From the Instituto de Microbiologia and
§ Faculdade de Farmácia, CCS-Bloco I, Universidade
Federal do Rio de Janeiro, 21944 970 Cidade Universitária, Rio de
Janeiro-RJ, Brazil, ¶ Laboratory for Molecular Structure, National
Institute for Biological Standard and Control, Potters Bar, Herts EN6
3QG, United Kingdom,
Department of Molecular Biology, Vanderbilt
University, Nashville, Tennessee 37235, and ** Disciplina de Biologia
Celular, Universidade Federal de São Paulo,
04023 062, São Paulo-SP, Brazil
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we have characterized the activity
of a uridine
diphospho-N-acetylglucosamine:polypeptide--N-acetylglucosaminyltransferase (O-
-GlcNAc-transferase) from Trypanosoma
cruzi. The activity is present in microsomal membranes and is
responsible for the addition of O-linked
-N-acetylglucosamine to cell surface proteins. This
preparation adds N-acetylglucosamine to a synthetic peptide KPPTTTTTTTTKPP containing the consensus threonine-rich
dodecapeptide encoded by T. cruzi MUC gene (Di Noia,
J. M., Sánchez D. O., and Frasch, A. C. C. (1995) J. Biol. Chem. 270, 24146-24149). Incorporation of
N-[3H]acetylglucosamine is linearly dependent
on incubation time and concentration of enzyme and substrate. The
transferase activity has an optimal pH of 7.5- 8.5, requires
Mn2+, is unaffected by tunicamycin or amphomycin, and is
strongly inhibited by UDP. The optimized synthetic peptide acceptor for the cytosolic O-GlcNAc-transferase (YSDSPSTST)
(Haltiwanger, R. S., Holt, G. D., and Hart, G. W. (1990)
J. Biol. Chem. 265, 2563-2568) is not a substrate for
this enzyme. The glycosylated KPPTTTTTTTTKPP product is susceptible to
base-catalyzed
-elimination, and the presence of
N-acetylglucosamine
-linked to threonine is supported by
enzymatic digestion and nuclear magnetic resonance data. These results
describe a unique biosynthetic pathway for T. cruzi surface mucin-like molecules, with potential chemotherapeutic implications.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Trypanosoma cruzi is the causative agent of Chagas' disease, a multisystemic disorder endemic in much of Latin America. This protozoan has a complex life cycle involving morphologically distinct stages in mammalian and insect hosts. Blood-sucking triatomine bugs transmit infective trypomastigotes to the mammalian host, which multiply intracellularly as amastigotes prior to differentiation into trypomastigotes, which, after rupture of the host cell, enter the blood stream, enabling infection of fresh cells or ingestion by a feeding triatomine bug, thus completing the biological cycle of transmission (1). Antigenic glycoconjugates, including the highly O-glycosylated sialoglycoproteins, known as mucin-like molecules, have been strongly implicated in the molecular mechanism of attachment to and invasion of mammalian host cells (2). The sialic acid residues present in these molecules are derived from host sialoglycoconjugates (3) and are transferred to the T. cruzi surface glycoproteins by a unique trans-sialidase (4). The first evidence for O-glycosylation of serine and/or threonine in trypanosomal glycoproteins came from studies of T. cruzi GP-25 (5), a glycoprotein corresponding to the C-terminal domain of cruzipain (6). More recently, O-glycosylated mucin-like proteins were demonstrated in metacyclic (7) and cell-derived trypomastigotes (8) and in epimastigotes (9, 10). Structural analyses have shown that these O-glycans vary between strains and developmental stages (9-11).
The striking feature of these O-glycans is that they are linked to the peptide backbone through an N-acetylglucosamine (GlcNAc) unit, with threonine (Thr) rather than serine (Ser) being the usual site of attachment (10, 12). The GlcNAc-Thr core can be extended by addition of Galp, Galf, and sialic acid residues. These were the first O-GlcNAc-linked oligosaccharides reported in surface glycoproteins. Previously, single O-GlcNAc units linked to Ser and/or Thr have been described on nuclear and cytosolic glycoproteins (13). This post-translational modification on the nuclear and cytoplasmic proteins is catalyzed by a cytosolic O-GlcNAc-transferase (14).
The unusual addition of O-GlcNAc to T. cruzi
surface glycoproteins prompted us to investigate this
post-translational modification in more detail. In this paper, we
describe a novel UDP-GlcNAc:polypeptide -N-acetylglucosaminyltransferase from T. cruzi
and specify the optimal conditions for its activity. We have also
characterized the in vitro glycosylation products and have
established the anomeric configuration of GlcNAc O-linked to
Thr.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials--
Radioactively labeled
UDP-[6-3H]GlcNAc (40-60 Ci/mmol),
UDP-[6-3H]GalNAc (5-15 Ci/mmol),
NaB[3H]4 (100-500 mCi/mmol), and
[1-3H]glucose (10-30 Ci/mmol) were purchased from
American Radiolabeled Chemicals, Inc. The radioactive sugar
alcohols N-[1-3H]acetylglucosaminitol
([3H]GlcNAcO),1
N-[1-3H]acetylgalactosaminitol
([3H]GalNAcO),
Galp1
4GlcNAcO[3H], and
Galp
1
6(Galp
1
4)[3H]GlcNAcO
were prepared by reduction of 10 µmol of saccharides with 10 µCi of
NaB[3H]4 in water for 1 h at room
temperature, followed by addition of 100 µmol of unlabeled
NaBH4. Borate salts were removed by repeated addition of
methanol and evaporation, passage through a mixed-bed ion exchange
resin, and gel filtration on Bio-Gel P-4 (extra fine). The radioactive
monosaccharides [6-3H]GlcN and [6-3H]GalN
were prepared by hydrolysis of radioactive sugar nucleotides in 3 M HCl at 100 °C for 4 h. Unlabeled UDP-GlcNAc, SP
Sephadex (SP-C25-120), and
-N-acetylglucosaminidase from
jack beans were obtained from Sigma. The peptides KPPTTTTTTTTKPP,
YSDSPSTST, and YSPTSPSK (with the O-
-GlcNAc on the serine
at position 5) used in this study were kindly supplied by A. C. C. Frasch, R. S. Haltiwanger, and G. W. Hart,
respectively. All other reagents were of the highest available
quality.
Parasites-- The Y strain of T. cruzi was used in all enzymatic experiments. Native sialoglycoproteins were purified from epimastigotes of the Y, CL-Brener, and Dm28c strains. Epimastigotes were cultured at 28 °C in brain-heart infusion medium supplemented with hemin and 5% of fetal calf serum, and harvested in the exponential phase of growth (15). Trypomastigotes were obtained from LLC-MK2 cells infected with tissue culture-derived trypomastigotes for 6 days, maintained at 37 °C in RPMI medium containing 10% fetal calf serum, under 5% CO2 (8).
Microsomal Membrane Preparation-- Pellets of 2 × 1011 epimastigotes or 1.12 × 109 cell-derived trypomastigotes were ground in liquid nitrogen. The homogenate was diluted with 10-20 ml of 250 mM sucrose and 25 mM Tris/HCl, pH 7.4 (Tris/sucrose buffer), and centrifuged for 10 min at 12,000 × g. The supernatant was then ultracentrifuged for 1 h at 120,000 × g, and the resulting pellet was resuspended in Tris/sucrose buffer with a glass-Teflon homogenizer and ultracentrifuged as above. The pellet was then resuspended in Tris/sucrose buffer and appropriately diluted for protein assay (16).
Enzymatic Assay: O--GlcNAc-transferase Activity--
The
standard assay contained, in a final volume of 50 µl, 25 mM Tris/HCl buffer (pH 7.4), 5 mM
MgCl2, 5 mM MnCl2, 0.1% Triton X-100, 1.5 µCi of UDP-[3H]GlcNAc (40-60 Ci/mmol), and
6.8 nmol of synthetic peptide acceptor substrate (KPPTTTTTTTTKPP). The
reaction was initiated by addition of microsomal membranes (250 µg of
protein). Control assays without the acceptor peptide were used to
correct for endogenous activity. The mixture was incubated at 28 °C
for 30 min, and the reaction terminated by addition of 950 µl of 50 mM formic acid. The reaction mixture was loaded onto a 1-ml
sulfopropyl-Sephadex column (SP-C 25-120) equilibrated in 50 mM formic acid. The column was washed with 5 ml of 50 mM formic acid, and the peptide and labeled glycopeptide were eluted with 4 ml of 0.5 M NaCl. Incorporation of
[3H]GlcNAc into peptide was determined on aliquots of the
eluate by liquid scintillation counting after addition of Bray solution (5 ml). In experiments to increase radiolabel incorporation, the reaction mixture (100 µl) contained 25 mM Tris-HCl (pH
7.4), 5 mM MgCl2, 5 mM
MnCl2, 15 µCi of UDP-[3H]GlcNAc, 6.8 nmol
of acceptor peptide, and microsomal membranes (1 mg of protein). Other
conditions and the method for the isolation of the labeled peptide were
as described above. For testing of trypomastigote microsomal fraction,
the reaction mixture (50 µl) contained 25 mM Tris-HCl (pH
7.4), 5 mM MgCl2, 5 mM
MnCl2, 1.5 µCi of UDP-[3H]GlcNAc, 3.4 nmol
of acceptor peptide, and 25 µg (protein) of the microsome
preparation. Other conditions were as described above.
Optimization of the O--GlcNAc-transferase
Assay--
Incorporation of [3H]GlcNAc was measured at
different temperatures (4, 20, 28, and 37 °C), different incubation
times (10, 20, 30, 60 and 120 min), and in the presence of increasing
concentrations of Triton (0.01-0.5%), the peptide substrate (0-34
nmol), and microsomes (100-300 µg of protein). The effect of pH on
GlcNAc-transferase activity was assayed in Tris-MES buffer at pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0 (final concentration in the
reaction mixture, 50 mM) under standard incubation
conditions. The synthetic peptide YSDSPSTST was used at 450 nmol and at
20 or 28 °C.
HPLC Purification of O-Glycosylated Peptide-- Incorporation of [3H]GlcNAc into synthetic peptide was confirmed by RP-HPLC on a C18 column (5 µm, 4 × 250 mm, Amersham Pharmacia Biotech) eluted with an H2O/acetonitrile/trifluoroacetic acid gradient. Solvent A was 0.1% aqueous trifluoroacetic acid, and solvent B was 0.089% trifluoroacetic acid in acetonitrile. The linear gradient was started 5 min after injection from 0 to 15% solvent B over 40 min (held until 50 min). The flow rate was 1 ml/min, and 0.8- or 1.0-ml fractions were collected. Aliquots were mixed with 5 ml of Bray solution, and the radioactivity was determined by liquid scintillation counting. Elution of unlabeled peptide was monitored by absorbance at 206 nm.
Analysis of the Reaction
Product--
[3H]GlcNAc-labeled peptide fraction
obtained by RP-HPLC was rechromatographed on Bio-Gel P-4 (extra fine)
(120 cm × 0.5 cm) at 0.6 ml/h with 0.2 M ammonium
acetate as the eluent. The column was calibrated using bovine serum
albumin and [3H]glucose as marker for the void
(vo) and included volumes, respectively. The elution positions of authentic standards of
[3H]GlcNAcO,
Galp1
4[3H]GlcNAcO, and
Galp
1
6(Galp
1
4)[3H] GlcNAcO
were also determined. Fractions of 300 µl were collected and assayed
for radioactivity. The labeled peptide fraction was desalted,
lyophilized, and dissolved in 10 mM NaOH, containing 0.3 M NaBH4, at 37 °C for 48 h to
-eliminate and reduce saccharides linked to threonine (17). The
solution was neutralized with 2 M acetic acid and passed
through Dowex 50W-X8 (25-50 mesh H+ form). Boric acid was
removed by repeated evaporation with methanol. The residue was
dissolved in distilled water and analyzed by gel filtration on Bio-Gel
P-4 column (as described above) and by descending paper chromatography
in ethyl acetate:pyridine:water (8:2:1) for 48 h (18). The
distribution of radioactivity on the paper chromatogram was determined
by cutting the paper strips into 1-cm sections and liquid scintillation
counting.
Digestion with Jack Bean -N-Acetylglucosaminidase--
The
reaction mixture for jack bean
-N-acetylglucosaminidase
digestion contained HPLC-purified [3H]GlcNAc-labeled
peptide (approximately 5 × 104 cpm), 50 mM phosphate-citrate buffer, pH 5.0, and 0.8 units of enzyme in a final volume of 100 µl. The mixture was incubated at
37 °C for 18 h (20, 21), the reaction was terminated by addition of 900 µl of 50 mM formic acid, and the solution
was loaded onto a 1-ml SP-Sephadex (25-120) column equilibrated in 50 mM formic acid. Released [3H]GlcNAc was
eluted with 50 mM formic acid (5 ml), lyophilized, and the
radioactivity was determined by liquid scintillation counting. p-Nitrophenyl
N-acetyl-
-D-glucosaminide was used as
substrate control. As a further positive control, 100 µmol of the
unlabeled synthetic glycopeptide YSPTSPSK, with the
O-
-GlcNAc on the serine at position 5 (kindly
provided by G. W. Hart) was digested under identical conditions.
The reaction was terminated by addition of 50 mM formic
acid, and the liberated GlcNAc was recovered on SP-Sephadex as above
and quantified using the Morgan-Elson reaction (22). A standard curve
was constructed using D-GlcNAc similarly chromatographed on SP-Sephadex.
Preparation of GlcNAc-rich Glycopeptides from Native Sialoglycoproteins for NMR Spectroscopy-- Sialoglycoproteins from epimastigote forms were purified as described by Previato et al. (4). The sialoglycoproteins from T. cruzi CL-Brener strain were subjected to partial acid hydrolysis (0.2 M trifluoroacetic acid for 2 h at 100 °C). The GlcNAc-rich glycopeptides were recovered by gel filtration on a column of Sephadex G25 SF (1 × 10 cm). Also, GlcNAc-rich glycopeptides were obtained from Y, CL-Brener, and Dm28c sialoglycoproteins by Smith degradation (23). Briefly, to 2 ml of a solution containing 30 mg of T. cruzi sialoglycoproteins in 0.1 M sodium acetate (pH 4.6), an equal volume of 0.2 M NaIO4 was added. After 24 h at 4 °C, oxidation was terminated by addition of glycerol. The oxidized products were recovered by gel filtration on Sephadex G25 SF (1 × 10 cm) column, using water as eluent, at a flow rate of 1 ml/min. The oxidized glycoproteins were reduced with sodium borohydride for 3 h at room temperature. Boric acid was removed by repeated evaporation with methanol. The material, oxidized and reduced, was partially hydrolyzed with 20 mM trifluoroacetic acid for 30 min at 100 °C. The resulting GlcNAc-rich glycopeptides were recovered by gel filtration on Sephadex G25 SF as above.
NMR Analysis of the GlcNAc-rich Glycopeptides--
NMR spectra
were acquired on a Varian Unity 500 NMR spectrometer equipped with a
5-mm PFG (pulsed field gradient) triple resonance probe and at
indicated probe temperatures of 30 or 40 °C. Standard pulse
sequences were used except for the introduction of an echo sequence
into the ROESY and TOCSY spectra and WHSQC pulse sequence, which is an
implementation of the method of Wider and Wüthrich (24). The
mixing time in the TOCSY spectra was 80 and 150 ms in the ROESY
spectra. Presaturation of the residual water signal was achieved using
a low power pulse from the transmitter. In the WHSQC spectra, obtained
at 500 MHz, heteronuclear decoupling was achieved using the GARP
sequence. 1H and 13C chemical shifts were
referenced to internal 3-(trimethylsilyl)tetra-deuteropropionic acid at
zero ppm (1H) and 1.8 ppm (13C, to
tetramethylsilane at zero) (25).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Development of an Assay for T. cruzi O--GlcNAc-transferase:
Optimization of Assay Conditions--
An assay for
O-GlcNAc-transferase activity was developed using microsomal
membranes prepared from epimastigotes of the Y strain of T. cruzi. To measure the transfer of GlcNAc in vitro,
UDP-[6-3H]GlcNAc was used as the GlcNAc donor and the
synthetic peptide KPPTTTTTTTTKPP as acceptor. This peptide was chosen
due to its similarity with a common motif in the peptide sequence of
deglycosylated sialoglycoproteins from the Y
strain2 and the MUC gene
family products from T. cruzi (26). KPPTTTTTTTTKPP proved to
be successful as a substrate for T. cruzi GlcNAc-transferase activity. As shown in Fig. 1, the
incorporation of [3H]GlcNAc into the peptide was time
dependent for a period up to 2 h (Fig. 1A) and was
proportional to increasing amounts of protein (using a microsomal
membrane preparation as the enzyme source) (Fig. 1B) and of
the peptide acceptor (Fig. 1C). The standard assay was
performed at 28 °C, although a similar rate was observed at
20 °C. At 37 °C, the activity was slightly less than that
observed at 4 °C (Fig. 1A).
|
Effect of Inhibitors-- Addition of 5 mM UDP reduced incorporation of [3H]GlcNAc into the synthetic peptide by 90%. Transferase activity was not reduced in the presence of tunicamycin or amphomycin, even when added at 10 µg/ml.
Ion Dependence and Optimum pH-- The transferase was inactive in the absence of metal ions, but activity was regained by adding Mn2+. Table I shows that among seven divalent cations tested, Mn2+ was the most effective in restoring activity. Co2+ and Ca2+ were able to restore 20 and 15% of the activity observed in the presence of Mn2+. The O-GlcNAc-transferase activity had a pH optimum between 7.5 and 8.5, with maximum activity at pH 8.5. Activity decreased gradually below pH 7.0 and above pH 8.5, with only 50% of the activity at pH 8.5 present at pH 9.0 (Fig. 1D).
|
Characterization of the Glycosylated Peptide Product--
The
radiolabeled material recovered from the SP-Sephadex column was
characterized by several techniques. A single peak of radioactivity was
obtained after reversed phase HPLC on a C18 column with an elution
volume distinct from that of the unlabeled peptide acceptor substrate
(Fig. 2A). Bio-Gel P-4
chromatography of this radiolabeled material indicated that its
apparent molecular mass was greater than that of the unlabeled peptide
acceptor (Fig. 2B). After base-catalyzed -elimination and
reduction of the purified radiolabeled glycopeptide, radioactive
material eluted at the same volume as authentic
N-acetylhexosaminitol on the P-4 column (Fig.
2C), and its identity was confirmed as
[3H]GlcNAcO by descending paper chromatography (Fig.
2D). In assays using larger amounts of
UDP-[3H]GlcNAc, two radiolabeled fractions were observed
in RP-HPLC (Fig. 3A) and on
the P-4 column (Fig. 3B). One fraction showed a
chromatographic profile identical with that of the glycopeptide obtained under standard conditions (Fig. 2, A and
B). The other fraction was assumed to be a glycopeptide
substituted with more than one GlcNAc residue (Fig. 3,
A and B), as both fractions liberated [3H]GlcNAcO (identified by descending paper
chromatography) on reductive
-elimination (Fig. 3C).
|
|
|
Determination of the Anomeric Configuration of the GlcNAc O-Linked
to Thr--
The anomeric configuration of the GlcNAc-Thr linkage was
determined by enzymatic and NMR experiments. Treatment of the
HPLC-purified [3H]GlcNAc-labeled peptide with
-N-acetylglucosaminidase from jack beans failed to
release [3H]GlcNAc from the glycopeptide, indicating that
GlcNAc is not
-linked to Thr. A glycopeptide YSPTSPSK with the
O-
-GlcNAc on the Ser at position 5 was used as a positive
control and liberated between 50 and 55% GlcNAc when digested under
identical conditions.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have previously characterized a novel series of
O-GlcNAc-linked oligosaccharides from surface
sialoglycoproteins of T. cruzi (9, 10). The first step in
the biosynthesis of these O-glycan chains is attachment of
GlcNAc to the peptide backbone. In the present study, we show that
microsomal membrane preparations from epimastigotes and trypomastigotes
of T. cruzi Y strain have an
O--GlcNAc-transferase that attaches GlcNAc to threonine
in a suitable acceptor peptide. We used several approaches to define the activity of this enzyme and to assess its relationship to the
cytosolic O-GlcNAc-transferase (14, 38).
We demonstrate that the synthetic peptide KPPTTTTTTTTKPP can function as acceptor for this novel transferase. This peptide incorporates a threonine-rich sequence, originally reported by DiNoia et al. (26), who showed that TTTTTTTTKPP is a common repeating motif in T. cruzi MUC gene products. The locations of the glycosylated threonines were not identified, but the peptide was a good substrate for in vitro glycosylation, at least two GlcNAc residues being incorporated in heavily labeled experiments. The nonapeptide YSDSPSTST, described by Haltiwanger et al. (38) as an optimum substrate for the cytosolic O-GlcNAc-transferase, was not glycosylated by the T. cruzi enzyme. UDP-[3H]GlcNAc was the activated GlcNAc donor in the T. cruzi system. Potentially, the mechanism of the reaction could be either direct transfer from UDP-GlcNAc or via formation of activated dolichol donors. To distinguish between these possibilities, the transferase activity was assayed in the presence of excess UDP and with the antibiotics tunicamycin or amphomycin, which are potent inhibitors of Dol-P-dependent glycosylation (39, 40). Only UDP abolished incorporation of [3H]GlcNAc into the acceptor peptide, indicating that UDP-GlcNAc acts directly as the GlcNAc donor. The cytosolic O-GlcNAc-transferase (38) also uses sugar nucleotides directly as sugar donors.
The T. cruzi enzyme, in common with most
glycosyltransferases (41), requires divalent metal cations for
activity, with Mn2+ being the most effective. This is in
contrast to the cytosolic O-GlcNAc-transferase, which shows
no metal ion dependence (38). Other differences between the T. cruzi microsomal enzyme and the ubiquitous cytosolic
O-GlcNAc-transferase are that the former has an optimal pH
range of 7.5-8.5, remains active at 37 °C, and has increased
activity when treated with Triton X-100. Most strikingly, the T. cruzi enzyme attaches GlcNAc to the hydroxylated amino acid via an
-linkage, whereas the anomeric specificity of the cytosolic enzyme
is
. In support of this, the [3H]GlcNAc peptide
(produced in vitro) was not susceptible to digestion of with
-N-acetylglucosaminidase from jack beans,
although under identical digestion conditions GlcNAc was readily
liberated from unlabeled synthetic YSPTSPSK (with the
O-
-GlcNAc on the serine at position 5), corresponding to
a sequence from the C-terminal repeat domain of the large subunit of
RNA polymerase II (42), which in vivo is glycosylated by the
cytosolic transferase. It is unlikely that the lack of susceptibility
of the
[3H]GlcNAc-K2P4T8
peptide to
-N-acetylglucosaminidase is attributable to
its amino acid sequence, as the jack bean enzyme is able to deglycosylate a diverse range of structurally distinct glycoproteins and glycopeptides, including sequences from nuclear pore protein, human
erythrocyte band 4.1 protein, and the 65-kDa erythrocyte cytosolic
protein (38, 42). More compellingly, NMR analysis of GlcNAc-rich
glycopeptides from the native sialoglycoconjugates of T. cruzi showed unambiguously that the GlcNAc residue linked to Thr
has the
-anomeric configuration, the 1JC, H,
NOE, and chemical shift data all being consistent with the
- rather
than the
-anomer.
O-GlcNAc has previously been reported in surface glycoproteins from Plasmodium falciparum (43) and Giardia lamblia (44). Subsequent studies, however, showed that O-linked GlcNAc is either absent or present only at very low levels in P. falciparum (45) and that glucose rather than N-acetylglucosamine is the O-linked sugar in G. lamblia (46). Because the O-GlcNAc-transferase activity of T. cruzi is associated with the microsomal fraction, and because its known natural substrates are GPI-anchored N-linked surface glycoproteins (10), it seems likely that it is associated with some compartment of the secretory pathway, although confirmation of this must await completion of detailed localization studies. Because the enzyme differs in its anomeric specificity, kinetic properties, and possibly in cellular location from the cytosolic enzyme described in higher eukaryotes, it may prove to be unique to T. cruzi. If so, it constitutes an exciting potential target for the rational design of novel chemotherapeutic agents. Purification of the transferase is currently in progress and should soon enable these questions to be addressed.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. A. C. C. Frasch and Dr. J. M. DiNoia (Instituto de Investigaciones Biotecnologicas, UNSAM, San Martin, Provincia de Buenos Aires, Argentina), Dr. R. Haltiwanger (Dept. of Biochemistry, SUNY, Stony Brook, NY), and Prof. G. W. Hart (Johns Hopkins University School of Medicine, Baltimore) for the synthetic peptides. Also, we thank Dr. R. Wait for critical reading of the manuscript, X. Lemercinier for the 13C-coupled WHSQC spectrum, the MRC Biomedical NMR Center for access to the Unity 600 NMR spectrometer, and the Sir Halley Stewart Trust for funding.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from Programa Núcleo de Excelência, Financiadora de Estudos e Projetos, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Conselho de Ensino para Graduados, and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
A Howard Hughes International Research Scholar. To whom
correspondence should be addressed. Tel.: 55-21-5903093; Fax:
55-21-2708647; E-mail: immglup{at}microbio.ufrj.br.
1 The abbreviations used are: [3H]GlcNAcO, N-[1-3H]acetylglucosaminitol; [3H]GalNAcO, N-[1-3H]acetylgalactosaminitol; MES, 2-(N-morpholino)ethanesulfonic acid; Galf, galactofuranose; Galp, galactopyranose; HPLC, high performance liquid chromatography; ROESY, rotating frame nuclear Overhauser enhancement sprectroscopy; RP, reverse phase; TOCSY, total correlation spectroscopy; NOE, nuclear Overhauser enhancement.
2 J. O. Previato and L. Mendonça-Previato, unpublished results.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|