioblastoma line U-373 MG on a
fibronectin substratum [21] and the stimulation of motil-
ity/migration in malignant astrocytes by galectin-1 [63,86],
the general perspective arises relating changes in this
aspect of tumor glycan expression to bioaffinity modula-
tion in the interaction with tissue lectins and thus to the
pattern of expressed lectins (lectinome). Combined studies
on glycan and lectin expression are thus proposed to
define new functional correlations. The path to the
presented results and this perspective has been paved by
a combination of synthetic and computational chemistry
with a test panel using biochemical, cell biological,
histopathological and animal studies. This insight into
the role of distant substituents in altering affinity for
certain lectins adds a new aspect to the discussion of the
range of physiological importance of the bisecting Glc-

NAc modification. It also brings a promising perspective
for medical applications in drug targeting. Systematic
glycan modeling can guide chemoenzymatic synthesis
towards the preparation of substituted N-glycans not
available from natural sources. This opens up a new route
to achieving affinity optimization of lectin-targeted thera-
peutics other than tailoring their actual ligand sites.
Acknowledgements
We are indebted to Professor J. C. Paulson for his gift of
a2–3- sialyltransferase, to Professor H. Kessler for his support, to Dr
S. Namirha for helpful discussion, and to B. Hofer for skilful technical
assistance. Generous financial support from the Deutsche Forschungsg-
emeinschaft andthe Wilhelm Sander-Stiftung is gratefully acknowledged.
References
1. Apweiler, R., Hermjakob, H. & Sharon, N. (1999) On the fre-
quency of protein glycosylation, as deduced from analysis of the
SWISS-PROT database. Biochim. Biophys. Acta 1473, 4–8.
2. Brockhausen, I. & Schachter, H. (1997) Glycosyltransferases
involved in N- and O-glycan biosynthesis. In Glycosciences: Status
and Perspectives (Gabius, H J. & Gabius, S., eds), pp. 79–113.
Chapman & Hall, Weinheim.
3. Reuter, G. & Gabius, H J. (1999) Eukaryotic glycosylation: whim
of nature or multipurpose tool? Cell. Mol. Life Sci. 55, 368–422.
4. Gabius, H J., Andre
´
, S., Kaltner, H. & Siebert, H C. (2002) The
sugar code: functional lectinomics. Biochim. Biophys. Acta 1572,
165–177.
5. Brockhausen, I., Schutzbach, J. & Kuhns, W. (1998) Glycopro-
teins and their relationship to human disease. Acta Anat. 161,

36–78.
6. Brisson, J R. & Carver, J.P. (1983) Solution conformation of
asparagine-linked oligosaccharides: a(1–2)-, a(1–3)-, b(1–2)-, and
b(1–4)-linked units. Biochemistry 22, 3671–3680.
7. Brisson, J R. & Carver, J.P. (1983) Solution conformation
of asparagine-linked oligosaccharides: a(1–6)-linked moiety.
Biochemistry 22, 3680–3686.
8. Homans, S.W., Dwek, R.A. & Rademacher, T.W. (1987) Solution
conformations of N-linked oligosaccharides. Biochemistry 26,
6571–6578.
9. Rutherford, T.J. & Homans, S.W. (1994) Restrained vs free
dynamics simulations of oligosaccharides: application to solution
dynamics of biantennary and bisected biantennary N-linked
glycans. Biochemistry 33, 9606–9614.
10. Balaji, P.V., Qasba, P.K. & Rao, V.S.R. (1996) Molecular
dynamics simulations of hybrid and complex type oligosacchar-
ides. Int. J. Biol. Macromol. 18, 101–114.
11. Qasba, P.K., Balaji, P.V. & Rao, V.S.R. (1997) Conformational
analysis of Asn-linked oligosaccharides: implications in biological
processes. J. Mol. Struct. (Theochem.) 395–396, 333–360.
12. Mazurier,J.,Dauchez,M.,Vergoten,G.,Montreuil,J.&Spik,G.
(1991) Molecular modeling of a disialylated monofucosylated
biantennary glycan of the N-acetyllactosamine type. Glycoconju-
gate J. 8, 390–399.
13. Naidoo, K.J., Denysyk, D. & Brady, J.W. (1997) Molecular
dynamics simulations of the N-linked oligosaccharide of the lectin
from Erythrina corallodendron. Protein Eng. 10, 1249–1261.
14. Qasba, P.K. (2000) Involvement of sugars in protein–protein
interactions. Carbohydrate Polym. 41, 293–309.
15. Campbell, C. & Stanley, P. (1984) A dominant mutation to ricin

resistance in Chinese hamster ovary cells induces UDP-GlcNAc:
glycopeptide b-4-N-acetylglucosaminyltransferase III activity.
J. Biol. Chem. 259, 13370–13378.
16. Stanley, P., Sundaram, S. & Sallustio, S. (1991) A subclass of cell
surface carbohydrates revealed by a CHO mutant with two gly-
cosylation mutations. Glycobiology 1, 307–314.
17. Sultan, A.S., Miyoshi, E., Ihara, Y., Nishikawa, A., Tsukada, Y.
& Taniguchi, N. (1997) Bisecting GlcNAc structures act as nega-
tive sorting signals for cell surface glycoproteins in forskolin-
treated rat hepatoma cells. J. Biol. Chem. 272, 2866–2872.
18. Taniguchi, N., Yoshimura, M., Miyoshi, E., Ihara, Y., Nishikawa,
A. & Fujii, S. (1996) Remodeling of cell surface glycoproteins by
N-acetylglucosaminyltransferase III gene transfection: modulation
of metastatic potentials and downregulation of hepatitis B virus
replication. Glycobiology 6, 691–694.
19. Yoshimura, M., Nishikawa, A., Ihara, Y., Taniguchi, S. & Tani-
guchi, N. (1995) Suppression of lung metastasis of B16 mouse
melanoma by N-acetylglucosaminyltransferase III gene transfec-
tion. Proc. Natl Acad. Sci. USA 92, 8754–8758.
20. Yoshimura,M.,Ihara,Y.,Ohnishi,A.,Ijuhin,N.,Nishiura,T.,
Kanakura, Y., Matsuzawa, Y. & Taniguchi, N. (1996) Bisecting
N-acetylglucosamine on K562 cells suppresses natural killer
cytotoxicity and promotes spleen colonization. Cancer Res. 56,
412–418.
21. Yamamoto,H.,Swoger,J.,Greene,S.,Saito,T.,Hurh,J.,Sweeley,
C., Leestma, J.L., Mkrdichian, E., Cerullo, L., Nishikawa, A.,
Ihara, Y., Taniguchi, N. & Moskal, J.R. (2000) b1,6-N-acetylg-
lucosamine-bearing N-glycans in human gliomas: implications for
a role in regulating invasivity. Cancer Res. 60, 134–142.
22. Gabius, S. & Gabius, H J. (1990) Sugar receptors of the stromal

cell layer in human long-term bone marrow cultures: their pre-
sence, modulatory responses to changes in the microenvironment
and potential role in cellular adhesion. Blut 61, 232–239.
23. Yoshimura, M., Ihara, Y., Nishiura, T., Okajima, Y., Ogawa, M.,
Yoshida, H., Suzuki, M., Yamamura, K i., Kanakura, Y.,
Matsuzawa, Y. & Taniguchi, N. (1998) Bisecting GlcNAc struc-
ture is implicated in suppression of stroma-dependent haemopoi-
esis in transgenic mice expressing N-acetylglucosaminyltransferase
III. Biochem. J. 331, 733–742.
24. Winterburn, P.J. & Phelps, C.F. (1972) The significance of
glycosylated proteins. Nature (London) 236, 147–151.
25. Gabius, H J. (1997) Animal lectins. Eur. J. Biochem. 243,
543–576.
26. Kaltner, H. & Stierstorfer, B. (1998) Animal lectins as cell adhe-
sion molecules. Acta Anat. 161, 162–179.
27. Gabius, H J. (2001) Probing the cons and pros of lectin-induced
immunomodulation: case studies for the mistletoe lectin and
galectin-1. Biochimie 83, 659–666.
132 S. Andre
´
et al.(Eur. J. Biochem. 271) Ó FEBS 2003
28. Danguy, A., Camby, I. & Kiss, R. (2002) Galectins and cancer.
Biochim. Biophys. Acta 1572, 285–293.
29. Rabinovich, G.A., Rubinstein, N. & Toscano, M.A. (2002) Role
of galectins in inflammatory and immunomodulatory processes.
Biochim. Biophys. Acta 1572, 274–284.
30. Weigel, P.H. & Yik, J.H.N. (2002) Glycans as endocytosis signals:
the cases of the asialoglycoprotein and hyaluronan/chondroitin
sulfate receptors. Biochim. Biophys. Acta 1572, 341–363.
31. Cummings, R.D. & Kornfeld, S. (1982) Characterization of the

structural determinants required for the high affinity interaction of
asparagine-linked oligosaccharides with immobilized Phaseolus
vulgaris leukoagglutinating and erythroagglutinating lectins.
J. Biol. Chem. 257, 11230–11234.
32. Yamashita, K., Hitoi, A. & Kobata, A. (1983) Structural
determinants of Phaseolus vulgaris erythroagglutinating lectin for
oligosaccharides. J. Biol. Chem. 258, 14753–14755.
33. Yamashita, K., Kochibe, N., Ohkura, T., Ueda, I. & Kobata, A.
(1985) Fractionation of
L
-fucose-containing oligosaccharides on
immobilized Aleuria aurantia lectin. J. Biol. Chem. 260, 4688–
4693.
34. Narasimhan, S., Freed, J.C. & Schachter, H. (1986) The effect of a
ÔbisectingÕ N-acetylglucosaminyl group on the binding of bian-
tennary, complex oligosaccharides to concanavalin A, Phaseolus
vulgaris erythroagglutinin (E-PHA), and Ricinus communis
agglutinin (RCA-120) immobilized on agarose. Carbohydr. Res.
149, 65–83.
35. Green, E.D. & Baenziger, J.U. (1987) Oligosaccharide specificities
of Phaseolus vulgaris leukoagglutinating and erythroagglutinating
phytohemagglutinins. Interactions with N-glycanase-released oli-
gosaccharides. J. Biol. Chem. 262, 12018–12029.
36. Gao-Uozumi,C X.,Uozumi,N.,Miyoshi,E.,Nagai,K.,Ikeda,
Y.,Teshima,T.,Noda,K.,Shiba,T.,Honke,K.&Taniguchi,N.
(2000) A novel carbohydrate binding activity of annexin V toward
abisectingN-acetylglucosamine. Glycobiology 10, 1209–1216.
37. Andre
´
,S.,Unverzagt,C.,Kojima,S.,Dong,X.,Fink,C.,Kayser,

K. & Gabius, H J. (1997) Neoglycoproteins with the synthetic
complex biantennary nonasaccharide or its a2,3/a2,6-sialylated
derivatives: their preparation, assessment of their ligand properties
for purified lectins, for tumor cells in vitro, and in tissue sections,
and their biodistribution in tumor-bearing mice. Bioconjugate
Chem. 8, 845–855.
38. Unverzagt, C., Andre
´
,S.,Seifert,J.,Kojima,S.,Fink,C.,
Srikrishna, G., Freeze, H., Kayser, K. & Gabius, H J. (2002)
Structure-activity profiles of complex biantennary glycans
with core fucosylation and with/without additional a2,3/a2,6
sialylation: synthesis of neoglycoproteins and their properties in
lectin assays, cell binding, and organ uptake. J. Med. Chem. 45,
478–491.
39. Bayley, H., Standring, D.N. & Knowles, J.B. (1978) Propane-1,3-
dithiol: a selective reagent for the efficient reduction of alkyl and
aryl azides to amines. Tetrahedron Lett. 19, 3633–3634.
40. Knorr, R., Treciak, A., Bannwarth, W. & Gillessen, D. (1989)
New coupling reagents in peptide chemistry. Tetrahedron Lett. 30,
1927–1930.
41. McBroom, C.R., Samanen, C.H. & Goldstein, I.J. (1972) Car-
bohydrate antigens: coupling of carbohydrates to proteins by
diazonium and phenylisothiocyanate reactions. Methods Enzymol.
28, 212–219.
42. Gabius, H J. & Bardosi, A. (1991) Neoglycoproteins as tools in
glycohistochemistry. Progr. Histochem. Cytochem. 22, 1–66.
43.Monsigny,M.,Petit,C.&Roche,A C.(1988)Colorimetric
determination of neutral sugars by a resorcinol sulfuric acid
micromethod. Anal. Biochem. 175, 525–530.

44. Siebert, H C., Andre
´
,S.,Asensio,J.L.,Can
˜
ada, F.J., Dong, X.,
Espinosa,J F.,Frank,M.,Gilleron,M.,Kaltner,H.,Koza
´
r, T.,
Bovin, N.V., von der Lieth, C W., Vliegenthart, J.F.G., Jime
´
nez-
Barbero, J. & Gabius, H J. (2000) A new combined computa-
tional and NMR-spectroscopical strategy for the identification of
additional conformational constraints of the bound ligand in an
aprotic solvent. Chembiochem 1, 181–195.
45. Siebert, H C., Lu
¨
,S Y.,Frank,M.,Kramer,J.,Wechselberger,
R.,Joosten,J.,Andre
´
,S.,Rittenhouse-Olson,K.,Roy,R.,vonder
Lieth, C W., Kaptein, R., Vliegenthart, J.F.G., Heck, A.J.R. &
Gabius, H J. (2002) Analysis of protein–carbohydrate interaction
at the lower size limit of the protein part (15-mer peptide) by NMR
spectroscopy, electrospray ionization mass spectrometry, and
molecular modeling. Biochemistry 41, 9707–9717.
46. Andre
´
,S.,Pieters,R.J.,Vrasidas,I.,Kaltner,H.,Kuwabara,I.,
Liu, F T., Liskamp, R.M.J. & Gabius, H J. (2001) Wedgelike

glycodendrimers as inhibitors of binding of mammalian galectins
to glycoproteins, lactose maxiclusters, and cell surface glyco-
conjugates. Chembiochem 2, 822–830.
47. Danguy,A.,Kayser,K.,Bovin,N.V.&Gabius,H J.(1995)The
relevance of neoglycoconjugates for histology and pathology.
Trends Glycosci. Glycotechnol. 7, 261–275.
48. Kayser, K., Bo
¨
hm,G.,Blum,S.,Beyer,M.,Zink,S.,Andre
´
,S.&
Gabius, H J. (2001) Glyco- and immunohistochemical refinement
of the differential diagnosis between mesothelioma and metastatic
carcinoma and survival analysis of patients. J. Pathol. 193,
175–180.
49. Kuchler,S.,Zanetta,J P.,Vincendon,G.&Gabius,H J.(1990)
Detection of binding sites for biotinylated neoglycoproteins and
heparin (endogenous lectins) during cerebellar ontogenesis in the
rat. Eur. J. Cell Biol. 52, 87–97.
50. Kojima,S.,Shimura,N.,Kubodera,A.,Takahashi,T.&Oya-
mada, H. (1991) Radioimmunodetection of human colon cancer
in nude mice by a new monoclonal antibody A7 against human
colorectal cancer. Nucl. Med. Biol. 18, 847–853.
51. Kojima, S. & Gabius, H J. (1988) Biodistribution of neoglyco-
proteins in mice bearing solid Ehrlich tumor. J. Cancer Res. Clin.
Oncol. 114, 468–472.
52. Kojima, S., Andre
´
, S., Korchagina, E.Y., Bovin, N.V. & Gabius,
H J. (1997) Tyramine-containing poly(4-nitrophenylacrylate) as

iodinatable ligand carrier in biodistribution analysis. Pharmaceut.
Res. 14, 879–886.
53. Unverzagt, C. (1996) Chemoenzymatic synthesis of a sialylated
undecasaccharide-asparagine. Angew. Chem. Int. Ed. 35, 2350–
2353.
54. Unverzagt, C. & Seifert, J. (2000) Chemoenzymatic synthesis of
deca and dodecasaccharide N-glycans of the ÔbisectingÕ type.
Tetrahedron Lett. 41, 4549–4553.
55. Unverzagt, C., Kunz, H. & Paulson, J.C. (1990) High-efficiency
synthesis of sialyloligosaccharides and sialoglycopeptides. J. Am.
Chem. Soc. 112, 9308–9309.
56. Narasimhan, S., Freed, J.C. & Schachter, H. (1985) Control of
glycoprotein synthesis. Bovine milk UDP-galactose: N-acetyl-
glucosamine b-4-galactosyltransferase catalyzes the preferential
transfer of galactose to the GlcNAc b1,2Mana1,3-branch of both
bisected and nonbisected complex biantennary asparagine-linked
oligosaccharides. Biochemistry 24, 1694–1700.
57. Berman, E., Walters, E. & Allerhand, A. (1981) Structure and
dynamic behavior of the oligosaccharide side chain of bovine
pancreatic ribonuclease B. Application of
13
C-nuclear magnetic
resonance spectroscopy. J. Biol. Chem. 256, 3853–3857.
58. Gonza
´
lez, L., Bruix, M., Dı
´
az-Maurin
˜
o,T.,Feizi,T.,Rico,M.,

Solı
´
s, D. & Jime
´
nez-Barbero, J. (2000) Conformational studies of
the Man8 oligosaccharide on native ribonuclease B and on the
reduced and denatured protein. Arch. Biochem. Biophys. 383,
17–27.
59. Koyota, S., Ikeda, Y., Miyagawa, S., Ihara, H., Koma, M.,
Honke, K., Shirakura, R. & Taniguchi, N. (2001) Down-regula-
tion of the a-Gal epitope expression in N-glycans of swine
Ó FEBS 2003 N-glycans with bisecting GlcNAc as ligands (Eur. J. Biochem. 271) 133
endothelial cells by transfection with the N-acetylglucosaminyl-
transferase III gene. Modulation of the biosynthesis of terminal
structures by a bisecting GlcNAc. J. Biol. Chem. 276, 32867–
32874.
60. Gabius, H J. (1997) Concepts of tumor lectinology. Cancer Invest.
15, 454–464.
61. 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.
62. Kopitz, J., von Reitzenstein, C., Andre
´
,S.,Kaltner,H.,Uhl,J.,
Ehemann, V., Cantz, M. & Gabius, H J. (2001) Negative
regulation of neuroblastoma cell growth by carbohydrate-depen-
dent surface binding of galectin-1 and functional divergence from
galectin-3. J. Biol. Chem. 276, 35917–35923.

63. Rorive,S.,Belot,N.,Decaestecker,C.,Lefranc,F.,Gordower,L.,
Micik, S., Maurage, C A., Kaltner, H., Ruchoux, M M., Dan-
guy, A., Gabius, H J., Salmon, I., Kiss, R. & Camby, I. (2001)
Galectin-1 is highly expressed in human gliomas with relevance for
modulation of invasion of tumor astrocytes into the brain par-
enchyma. Glia 33, 241–255.
64. Brewer, C.F. (2002) Binding and cross-linking properties of
galectins. Biochim. Biophys. Acta 1572, 255–262.
65. Rappl, G., Abken, H., Muche, J.M., Sterry, W., Tilgen, W.,
Andre
´
,S.,Kaltner,H.,Ugurel,S.,Gabius,H J.&Reinhold,U.
(2002) CD4
+
CD7
–
leukemic T cells from patients with Se
´
zary
syndrome are protected from galectin-1-triggered T cell death.
Leukemia 16, 840–846.
66. Hittelet, A., Legendre, H., Nagy, N., Bronckart, Y., Pector,
J C., Salmon, I., Yeaton, P., Gabius, H J., Kiss, R. & Camby, I.
(2003) Upregulation of galectins-1 and -3 in human colon cancer
and their role in regulating cell migration. Int. J. Cancer 103,
370–379.
67. Nagy, N., Legendre, H., Engels, O., Andre
´
,S.,Kaltner,H.,
Wasano,K.,Zick,Y.,Pector,J C.,Decaestecker,C.,Gabius,

H J., Salmon, I. & Kiss, R. (2003) Refined prognostic evaluation
in colon carcinoma using immunohistochemical galectin finger-
printing. Cancer 97, 1849–1858.
68. Ru
¨
diger, H. & Gabius, H J. (2001) Plant lectins. Glycoconjugate
J. 18, 589–613.
69. Lee, R.T., Gabius, H J. & Lee, Y.C. (1994) The sugar-combining
area of the galactose-specific toxic lectin of mistletoe extends
beyond the terminal sugar residue: comparison with a homo-
logous toxic lectin, ricin. Carbohydr. Res. 254, 269–276.
70. Ru
¨
diger, H., Siebert, H C., Solı
´
s, D., Jime
´
nez-Barbero, J.,
Romero,A.,vonderLieth,C W.,Dı
´
az-Maurin
˜
o, T. & Gabius,
H J. (2000) Medicinal chemistry based on the sugar code: fun-
damentals of lectinology and experimental strategies with lectins
as targets. Curr. Med. Chem. 7, 389–416.
71. Green, E.D., Brodbeck, R.M. & Baenziger, J.U. (1987) Lectin
affinity high-performance liquid chromatography. Interactions of
N-glycanase-released oligosaccharides with Ricinus communis
agglutinin I and Ricinus communis agglutinin II. J. Biol. Chem.

262, 12030–12039.
72. Angata, T. & Brinkman-van der Linden, E.C.M. (2002) I-type
lectins. Biochim. Biophys. Acta 1572, 294–316.
73. Gabius, H J., Bodanowitz, S. & Schauer, A. (1988) Endogenous
sugar-binding proteins in human breast tissue and benign and
malignant breast lesions. Cancer 61, 1125–1131.
74. Gabius, H J., Unverzagt, C. & Kayser, K. (1998) Beyond plant
lectin histochemistry: preparation and application of markers to
visualize the cellular capacity for protein-carbohydrate recogni-
tion. Biotech. Histochem. 73, 263–277.
75. Gabius, H J. (2001) Glycohistochemistry: the why and how of
detection and localization of endogenous lectins. Anat. Histol.
Embryol. 30, 3–31.
76. Yamazaki, N., Kojima, S., Bovin, N.V., Andre
´
,S.,Gabius,S.&
Gabius, H J. (2000) Endogenous lectins as targets for drug
delivery. Adv. Drug Deliv. Rev. 43, 225–244.
77. Davis, B.J. & Robinson, M.A. (2002) Drug delivery systems based
on sugar–macromolecule conjugates. Curr. Opin. Drug Discov.
Dev. 5, 279–288.
78. Chiu, M.H., Tamura, T., Wadhwa, M.S. & Rice, K.G. (1994)
In vivo targeting function of N-linked oligosaccharides with ter-
minating galactose and N-acetylgalactosamine residues. J. Biol.
Chem. 269, 16195–16202.
79. Gupta, D. & Surolia, A. (1994) Synthesis of neoglycopeptides and
analyses of their biodistribution in vivo to identify tissue specific
uptake and novel putative membrane lectins. Glycoconjugate J. 11,
558–571.
80. Hounsell, E.F. (1997) Methods of glycoconjugate analysis. In

Glycosciences: Status and Perspectives (Gabius, H J. & Gabius, S.,
eds), pp. 15–29. Chapman & Hall, Weinheim.
81. Geyer, H. & Geyer, R. (1998) Strategies for glycoconjugate ana-
lysis. Acta Anat. 161, 18–35.
82. Wu,A.M.,Wu,J.H.,Tsai,M S.,Kaltner,H.&Gabius,H J.
(2001) Carbohydrate specificity of a galectin from chicken liver
(CG-16). Biochem. J. 358, 529–538.
83. Wu,A.M.,Wu,J.H.,Tsai,M S.,Liu,J H.,Andre
´
,S.,Wasano,
K., Kaltner, H. & Gabius, H J. (2002) Fine specificity of domain-I
of recombinant tandem-repeat-type galectin-4 from rat gastro-
intestinal tract. Biochem. J. 367, 653–664.
84. Sasai,K.,Ikeda,Y.,Eguchi,H.,Tsuda,T.,Honke,K.&Tani-
guchi, N. (2002) The action of N-acetylglucosaminyltransferase-V
is prevented by the bisecting GlcNAc residue at the catalytic step.
FEBS Lett. 522, 151–155.
85.Lahm,H.,Andre
´
,S.,Hoeflich,A.,Fischer,J.R.,Sordat,B.,
Kaltner, H., Wolf, E. & Gabius, H J. (2001) Comprehensive
galectin fingerprinting in a panel of 61 human tumor cell lines by
RT-PCR and its implications for diagnostic and therapeutic pro-
cedures. J. Cancer Res. Clin. Oncol. 127, 375–386.
86. Camby,I.,Belot,N.,Lefranc,F.,Sadeghi,N.,DeLaunoit,Y.,
Kaltner,H.,Musette,S.,Darro,F.,Danguy,A.,Salmon,I.,
Gabius, H J. & Kiss, R. (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.

134 S. Andre
´
et al.(Eur. J. Biochem. 271) Ó FEBS 2003