Báo cáo khoa học: Introduction of extended LEC14-type branching into core-fucosylated biantennary N-glycan Glycoengineering for enhanced cell binding and serum clearance of the neoglycoprotein pot

Introduction of extended LEC14-type branching into
core-fucosylated biantennary N-glycan
Glycoengineering for enhanced cell binding and serum clearance
of the neoglycoprotein
Sabine Andre
´
1
, Shuji Kojima
2
, Ingo Prahl
3
, Martin Lensch
1
, Carlo Unverzagt
3
and Hans-Joachim Gabius
1
1 Institut fu
¨
r Physiologische Chemie, Tiera
¨
rztliche Fakulta
¨
t, Ludwig-Maximilians-Universita
¨
tMu
¨
nchen, Germany
2 Faculty of Pharmaceutical Sciences, Tokyo University of Science, Japan
3 Bioorganische Chemie, Universita
¨

t Bayreuth, Germany
N-Glycosylation is the most frequent and structurally
most variegated form of post-translational modifica-
tion [1–3]. Ironically, it is exactly due to this unsur-
passed molecular complexity that progress to assign
functional significance to distinct glycan epitope lags
behind the advances of work on other types of protein
modifications. Taking a step to change this situation
was the driving force for our study. At first glance, we
consider it reasonable to interpret the enormous struc-
tural complexity of the carbohydrate part of glycopro-
teins as a wide array of signals; this concept provides
direction for research [4,5]. As documented already at
the level of nascent glycoproteins, their N-glycan struc-
ture is relevant for quality control, underscoring the
Keywords
drug targeting; galectin; glycosylation; lectin;
tumor imaging
Correspondence
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 80 2180 2508
Tel: +49 89 2180 2290
E-mail:
(Received 16 December 2004, revised 23
February 2005, accepted 2 March 2005)
doi:10.1111/j.1742-4658.2005.04637.x
A series of enzymatic substitutions modifies the basic structure of
complex-type biantennary N-glycans. Among them, a b1,2-linked N-ace-
tylglucosamine residue is introduced to the central mannose moiety of the
core-fucosylated oligosaccharide by N-acetylglucosaminyltransferase VII.
This so-called LEC14 epitope can undergo galactosylation at the b1,2-
linked N-acetylglucosamine residue. Guided by the hypothesis that struc-
tural modifications in the N-glycan alter its capacity to serve as ligand for
lectins, we prepared a neoglycoprotein with the extended LEC14 N-glycan
and tested its properties in three different assays. In order to allow compar-
ison to previous results on other types of biantennary N-glycans the func-
tionalization of the glycans for coupling and assay conditions were
deliberately kept constant. Compared to the core-fucosylated N-glycan no
significant change in affinity was seen when testing three galactoside-speci-
fic proteins. However, cell positivity in flow cytofluorimetry was enhanced
in six of eight human tumor lines. Analysis of biodistribution in tumor-

bearing mice revealed an increase of blood clearance by about 40%, yield-
ing a favorable tumor ⁄ blood ratio. Thus, the extended LEC14 motif affects
binding properties to cellular lectins on cell surfaces and organs when com-
pared to the core-fucosylated biantennary N-glycan. The results argue in
favor of the concept of viewing substitutions as molecular switches for
lectin-binding affinity. Moreover, they have potential relevance for glyco-
engineering of reagents in tumor imaging.
Abbreviations
CHO, Chinese hamster ovary; GlcNAc, N-acetylglucosamine; GlcNAc-TVII, N-acetylglucosaminyltransferase VII; LacNAc, N-acetyllactosamine;
LCA, Lens culinaris agglutinin; PSA, Pisum sativum agglutinin; TFA, trifluoroacetic acid.
1986 FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS
notion that the glycan’s intimate interplay with specific
lectins can be central to realization of its functional
potential [6–8]. Because glycans differing in structure
by specific substitutions might react with cell lectins in
a characteristic manner, a route of translating struc-
tural differences into effects, e.g. leading to altered cell
adhesion, growth control or endocytic uptake, is envi-
sioned. Fittingly, the complexity of glycan structures is
matched by expression of lectin families [5,9–11]. Our
aim, in essence, is to systematically measure ligand
properties of N-glycans with different substitutions. In
so doing we combine the emerging technology for
chemoenzymatic tailoring of complex-type N-glycans
on a preparative scale with biochemical ⁄ cell biological
methods, starting with the preparation of neoglycopro-
teins with a homogeneous sugar part as suitable test
substances. Our initial studies with complex-type bian-
tennary N-glycans bearing either a bisecting N-acetyl-
glucosamine (GlcNAc) or a core-fucose (Fuc) residue

have lent support to the validity of our hypothesis
[12–14]. These two substitutions act like switches on lig-
and properties. With these data in hand, the description
of the naturally occurring core-fucosylated N-glycan
containing an additional b1,2-linked GlcNAc moiety
attached to the central Man residue (LEC14) ([15,16];
for glycan structures see lower part of Fig. 1) promp-
ted us to take the next step in our program by examin-
ing its properties as ligand.
The approach to track down the LEC14 N-glycan
variant actually exploited an impact of N-glycan sub-
stitutions on lectin binding. Development of resistance
to two agglutinins with dual affinity to the core-fucose
unit and the trimannosyl core region, i.e. Pisum sati-
vum and Lens culinaris agglutinins (PSA, LCA) [17],
led to the selection of the LEC14 mutant of Chinese
hamster ovary cells (CHO) [15,16]. Besides substantial
branching in poly(N-acetyllactosamine) chains implied
by the occurrence of 3,4-disubstituted GlcNAc in
methylation analysis the presence of a b1,2-linked Glc-
NAc moiety attached to the central Man residue in the
trimannoside core was defined [18]. It apparently per-
turbs binding to PSA ⁄ LCA, the likely cause for the
enhanced lectin resistance, and contributes to the com-
plex changes in the glycoproteomic profile of the
LEC14 mutant vs. wild-type cells [18]. The enzymatic
introduction of the b1,2-linked GlcNAc moiety into
the biantennary N-glycan by N-acetylglucosaminyl-
transferase VII (GlcNAc-TVII) depends critically on
the presence of the core-fucose unit so that the LEC14

type of glycan will invariably harbor two core substitu-
tions [19]. An immediate question concerns the possi-
bility of further processing.
While the bisecting GlcNAc residue in b1,4-linkage
to the central mannose unit has only been described as
an acceptor for chain elongation in glycans of Mgat2-
null mice [20,21], the question on branch elongation of
the LEC14-specific b1,2-linked GlcNAc could initially
not be answered unequivocally. The extensive treat-
ment of glycopeptides by b-galactosidase and N-acetyl-
b-d-glucosaminidase to trim the glycan antennae prior
to structural analysis of the core may well have also
impaired this branch elongation [18]. This situation
was resolved by total synthesis of a complete LEC14
N-glycan. The availability of material derived from
chemical synthesis not only unambiguously confirmed
this particular core structure but also provided suffi-
cient quantities for glycosyltransferase assays [22].
Galactosyltransferase was found to elongate the unu-
sual b1,2-linked GlcNAc residue ([23]; for structure of
the glycan with the new branch see Fig. 1). In accord-
ance with the high level of resistance to glycosidase
treatment of this GlcNAc residue (an indication for
rather poor spatial accessibility compared with the
GlcNAc moieties in the a1,3- and a1,6-arms) its reac-
tivity towards galactosyltransferase was lower than for
terminal GlcNAc residues in the antennae [23]. Thus,
the demonstration of substrate properties of the
LEC14 core for galactosylation suggests, but does not
prove the presence of this type of triantennary N-gly-

can in the complex profile observed for CHO cell
glycans. Its occurrence might account for enhanced
glycopeptide binding to immobilized Ricinus communis
agglutinin I relative to wild-type glycans [18]. Conse-
quently, we addressed the ensuing question whether
the addition of an N-acetyllactosamine (LacNAc) unit
to a core-fucosylated biantennary N-glycan at the cen-
tral Man residue in b1,2-linkage will alter the ligand
properties especially for endogenous receptors. Because
chemical conjugation of the synthetic oligosaccharides
to a carrier protein is feasible for a spacered N-glycan
[23], we prepared a neoglycoprotein from an extended
LEC14 N-glycan after suitable linker design. The lig-
and properties of the resulting neoglycoprotein (A )
were tested in three different systems: (a) tested as lig-
and immobilized to a plastic surface with five sugar re-
ceptors, among them growth-regulatory galectins [24];
(b) tested as ligand for surface receptors of different
types of tumor cells; and (c) injected into circulation
with monitoring of the time course of biodistribution.
The data set on the neoglycoprotein prepared from a
core-fucosylated N-glycan devoid of the b1,2-substitu-
tion (C) and tested previously under identical condi-
tions [13] allowed direct comparison to pinpoint any
detectable influence of the new b1,2-branch.
S. Andre
´
et al. Extended LEC14-type N-glycan as lectin ligand
FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS 1987
Extended LEC14-type N-glycan as lectin ligand S. Andre

´
et al.
1988 FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS
Results
Synthetic background
We have set ourselves the task of systematically deter-
mining ligand properties of N-glycans, here the per-
galactosylated LEC14 dodecasaccharide (Fig. 1,
compound 7), by combining chemoenzymatic synthesis
with biochemical ⁄ cell biological techniques. In order to
imitate the natural presentation of N-glycans on a gly-
coprotein, conjugation of the synthetic product to the
carrier protein, which is otherwise free of ligand prop-
erties, was necessary. Toward this end we started from
the protected nonasaccharide 1 [22] to reach the per-
galactosylated LEC14 epitope [23] as strategically
outlined in Fig. 1. The resulting spacered dodecasac-
charide was conjugated to BSA after the spacer’s
amino function was activated by reaction with thio-
phosgene to its isothiocyanate. N-glycan attachment to
the carrier protein was visualized by gel electrophoretic
analysis revealing the N-glycan-dependent shift of the
molecular mass (lower part of Fig. 1). The protein was
turned totally into a glycan carrier, because no staining
was visible at the position of unsubstituted albumin.
To determine the incorporation yield, additional ana-
lytical procedures were performed. MS gave evidence
for a spectrum of neoglycoproteins with one to four
attached N-glycan chains, and the colorimetric assay
determined an average of 3.2 N-glycans per carrier

molecule [23]. Of note for the intended comparison to
the other so far tested complex-type biantennary
N-glycans without ⁄ with substitutions, the linker design
could thus be kept constant. Even more important, the
incorporation yield of this reaction was only slightly
lower than for the N-glycan with a bisecting GlcNAc
moiety (B) or the unsubstituted form (D) at 3.6 N-gly-
cans per carrier protein and the core-fucosylated
N-glycan at 3.9 oligosaccharide chains (C) [12–14].
This result, ensuring rather similar glycan density, was
the prerequisite to proceed to testing the ligand prop-
erties of the extended LEC14 dodecasaccharide using
neoglycoprotein A in three different assay systems
with: (a) purified sugar receptors; (b) tumor cell surfa-
ces in vitro; and (c) organ lectins in vivo.
Affinity to purified lectins/antibodies
The first assay system was designed to simulate proper-
ties of glycoproteins presented on a cell surface by
adsorbing the neoglycoprotein to the plastic surface of
microtiter plate wells. The homogeneity of the structure
of the synthetic N-glycan, rigorously controlled by our
analytical procedures (see Experimental procedures)
and definitively excluding the presence of contaminating
glycoforms, will account for a clear-cut correlation
between structure and ligand properties. Also, the assay
deliberately avoided surface immobilization of the car-
bohydrate-binding proteins, which might affect their
binding properties. Under these conditions, carbohy-
drate-dependent and saturable binding of toxic mistletoe
lectin, the growth⁄ invasion-regulatory galectin-1 and

the natural autoantibody was invariably detected
(Fig. 2). The calculated Scatchard plots gave straight
lines in all cases, evidence for a single class of binding
sites. Although the different sugar receptors home in on
the same basic unit, i.e. terminal galactose, their affinity
is clearly disparate (Fig. 2A–C). The IgG subfraction
bound with the highest affinity, followed by the plant
agglutinin with two binding sites per B-subunit in the
(AB)
2
-tetramer and the homodimeric endogenous lectin
with its two binding sites at opposing sides of the pro-
tein. Galectins afford the opportunity to further exam-
ine the relationship between the spatial presentation of
carbohydrate recognition domains and ligand affinity.
We thus tested two further members of the galectin fam-
ily, i.e. galectins-3 and -5. These two monomeric pro-
teins share ligand specificity with galectin-1 but not its
homodimeric cross-linking design. Due to their mono-
meric constitution in solution no affinity enhancement
by ligand clustering through a bivalent module is expec-
ted. Indeed, these two lectins were inferior in terms of
binding affinity to galectin-1, their K
D
values at
820 ± 71 nm (galectin-3) and 734 ± 157 nm (galectin-
5; Fig. 2D) with about three to fivefold increases in B
max
Fig. 1. Chemical and enzymatic steps to produce the LEC14-type N-glycan with the LacNAc branch in b1,2-linkage at the central Man unit
starting from the protected nonasaccharide 1 [34,35]. (a) (NH

4
)
2
Ce(NO
3
)
6
,CH
3
CN-H
2
O, 80 °C (71%); (b) 1 Ethylenediamine, n-BuOH, 80 °C;
2. Ac
2
O, pyridine; 3 MeNH
2
(41%) in H
2
O (96% for steps 1–3); (c) 1 Propanedithiol, NEt
3
, MeOH; 2 N-carbobenzoxy-6-amino hexanoic acid
4, TBTU, HOBt, N-methylpyrrolidone (31% for steps 1–2 after RP-HPLC); (d) PdO-H
2
O ⁄ H
2
, MeOH-AcOH (95%); (e) UDP-Gal (4 eq.), b1,4-ga-
lactosyltransferase, alkaline phosphatase (75%); (f) 1 Thiophosgene, CH
2
Cl
2

-H
2
O, NaHCO
3
; 2 BSA, H
2
O, NaHCO
3
; 6 days. The last two
schemes for N-glycan sequences allow structural comparison between the N-glycan of neoglycoprotein A and the previously studied N-gly-
cans in neoglycoproteins B–D (upper panel). Gel documentation is added in the bottom panel for visualization of the gel electrophoretic
mobility of the carbohydrate-free carrier protein BSA (lanes a, c, e; 0.15 lgÆlane
)1
) relative to that of the neoglycoprotein A (lanes b, d, f;
0.2 lgÆper lane, 0.15 lgÆper lane and 0.1 lgÆper lane, respectively). Positions of two marker proteins for molecular mass designation (in kDa)
are indicated by arrowheads.
S. Andre
´
et al. Extended LEC14-type N-glycan as lectin ligand
FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS 1989
values. These results confirm a striking effect of receptor
topology at constant spatial features of the ligand. They
also enable us to set the ligand properties of the exten-
ded LEC14 dodecasaccharide in relation to the so far
studied N-glycans, especially the N-glycans without any
substitution or with core-fucosylation.
The presence of the new glycan branch in the exten-
ded LEC14 neoglycoprotein appeared to enhance
affinities toward human proteins and reduce affinity
toward the plant lectin relative to the properties of the

complex-type biantennary N-glycan lacking a substitu-
tion. However, the strict dependence of GlcNAc-TVII
activity on core-fucosylation in its substrate [19], and
therefore the presence of this substitution in the
LEC14 epitope, must not be neglected. The ensuing
comparison between the properties of the LEC14
dodecasaccharide epitope and the core-fucosylated
decasaccharide clearly revealed that the b1,2-linked
glycan branch did not significantly affect affinity to the
three tested types of sugar receptor in this assay sys-
tem. In contrast, the consideration of the data for the
N-glycan with bisecting GlcNAc (B) with reductions in
affinity underscores the sensitivity of this parameter to
other structural alterations in the N-glycan. Because
the nature of the carbohydrate-binding protein matters
notably in this respect, it is mandatory to proceed to
test the pergalactosylated LEC14 N-glycan against a
complex panel of binding partners. In order to extend
mapping the ligand properties of the LEC14 dodeca-
saccharide, we thus moved in our analysis from a test
system with purified sugar receptors to cell surfaces
with an array of lectins. To add potential clinical rele-
vance we selected tumor cells of different histogenesis
representing common cancer types. In the same way as
isolated lectins these established cell models also offer
the advantage for comparative analysis when ade-
quately controlled for constant surface properties.
Affinity to tumor cell surfaces
The first step in the cytofluorimetric analysis was dedi-
cated to documenting the carbohydrate-dependent and

saturable binding of the labeled neoglycoprotein to cell
surfaces (Fig. 3). Biotinylated carrier protein without
the N-glycan failed to produce a signal above back-
ground, excluding interactions by the protein part or
its label. Mimicking the situation when a glycoprotein
encounters a cell surface, the neoglycoprotein A reacted
with cell surfaces in a cell type-specific manner
(Fig. 4). As highlighted by these results, distinct pre-
ference of binding was determined for the B- and
T-lymphoblastoid cells among the set of leukemia ⁄
lymphoma lines and to the mammary carcinoma cells
among the carcinoma lines, when measuring staining
Fig. 2. Illustration of Scatchard plot analysis of carbohydrate-dependent interaction between the carrier-immobilized N-glycan (A) and the
mistletoe lectin (VAA; A), human galectin-1 (B), the lactoside-binding IgG subfraction (C) and rat galectin-5 (D) in a representative experimen-
tal series. K
D
values (mean ± SD) are given for the complete set of analytical data with at least four different experimental series for each
sugar receptor. The extent of total binding (s) was reduced by that of binding which was not inhibitable by glycoinhibitors (h,75m
M lactose
and 1 mg asialofetuinÆmL
)1
) to calculate the level of carbohydrate-dependent binding (n) (see inset).
Extended LEC14-type N-glycan as lectin ligand S. Andre
´
et al.
1990 FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS
intensity. Extending the data of the solid-phase assay,
the LEC14 dodecasaccharide is a ligand for cellular
lectins. To give potential reason to the presence of the
GlcNAc-TVII, as detected in the LEC14 mutant [19],

the comparison between binding data of this neoglyco-
protein (A) and that presenting the core-fucosylated
N-glycan (C) without the b1,2-branch is expedient.
In general, comparison to the other, so far tested
10
0
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
10
0
0 128
number of events
0 128
0 128

0 128
10
1
10
2
10
3
10
4
10
0
10
1
10
2
10
3
10
4
AB
CD
Fig. 3. Semilogarithmic representation of
the fluorescent surface staining of cells of
the human T-lymphoblastoid line CCRF-CEM
in the absence of incubation with the biotin-
ylated neoglycoprotein (negative control;
shaded) and after incubation with increasing
concentrations of neoglycoprotein in two
steps: up to 2 lgÆmL
)1

(lines with
0.5 lgÆmL
)1
,1lgÆmL
)1
and 2 lgÆmL
)1
from
left to right); (A) and up to 50 lgÆmL
)1
(lines
with 2 lgÆmL
)1
,5lgÆmL
)1
,10lgÆmL
)1
,
25 lgÆmL
)1
and 50 lgÆmL
)1
from left to
right); (B). Controls with an incubation step
using biotinylated carrier protein
(25 lgÆmL
)1
); (C) instead of the neoglyco-
protein and an inhibition of staining using
glycoinhibitors (75 m

M lactose and 1 mg
asialofetuinÆmL
)1
); (D) document lack of
label ⁄ carrier protein-dependent binding and
the carbohydrate dependence of binding.
number of events
0128
10
0
10
1
10
2
10
3
10
4
0128
10
0
10
1
10
2
10
3
10
4
0128

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0
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1
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2
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3
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4
0128
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4
0128
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3

10
4
70.3 %/252.2
98.2 %/177.8
16.3 %/15.3 55.0 %/45.1
64.4 %/62.3
88.4 %/56.1
74.4 %/29.483.3 %/420.6
0128
10
0
10
1
10
2
10
3
10
4
0128
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0
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1
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3
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4

0128
10
0
10
1
10
2
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3
10
4
ABCD
EFGH
Fig. 4. Semilogarithmic representation of the binding of the fluorescent indicator (streptavidin ⁄ R-phycoerythrin conjugate) in the absence of
the probe during processing (negative control; shaded) and after the incubation step with the biotinylated neoglycoprotein (25 lgÆmL
)1
; black
line) for the B-lymphoblastoid line Croco II (A), the T-lymphoblastoid line CCRF-CEM (B), the erythroleukemia line K-562 (C), the acute myelo-
genous leukemia line KG-1 (D), the mammary carcinoma line DU4475 (E) and the colon adenocarcinoma lines C205 (F), SW480 (G) and
SW620 (H). Quantitative data on percentage of positive cells (%) and mean channel fluorescence are given in each panel.
S. Andre
´
et al. Extended LEC14-type N-glycan as lectin ligand
FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS 1991
carrier-immobilized N-glycans revealed rather favora-
ble ligand properties (Fig. 5). A tendency for enhanced
binding relative to the core-fucosylated N-glycan indi-
cated that the new branch is not an inert modification
at this level of testing. The decrease of cell positivity
for KG-1 cells and rather constant results for K-562

cells can be judged as internal controls for cell-type
specificity in the direct comparison to the core-fucosyl-
ated N-glycan. Thus, this assay system revealed several
cases with an improvement of ligand properties with
cell type-dependent characteristics. Because the N-gly-
can profile of glycoproteins not only governs cell
surface binding in vitro but also serum survival in
circulation, a parameter of interest for prolonging
bioavailability of pharmaproteins, we next tested the
influence of this N-glycan in biodistribution analysis
in vivo.
Biodistribution in vivo
Organ retention and blood content of the iodinated
neoglycoprotein (A) were monitored after intravenous
injection. Six time points from 15 min to 12 h were set
to determine the time course of these parameters. Hep-
atic uptake was rapid and the major route of blood
clearance (Table 1). When comparing blood ⁄ organ
retention of this neoglycoprotein for the four major
sites to the cases of the so far tested N-glycans (B–D),
blood clearance was best for the neoglycoprotein bear-
ing the LEC14 dodecasaccharide (A) (Fig. 6). We have
deliberately run these experiments in tumor-bearing
mice to look at tumor uptake relative to blood back-
ground, a factor with impact on sensitivity of tumor
imaging. The tumor ⁄ blood ratio after 1 h was 0.7 for
neoglycoprotein (A) but 0.53 for C bearing a core-
fucosylated N-glycan with 3.11 ± 0.17% in blood and
1.65 ± 0.06% in the tumor. The detected difference
adds to the evidence for modulation of ligand proper-

ties by the extended LEC14 motif. Placing this glycan
at best position in this respect, the ratio for the unsub-
stituted nonasaccharide N-glycan (D) was 0.38 (tumor:
1.16 ± 0.09; blood: 3.07 ± 0.06%) and 0.47 (tumor:
1.47 ± 0.09; blood: 3.11 ± 0.14%) for the decasac-
charide with the bisecting GlcNAc moiety (B). Regard-
ing the individual organ sites no major alteration of
uptake and retention after 1 h was detectable except
for the N-glycan with bisecting GlcNAc (Fig. 6).
Discussion
The basic complex-type biantennary N-glycan is sub-
ject to enzymatic substitutions. Structural aspects have
been mostly clarified but the functional significance of
their presence is still a matter of debate. Our hypothe-
sis interprets occurrence of substitutions as a means to
modulate ligand properties in interactions with endo-
genous lectins. The versatile potential for fine-tuning a
respective information transfer would then clearly out-
weigh the required investment in coding for the diver-
sity of glycosyltransferases and regulation of their
activity. In other words, glycosyltransferase activities
produce lectin-binding epitopes. By virtue of adding
substitutions, which may not even directly participate
in binding, they might also affect the affinity of the
binding sites. To demonstrate that a structural alter-
ation in an N-glycan changes its binding parameters
requires experimental evidence difficult to collect with
natural glycoproteins. They generally present more
than one type of glycan chain and exhibit microhetero-
geneity, confounding efforts to establish a direct struc-

ture ⁄ activity profile. To address this issue, we turned
Fig. 5. Comparison of the percentage of positive cells (upper panel)
and mean channel fluorescence (bottom panel) in flow cytofluori-
metric analysis using the LEC14-dodecasaccharide-bearing neogly-
coprotein A and neoglycoproteins with complex-type biantennary
N-glycan ligand parts substituted by bisecting GlcNAc (B) or core-
fucosylation (C) or without any substitution (D) (see Fig. 1 for struc-
tural comparison). Data for neoglycoproteins B, C, D have
previously been published [12–14] and are shown for comparison.
The standard deviations within experimental series are generally
below 7.5%.
Extended LEC14-type N-glycan as lectin ligand S. Andre
´
et al.
1992 FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS
to the synthesis of neoglycoproteins. In contrast to free
N-glycans they harbor a homogeneous sugar part and
favorably maintain a local density akin to natural gly-
coproteins. In fact, affinity to galectins is sensitive to
changes in local density of glycan chains and improved
by certain modes of clustering [25–27].
As stated above, it is our aim to delineate struc-
ture ⁄ activity profiles for N-glycans. Toward this end, we
have so far tested three types of biantennary N-glycans
in different assay systems [12–14]. They were confronted
with different situations in vitro and in vivo, i.e. the N-
glycan as ligand in a matrix simulating a cell membrane
or in solution ⁄ serum confronted with lectin-presenting
cell surfaces. Evidently, monitoring tumor cells and bio-
distribution has relevance to the glycan’s suitability for

drug targeting or imaging [28–30]. Our previous results
with the neoglycoprotein probes, which were kept rather
constant in all relevant features (nature of carrier pro-
tein, linker chemistry, yield of glycan incorporation),
supported the hypothesis given above [12–14]. In this
report, we examined the impact of the pergalactosylated
LEC14 motif, a b1,2-linked LacNAc disaccharide emer-
ging from the central bMan unit of a core-fucosylated
N-glycan [7] (Fig. 1). The bMan moiety of a
LEC14 N-glycan is substituted in positions 2, 3 and 6, a
remarkable illustration of the capacity of glycan units to
engender branching, in contrast with amino acids and
nucleotides. As outlined in the introduction, a dis-
tinct N-acetylglucosaminyltransferase (GlcNAc-TVII) is
responsible for the introduction of the b1,2-GlcNAc
into mammalian N-glycans [19]. Of note, a b1,2-substi-
tution at this site of the core is also found in nonmam-
malian N-glycan chains, here with xylose as added sugar
unit [31–33]. This position is thus apparently predis-
posed for enzymatic modification. Immunologically, this
residue is relevant due to its allergenic activity, an indi-
cation for accessibility to interactions with immunoglo-
bulin E [33]. Likewise, the b1,2-GlcNAc residue at this
position of a mammalian-type core-fucosylated N-gly-
can is a contact point, as shown by its acceptor capacity
in enzymatic galactosylation [23]. Moreover, the selec-
tion process to isolate the LEC14 mutant cells exploited
the detrimental effect of this core substitution on an
interaction with a receptor protein, i.e. reduction of
binding of the plant agglutinins PSA ⁄ LCA [15,16].

Our results with purified lectins ⁄ antibodies reveal
no major influence of this enzymatically elongated
branch on ligand properties, when compared with
the core-fucosylated N-glycan lacking this branch.
Table 1. Biodistribution of LEC14-dodecasaccharide-bearing neoglycoprotein A in Ehrlich-solid-tumor-bearing mice (% injected doseÆg
)1
tissue). Each value indicates the mean ± SD for four to five mice.
Time (h) 1 ⁄ 4h 1⁄ 2h 1h 3h 6h 12h
Blood 5.15 ± 0.36 4.49 ± 0.22 2.19 ± 0.33 0.92 ± 0.13 0.62 ± 0.06 0.45 ± 0.10
Liver 34.45 ± 4.49 6.51 ± 0.74 2.52 ± 0.14 1.29 ± 0.17 0.95 ± 0.07 0.71 ± 0.03
Kidneys 1.53 ± 0.23 4.60 ± 0.56 2.96 ± 0.64 1.45 ± 0.33 0.81 ± 0.28 0.51 ± 0.22
Spleen 0.73 ± 0.10 2.37 ± 0.27 1.23 ± 0.20 0.58 ± 0.10 0.33 ± 0.04 0.25 ± 0.05
Heart 0.24 ± 0.02 1.50 ± 0.11 0.69 ± 0.13 0.29 ± 0.04 0.18 ± 0.02 0.15 ± 0.01
Lungs 0.55 ± 0.07 1.99 ± 0.14 1.10 ± 0.17 0.48 ± 0.09 0.38 ± 0.08 0.26 ± 0.01
Thymus 0.08 ± 0.01 1.71 ± 0.14 1.01 ± 0.16 0.47 ± 0.16 0.23 ± 0.07 0.27 ± 0.12
Pancreas 0.36 ± 0.07 1.87 ± 0.21 1.05 ± 0.20 0.40 ± 0.06 0.20 ± 0.04 0.17 ± 0.12
Lymph node 0.09 ± 0.02 1.25 ± 0.12 0.69 ± 0.13 0.38 ± 0.09 0.18 ± 0.07 0.19 ± 0.03
Muscle 0.28 ± 0.02 0.59 ± 0.06 0.36 ± 0.04 0.16 ± 0.04 0.11 ± 0.01 0.07 ± 0.01
Brain 0.07 ± 0.01 0.18 ± 0.02 0.10 ± 0.01 0.04 ± 0.01 0.02 ± 0.01 0.03 ± 0.00
Vertebrae 0.21 ± 0.03 1.04 ± 0.09 0.67 ± 0.15 0.32 ± 0.10 0.16 ± 0.03 0.16 ± 0.03
Tumor 0.36 ± 0.02 1.97 ± 0.22 1.54 ± 0.21 0.58 ± 0.07 0.37 ± 0.08 0.31 ± 0.02
Fig. 6. Comparison of aspects of the biodistribution patterns of iodi-
nated neoglycoproteins with the following complex-type N-glycan
ligand parts 1 h afer injection: LEC14 dodecasaccharide (A)and
complex-type biantennary N-glycans substituted by bisecting Glc-
NAc (B) or core-fucosylation (C) or without any substitution (D) (see
Fig. 1 for structural comparison). Data for neoglycoproteins B, C, D
have previously been published [12–14] and are shown for compar-
ison. The range of the standard deviation shown for each result by
bars was between 1.95 and 21.6%.

S. Andre
´
et al. Extended LEC14-type N-glycan as lectin ligand
FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS 1993
Looking at the growth ⁄ invasion-regulatory galectin-1,
it might be that the necessary contact to the subter-
minal GlcNAc residue during binding, a factor contri-
buting to ligand selection [34], is spatially hindered.
No affinity enhancement relative to the core-fucosyl-
ated decasaccharide was detectable. The affinity of
binding of the monomeric galectins-3 and -5 was con-
siderably reduced, arguing in favor of an influence of
the strong cross-linking activity of galectin-1 as a clue
for the functional divergence noted in a tumor cell
system [35,36]. No indication for positive cooperati-
vity of galectin-3 binding was observed. This binding
mode was operative with laminin as substratum for
this generally monomeric lectin which can form a
small extent of pentamer in solution [37,38]. When
testing cell surfaces with their full array of carbohy-
drate-binding proteins, a clear impact of presence of
the new branch was determined. This effect hinged
on the cell type, preferentially leading to increased
binding relative to the core-fucosylated decasaccharide
as ligand. In addition to its principal value to delin-
eate evidence for a structure ⁄ activity correlation this
result signifies that cell-presented lectins in most of
these tumor lines do not share the core specificity
with the plant agglutinins PSA ⁄ LCA which would
have been impaired by introducing the b1,2-branch.

Our result underscores differences between plant and
mammalian lectins and recommends using endo-
genous lectins for functional glycoproteomic profiling
of clinical samples [39].
The evidence for a contribution of this b1,2-branch
in the LEC14-type dodecasaccharide to overall ligand
properties was supported by the biodistribution analy-
sis, revealing rapid clearance elicited by pergalactosyl-
ated LEC14 epitope. In contrast to galectins the
C-type endocytic receptor of hepatocytes accommo-
dates galactose as central contact point [40]. This result
can be relevant for an application. Actually, tailoring
of the glycan part of pharmaproteins (glycoengineer-
ing) has become a fertile field of research in order to
manipulate cellular uptake and serum half-life [41–47].
The measured rapid clearance of the respective neogly-
coprotein bearing a b1,2-branch constituted by a Lac-
NAc disaccharide can be advantageous when using an
iodinated glycoprotein for imaging, as it lowers the
background. The detection of this property immedi-
ately raises the question of how this parameter will be
altered when the b1,2-branch is shifted away from the
central Man unit to the Man residues in the branch
extensions by GlcNAc-TIV or GlcNAc-TV. Indeed,
the consequences of hereby generating the two natural
versions of triantennary N-glycans as part of neoglyco-
proteins have not yet been rigorously determined using
our panel of assays. Thus, it is our next challenge to
address this issue.
Experimental procedures

Synthetic and analytical procedures
NMR spectra were recorded on a Bruker AMX 500 spectro-
meter (Karlsruhe, Germany). HPLC separations were per-
formed 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 (Du
¨
ren, Germany, 300 · 25 mm).
Carbohydrate-free BSA and bovine b1,4-galactosyltrans-
ferase were purchased from Sigma (Munich, Germany), alka-
line phosphatase (calf intestine, molecular biology grade)
from Roche Diagnostics (Heidelberg, Germany). UDP-
galactose was a generous donation from Roche Diagnostics.
ESI-TOF mass spectra were recorded with methanol ⁄ water
as solvent on a Micromass LCT spectrometer connected to
an Agilent HP 1100 HPLC apparatus. MALDI-TOF mass
spectra were recorded on a Bruker Reflex III using linear
mode and an acceleration voltage of 20 kV. For sample
preparation in MALDI-TOF-MS a solution of the neogly-
coprotein (1 lL, 7 mgÆmL
)1
) in 0.1% (v ⁄ v) trifluoroacetic
acid (TFA) was mixed with 1.5 lL of 33% acetonitrile in
0.1% (v ⁄ v) TFA and 2.5 lL of a saturated solution of
sinapinic acid in 0.1% (v ⁄ v) TFA and dried in high vacuum.
The structures of the synthetic N-glycans were routinely
confirmed by the following 2D-NMR-experiments: TOCSY,

NOESY, HMQC, HMQC-COSY, HMQC-DEPT, and
HMQC-TOCSY. Signals of NMR spectra were assigned
according to the following convention including designation
of spacer atoms illustrated for compound 7 in Fig. 1.
Preparation of neoglycoprotein A
For conjugation of the derivatized dodecasaccharide to the
carrier protein the amino group was transformed into its
isothiocyanate. In a 1.5 mL plastic vial 6-aminohexanoyl-
N-glycan 7 (0.77 mg, 0.34 lmol) was dissolved in sodium
hydrogencarbonate (200 lL, 10 mgÆmL
)1
) followed by addi-
tion of dichloromethane (200 lL) and thiophosgene (5 lL,
19.7 lmol). The biphasic mixture was vigorously stirred.
After 4 h (TLC: isopropanol ⁄ 1 m ammonium acetate, 2 : 1)
the mixture was centrifuged and the aqueous phase was sep-
arated. Subsequently, the organic phase was extracted twice
with sodium hydrogencarbonate (100 lL, 10 mgÆmL
)1
). The
combined aqueous phases were extracted twice with dichlo-
romethane (500 lL). Carbohydrate-free BSA (2 mg) was dis-
solved in the aqueous solution of the isothiocyanate, and the
reaction vial was kept for 6 days at ambient temperature.
Extended LEC14-type N-glycan as lectin ligand S. Andre
´
et al.
1994 FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS
The reaction mixture was centrifuged, and the clear superna-
tant was fractionated by gel filtration (Pharmacia Hi Load

Superdex 30 (600 · 16 mm); eluent: 0.1 m ammonium hydro-
gencarbonate; flow rate 0.75 mLÆmin
)1
; detection: 214 and
254 nm). Further quality controls were performed by gel
electrophoretic analysis and colorimetric determination of
the average glycan content as described previously [12–14]:
yield, 2.07 mg; R
f
amine 7 ¼ 0.14 (i-propanol ⁄ 1 m ammo-
nium acetate, 2 : 1); R
f
isothiocyanate ¼ 0.50 (i-propanol ⁄
1 m ammonium acetate, 2 : 1). MALDI-MS: M
calcd
¼ 68694,
70957, 73220, 75484 (1, 2, 3, 4 N-glycans per BSA molecule);
M
found
¼ 68649, 70971, 73242, 75475 (1, 2, 3, 4 N-glycans
per BSA molecule).
The neoglycoprotein A was then used in solid-phase and
cell-binding assays either free of label or for labeling with
the N-hydroxysuccinimide ester derivative of biotin under
conditions identical to the preparation of the other N-gly-
can-bearing probes tested previously [12–14].
Solid-phase assay
The matrix for the assay was established by adsorption of
neoglycoprotein to the surface of plastic microtiter plate
wells under conditions used previously [12–14]. Controls for

standardizing coating density were performed with biotinyl-
ated neoglycoprotein using streptavidin–peroxidase conju-
gate as indicator. Ligand properties of the N-glycan were
probed with different types of carbohydrate-binding pro-
teins. The galactoside-specific agglutinin from mistletoe
(Viscum album L. agglutinin, VAA), human galectin-1,
murine galectin-3 and rat galectin-5 as well as the immuno-
globulin G subfraction with preferential affinity to b-gal-
actosides from human serum were isolated and checked for
purity and quaternary structure by gel electrophoresis and
filtration, electrospray ionization MS, ultracentrifugation
and haemagglutination [48–54]. Biotinylation was carried
out under activity-preserving conditions, and label incor-
poration was assessed by binding assays with streptavidin–
peroxidase conjugate or a proteomics protocol [48,55].
Binding studies of the sugar receptors to the glycan-present-
ing matrix were performed by stepwise increases of probe
concentration up to saturation with duplicates at each con-
centration and at least four independent series including
controls to determine extent of carbohydrate-dependent
binding by its inhibition using a mixture of 75 mm lactose
and 1 mg asialofetuinÆmL
)1
, and the data sets were algebra-
ically transformed to obtain K
D
values and the number of
bound sugar receptor molecules at saturation (B
max
), fol-

lowing the protocol of our previous reports on neoglyco-
proteins with synthetic N-glycans [12–14].
Cell-binding assay
Using the biotinylated neoglycoprotein as probe, automated
flow cytofluorimetric analysis of carbohydrate-dependent
cell surface binding was performed with the following
human tumor lines: Croco II (B-lymphoblastoid cell line),
CCRF-CEM (T-lymphoblastoid cell line), K-562 (erythro-
leukemia cell line), KG-1 (acute myelogenous leukemia cell
line), DU4475 (mammary carcinoma cell line) as well as
C205, SW480 and SW620 (colon adenocarcinoma cell
lines). Except for the Croco II line established in our labor-
atory [56] the cells were commercially available (American
Type Culture Collection, Rockville, MD, USA) and rou-
tinely cultured under the recommended conditions. The
adherent colon carcinoma cells were detached by exposing
them to NaCl ⁄ P
i
containing 2 mm EDTA. Prior to the ana-
lysis cells were routinely washed carefully with Dulbeccos’s
NaCl ⁄ P
i
solution containing 0.1% (w ⁄ v) carbohydrate-free
BSA to remove any inhibitory serum glycoproteins and to
saturate nonspecific protein-binding sites. For this purpose,
an incubation step with ligand-free carrier protein for
30 min at 4 °C was added prior to the incubation with the
labeled neoglycoprotein at this temperature to minimize
uptake by endocytosis. Carbohydrate-dependent binding of
the neoglycoprotein (25 lgÆmL

)1
) to the cells (8 · 10
6
cellsÆmL
)1
) was assessed in a FACScan instrument (Becton-
Dickinson, Heidelberg, Germany) with the fluorescent
indicator conjugate streptavidin ⁄ R-phycoerythrin (1 : 40;
Sigma). Controls to assess carbohydrate-independent bind-
ing of the carrier via its protein part or label and to docu-
ment sugar inhibition were run in each series, as previously
described [12–14].
Analysis of in vivo biodistribution
Radiolabeling of the neoglycoprotein was performed by the
chloramine-T method [57]. Briefly, fresh chloramine-T and
sodium metabisulfite solutions were prepared, and the neo-
glycoprotein was dissolved in NaCl ⁄ P
i
(pH 7.2) at a con-
centration of 1 mg proteinÆmL
)1
.A10lL portion of
125
I-labelled NaI (74 MBqÆmL
)1
NaCl ⁄ P
i
) solution was
added to 50 lL of the neoglycoprotein-containing solution,
subsequently 10 lL of chloramine-T (3 mgÆ mL

)1
H
2
O) solu-
tion were added, and the mixture was incubated at room
temperature for 3 min. Thereafter, chloramine-T solution
was pipetted to the above mixture in two further portions at
intervals of 3 min, and then the reaction was stopped by add-
ing 30 lL of freshly prepared sodium metabisulfite solution
(5 mgÆmL
)1
). Label-free neoglycoprotein (50 lg) was added
as a carrier prior to the separation step by Sephadex G-50
(Pharmacia Biotech, Freiburg, Germany) gel permeation
chromatography to remove any reagents from the radioiodi-
nated product. The specific radioactivity of batches of
125
I-labeled neoglycoprotein was in the range between 8 and
10 MBqÆmg
)1
protein. To monitor biodistribution of the
iodinated product tumor-bearing mice were used [58,59].
Approximately 5 · 10
6
Ehrlich ascites tumor (EAT) cells had
been injected subcutaneously into the right rear leg of male
ddY mice of the age of 7 weeks for tumor inoculation. On
S. Andre
´
et al. Extended LEC14-type N-glycan as lectin ligand

FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS 1995
the sixth to eighth day after inoculation, when the tumor had
grown to 0.3–0.6 g in weight, radioiodinated neoglycoprotein
was injected intravenously at a dose of 80–100 kBq (equival-
ent to 10 lg of protein) via the tail vein. Tissues including
blood samples were obtained after the indicated periods,
weighed, and the radioactivity level was assessed with an
Auto Well gamma System (Aloka ARC 300, Tokyo, Japan).
The percentage of injected dose per gram of wet tissue or per
ml of blood was calculated in each case as described previ-
ously [12].
Acknowledgements
We express our gratitude to B. Hofer and L. Mantel
for skillful technical assistance, to Dr S. Namirha for
helpful discussion and to the Deutsche Forschungsge-
meinschaft, the Dr M Scheel-Stiftung fu
¨
r Krebs-
forschung, the Fonds der Deutschen Chemischen
Industrie, Roche Diagnostics and the Mizutani
Foundation for Glycoscience for generous financial
support.
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