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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.