The trans-sialidase from Trypanosoma cruzi efficiently transfers α-(2→3)-linked N-glycolylneuraminic acid to terminal β-galactosyl units

Article abstract of DOI:10.1016/j.carres.2007.07.018

The trans-sialidase from Trypanosoma cruzi (TcTS), the agent of Chagas' disease, is a unique enzyme involved in mammalian host-cell invasion. Since T. cruzi is unable to synthesize sialic acids de novo, TcTS catalyzes the transfer of α-(2→3)-sialyl residues from the glycoconjugates of the host to terminal β-galactopyranosyl units present on the surface of the parasite. TcTS also plays a key role in the immunomodulation of the infected host. Chronic Chagas' disease patients elicit TcTS-neutralizing antibodies that are able to inhibit the enzyme. N-Glycolylneuraminic acid has been detected in T. cruzi, and the trans-sialidase was pointed out as the enzyme involved in its incorporation from host glycoconjugates. However, N-glycolylneuraminic acid α-(2→3)-linked-containing oligosaccharides have not been analyzed as donors in the T. cruzi trans-sialidase reaction. In this paper we studied the ability of TcTS to transfer N-glycolylneuraminic acid from Neu5Gc(α2→3)Gal(β1→4)GlcβOCH₂CH₂N₃ (1) and Neu5Gc(α2→3)Gal(β1→3)GlcNAcβOCH₂CH₂N₃ (2) to lactitol, N-acetyllactosamine and lactose as acceptor substrates. Transfer from 1 was more efficient (50-65%) than from 2 (20-30%) for the three acceptors. The reactions were inhibited when the enzyme was preincubated with a neutralizing antibody. K_m values were calculated for 1 and 2 and compared with 3′-sialyllactose using lactitol as acceptor substrate. Analysis was performed by high-performance anion-exchange (HPAEC) chromatography. A competitive transfer reaction of compound 1 in the presence of 3′-sialyllactose and N-acetyllactosamine showed a better transfer of Neu5Gc than of Neu5Ac.

Full text of DOI:10.1016/j.carres.2007.07.018

Carbohydrate Research 342 (2007) 2465–2469
Note
The trans-sialidase from Trypanosoma cruzi efficiently transfers
a-(2!3)-linked N-glycolylneuraminic acid to terminal
b-galactosyl units
*
´
´
´
Rosalıa Agustı, Marıa Eugenia Giorgi and Rosa M. de Lederkremer
CIHIDECAR, Departamento de Quı´mica Orga´nica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
Ciudad Universitaria, Pabello´n II, 1428 Buenos Aires, Argentina
Received 12 June 2007; received in revised form 20 July 2007; accepted 24 July 2007
Available online 2 August 2007
Abstract—The trans-sialidase from Trypanosoma cruzi (TcTS), the agent of Chagas’ disease, is a unique enzyme involved in mam-
malian host-cell invasion. Since T. cruzi is unable to synthesize sialic acids de novo, TcTS catalyzes the transfer of a-(2!3)-sialyl
residues from the glycoconjugates of the host to terminal b-galactopyranosyl units present on the surface of the parasite. TcTS also
plays a key role in the immunomodulation of the infected host. Chronic Chagas’ disease patients elicit TcTS-neutralizing antibodies
that are able to inhibit the enzyme. N-Glycolylneuraminic acid has been detected in T. cruzi, and the trans-sialidase was pointed out
as the enzyme involved in its incorporation from host glycoconjugates. However, N-glycolylneuraminic acid a-(2!3)-linked-con-
taining oligosaccharides have not been analyzed as donors in the T. cruzi trans-sialidase reaction. In this paper we studied the abil-
ity of TcTS to transfer N-glycolylneuraminic acid from Neu5Gc(a2!3)Gal(b1!4)GlcbOCH2CH2N3 (1) and Neu5Gc(a2!3)
Gal(b1!3)GlcNAcbOCH2CH2N3 (2) to lactitol, N-acetyllactosamine and lactose as acceptor substrates. Transfer from 1 was more
efficient (50–65%) than from 2 (20–30%) for the three acceptors. The reactions were inhibited when the enzyme was preincubated
with a neutralizing antibody. Km values were calculated for 1 and 2 and compared with 30-sialyllactose using lactitol as acceptor
substrate. Analysis was performed by high-performance anion-exchange (HPAEC) chromatography. A competitive transfer reac-
tion of compound 1 in the presence of 30-sialyllactose and N-acetyllactosamine showed a better transfer of Neu5Gc than of Neu5Ac.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: HPAEC; Neu5Gc transfer; trans-Sialidase; Trypanosoma cruzi
The trans-sialidase from Trypanosoma cruzi (TcTS), the
agent of Chagas’ disease, is a unique enzyme important
for the survival of the parasite in the mammalian or in-
sect host.1,2 This process involves the transfer of a-
(2!3)-linked sialic acid from host glycoconjugates to
terminal b-galactopyranosyl units of parasite mucins.3
This is the pathway that T. cruzi utilizes to incorporate
sialic acid, since it is unable to synthesize it. The TcTS
not only acts on the surface of the parasite, but it is also
shed into the blood during infection.4 Animals that sur-
vive infection, as well as chronic Chagas’ disease pa-
tients, elicit TS-neutralizing antibodies that are able to
inhibit TS enzymatic activity, thus limiting the period
in which the TS can systemically operate to the early
steps of the infection.5
In strains belonging to the less virulent lineage 1, ter-
minal b-D-galactofuranosyl units are also present in the
oligosaccharides.6–9 We have recently proved that the
presence of the furanosyl units does not hamper sialyla-
tion of the terminal galactopyranose in the oligosac-
charides.10 We have also described that lactose derivatives
could act as inhibitors of the enzyme, proving that the
sugar b-linked to galactopyranose is not critical for the
acceptor properties. In fact, lactitol and lactobionic
acid, which are open-chain derivatives of lactose, are
very good acceptors of sialic acid. Lactitol, which was
the best of the ones tested, effectively inhibited the
*
Corresponding author. Tel./fax: +54 11 45763352; e-mail:
lederk@qo.fcen.uba.ar
0008-6215/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.07.018

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R. Agustı´ et al. / Carbohydrate Research 342 (2007) 2465–2469
transfer of sialic acid to N-acetyllactosamine and inhib-
ited parasite resialylation.11 Lactitol also prevented the
apoptosis caused by the TcTS.12
the enrichment of Neu5Gc in carcinomas and fetuses
to a higher uptake by these fast-growing tissues. Traces
of Neu5Gc could be incorporated from dietary
Recombinant TS has also been used for the prepara-
tion of sialylated oligosaccharides using a phenolic
sialoside13–16 or sialyllactose obtained from bovine
colostrum17,18 as donors.
TcTS
Neu5Gc(α2→3)Gal(β1→4)GlcβR
Gal(β1→4)GlcβR
1
N-Glycolylneuraminic acid (Neu5Gc) occurs fre-
quently in the animal kingdom, but is presumed absent
from normal human tissues.19 Its presence in human tu-
mor cells has been described.20 The authors attributed
Lactitol
Neu5Gc(α2→3)lactitol
R= OCH2CH2N3
Figure 1. N-Glycolylneuraminic acid transfer by Trypanosoma cruzi
trans-sialidase from donor 1 to lactitol.
Figure 2. Transfer analysis of Neu5Gc from Neu5Gc-oligosaccharides to acceptor substrates by the trans-sialidase from Trypanosoma cruzi. A(1–4),
acceptor substrate lactitol; B(1–4), acceptor substrate N-acetyllactosamine; C(1–4), acceptor substrate lactose. A1–C1 donor 1 was incubated with the
acceptor and trans-sialidase for 15 min at 25 ꢁC, and the reaction mixtures were analyzed by HPAEC-PAD as described under Section 1. A2–C2 is
the same as above but the trans-sialidase was preincubated with a neutralizing antibody. A3–C3 donor 2 was used and incubated as for donor 1. A4–
C4 donor 2 and TcTS preincubated with the antibody were used. Neu5GcLol, N-glycolylneuraminyllactitol; Neu5GcLNAc, N-glycolylneuraminyl
N-acetyllactosamine; Neu5GcL, N-glycolylneuraminyllactose; 1, Neu5Gca2!3Galb1!4GlcbOCH2CH2N3; 2, Neu5Gca2!3Galb1!3GlcNAc-
bOCH2CH2N3.

R. Agustı´ et al. / Carbohydrate Research 342 (2007) 2465–2469
2467
sources,21 as humans are genetically unable to produce
it, and circulating anti-Neu5Gc antibodies are present
in most normal humans.
rides in which Gal is b-(1!4)-linked to GlcNAc were
better donors of Neu5Ac than those in which Gal is b-
(1!3)-linked.26 The Km and Vm values we obtained for
30-sialyllactose were 3.21 mM and 1.70 pmol/min ng,
respectively. Hydroxylation of the N-acyl moiety in the
sialic acid offers the possibility of another interaction
with a conveniently located amino acid of the TcTS.
It has been determined that the neutralization of TcTS
circulating activity by monoclonal neutralizing antibod-
ies (mNtAb) reduced the sialic acid content in platelets27
N-Glycolylneuraminic acid has been detected in T.
cruzi by selective acid hydrolysis of epimastigote cells.
It was rightly suggested by the authors that the
Neu5Ac/Neu5Gc ratios obtained from different strains
reflected the composition of the culture medium.22
Accordingly, it was reported that blood-stream trypo-
mastigote forms obtained from infected mice contain
mainly Neu5Gc, and a trans-sialidase was suggested as
responsible for its incorporation.23 N-Glycolylneurami-
nic acid is found in 98% of the total sialic acids in mouse
serum.24 However, as far as we know, Neu5Gca(2!3)-
linked oligosaccharides have only been tested as donors
in the trans-sialidase reaction of Trypanosoma congo-
lense. In this case, a culture supernatant of procyclic
cells was used as a source for the enzyme, with a labeled
substrate as acceptor.25
In the present work, we studied for the first time the
ability of recombinant TcTS to transfer Neu5Gc from
Neu5Gca(2!3)Galb1-R trisaccharides to N-acetyllac-
tosamine, lactose or lactitol, which are all good acceptor
substrates for the enzyme.
The transfer to terminal b-galactopyranosyl units was
studied
using
Neu5Gca2!3Galb1!4GlcbOCH2-
CH2N3 (1) and Neu5Gca2!3Galb1!3GlcNAcbO-
CH2CH2N3 (2) as donor substrates.
As shown in Figure 1 the reaction is illustrated for do-
nor 1 and lactitol as acceptor. Analysis was performed
using high-performance anion-exchange chromatogra-
phy with pulse amperometric detection (HPAEC-
PAD). Under the conditions used (see Section 1), the
retention times for compounds 1 (tR 18.00 min) and 2
(tR 19.70 min) were similar but well separated from the
substrates and products in each case. For the three
acceptor substrates, the transfer from trisaccharide 1
was more efficient (transfer values 50–65%) than from
trisaccharide 2 (20–30%) (Fig. 2). As expected, the pres-
ence of an additional OH, in the sialic acid N-substitu-
ent, increases the retention time of the glycolylated
derivatives with respect to the acetylated ones. For exam-
ple, Neu5GcLactitol (tR 22.6 min) elutes more than three
times later than Neu5AcLactitol (tR 6.1 min) (not
shown). Free Neu5Gc was eluted at 26.1 min and was
not detected, excluding hydrolase activity for the TcTS.
To compare the affinity of the N-glycolylneuraminic acid
oligosaccharides to the enzyme, Km values were calcu-
lated for 1, 2 and 30-sialyllactose using lactitol as the
acceptor substrate, under the same conditions, show-
ing that both Neu5Gc-disaccharides were better sub-
strates than 30-sialyllactose having 1 (Km = 0.84 mM,
Vm = 0.48 pmol/min ng) higher affinity than 2 (Km =
1.53 mM, Vm = 0.33 pmol/min ng). In agreement with
our results, in a comparative study using Neu5Aca(2!3)
linked oligosaccharides, it was shown that the saccha-
Figure 3. Competitive transfer of N-acetylneuraminic acid and N-
glycolylneuraminic acid by TcTS. 30-Sialyllactose was incubated with
N-acetyllactosamine as acceptor substrate, the TcTS (panel A) and in
the presence of donors 1 (panel B) or 2 (panel C) and analyzed by
HPAEC-PAD. Abbreviations and conditions as for Figure 2.

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R. Agustı´ et al. / Carbohydrate Research 342 (2007) 2465–2469
and prevented the immune system damage, even in
extremely virulent models.5 In our case, transfer of
Neu5Gc was almost completely inhibited when the
enzyme was incubated with the monoclonal antibody
previous to incubation with the acceptor substrates
(Fig. 2). This result confirms that the same active site
is being used for the transfer of sialic acid from Neu5Gc
and Neu5Ac-containing glycoconjugates.
Analysis by HPAEC-PAD was performed using a
Dionex ICS-3000 HPLC system equipped with a pulse
amperometric detector (PAD). A CarboPac PA-100
ion-exchange analytical column (4 · 250 mm) equipped
with a guard column PA-100 (4 · 50 mm) was eluted
with a linear gradient over 60 min from 50 to 300 mM
NaAcO in 100 mM NaOH at a flow rate of 1.0 mL/
min at room temperature.
Competitive transfer of Neu5Gc with respect to
Neu5Ac was studied for oligosaccharides 1 and 2 by
incubating each with sialyllactose, TcTS and N-acetyl-
lactosamine as acceptor substrate, with all substrates
in 1 mM concentrations (Fig. 3). The peak at 8.40 min
corresponds to free sialic acid present in our sample of
Neu5AcL, but does not interfere with the experiment.
Comparison with the incubation in the absence of the
Neu5Gc oligosaccharides (Fig. 3A) showed that, appar-
ently, Neu5GcLNAc was formed in a larger amount
than Neu5AcLNAc in the presence of compound 1
and sialyllactose as donors. Moreover, the lactose
obtained when Neu5Ac is transferred from Neu5Aclac-
tose to N-acetyllactosamine is readily used by TcTS as
an acceptor substrate forming Neu5Gclactose (Fig. 3,
panels B and C). Neu5GcL is co-chromatographed with
a sample obtained from bovine colostrum (not shown).
In conclusion, we describe for the first time that
N-glycolylneuraminic acid is efficiently transferred by a
recombinant TcTS from synthetic Neu5Gc-contain-
ing trisaccharides to the common N-acetyllactos-
amine unit of glycoconjugates. The reaction could be
used for preparative purposes of Neu5Gc-containing
glycoconjugates.
1.3. Enzyme kinetics
Reaction mixtures of 20 lL containing 20 mM Tris buf-
fer, pH 7, 30 mM NaCl, 1 mM Neu5Gca2!3Galb1!
4GlcbOCH2CH2N3 (1) or Neu5Gca2!3Galb1!3Glc-
NAcbOCH2CH2N3 (2) as donors and 1 mM lactitol,
N-acetyllactosamine or lactose as acceptor substrates
were incubated with 300 ng purified trans-sialidase.
After incubation, reaction mixtures were diluted with
40 lL deionized water and frozen. Sialylation of accep-
tors was calculated as the percentage of the sialylated
product obtained over the total amount of sialic acid
(free or linked to a saccharide).
For Km calculations, 1 mM lactitol and different con-
centrations of the donor substrates were incubated for
15 min at 25 ꢁC as before. Samples were then diluted 5
times, and 20 lL of each were analyzed by HPAEC.
The extent of sialylation of substrates was calculated
from the decrease in the concentration of the donor sub-
strate using galacturonic acid as internal standard, in
comparison with the corresponding control without en-
zyme. The Km values were obtained graphically by the
Lineweaver–Burk method.29
1.4. Inhibition of TcTS by neutralizing monoclonal
antibody
1. Experimental
1.1. Oligosaccharide substrates
Reaction mixtures of 20 lL containing 20 mM Tris buf-
fer, pH 7, 30 mM NaCl, 1 mM Neu5Gca2!3Galb1!
4GlcbOCH2CH2N3 (1) or Neu5Gca2!3Galb1!3Glc-
NAcbOCH2CH2N3 (2) as donors and 1 mM lactitol,
N-acetyllactosamine or lactose as acceptor substrates
were incubated with 300 ng purified trans-sialidase pre-
viously incubated for 15 min with the neutralizing
monoclonal antibody. After incubation, reaction mix-
tures were diluted with 40 lL of deionized water and
frozen.
The donor substrates, Neu5Gca2!3Galb1!3GlcNAc-
bOCH2CH2N3 and Neu5Gca2!3Galb1!4Glcb OCH2-
CH2N3. were kindly provided by the Consortium for
Functional Glycomics. Lactose was from Calbiochem
(San Diego, CA). Lactitol and N-acetyllactosamine were
purchased from Sigma Chemical Co. Neu5AcLactose
(30-Sialyllactose) was obtained from bovine colostrum
by an adaptation of a reported method.28
1.2. General procedures
1.5. Competitive transfer of Neu5Gc and Neu5Ac to
N-acetyllactosamine
For the sialylation experiments a recombinant TcTS
expressed in Escherichia coli was kindly provided by
A. C. C. Frasch, and a TS-neutralizing mouse monoclo-
nal antibody (immunoglobulin G2a) directed to the ac-
tive site of the enzyme was from O. Campetella
(UNSAM, Universidad Nacional de General San Mar-
tin, Buenos Aires, Argentina).
Reaction mixtures of 20 lL containing 20 mM Tris buf-
fer, pH 7, 30 mM NaCl, 1 mM 30-sialyllactose, 1 mM
N-acetyllactosamine, and 1 mM disaccharides 1 or 2
were incubated with purified trans-sialidase for 15 min
at room temperature. Samples were then diluted 5 times
with deionized water and analyzed by HPAEC.

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2469
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Acknowledgments
We thank A. C. C. Frasch and O. Campetella from Uni-
versidad Nacional General San Martın, Argentina, for
´
the kind gift of trans-sialidase from T. cruzi and the
neutralizing antibody, respectively. The carbohy-
drate compounds Neu5Gca2!3Galb1!3GlcNAc-
´
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bOCH2CH2N3
and
Neu5Gca2!3Galb1!4Glcb
OCH2CH2N3 were provided by the Carbohydrate Syn-
thesis/Protein Expression Core of The Consortium for
Functional Glycomics funded by the National Institute
of General Medical Sciences Grant GM62116. We also
thank Ola Blixt and James Paulson for the samples
(The Scripps Research Institute, La Jolla, CA, USA).
This work was supported by grants from Agencia Nac-
´
´
´
ional de Promocion Cientıfica y Tecnologica (ANPCyT)
and Universidad de Buenos Aires, Argentina. R. M. de
Lederkremer is a research member of CONICET. M. E.
Giorgi is a fellow from ANPCyT.
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