Specificity of β1,4-galactosyltransferase inhibition by 2-naphthyl 2-butanamido-2-deoxy-1-thio-β-D-glucopyranoside

Article abstract of DOI:10.1007/s10719-010-9312-3

Inhibitors of Galactosyltransferase (GalT) have the potential of reducing the amounts of adhesive carbohydrates on secreted and cell surface-bound glycoproteins. We recently found a potent inhibitor of β4GalT, 2-naphthyl 2-butanamido-2-deoxy-1-thio-β-D-glucopyranoside (compound 612). In this work, we have tested compound 612 for the specificity of its inhibition and examined its effect on GalT, and on GlcNAc- and GalNAc-transferases in homogenates of different cell lines, as well as on recombinant glycosyltransferases. Compound 612 was found to be a specific inhibitor of β4GalT. The specificity of recombinant human β3GalT5 that also acts on GlcNAc-R substrates, revealed similarities to bovine milk β4GalT. However, 612 was a poor substrate and not an inhibitor for β3GalT5. To further determine the specific structures responsible for the inhibitory property of 612, we synthesized (2-naphthyl)-2-butanamido-2-deoxy-β-D- glucopyranosylamine (compound 629) containing nitrogen in the glycosidic linkage, and compared it to other naphthyl and quinolinyl derivatives of GlcNAc as substrates and inhibitors. Compound 629 was a substrate for both β4GalT and β3GalT5. This suggests that properties of 612 other than the presence of the naphthyl ring alone were responsible for its inhibitory action. The results suggest a usefulness of 612 in specifically blocking the synthesis of type 2 chains and thus epitopes attached to type 2 chains. In addition, 612 potently inhibits β4GalT in cell homogenates and thus allows assaying β3GalT activity in the presence of β4GalT.

Full text of DOI:10.1007/s10719-010-9312-3

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Glycoconj J (2010) 27:673–684
DOI 10.1007/s10719-010-9312-3
Specificity of β1,4-galactosyltransferase inhibition
by 2-naphthyl 2-butanamido-2-deoxy-1-thio-β-D-
glucopyranoside
Yin Gao & Carmen Lazar & Walter A. Szarek &
Inka Brockhausen
Received: 23 June 2010 /Revised: 8 October 2010 /Accepted: 11 October 2010 /Published online: 26 October 2010
#
Springer Science+Business Media, LLC 2010
Abstract Inhibitors of Galactosyltransferase (GalT) have
the potential of reducing the amounts of adhesive carbohy-
drates on secreted and cell surface-bound glycoproteins. We
recently found a potent inhibitor of β4GalT, 2-naphthyl 2-
butanamido-2-deoxy-1-thio-β-D-glucopyranoside (com-
pound 612). In this work, we have tested compound 612
for the specificity of its inhibition and examined its effect
on GalT, and on GlcNAc- and GalNAc-transferases in
homogenates of different cell lines, as well as on
recombinant glycosyltransferases. Compound 612 was
found to be a specific inhibitor of β4GalT. The specificity
of recombinant human β3GalT5 that also acts on GlcNAc-R
substrates, revealed similarities to bovine milk β4GalT.
However, 612 was a poor substrate and not an inhibitor for
β3GalT5. To further determine the specific structures
responsible for the inhibitory property of 612, we synthe-
sized (2-naphthyl)-2-butanamido-2-deoxy-β-D-glucopyrano-
sylamine (compound 629) containing nitrogen in the
glycosidic linkage, and compared it to other naphthyl and
quinolinyl derivatives of GlcNAc as substrates and inhib-
itors. Compound 629 was a substrate for both β4GalT and
β3GalT5. This suggests that properties of 612 other than the
presence of the naphthyl ring alone were responsible for its
inhibitory action. The results suggest a usefulness of 612 in
specifically blocking the synthesis of type 2 chains and thus
epitopes attached to type 2 chains. In addition, 612 potently
inhibits β4GalT in cell homogenates and thus allows
assaying β3GalT activity in the presence of β4GalT.
.
.
Keywords Galactosyltransferase Inhibition Substrate
.
.
specificity Glycosyltransferases Chemical synthesis
Abbreviations
Bn
benzyl
FBS
GalT
Me
fetal bovine serum
galactosyltransferase
methyl
PBS
Ph
phosphate buffered saline
phenyl
Pnp
HMBC
p-nitrophenyl
Heteronuclear Multiple Bond Correlation
Spectroscopy
:
:
Y. Gao C. Lazar I. Brockhausen
Department of Medicine, Division of Rheumatology,
Queen’s University,
NOESY
Nuclear Overhauser Effect Spectroscopy
Kingston, ON K7L 3N6, Canada
:
Y. Gao C. Lazar
Department of Biochemistry, Queen’s University,
Kingston, ON K7L 3N6, Canada
Introduction
I. Brockhausen (*)
Department of Medicine, Queen’s University,
Kingston, ON K7L 3N6, Canada
e-mail: brockhau@queensu.ca
The synthesis of glycoconjugates by glycosyltransferases
plays a critical role in mediating biological recognition
processes [1, 2]. Galactosyltransferases (GalT) add a Gal
residue to glycoprotein-bound glycans, which can be
further extended to form sialylated, fucosylated and other
W. A. Szarek
Department of Chemistry, Queen’s University,
Kingston, ON K7L 3N6, Canada

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674
Glycoconj J (2010) 27:673–684
complex carbohydrate epitopes. These epitopes play im-
portant roles in cell-cell and cell-lectin interactions, in
homing of lymphocytes, the immune system, inflammation,
bacterial adhesion, fertilization and other biological pro-
cesses. Gal residues are recognized by galectins that control
cell-cell or cell-matrix interactions, apoptosis and signal
transduction [3–6]. In cancer, these interactions can regulate
the invasive and metastatic behaviour of cells [7–10].
Members of the β3GalT and β4GalT families [11–13]
are involved in the extension of glycan chains. β4GalT
synthesize the common type 2 chain backbone, Galβ1-
4GlcNAcβ1-3, which can be extended and branched, and
serves as a scaffold for subsequent modifications and the
attachment of blood group and Lewis x and y antigens.
β3GalT catalyze the biosynthesis of type 1 chains, Galβ1-
3GlcNAcβ1-3, that can carry Lewis a and b antigens. The
abundance of these epitopes is often altered in cancer and is
related to the metastatic potential of cancer cells [7, 10, 14,
15].
β3GalT5 is the key enzyme involved in the synthesis of
type 1 chains [16–20]. The activity and transcript levels of
β3GalT5 were shown to be decreased in tissue samples of
human colon adenocarcinoma, relative to the surrounding
normal mucosa [15–17]. In contrast, the activity of β4GalT
has been found to be increased in cancer [21–23]. The
activity or mRNA levels of β4GalT also increase in
endothelial and cartilage derived cells after treatment of
cells with tumor necrosis factor (TNF)α [24, 25]. These
alterations are expected to cause a shift towards type 2
chains in inflamed and tumor tissues. We have therefore
developed inhibitors for β4GalT that may serve to decrease
the abundance of type 2 chains, and reduce the levels of
sialyl-Lewis x determinants involved in selectin binding,
cancer invasiveness and metastasis [26–28].
GlcNAc-naphthyl derivatives have previously been
shown to be inhibitors of bovine milk β4GalT and did
not act as acceptor substrates [26]. In particular, 2-
naphthyl 2-butanamido-2-deoxy-1-thio-β-D-glucopyrano-
side (compound 612) has been used in airway cell cultures
to decrease cell surface galactosylation and receptor sites
for Pseudomonas aeruginosa [27]. However, it has not
been determined if these inhibitors are specific for
β4GalT. In the present work, we determined the nature
and specificity of 612, and examined the inhibition of a
number of recombinant glycosyltransferases and enzymes
from cell homogenates. We focused on β4GalT1 and
β3GalT5, two enzymes that are important for the
extension of glycoprotein-bound glycans. We found that
612 is a specific inhibitor for β4GalT and thus has the
potential to reduce the synthesis of type 2 glycan chains.
Replacement of the sulfur atom with nitrogen in the
glycosidic linkage of 612 abrogated the inhibitory prop-
erties of 612.
Materials and methods
Materials
Materials were purchased from Sigma unless stated otherwise.
GlcNAc-analogs, including compounds were synthesized or
obtained as previously described [26, 29–33]. Insect cell
supernatants containing soluble human GlcNAc-transferases
I and II (GnT I or GnT II) were kindly donated by H.
Schachter, Hospital for Sick Children, Toronto. GnT II
substrate Manα1-6(GlcNAcβ1-2Manα1-3)Manβ-octyl was
produced from Manα1-6(Manα1-3)Manβ-octyl (Man3-
octyl, from Toronto Research Chemicals) using GnT I [30].
The GnT I product was purified by anion exchange
chromatography and reverse phase HPLC, and was free of
GnT I substrate as judged by GnT I assays and mass
spectrometry [31]. Recombinant soluble bovine polypeptide
GalNAc-transferase T1 (ppGalNAcT1) was donated by A.
Elhammer [34] and stored at −20°C. The enzyme that
synthesizes O-glycan core 2, soluble human core 2 GlcNAc-
transferase (C2GnT1), was produced in insect cells and
stored at −80°C as described [35]. NMR and mass
spectrometry was carried out in the Department of Chemis-
try, Queen’s University, as described [26, 32].
Genes encoding soluble human β3GalT1 and 2 [12],
cloned into baculovirus vectors were obtained from Henrik
Clausen, University of Denmark, Copenhagen. Sf9 insect
cells were grown by Don Jarvis, University of Wyoming, in
complete TNM-FH medium containing 10% fetal bovine
serum (FBS) [36]. Cells (2 million cells/5 ml medium) were
infected with baculovirus (E2) and the culture supernatants
were harvested at 24, 48 and 72 hpi. Soluble human
β3GalT5 was expressed in insect cells in the absence of
FBS and in the presence of BSA. The cell supernatant, as
well as the monoclonal antibody MAb5B8, which is
specific to β3GalT5, were kindly provided by Henrik
Clausen. Enzymes were stored at −80°C or for several
weeks at 4°C.
Purification of β3GalT5
β3GalT5 from Sf9 insect cell supernatants with a specific
activity of 19.3 nmol/h/mg using 0.5 mM GlcNAcβ-Bn
substrate was purified as follows. Enzyme was applied to
Q-Sepharose and eluted with 25 mM Tris–HCl, pH 7.2,
10 mM NaCl, 1 mM MnCl2. Subsequently, eluted enzyme
was applied to S-Sepharose and eluted with 35 mM MES,
pH 6.0, 1 mM MnCl2, and a gradient of 0.05 to 1 M NaCl.
Fractions were assayed for β3GalT5 activity and analyzed
by SDS-PAGE. Fractions that contained the highest
β3GalT5 activity were pooled and stored at −80°C. This
purified enzyme solution had an activity of 6.5 μmol/h/mg
using 0.5 mM GlcNAcβ-Bn as the acceptor substrate.

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Glycoconj J (2010) 27:673–684
675
ELISA assay
GlcNAc-transferases were assayed using 1 mM UDP-[3H]
GlcNAc as the donor and 2 mM Galβ1-3GalNAcα-Bn
acceptor (for core 2 β6-GlcNAc-transferase), 2 mM
GlcNAcβ1-3GalNAcα-pnp (for core 4 β6-GlcNAc-
transferase), 2 mM GalNAcα−Bn (for core 3 β3-
GlcNAc-transferase), 0.5 mM Man3octyl (for GnT I) or
1.7 mM Manα1-6(GlcNAcβ1-2Manα1-3)Manβ-octyl
(for GnT II). Mn2+ was omitted in β6-GlcNAc-
transferase assays. GlcNAc-transferase V (Gn-TV) was
assayed as described [33], followed by HPLC separation
of substrates and products. For inhibition studies, com-
pound 612 was present at 0.5 mM concentration with 10%
methanol in the assay, or as indicated in the tables.
The confirmation of β3GalT5 expression was made with an
ELISA assay using antibody MAb5B8. Briefly, purified
β3GalT5 was diluted 1:5, 1:10 and 1:50 in coating buffer
(0.05 M carbonate-bicarbonate, pH 9.7). 100 μl of
β3GalT5 preparation were transferred into each well of
24 well plates and incubated at 4°C overnight. After
incubation, supernatants were removed and wells washed
3 times with 200 μl PBS containing 10% Tween, followed
by blocking at 4°C overnight with 200 μl PBS containing
1% BSA. Wells were then washed 3 times before transfer
into each well of 100 μl primary antibody MAb 5B8 (1:4
dilution in PBS containing 10% Tween and 1% BSA).
After incubation for 1 h at room temperature (rt), wells
were washed and 100 μl of secondary antibody (donkey
anti-mouse IgG-HPR at 1:1000 dilution in PBS containing
10% Tween and 1% BSA) were applied and incubated for
1 h at rt. Each well was washed carefully before adding
100 μl of chromogenic TMB substrate solution (3,3′, 5,5′-
tetramethylbenzidine). The reaction was allowed to proceed
for 30 min; the reaction was then stopped with 1% SDS.
The absorbance at 370 nm was read with a microplate
reader.
Large scale preparation of GalT products
For the large scale preparation of non-radioactive GalT
enzyme products for structural analysis, GlcNAcβ-benzyl
was used as acceptor substrate and non-radioactive UDP-
Gal, and the assay was scaled up 100-fold. Products were
purified by ion exchange chromatography (using 10 ml
AG1x8, 100–200 mesh), followed by HPLC, using a C18
column and 7% acetonitrile/93% water as the eluant [26].
The enzyme products, Galβ1-3/4GlcNAcβ-benzyl were
exchanged with 99.96% D2O and analyzed by 600 MHz
1H-NMR spectroscopy, using a Bruker spectrometer at
Queen’s University, Department of Chemistry.
Glycosyltransferase assays
Glycosyltransferases were assayed as previously described
[26, 28, 29, 31–33, 35]. Enzyme products were isolated by
the AG1x8 or C18 Sep-Pak method, and analyzed by
reverse phase HPLC using a C18 column and acetonitrile/
water mixtures as the mobile phase. Assays were carried
out at least in duplicate determinations. GalT were assayed
in a total volume of 40 μl containing 0.5 mM acceptor
substrate (or as indicated in the tables), recombinant
enzyme preparation or cell homogenate (0.1 to 0.2 mg
protein), 0.5 mM UDP-[3H]Gal (1600–1800 cpm/nmol),
0.125 M MES buffer (pH 7), 12.5 mM MnCl2, 10 mM
AMP, and 5 mM γ-galactonolactone. In assays of cell
homogenates, 0.125% Triton X-100 was present. The
standard GalT assays contained GlcNAcβ-Bn as the acceptor
substrate, and control assays lacked the acceptor. Reaction
mixtures were incubated for 1 h at 37°C, and products isolated
using 0.2 ml columns of AG1×8 (100–200 mesh). Radioac-
tivity of the eluate was determined by scintillation counting.
For further analysis of products, mixtures were separated by
reverse phase HPLC [26]. Kinetic parameters were deter-
mined using the program OriginPro [26, 28].
Synthesis of (2-naphthyl)-2-butanamido-2-deoxy-β-D-
glucopyranosylamine (629)
To a solution of 2-butanamido-2-deoxy-D-glucopyranose
[37] (500 mg, 2.0 mmol) and 2-naphthylamine (200 mg,
1.4 mmol) in methanol–water (9:1 v/v, 10 ml) were added
~3 drops of 0.5 N HCl and the reaction mixture was stirred
at rt for 6 h, then heated at 60°C for 10 h. The mixture was
neutralized with NaHCO3, washed with ethyl acetate and
filtered. The filtrate was concentrated, and the residue was
washed with ethyl acetate–hexane (1:3 v/v) to give the title
compound as a white solid (340 mg, 45%): mp 196–197°C;
25
1
½aꢀD ꢁ18:8ꢂðc 0:2; CH3OHÞ; H NMR (400 MHz, DMSO-
d6): δ 0.79 (t, J=7.4 Hz, 3 H), 1.43–1.58 (m, 2 H), 2.01–
2.16 (m, 2 H), 3.16–3.25 (m, 1 H), 3.25–3.30 (m, 1 H),
3.40–3.57 (m, 2 H), 3.64–3.77 (m, 2 H), 4.47–4.56 (m,
2 H), 4.97 (app d, J=5.6 Hz, 1 H), 5.04 (app d, J=5.2 Hz,
1 H), 6.27 (d, J=6.8 Hz, 1 H, H-1), 6.84–6.93 (m, 2 H),
7.16 (app t, J=7.0 Hz, 1 H), 7.32 (app t, J=7.0 Hz, 1 H),
7.54–7.71 (m, 3 H), 7.98 (d, J=8.0 Hz, 1 H); 13C NMR
(100 MHz, DMSO-d6): δ 13.4, 18.7, 37.4, 55.2, 60.9, 70.8,
74.4, 77.4, 85.7 (C-1), 105.4, 117.5, 121.8, 125.8, 126.1,
127.2, 127.5, 128.5, 134.7, 144.6, 174.4; HRMS (EI)
Calculated for C20H27N2O5: 375.1919 [M+H]+. Found:
375.1909.
Core 1 β3GalT was assayed similarly using 0.5 or
1 mM GalNAcα-Bn as the acceptor. Polypeptide
GalNAc-transferase activities were assayed using 1 mM
UDP-[3H]GalNAc as the donor and 1 mM 362
(TTTVTPTPTG) or 2.5 mM AQPTPPP as the acceptor.

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676
Glycoconj J (2010) 27:673–684
Cell cultures and treatments
be inhibited by 612. Utilizing GlcNAcβ-Bn as the
acceptor substrate, GalT activities were high in all
homogenates tested, including those from lung cancer
cells H292, intestinal cancer cells Caco-2, mouse lym-
phocytic cells MOPC and P388, and prostate cell lines
RWPE-1, PC-3, DU145, LNCap and VCap. The addition
of 0.5 mM compound 612 inhibited GalT activities by
68% in P388 cells, and by more than 93% in all other cell
homogenates.
In order to determine the specificity of inhibition in cell
homogenates, we tested a number of cell-derived glycosyl-
transferases in the presence and absence of 612 (Table 1).
In the absence of the inhibitor, the activity of polypeptide
GalNAc-transferase (ppGalNAcT), using 2.5 mM
AQPTPPP as the acceptor substrate, was high in H292 cell
homogenates (115.3 nmol/h/mg). The presence of 1 mM
612 in the assay reduced ppGalNAcT activity in lung and
other cell homogenates by <4% (Table 1). No inhibition by
compound 612 of core 1 β3Gal-transferase (C1GalT) was
observed in cell homogenates from lung and lymphocytic
cells. While homogenates from P388 cells did not show
detectable activity of core 2 β6GlcNAc-transferase
(C2GnT), MOPC cell homogenates showed low activities,
which were not inhibited by 612. HPLC assays showed that
C2GnT activity in lung cells (2.8 nmol/h/mg) was inhibited
by less than 14%. There was no detectable activity of core 3
β3GlcNAc-transferase (C3GnT), and core 4 β6-GlcNAc-
All of the cell growth media contained 100 U/ml Penicillin
and 0.1 mg/ml Streptomycin. Human colonic cancer cells
HT29 and mouse lymphocytic cells MOPC and P388 were
grown in DMEM medium (GIBCO) containing 10% FBS.
Human airway epithelial cancer cells NCI-H292 were
propagated in RPMI 1640 medium (GIBCO) containing
10% FBS. Human colon cancer cells Caco-2 were grown in
DMEM medium (GIBCO) containing 10% FBS. Prostate
cancer cells PC-3, DU145, LNCap and VCap, as well as
normal prostate cells (ATCC) were grown as recommended
by ATCC. Cell homogenates were prepared by hand
homogenizing in 0.25 M sucrose of cells washed with
PBS. The protein content was determined by the BioRad
(Bradford) method.
Results
Inhibition of glycosyltransferases in cell homogenates
by 612
Mammalian cells express a combination of different
members of the β3GalT and β4GalT families which
are Golgi membrane-bound. We used cell homogenates
to test whether these membrane-bound enzymes would
Table 1 Inhibition by compound 612 of glycosyltransferase activities in cell homogenates
Enzyme
Cell type
Substrate
Inhibitor
Activity without inhibitor
(nmol/h/mg)
Inhibition (%)
β3/4GalT
β3/4GalT
β3/4GalT
β3/4GalT
ppGalNAcT
ppGalNAcT
C1GalT
C1GalT
C2GnT
H292
CaCo-2
MOPC
P388
0.5 mM GlcNAcβ-Bn
0.5 mM GlcNAcβ-Bn
1.0 mM GlcNAcβ-Bn
1.0 mM GlcNAcβ-Bn
2.5 mM AQPTPPP
0.5 mM 612
0.5 mM 612
1.0 mM 612
1.0 mM 612
1.0 mM 612
1.0 mM 612
0.5 mM 612
1.0 mM 612
0.5 mM 612
0.5 mM 612
0.5 mM 612
0.5 mM 612
0.5 mM 612
0.5 mM 612
34.9
38.4
2.3
95
98
93
167.0
115.3
0.7
68
H292
MOPC
H292
P388
< 4
< 1
< 1
< 1
< 14
nd
1.0 mM AQPTPPP
0.5 mM GalNAcα-Bn
1.0 mM GalNAcα-Bn
2.0 mM Galβ1-3GalNAcα-Bn
2.0 mM GalNAcα-Bn
2.0 mM GlcNAcβ1-3GalNAcα-pnp
0.5 mM Man3Oct
1.4
15.8
2.8
H292
H292
H292
H292
H292
PC-3
C3GnT
<1
C4GnT
<1
nd
GnT I
1.4
<1
GnT II
1.7 mM GnMan3Oct
7.7
<1
GnT V
0.5 mM GnMan(Gn4Man)4ManOct
0.6
<1
Glycosyltransferase activities in cell homogenates were assayed as described in Materials and Methods. Compound 612 (2-naphthyl-2-
butanamido-2-deoxy-1-thio-β-D-glucopyranoside) was added to the complete assay at a final concentration indicated in the table. The methanol
concentration in assays was 10%. Homogenates were prepared from human airway cells H292, human colonic cancer cells Caco-2, and mouse
lymphocytic cells MOPC and P388. ppGalNAcT, polypeptide GalNAc-transferase; C1GalT, core 1 β3Gal-transferase; C2GnT, core 2 β6GlcNAc-
transferase; C3GnT, core 3 β3GlcNAc-transferase; C4GnT, core 4 β6-GlcNAc-transferase; GnT I, GlcNAc-transferase I; GnT II, GlcNAc-
transferase II. Man3Oct, Manα1-6(Manα1-3)Manβ-octyl; GnMan3Oct, Manα1-6(GlcNAcβ1-2Manα1-3)Manβ-octyl; GnMan(Gn4Man)4Man-
Oct, GlcNAcβ1-2Manα1-6(GlcNAcβ1-2[4-deoxy-Manα1-3]4-O-methylManβ-octyl; Bn, benzyl; nd, not determined; pnp, p-nitrophenyl