Hydrolase and sialyltransferase activities of Trypanosoma cruzi trans-sialidase towards NeuAc-α-2,3-Gal-β-O-PNP

Article abstract of DOI:10.1016/S0960-894X(00)00611-9

NeuAc-α-2,3-Gal-β-O-PNP has been synthesised and its ability to act as a substrate for the hydrolase and transferase activities of Trypanosoma cruzi trans-sialidase have been investigated. The turn-over of this compound shows marked differences from the behaviour of NeuAc-MU. In addition, distinct differences in the action of T. cruzi trans-sialidase and Clostridium perfringens neuraminidase on NeuAc-α-2,3-Gal-β-O-PNP were apparent.

Full text of DOI:10.1016/S0960-894X(00)00611-9

Bioorganic & Medicinal Chemistry Letters 11 (2001) 141±144
Hydrolase and Sialyltransferase Activities of Trypanosoma cruzi
trans-Sialidase Towards NeuAc-ꢀ-2,3-Gal-ꢁ-O-PNPy
Jennifer A. Harrison,a K. P. Ravindranathan Kartha,a,{ W. Bruce Turnbull,a
Shona L. Scheuerl,a James H. Naismith,a Sergio Schenkmanb and Robert A. Fielda,Ã,{
aSchool of Chemistry, University of St Andrews, St Andrews, KY16 9ST, UK
bDisc. de Biologia Celular, Escola Paulista de Medicina, 04023-062 Sao Paulo, Brazil
Received 22 June 2000; accepted 26 October 2000
AbstractÐNeuAc-a-2,3-Gal-b-O-PNP has been synthesised and its ability to act as a substrate for the hydrolase and transferase
activities of Trypanosoma cruzi trans-sialidase have been investigated. The turn-over of this compound shows marked di€erences
from the behaviour of NeuAc-MU. In addition, distinct di€erences in the action of T. cruzi trans-sialidase and Clostridium per-
fringens neuraminidase on NeuAc-a-2,3-Gal-b-O-PNP were apparent. # 2001 Elsevier Science Ltd. All rights reserved.
The South American trypanosome, Trypanosoma cruzi,
is the etiological agent responsible for Chagas' disease, a
debilitating and often fatal condition prevalent in South
and Central American populations.2 This motile, blood-
borne parasite needs to invade mammalian cells to
undergo cell division and hence complete its life cycle.3
To do this, the parasite must ®rst adhere to the surface
of host cells. This it achieves by the generation of a
negatively charged glycopeptide coat on its surface. The
charged moieties concerned contain sialic acid (NeuAc).
However, the parasite does not in fact produce sialic
acid itself; with the aid of a cell surface trans-sialidase, it
scavenges this sugar from mammalian glycoconjugates
and transfers it onto mucin glycopeptides on the para-
site cell surface in a regio- and stereo-controlled manner
(i.e. it speci®cally uses and makes a-2,3-linked sialo-
sides).4 As such, T. cruzi trans-sialidase represents a
target for therapeutic intervention.
perspective, the structural and functional properties of
trans-sialidase have been reviewed.6 More recent studies
employing site-directed mutagenesis and selective peptide
deletions have suggested a role for two protein domains
in the sialyltransferase activity of trans-sialidase7 and
have led to proposals about the location of potential
galactose binding sites on the enzyme.8 The recently
reported crystal structure of the T. rangeli sialidase,9
which is approx. 70% identical to the core globular region
of T. cruzi trans-sialidase, supports the presence of a
distinct acceptor substrate binding site in trans-sialidase.
Assays for sialidase activity routinely rely on the clea-
vage of para-nitrophenyl (PNP) or 4-methylumbelliferyl
(MU) sialosides, which give rise to UV±vis active and
¯uorescent products, respectively. We have been unable
to determine reliable kinetic parameters for the hydro-
lysis or transfer of NeuAc from NeuAc-a-PNP by trans-
sialidase.10 However, we note that this substrate, which
is straightforward to prepare on a gram scale, is very
e€ective for milligram-scale biotransformations, which
proceed with high eciency when stoichiometric accep-
tor is present.11 A more detailed account of this obser-
vation can be seen in the recent work of Crout and co-
workers.12 We note that Scudder and co-workers
reported the rate of NeuAc transfer to acceptor from
NeuAc-PNP was some 25-fold less than from NeuAc-a-
2,3-lactose.13 We were therefore drawn to consider the
development of a trans-sialidase assay that would monitor
cleavage of the NeuAc-a-2,3-Gal glycosidic linkage, but
The aim of this study reported was to identify features
of the structure and/or mechanism of trans-sialidase
that mark it out as di€erent from the purely hydrolytic
sialidases, which have been well studied and for which
there are several crystal structures.5 From a biological
ÃCorresponding author. Fax: +44-1603-592003; e-mail: r.a.®eld@
uea.ac.uk
ySee ref. 1
{Present address: School of Chemical Sciences, University of East
Anglia, Norwich, NR4 7TJ, UK
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00611-9

142
J. A. Harrison et al. / Bioorg. Med. Chem. Lett. 11 (2001) 141±144
which would give a simple optical readout. This would
obviate the need for HPAEC analysis, as required with
NeuAc-a-2,3-lactose.13
was therefore investigated. Clearly this enzyme is
designed to hydrolyse lactose, so an alternative acceptor
substrate was required. Gal-b-1,3-GlcNAc-b-O-octyl19
proved e€ective as a trans-sialidase acceptor and was
not cleaved by the E. coli b-galactosidase.17 The revised
assay, operating at pH 7.5 with the E. coli b-galactosi-
dase and Gal-b-1,3-GlcNAc-b-O-octyl as the acceptor
substrate, was used in further studies.20
The proposed assay (outlined in Scheme 1) employs
NeuAc-a-2,3-Gal-PNP (synthesised as outlined in
Figure 1)14 as a substrate. The basis of the assay
relies on trans-sialidase to transfer NeuAc to either
water (hydrolase) or an acceptor sugar (transferase), with
the Gal-PNP released in this step being cleaved in situ
by b-galactosidase to liberate PNP, the anion of
which can be monitored spectroscopically (ÁA400 m;
e=18,300 M 1).15,16
The addition of acceptor substrate was shown to stimulate
the release of PNP in this assay (Figure 2, entries 1 and
2), which contrasts to the situation where NeuAc-MU is
used as the donor.21 In addition, the same assay per-
formed with the hydrolytic Clostridium perfringens
sialidase showed no such stimulation (Figure 2, entries 4
and 5). In keeping with earlier studies,22 trans-sialidase
proved to be insensitive to the standard glycal neur-
aminidase inhibitor DANA, whilst the Clostridium
neuraminidase was sensitive (Figure 2, entries 3 and 6).
Clearly the choice of b-galactosidase is critical, since it
could also cleave b-galactoside acceptors present in the
assay. In the ®rst instance, we chose to work with sweet
almond b-glucosidase, which has residual b-galactosi-
dase activity but does not cleave inter-sugar glycosidic
linkages with any signi®cant eciency (i.e. lactose is not
a substrate for this enzyme).15,17 However, the pH opti-
mum for this enzyme (5.6)15 is signi®cantly lower than
that of trans-sialidase (7.5).13 As a compromise, assays
were conducted at pH 6.5, although it became apparent
that NeuAc-a-2,3-Gal-PNP was not particularly stable
at this pH. E. coli b-galactosidase (pH optimum 7.4)18
With a view to indentifying potential trans-sialidase
inhibitors, we wondered whether S-linked NeuAc-Gal
disaccharides, with a greater distance between the
NeuAc and Gal moieties by virtue of the greater C±S
versus C±O bond length, might be inhibitory. Such
compounds have previously been investigated as sialidase
Scheme 1. Outline of coupled spectrophotometric assay for trans-sialidase.
Figure 1. Synthesis of disaccharide donor substrate NeuAc-a-2,3-Gal-b-O-PNP. (i) 2,2-Dimethoxypropane, TsOH, rt, 24 h followed by aq TFA/
DCM, rt, 10 min; (ii) BzCN, pyr/DCM, ice bath to 10ꢀC, 16 h; (iii) aq TFA/DCM, ice bath, 10 min (65% over 3 steps); (iv) Dowex 50W-X8-
200(H+), MeOH, rt, 2 h, quant.; (v) Ac2O, pyr, rt, 48 h, quant.; (vi) AcCl, HCl(g)
,
50 ꢀC to rt, 20 h, quant.; (vii) KSAc, DCM, rt, 20 h, 90%; (viii)
Na, MeOH, 40 ꢀC, 40 min, followed by MeI, DMF, rt, 20 h, 91%; (ix) NIS, TfOH, 3 A mol. sieves, DCM/MeCN, 45 ꢀ to 20 ꢀC, 4 h, 55%; (x)
NaOMe, MeOH, ice bath, 20 h, quant.; (xi) 0.1 M NaOH, ice bath, 4 h, quant.

J. A. Harrison et al. / Bioorg. Med. Chem. Lett. 11 (2001) 141±144
143
A number of groups have investigated the mechanism of
trans-sialidase but it is clear that the conclusions drawn
are heavily dependent on the substrates used and the
temperature of the reaction, in particular.13,21,22,26 The
ratio of hydrolysis to transfer for NeuAc-a-2,3-lactose
(1:13) is substantial.13 The relative rates of hydrolysis of
NeuAc-a-2,3-lactose (1) and NeuAc-MU (10)21 contrast
with the relative rates of transfer of NeuAc from NeuAc-
a-2,3-lactose (25), NeuAc-MU (1) and NeuAc-PNP
(1).13 In the current study, we observe a modest hydro-
lysis:transfer selectivity with NeuAc-a-2,3-Gal-PNP
(approx 1:4) which is mid-way between that observed
with NeuAc-a-2,3-lactose and NeuAc-MU.
Studies with NeuAc-MU conclude that aglycone release
is rate limiting21,22 since the addition of acceptor does
not in¯uence the rate of release of the MU aglycone. In
contrast, with NeuAc-a-2,3-Gal-PNP as a donor sub-
strate we have been able to demonstrate that the pre-
sence of acceptor does in¯uence the rate of NeuAc
transfer. We note that NeuAc-a-2,3-Gal is a poorer
trans-sialidase substrate than NeuAc-a-2,3-Gal-b-1,4-
Glc by some 200 fold.26 NeuAc-a-2,3-Gal-PNP appears
to be recognised and acted upon by trans-sialidase bet-
ter than do `simple' synthetic substrates (e.g. NeuAc-
MU) but less well than `more natural' substrates (e.g.
NeuAc-a-2,3-Gal-b-1,4-Glc). It would appear that
comparison of mechanistic information obtained with
di€erent types of trans-sialidase donor substrate should
be made with caution.
Figure 2. E€ect of acceptor substrate and putative inhibitor on the
activity of Trypanosoma cruzi trans-sialidase and Clostridium perfrin-
gens sialidase.20
inhibitors,23 and have been found not to serve as sub-
strates for Vibrio cholerae sialidase.24
In conclusion, we have developed a straightforward
spectrophotometric assay capable of monitoring both
the hydrolase and sialyltransferase activities of trans-
sialidase. Whilst NeuAc-a-2,3-Gal-PNP is a useful alter-
native to radiochemical substrates for routine monitoring
of trans-sialidase activity, it seems likely that it is not
suitable for mechanistic studies aimed at understanding
the action of trans-sialidase on naturally occurring
parasite and mammalian glycoconjugates.
However, NeuAc-a-2,3-S-Gal-b-O-octyl25 was found to
show no signi®cant inhibition of trans-sialidase at milli-
molar concentrations.
For both hydrolase ( acceptor) and transferase (+
acceptor) assays, NeuAc-a-2,3-Gal-PNP gave Km values
in excess of 5 mM, in keeping with data from Horen-
stein and co-workers for the reaction of trans-sialidase
with NeuAc-a-2,3-Gal.26 In the 1±5 mM range, the
transfer:hydrolysis ratio for NeuAc-a-2,3-Gal-PNP was
approximately 4 (Fig. 3).
Acknowledgements
This work was supported by the BBSRC, the Wellcome
Trust (Grant refs: 042472 and 040331), GlaxoWellcome,
and the Association for International Cancer Research.
References and Notes
1. A preliminary account of this work appeared in: Harrison,
J. A.; Kartha, K. P. R.; Smith, S. L.; Naismith, J. H.;
Schenkman, S.; Field, R. A. Biochem. Soc. Trans. 1997, 25,
363S.
2. Chance, M. L.; Molyneux, D. H. Curr. Opin. Infect. Dis.
1995, 8, 328.
3. Burleigh, B. A.; Andrews, N. W. Annu. Rev. Microbiol.
1995, 49, 175.
4. Colli, W. FASEB J. 1993, 7, 1257.
5. For an overview of the biological function of sialic acid and
sialidases see: Biology of the Sialic Acids; Rosenberg, A., Ed.;
Plenum Press: New York and London, 1995. Essentials of
Glycobiology; Varki, A., Cummings, R., Esko, J., Freeze, H.,
Figure 3. E€ect of acceptor substrate Gal-b-1,3-GlcNAc-b-O-octyl
(1 mM) on the activity of Trypanosoma cruzi trans-sialidase towards
NeuAc-a-2,3-Gal-PNP.

144
J. A. Harrison et al. / Bioorg. Med. Chem. Lett. 11 (2001) 141±144
Hart, G., Marth, J., Eds.; Cold Spring Harbor Laboratory
Press, 1999.
6. Schenkman, S.; Eichinger, D.; Pereira, M. E. A.; Nussenz-
weig, V. Annu. Rev. Microbiol. 1994, 48, 499.
7. Smith, L. E.; Eichinger, D. Glycobiology 1997, 7, 445.
8. Chuenkova, M.; Pereira, M. E. A.; Taylor, G. L. Biochem.
Biophys. Res. Commun. 1999, 262, 549.
9. Buschiazzo, A.; Tavares, G.; Campetella, O.; Spinelli, S.;
Cremona, M. L.; Paris, G.; Amaya, M. F.; Frasch, A. C. C.;
Alzari, P. M. EMBO J. 2000, 19, 16.
10. trans-Sialidase used in this study was a 70 kDa recombi-
nant material truncated to remove C-terminal repeats, but
which retained the catalytic N-terminal part of the enzyme.
The recombinant material was His-tagged to aid puri®cation.
Schenkman, S.; Chaves, L. B.; Pontes de Carvalho, L. C.;
Eichinger, D. J. Biol. Chem. 1994, 269, 7970.
11. Harrison, J. A. PhD Thesis, University of St Andrews,
UK, 1998.
12. Singh, S.; Scigelova, M.; Hallbery, M. L.; Howarth, O. W.;
Crout, D. H. G. Chem. Commun. 2000, 1013.
17. The inactivity of the almond enzyme towards lactose was
con®rmed by attempting to monitor the release of glucose
from lactose using a commerical glucose assay kit (Sigma
Chemical Co.). E. coli b-galactosidase was used as a positive
control. The same assay was used to con®rm that Gal-b-1,3-
GlcNAc-octyl is not a substrate for E. coli b-galactosidase.
18. Wallenfels, K.; Malhotra, O. P. Adv. Carbohydr. Chem.
Biochem. 1961, 16, 239.
19. Gal-b-1,3-GlcNAc-b-O-octyl was synthesised essentially
as described for the corresponding 8-ethoxycarbonyloctyl gly-
coside. Lemieux, R. U.; Bundle, D. R.; Baker, D. A. J. Am.
Chem. Soc. 1975, 97, 4076.
Selected characteristic analytical data for Gal-b-1,3-GlcNAc-
b-O-octyl: dH (D2O): 2.04 (3H, s, N-Ac), 3.92 (2H, m, 6a,b-H),
0
0
4.44 (1H, d, J1,2 7.6 Hz, H-1), 4.57 (1H, d, J11 ,22 7.6 Hz);
dc (D2O): 11.5, 20.1, 20.4, 23.2, 26.4, 26.6, 29.2, 52.7, 58.8,
59.1, 66.6, 66.8, 68.7, 68.8, 70.6, 73.4, 73.5, 80.6, 99.0, 101.6,
172.6. FAB-MS: Found [M+H]+ 496; C22H42NO11 requires
495.6.
20. Typical assay: 30mM HEPES pH 7.5, E. coli b-galactoside
(80 units), donor substrate (1±5 mM), acceptor substrate
(1 mM) and trans-sialidase in a total volume of 50 mL. This
mixture was incubated at 37 ꢀC for 30 mins, quenched by the
addition of 1 mL of Na2CO3 (100 mM, pH 10), and the A400
measured. Stopped assays proved more reliable than con-
tinuous assays.
21. Ribeirao, M.; Pereira-Chioccola, V. L.; Eichinger, D.;
Rodrigues, M. M.; Schenkman, S. Glycobiology 1997, 7, 1237.
22. Todeschini, A. R.; Mendoca-Previato, L.; Previato, J. O.;
Varki, A.; van Halbeek, H. Glycobiology 2000, 10, 213 and
references cited therein.
13. Scudder, P.; Doom, J. P.; Chuenkova, M.; Manger, I. D.;
Pereira, M. E. A. J. Biol. Chem. 1993, 268, 9886.
14. Selected characteristic analytical data for NeuAc-a-2,3-
0
0
0
0
Gal-PNP: dH (D2O): 1.72 (1H, t, J3 a,3 e=J3 a,4 12.3 Hz, H-
30a), 1.93 (3H, s, N-Ac), 2.69 (1H, dd, J3 a,3 e, J3 e,4 4.7 Hz, H-
30e), 4.16 (1H, dd, J2,3 9.8 Hz, J3,4 3.0 Hz, H-3), 5.20 (1H, d,
J1,2 7.8 Hz, H-1), 7.15 and 8.18 (2d, 4H, Ar); dC (D2O): 20.1
(NAc), 38.3 (30), 49.9 (50), 60.9 and 61.0 (6, 90), 66.2, 66.5, 68.3,
69.8, 70.4, 70.6, 72.0, 97.9 (1), 98.3 (20), 114.5 (Â2, Ph), 124.2
(Â2, Ph), 140.5 (Ph), 161.3 (Ph), 171.6 (10), 173.1 (NCO.Me);
ES-MS: Found [M H] 591; C23H32N2O16 requires 592.
15. Dale, M. P.; Ensley, H. E.; Kern, K.; Sastry, K. A. R.;
Byers, L. D. Biochemistry 1985, 24, 3530.
0
0
0
0
23. Kessler, J.; Heck, J.; Tanenbaum, S. W.; Flashner, M. J.
Biol. Chem. 1982, 257, 5056.
16. A variation on this assay, which relies on transfer of
NeuAc from a-2,3-sialyl-lactose onto ortho-nitrophenyl-b-
galactopyranoside, has recently been reported. By coupling
with b-galactosidase the assay measures removal of acceptor
as a function of time, whereas the assay reported herein mea-
sures cleavage of donor. Lee, S.-G.; Kim, B.-G. Biotechnol.
Lett. 2000, 22, 819.
24. Wilson, J. C.; Kiefel, M. J.; Angus, D. I.; von Itzstein, M.
Org. Lett. 1999, 1, 443 and references cited therein.
25. Turnbull, W. B.; Field, R. A. J. Chem. Soc., Perkin Trans.
1 2000, 1859.
26. Yang, J.; Schenkman, S.; Horenstein, B. A. Biochemistry
2000, 39, 5902.