The
trans
-sialidase from the African trypanosome
Trypanosoma brucei
Georgina Montagna
1
, M. Laura Cremona
1
, Gasto
´
n Paris
1
, M. Fernanda Amaya
2
, Alejandro Buschiazzo
2
,
Pedro M. Alzari
2
and Alberto C. C. Frasch
1
1
Instituto de Investigaciones Biotecnolo
´
gicas – Instituto Tecnolo
´
gico de Chascomu
´
s, Consejo Nacional de Investigaciones
Cientı
´

lysis. The enzymatic activity of mutants at key positions
involved in the transfer reaction revealed that the catalytic
sites of TcTS and TbTS are likely to be similar, but are not
identical. As in the case of TcTS and TrSA, the substitution
of a conserved tryptophanyl residue changed the substrate
specificity rendering a mutant protein capable of hydrolysing
both a-(2,3) and a-(2,6)-linked sialoconjugates.
Keywords: trans-sialidase; sialidase; T. brucei; procyclic
trypomastigotes.
African trypanosomiasis has re-emerged as a major health
threat, with an epidemic resulting in more than 100 000 new
infections per year. With 300 000 cases officially reported,
human trypanosomiasis, or sleeping sickness caused by
Trypanosoma brucei ssp. gambiense and ssp. rhodesiense,has
now returned to the epidemic levels of the 1930s in many
historic foci across Africa. T. brucei ssp. brucei causes the
Ôngana diseaseÕ in domestic animals, which can reduce food
production as much as 50%. The parasite, which lives and
multiplies in the blood of the infected host, eludes the
immune system by consecutively expressing structurally
different forms of variant surface glycoproteins (VSG) [1].
The VSG coat from the bloodstream form is replaced by the
invariant procyclin surface coat of the procyclic insect stage
when entering the tsetse insect vector (Glossina sp.) These
procyclins are a small family of very similar acid repetitive
proteins [2,3] that might protect procyclic cells from
digestion by the digestive enzymes in the fly [4].
Unable to synthesize sialic acids, trypanosomes use a
specific enzyme, the trans-sialidase, to scavenge the mono-
saccharide from host glycoconjugates and to sialylate

n, Pcia de Buenos Aires, Argentina.
Fax: + 54 11 4752 9639, Tel.: + 54 11 4580 7255,
E-mail:
Abbreviations:TrSA,T. rangeli sialidase; TcTS, T. cruzi
trans-sialidase; TbTS, T. brucei trans-sialidase; VSG,
variant surface glycoproteins; IMAC, iminodiacetic
acid metal affinity chromatography; MUNen5Ac, 2¢-(4-methylum-
belliferyl)-a-
D
-N-acetylneuraminic acid; 3¢SL, sialyl-a-(2,3)-lactose;
6¢SL, sialyl-a-(2,6)-lactose; GSS, Genome Sequence Survey.
(Received 10 January 2002, revised 26 April 2002,
accepted 30 April 2001)
Eur. J. Biochem. 269, 2941–2950 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02968.x
Y342H [11]. The overall structure of the TcTS comprises
an N-terminal globular region of 642 amino acids carrying
the catalytic activity (see below), followed by a C-terminal
extension of tandemly repeated sequences named SAPA
(shed acute phase antigen) that are not required for the
enzymatic activity. SAPA is highly antigenic and is
involved in the stabilization of the enzymatic activity once
released in the blood of the infected host [12]. Members in
the sialidase family of T. rangeli (TrSA) are about 70%
identicaltoTcTS[13],andsomeofthemalsolack
enzymatic activity.
The crystal structures of several microbial sialidases
have been determined. They share a similar catalytic
domain that displays a typical six-bladed b propeller
topology originally observed in influenza virus sialidase
[14]. Some sialidases are multidomain proteins and include

Trypanosomes
Procyclic forms of T. brucei brucei stock EATRO427 were
cultivated axenically in SDM-79 as described previously
[19]. The strain was kindly provided by F. R. Opperdoes
(Christian de Duve Institute of Cellular Pathology, Brussels,
Belgium).
Nucleic acid isolation
Total DNA from culture procyclic forms was isolated using
a conventional proteinase K/phenol/chloroform method as
described previously [20]. Total RNA was purified using
TRIzol reagent following manufacturer’s instructions (Life
Technologies Inc.).
Southern blot analysis
Total DNA was digested with the indicated restriction
enzymes and 2.5 lg of the sample per line was electro-
phoresed in 0.8% agarose gel and transferred for Southern
blot on Zeta-Probe nylon membranes (Bio-Rad) as des-
cribed previously [20].
PCR radiolabeling of probes was performed by substi-
tuting the nonradioactive dCTP by 30 lCi of [a-
32
P]dCTP
in a 30-cycle primer extension reaction after optimization of
thetemplateandMgCl
2
concentration. The TbTS probe
was made with oligonucleotide FRIP (5¢-ATAAGG
TAGAGCGCACTGTGCA-3¢) using clone TbTS digested
with EcoRV as template. Probe TbTS-like was made from
clone pGEM-TbTS-like using oligonucleotide (5¢-CTT

obtained from Genome Sequence Survey (GSS) AQ661000
for AminoMet and AQ656761 for STOP. Primers for
TbTS-like were obtained from a BAC clone: AC009463,
which contains the complete ORF. The PCR products
were cloned on pGEM-T Easy vector following the
A-tailing procedure. The clones were called pGEM-TbTS
and pGEM-TbTSlike. These clones were used as template
for automated (AbiPrism) or manual (dideoxy-chain
termination method with Sequenase-USB) sequencing or
for subcloning in the expression vector.
Cloning of TbTS 5¢ UTR
First strand cDNA was prepared with the Superscript II
system using an internal primer (5¢-TGAAAATCAACAG
CAGTCTC-3¢) that binds to position 58–40 of TbTS ORF.
RT-PCR was carried out with the primers for T. brucei
mini-exon as forward (5¢-AACGCTATTATTAGAACA
GTTTCTGTACT-3¢) and the one used for first strand
synthesis as reverse, using Vent DNA polymerase. The
product was cloned into pGEM-T Easy vector after
A-tailing and sequenced using the dideoxy-chain termin-
ation method with Sequenase (USB).
Site-directed mutagenesis
Site directed point mutagenesis was performed using the
QuikChange Site-directed mutagenesis kit (Stratagene),
2942 G. Montagna et al. (Eur. J. Biochem. 269) Ó FEBS 2002
according to the manufacturer’s instructions. All clones
were sequenced to confirm mutation of target sites.
Expression of
trans
-sialidase genes in bacteria

M
isopropyl
thio-b-
D
-galactoside (Sigma) and induction was maintained
at 18 °C for 12–16 h. Cells were harvested, washed with
NaCl/Tris (20 m
M
Tris/HCl pH 7.6 and 50 m
M
NaCl) and
frozen ()80 °C) until needed. After thawing, lysis was
carried out in the presence of 20 m
M
Tris/HCl pH 7.6,
30 m
M
NaCl,0.5%TritonX-100,1m
M
phenyl-
methylsulfonyl fluoride, 100 lgÆmL
)1
DNAse I. Superna-
tants were centrifuged at 21 000 g for 30 min and subjected
to iminodiacetic acid metal affinity chromatography
(IMAC) (HiTrap Chelating, Amersham Pharmacia Biotech
AB) Ni
2+
-charged equilibrated in 20 m
M

M
2¢-(4-methylumbelliferyl)-a-
D
-N-acetylneuraminic
acid (MUNen5Ac, Sigma). The assay was performed in
50 lLin20m
M
Pipes pH 6.9. After incubation at 35 °C,
the reaction was stopped by dilution in 0.2
M
sodium
carbonate pH 10, and fluorescence was measured with a
DYNAQuant
TM
200 fluorometer (Hoefer Pharmacia Inc).
Trans-sialidase activity was measured in 20 m
M
Pipes
pH 6.9, using 1 m
M
Neu5Ac-a-(2–3) lactose as sialic acid
donor and 12 l
M
[
D
-glucose-1-
14
C]lactose (55 mCiÆmmol
)1
)

at 37 °C for 30 min. Samples were then neutralized with
13 lL of sodium arsenite 2% w/v in HCl (0.5 N) by slow
addition of the reactive. Tubes were gently vortexed to
complete the reduction reaction. After the total disappear-
ance of yellow colour (5 min) 152 lL of thiobarbituric acid
(36 m
M
, pH 9.0) were added and then incubated in a boiling
water bath for 15 min Samples were then cooled in an ice-
water bath for 5 min, followed by room-temperature colour
stabilization. The samples were centrifuged, and 20 lLwere
separated by high-performance liquid chromatography
through a C
18
reverse phase column (Pharmacia Biotech)
using 2 : 3 : 5 water/methanol/buffer (buffer: 0.2% phos-
phoric acid; 0.23
M
sodium perchlorate). Absorbance was
measured at 549 nm. A sialic acid calibration curve was
previously set, and absorbance values were always read in
the linear range.
RESULTS
The
T. brucei trans
-sialidase primary sequence
conserves most of the structurally relevant amino-acid
residues of bacterial and protozoan sialidases
BLAST
searches were performed using sequences corres-

protein coded by these genes showed that TbTS is organized
into three putative regions (Fig. 2). An N-terminal region of
100 amino acids, which is absent in TcTS, a middle region of
372 amino acids, which is 45% identical to the catalytic
domain of the T. cruzi enzyme and a C-terminal region of
298 amino acids followed by an hydrophobic region likely
to correspond to a GPI-anchor signal. TbTS is probably
anchored by GPI to the surface membrane since native
procyclic trans-sialidase can be released from the parasite by
treatment with phospholipase D [24]. The 298 amino acids
in the C-terminal domain are 30% identical to the TcTS
lectin-like domain. TbTS does not have a repetitive domain
at the C-terminus that is homologous to the T. cruzi SAPA
domain.
The catalytic region revealed the conservation of most of
the structurally relevant residues displayed in bacterial and
protozoan sialidases and trans-sialidases (Fig. 2), such as an
arginine triad that binds to the carboxylate group common
to all the sialic acid derivatives (R133, R346, R431), a
glutamic acid (E473) that stabilizes one of the arginine side
chains, a negatively charged group (D157) proposed as a
possible proton donor in the hydrolytic reaction and two
essential residues at the bottom of the site (E331, Y457),
which are well positioned to stabilize a putative sialosyl
cation intermediate [25]. This tyrosine residue was found to
be a determinant for the catalytic activity of TcTS [11]
The comparison of the crystal structure of TrSA with the
homologous model of TcTS reveals a few amino acid
changes close to the substrate-binding cleft that might
modulate the sialyltransferase activity [17]. Most of these

designed to have this amino acid at position +1. The new
construct, which includes an N-terminal extension of 10
amino acids expressing a His-tag, codes for a 745 amino-
acidproteinwithapredictedmolecularmassof81.4kDa
and displaying both sialidase and trans-sialidase activity
(data not shown). All further work was performed with this
protein. To perform kinetic studies, the protein was purified
Fig. 2. Comparison of protein structure and sequence between TbTS and
TcTS. (A) Primary structure of TbTS and TcTS. The positions of the
FRIP, Asp boxes and TcTS superfamily motifs are underlined.
(B) Amino-acid sequence of the conserved region of the catalytic do-
main of TbTS and TcTS. The FRIP and the Asp boxes are underlined.
The identity in amino acids between the two primary sequences are
indicated with vertical bars and the boxes highlight the residues involved
in the catalytic centre of the sialidases of known crystal structure.
Fig. 1. Differences among TbTS clones. Eleven clones of TbTS were
sequenced and analysed. They could be classified in eight distinct
groups with differences in only nine positions. The nucleotide changes
in the triplet sequence are indicated (uppercase). The mutations that
cause amino-acid changes are boxed.
2944 G. Montagna et al. (Eur. J. Biochem. 269) Ó FEBS 2002
through passage on a iminodiacetic acid metal affinity
column followed by FPLC anionic exchange (see Experi-
mental procedures for details). After the anionic exchange
column, the protein was > 95% pure (Fig. 3). MUNen5Ac
was used as substrate to assay for sialidase activity, and a
mix of Neu5Ac-a-(2,3) and Neu5Ac-a-(2,6)-lactose as sialic
acid donor and lactose as acceptor for the trans-sialidase
activity (Fig. 3). The affinity for sialyl-lactose as substrate of
TbTS (2.27 m

Point mutations at critical amino-acid residues
revealed features of the catalytic site of African
trypanosomes
trans
-sialidase
Based on the crystal structure of TrSA [17], mutants of
TbTS at key positions involved in substrate binding and
specificity were constructed and characterized. These
mutants include (see Fig. 4A) the exposed aromatic side
chain that favours the sialyl-a-(2,3) substrate specificity
(W400 in TbTS mature protein), a tyrosine residue sugges-
ted to be part of a second carbohydrate-binding site in the
catalytic cleft (Y191 in TbTS), a proline residue that was
found to increase the sialidase activity in TrSA (P371 in
TbTS) and a tyrosine residue that is well positioned to
stabilize a putative sialosyl cation intermediate (Y430 in
TbTS) [17].
The mutant proteins were produced and purified with the
same criteria described for the wild-type in the previous
section. As shown in Fig. 4B, mutations at positions 371
and 430 of TbTS completely abolished both sialidase and
II
AB
I
1/v (nmol sialic acid
-1
.min.mg) x 10
-3
1/S (mM
-1

min
-1
.mg
-1
78
0
10
20
Elution (mL)
250
0
Citrate (mM)
kDa
78 91011
66
97.4
45
Absorbance 280 nm
91011
Fractions
Fractions
Fig. 3. Purification of recombinant TbTS pro-
tein. (A) TbTS protein was purified by anion-
exchange chromatography (Mono Q) after
IMAC chelating column. The elution profile
of Mono Q is shown. Fractions were collected
and analysed by SDS/PAGE as indicated in
Experimental procedures. (B) Lineweaver–
Burk plots of sialidase and trans-sialidase
activities. I, the sialidase activity was measured

371
400
430
TWY
461
TbTS wild type 8074.33 ± 691.52 (100) 100 933.8 ± 60.78 (100) 100
TbTS Y430-H
0000
TbTS T371-Q
0000
TbTS Y191-S
0 0.6 0 12.8
TbTS W400-A
00 0104.29 ± 8.3 (11.2)
trans-sialidase activity TcTS
a
b
TcTSsialidase activity
A
B
Fig. 4. Site-directed mutagenesis on TbTS. (A) Relative positions of
the site-directed mutagenesis on TbTS refer to the relevant amino acids
for trans-sialidase activity on TcTS. (B) Recombinant proteins were
expressed and purified as indicated in Experimental procedures.
Sialidase activity was measured using MUNen5Ac as substrate and
trans-sialidase activity was measured using sialyl-a-(2,3)-lactose and
lactose as the sialic acid donor and acceptor, respectively. Activities are
expressed as nmol sialic acid per min per mg (free sialic acid for sia-
lidase activity; amount of sialic acid transferred to lactose for trans-
sialidase acitivity). The percentage of activity referred to wild-type

the case of TbTS, the mutant protein W400A was obtained
and assayed for activity using sialyl-a-(2,3)-lactose (3¢SL)
and sialyl-a-(2,6)-lactose (6¢SL). The mutated enzyme was
now capable of hydrolysing the a-(2,6) regioisomer, losing
the strict specificity of the wild-type enzyme for the sialyl-
a-(2,3) substrate (Fig. 5).
The active sites of the
T. brucei
and
T. cruzi trans
-sialidases are highly conserved
As expected from their similar function and common
evolutionary origin, critical active site residues are largely
conserved in all trypanosomal sialidases and trans-siali-
dases. The 3D structure of the T. rangeli sialidase bound to
2,3-didehydro-2-deoxy-N-acetylneuraminic acid (Neu2-
en5Ac, a sialidase inhibitor) [17] showed 33 amino acids
that are positioned close to the inhibitor. They have at least
one atom at less than 7 A
˚
from Neu2en5Ac. Among these
positions, 26 amino acids (79%) are conserved between
TcTS and TbTS, 24 (73%) are conserved between TbTS
and TrSA, and 22 (67%) are conserved between TcTS and
TrSA. These relative similarities differ significantly from
those found when comparing the entire catalytic domains
(Fig. 4), thus revealing a functional constraint on the
evolution of trans-sialidases.
All amino-acid residues that have been found to be
important for the function in other viral and bacterial

studied the role of these residues using exchange mutagen-
Fig. 5. Activity of 3¢SL and 6¢SL hydrolysis of the amino-acid substi-
tution W400-A on TbTS. Sialidase activity of TbTS W400A mutant
protein was measured using sialyl-a-(2,3)-lactose (3¢SL) and sialyl-
a-(2,6)-lactose (6¢SL) as sialic acid donor substrates as described in
Experimental procedures.
Fig. 6. Amino-acid positions close to the inhibitor Neu2en5Ac (shown in
yellow) in the crystal structure of TrSA-Neu2en5Ac complex [17].
Amino-acid side-chains shown in blue are strictly conserved in
microbial sialidases, those shown in green are invariant in three
trypanosomal enzymes (TrSA, TcTS and TbTS), and those shown in
redareconservedinthetwotrypanosomaltrans-sialidases, but differ in
TrSA, and could be important for transglycosylation (see text).
2946 G. Montagna et al. (Eur. J. Biochem. 269) Ó FEBS 2002
esis between TrSA and TcTS. Along similar lines, Paris
et al. [18] demonstrated that the substitution Q284-P in
TrSA increased significantly the hydrolytic activity of the
enzyme. The three other positions in the neighbourhood of
the active site that differ between trypanosomal sialidase
and trans-sialidase are M96, F114 and V180 in TrSA,
substituted by valine, tyrosine and alanine residues in the
trans-sialidases, respectively (Fig. 6). Although it is difficult
to assess the functional role of these substitutions in the
absence of a crystal structure for trans-sialidase, they could
contribute to modulation of specific protein–sialic acid
interactions, which are important for the transfer reaction to
occur.
Genomic organization of TbTS genes
Southern blot analysis of total DNA from T. brucei brucei
strain probed with the catalytic region of the genes showed

and trans-sialidases (data not shown). Southern blot
analysis with a probe corresponding to the central part of
this gene (Fig. 7B) demonstrated that it is present in
one copy in T. brucei (Fig. 7A, panel II). We analysed
TbTS-like gene with the
IPSORT
program to subcloning and
tested its product for enzymatic activity. As expected, the
new construct coded for a protein of 703 amino acids
that displayed no sialidase/trans-sialidase activity when
expressed in bacteria (Fig. 7B).
DISCUSSION
We are describing for the first time the gene coding for an
active trans-sialidase of the African trypanosome Trypan-
osoma brucei brucei. Both sialidase and trans-sialidase
activities are mediated by the same protein, encoded by
the gene identified here. The trans-sialidase in African
trypanosomes is expressed in the procyclic form, the stage of
the parasite that replicates in the tsetse fly midgut. Procyclic
forms are characterized by the synthesis of a surface coat
composed of procyclins (otherwise known as procyclic acid
repetitive protein). Each cell is covered by approximately six
million procyclin molecules [29] that are attached to the
surface membrane by GPI anchors [4]. It has been shown
that isolated de-sialylated procyclin can be sialylated by
culture-purified trans-sialidase [30]. The unusual GPI
anchor of procyclin was known to contain five sialic acid
molecules on its structure, but it might be sialylated in
regions other than the GPI anchor, because the number of
sialic acid residues is about 10 per procyclin molecule [31].

FRIP
VIVxNVLLYNR
1
100 472 770
TbTS
LTIxNAMLYNR
5/5 4/5 4/5
2/5 2/5 3/5
YRSP
683
1
TbTS like
B
30% similarity
TbTs probe
TbTs like
p
robe
Fig. 7. Southern blot analysis of TbTS and TbTS-like. (A) Genomic
DNA of T. brucei digested with the indicated restriction enzymes,
hybridized with a TbTS probe (I) and TbTS-like probe (II). The filter
was washed at 65 °Cin0.1 · NaCl/Cit, 0.1% SDS. As controls, maize
DNA digested with EcoR1 and T. cruzi DNA digested with PstIwere
used. (B) Schematic representation of primary sequence of TbTS and
TbTS-like. Catalytic (open box) and lectin-like domains (shaded box)
are shown. The differences in FRIP, Asp boxes and trans-sialidase
superfamily motifs are also indicated. Dark bars indicate the position
of the region used as probe for Southern blot analysis.
Ó FEBS 2002 The trans-sialidase of African trypanosomes (Eur. J. Biochem. 269) 2947
trans-sialidase of American trypanosome T. cruzi (reviewed

The identity in the catalytic region of the two enzymes led
us to investigate whether the same architecture of the active
site is likely to be shared by both enzymes. There is growing
evidence suggesting the existence of distinct donor- and
acceptor-binding sites to account for the sialyl-transferase
activity of T. cruzi enzyme, supported by recent crystallo-
graphic data of enzyme–substrate analog complexes. An
inhibitor contacting residue (Y119) and a shallow depres-
sion (formed by P283, Y248 and W312) are favourably
positioned in the T. cruzi enzyme to be involved in binding
the acceptor molecule. P284 has been shown to be one of the
essential amino-acid residues for trans-sialylation, as a
TrSA-TcTS chimerical molecule displaying only sialidase
activity was able to trans-sialylate after mutation of Q284 to
a proline residue [28]. The mutation of the homologous
residue, P371Q, seems to induce the same effect on the
structure of the active site of African trans-sialidase.
Our previous results on the T. cruzi enzyme indicate a
crucial role for Y119 in binding the acceptor carbohydrate,
since the single substitution YfiS strongly affects the
transfer/hydrolysis ratio towards a more efficient hydrolase,
while the inverse substitution in TrSA retains a significant
sialidase activity [17]. The substitution of the homologous
residue in TbTS, Y191, causes a dramatic effect on this
enzyme, abolishing both sialidase and trans-sialidase activ-
ities. Many microbial sialidases, such as the enzymes from
Vibrio cholerae and influenza virus can cleave a-(2,3), a-(2,6)
and even a-(2,8)-linked sialic acid conjugates [14,37]. Both
trypanosome sialidase and trans-sialidases, as well as
Salmonella typhimurium (StSA) [25] and Macrobdella decora

bacteria
catalytic domain
lectin-like
domain
lectin-like
domain (wing-2)
lectin-like
domain (wing-1)
VcSA
44 %
43 %
27%
23 %
31 %
33 %
Fig. 8. Structural similarity between sialidases and trans-sialidases of different origins. Comparison of the primary structures of the different domains
(catalytic in light grey bars, lectin-like in black bars) of sialidases and trans-sialidases from trypanosomes (TrSA, T. rangeli sialidase GenBank
accession number U83180; TcTS, T. cruzi trans-sialidase, L26499; TbTS, T. brucei trans-sialidase, AF310232) and sialidases of bacterial origin
(StSA, Salmonella typhimurium sialidase, M55342; VcNA, Vibrio cholerae neuraminidase, M83562). Numbers indicate the percentage of identity.
The developmental stage where the proteins are present, in the case of Trypanosoma species, is indicated on the left. The consensus Asp-box
sequence and FRIP motif are shown with vertical bars.
2948 G. Montagna et al. (Eur. J. Biochem. 269) Ó FEBS 2002
trypanosomes as new alternatives for chemotherapy. These
compounds are needed urgently, because the available drugs
are only effective in 50% of the acute infections and their
usefulness for parasitological cure in chronic infections is
controversial [40,41]. Since the first years of the 20th
century, human and animal trypanosomiasis have been
recognized as a cause of morbidity and mortality through-
out sub-Saharan Africa and a major constraint on the use of

nCientı
´
fica y Tecnolo
´
gica, Argentina. The research from
ACCF was supported in part by an International Research Scholars
Grant from the Howard Hughes Medical Institute and a fellowship
from the John Simon Guggenheim Memorial Foundation.
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