The trans -sialidase from the african trypanosome Trypanosoma brucei

The trans-sialidase from the African trypanosomeTrypanosoma brucei

Georgina Montagna1, M. Laura Cremona1, Gaston Paris1, M. Fernanda Amaya2, Alejandro Buschiazzo2,Pedro M. Alzari2 and Alberto C. C. Frasch1

1Instituto de Investigaciones Biotecnologicas – Instituto Tecnologico de Chascomus, Consejo Nacional de Investigaciones

Cientıficas y Tecnicas, Universidad Nacional de General San Martın, Provincia de Buenos Aires, Argentina;2Unite de Biochimie Structurale, CNRS URA 2185, Institut Pasteur, Paris, France

Trypanosoma brucei is the cause of the diseases known assleeping sickness in humans (T. brucei ssp. gambiense andssp. rhodesiense) and ngana in domestic animals (T. bruceibrucei) inAfrica. Procyclic trypomastigotes, the tsetse vectorstage, express a surface-bound trans-sialidase that transferssialic acid to the glycosylphosphatidylinositol anchor ofprocyclin, a surface glycoprotein covering the parasite sur-face. Trans-sialidase is a unique enzyme expressed by a fewtrypanosomatids that allows them to scavenge sialic acidfrom sialylated compounds present in the infected host. Theonly enzyme extensively characterized is that of the Ameri-can trypanosomeT. cruzi (TcTS). In this work we identifiedand characterized the gene encoding the trans-sialidase fromT. brucei brucei (TbTS). TbTS genes are present at a smallcopy number, at variance with American trypanosomeswhere a large gene family is present. The recombinant TbTS

protein has both sialidase and trans-sialidase activity, but it isabout 10 times more efficient in transferring than in hydro-lysing sialic acid. Its N-terminus contains a region of 372amino acids that is 45% identical to the catalytic domain ofTcTS and contains the relevant residues required for cata-lysis. The enzymatic activity of mutants at key positionsinvolved in the transfer reaction revealed that the catalyticsites of TcTS and TbTS are likely to be similar, but are notidentical. As in the case of TcTS and TrSA, the substitutionof a conserved tryptophanyl residue changed the substratespecificity rendering amutant protein capable of hydrolysingboth a-(2,3) and a-(2,6)-linked sialoconjugates.

Keywords: trans-sialidase; sialidase; T. brucei; procyclictrypomastigotes.

African trypanosomiasis has re-emerged as a major healththreat, with an epidemic resulting in more than 100 000 newinfections per year. With 300 000 cases officially reported,human trypanosomiasis, or sleeping sickness caused byTrypanosoma brucei ssp. gambiense and ssp. rhodesiense, hasnow returned to the epidemic levels of the 1930s in manyhistoric foci across Africa. T. brucei ssp. brucei causes the�ngana disease� in domestic animals, which can reduce foodproduction as much as 50%. The parasite, which lives andmultiplies in the blood of the infected host, eludes theimmune system by consecutively expressing structurallydifferent 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 stagewhen entering the tsetse insect vector (Glossina sp.) Theseprocyclins are a small family of very similar acid repetitiveproteins [2,3] that might protect procyclic cells fromdigestion 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 sialylateacceptormolecules present on the surface of parasite plasmamembrane [5]. Indeed, the presence of trans-sialidaseactivity is unique to a few trypanosomes, being absent inall other cell types tested so far. Trans-sialidase is a modifiedsialidase that instead of hydrolysing sialic acid, transfers themonosaccharide to another sugar moiety. The only trans-sialidase extensively studied is the one from Trypanosomacruzi (TcTS). The enzyme is involved in sequestering sialicacid from sialoglycoconjugates present in the blood andother tissues in the infected vertebrate host. The sialic acid istransferred to terminal galactoses present in mucins, highlyO-glycosylated proteins that cover the parasite surface [5].Sialylated mucins have been suggested to be involved ininvasion of the mammalian host cells and in protectionagainst complement lysis [6–8].In T. cruzi and T. rangeli (a related American parasite

which only displays sialidase activity), trypanosomalsialidases are encoded by a multigenic family [9,10]. InT. cruzi, there are about 140 genes, half of them encodingproteins that display enzymatic activity. The other mem-bers code for proteins lacking activity due to a mutation

Correspondence to Instituto de Investigaciones Biotecnologicas,

Universidad Nacional de General San Martın, INTI,

Avemida. Gral Paz s/n, Edificio 24, Casilla de Correo 30,

1650 San Martın, Pcia de Buenos Aires, Argentina.

Fax: + 54 11 4752 9639, Tel.: + 54 11 4580 7255,

E-mail: [email protected]

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) � FEBS 2002 doi:10.1046/j.1432-1033.2002.02968.x

Y342H [11]. The overall structure of the TcTS comprisesan N-terminal globular region of 642 amino acids carryingthe catalytic activity (see below), followed by a C-terminalextension of tandemly repeated sequences named SAPA(shed acute phase antigen) that are not required for theenzymatic activity. SAPA is highly antigenic and isinvolved in the stabilization of the enzymatic activity oncereleased in the blood of the infected host [12]. Members inthe sialidase family of T. rangeli (TrSA) are about 70%identical to TcTS [13], and some of them also lackenzymatic activity.The crystal structures of several microbial sialidases

have been determined. They share a similar catalyticdomain that displays a typical six-bladed bpropellertopology originally observed in influenza virus sialidase[14]. Some sialidases are multidomain proteins and includeone or more noncatalytic domains, which may be involvedin carbohydrate recognition, as for the enzymes fromVibrio cholerae [15] and Micromonospora viridifaciens [16].The three-dimensional structure of TrSA [17] showed thattrypanosomal enzymes fold into two distinct structuraldomains: the bpropeller catalytic domain and a tightlyassociated C-terminal domain with the characteristicbbarrel topology of plant lectins. These crystallographicstudies also showed that they share a similar active sitearchitecture, where several amino-acid residues critical forenzyme function, are strictly conserved. In T. cruzi andT. rangeli, a conserved tryptophan residue (W313) wasrecently shown to be implicated in the binding of thesubstrate and to be determinant for the specificity fora-(2,3) linkages [18]. Other residues in the surrounding ofthe active site differ when the structures of sialidase andtrans-sialidase are compared. In particular, two residuesfrom TcTS, Y119 and P284, were found to be critical forthe transfer reaction and were proposed to modulate thesubstrate-binding cleft, providing trans-sialidase with thecapacity for transferring the monosaccharide.We report here the first gene coding for a trans-sialidase

belonging to African trypanosomes. The deduced trans-sialidase protein sequence is only 38% similar to the trans-sialidase of T. cruzi, but conserves all the amino-acidresidues that are relevant for the enzymatic activity. Singlepoint mutation at critical positions, revealed distinctfeatures between trans-sialidase active sites in Americanand African trypanosomes.

E X P E R I M E N T A L P R O C E D U R E S

Trypanosomes

Procyclic forms of T. brucei brucei stock EATRO427 werecultivated axenically in SDM-79 as described previously[19]. The strain was kindly provided by F. R. Opperdoes(Christian deDuve Institute of Cellular Pathology, Brussels,Belgium).

Nucleic acid isolation

Total DNA from culture procyclic forms was isolated usinga conventional proteinase K/phenol/chloroform method asdescribed previously [20]. Total RNA was purified usingTRIzol reagent following manufacturer’s instructions (LifeTechnologies Inc.).

Southern blot analysis

Total DNA was digested with the indicated restrictionenzymes and 2.5 lg of the sample per line was electro-phoresed in 0.8% agarose gel and transferred for Southernblot 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-32P]dCTPin a 30-cycle primer extension reaction after optimization ofthe template and MgCl2 concentration. The TbTS probewas made with oligonucleotide FRIP (5¢-ATAAGGTAGAGCGCACTGTGCA-3¢) using clone TbTS digestedwith EcoRV as template. Probe TbTS-like was made fromclone pGEM-TbTS-like using oligonucleotide (5¢-CTTGCTAGCCTCTGCAGCCGACAT-3¢). The filters werehybridized with the probes described using hybridizationsolution containing 0.5 M NaH2PO4, 7% SDS, 1 mM

EDTA and 1% BSA, at 62 �C.

Cloning of trans-sialidase genes

PCR was carried out using Vent DNA Polymerase (NewEngland Biolabs) on 100 ng of parasite DNA. PCRprimers contained restriction enzymes sites to facilitate thesubsequent cloning steps in the expression vector. ForTbTS the primers were as follows: AminoMet (5¢-ATGGAGGAACTCCACCAACAAAT-3¢, forward) andSTOP (5¢-TATAGATCTTCAAATCGCCAACACATACAT-3¢, reverse, underlined is the BglII restriction site).For TbTS-like: TbTSIIamino (5¢-CTTGCTAGCATGCGCGTTGTATACCAG-3¢, forward, underlined is theNheI restriction site) and TbTSIIStop (5¢-AGAGATCTAGAACGCGTGGTCTGC-3¢, reverse, underlined is theBglII restriction site). Primer sequences for TbTS wereobtained from Genome Sequence Survey (GSS) AQ661000for AminoMet and AQ656761 for STOP. Primers forTbTS-like were obtained from a BAC clone: AC009463,which contains the complete ORF. The PCR productswere cloned on pGEM-T Easy vector following theA-tailing procedure. The clones were called pGEM-TbTSand pGEM-TbTSlike. These clones were used as templatefor automated (AbiPrism) or manual (dideoxy-chaintermination method with Sequenase-USB) sequencing orfor subcloning in the expression vector.

Cloning of TbTS 5¢ UTR

First strand cDNA was prepared with the Superscript IIsystem using an internal primer (5¢-TGAAAATCAACAGCAGTCTC-3¢) that binds to position 58–40 of TbTS ORF.RT-PCR was carried out with the primers for T. bruceimini-exon as forward (5¢-AACGCTATTATTAGAACAGTTTCTGTACT-3¢) and the one used for first strandsynthesis as reverse, using Vent DNA polymerase. Theproduct was cloned into pGEM-T Easy vector afterA-tailing and sequenced using the dideoxy-chain termin-ation method with Sequenase (USB).

Site-directed mutagenesis

Site directed point mutagenesis was performed using theQuikChange Site-directed mutagenesis kit (Stratagene),

2942 G. Montagna et al. (Eur. J. Biochem. 269) � FEBS 2002

according to the manufacturer’s instructions. All cloneswere sequenced to confirm mutation of target sites.

Expression of trans-sialidase genes in bacteriaand protein purification

The plasmid containing the complete ORF of the TbTS(pGEM-TbTS) was cut with EcoRI and the DNA fragmentcorresponding to TbTS gene was ligated into the expressionvector pTrcHisC (Invitrogen). The His-tag encoded in theplasmid vector was used to purify the recombinant protein.A TbTS construct starting at the codon for leucine 28 wasobtained by PCR on the pGEM-TbTS plasmid using thefollowings primers: LTSK (5¢-TATGCTAGCTTGACTTCCAAGGCTGCGG-3¢, forward, underlined is the NheIrestriction site) and STOP (see above). After digestion withthe corresponding restriction enzymes, the fragment wasligated to pTrcHisC vector. A similar procedure was carriedout for TbTS-like gene, but the PCR reaction wasperformed using pGEM-TbTS-like as template and thefollowings primers: TbTSIILCS (5¢-CTTGCTAGCCTCTGCAGCCGACAT-3¢, forward, underlined is the NheIrestriction site) and TbTSIIcarboxi (5¢-TAGAGATCTTACATAAATAGGGAATA-3¢, reverse, underlined is theBglII restriction site). The constructs were introduced inE. coli BL21 (DE3) pLysS cells by the calcium chloridemethod. Overnight cultures were diluted 1 : 50 in TerrificBroth and grown at 37 �C up toD600 0.8–1.0, with constantagitation at 250 r.p.m. Bacteria were induced to overexpress recombinant protein by adding 0.5 mM isopropylthio-b-D-galactoside (Sigma) and induction was maintainedat 18 �C for 12–16 h. Cells were harvested, washed withNaCl/Tris (20 mM Tris/HCl pH 7.6 and 50 mM NaCl) andfrozen ()80 �C) until needed. After thawing, lysis wascarried out in the presence of 20 mM Tris/HCl pH 7.6,30 mM NaCl, 0.5% Triton X-100, 1 mM phenyl-methylsulfonyl fluoride, 100 lgÆmL)1 DNAse I. Superna-tants were centrifuged at 21 000 g for 30 min and subjectedto iminodiacetic acid metal affinity chromatography(IMAC) (HiTrap Chelating, Amersham Pharmacia BiotechAB) Ni2+-charged equilibrated in 20 mM Pipes pH 6.9 and0.5 M NaCl (buffer IMAC). The column was washed with30 mM imidazole in buffer IMAC. Elution was achievedusing a linear gradient 30–250 mM imidazole in bufferIMAC. The activity peak was pooled, dialyzed against20 mM Bistris pH 7.4 and further purified by FPLC anionexchange (Mono Q) equilibrated with the same buffer. Theprotein was eluted by applying a linear gradient of0–250 mM trisodium citrate. Purified proteins were analysedby SDS/PAGE under reducing conditions, stained withCoomasie Blue R250, and quantitated with Kodak 1D 3.0software using purified BSA as standard.

Enzyme activity assays

Enzyme activity assays were carried out using the purifiedproteins as described in the previous section. Neuraminidaseactivity was determined by measuring the fluorescence of4-methylumbelliferone released by the hydrolysis of0.2 mM 2¢-(4-methylumbelliferyl)-a-D-N-acetylneuraminicacid (MUNen5Ac, Sigma). The assay was performed in50 lL in 20 mM 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 aDYNAQuantTM 200 fluorometer (Hoefer Pharmacia Inc).Trans-sialidase activity was measured in 20 mM PipespH 6.9, using 1 mM Neu5Ac-a-(2–3) lactose as sialic aciddonor and 12 lM [D-glucose-1-14C]lactose (55 mCiÆmmol)1)(Amersham) as acceptor, in 30 lL final volume at 35 �C.The reaction was stopped by dilution, and sialyl-[14C]lactosewas quantitated with a b-scintillation counter as describedpreviously [21]. Suitable modifications were made to thestandard reaction to obtain the kinetic constants.MUNen5Ac is an unspecific substrate and it does not allowa distinction between hydrolysis of a-(2,3)- and a-(2,6)-linked sialic acid. Therefore, in order to determine thesubstrate specificity of wild-type and mutant proteins,sialidase activity was measured using sialyl-a-(2,3)-lactose(3¢SL) or sialyl-a-(2,6)-lactose (6¢SL) as substrates. Quanti-tation of 3¢SL and 6¢SL hydrolysis was carried out by thethiobarbituric method [22]. Predefined quantities of wild-type or mutant proteins were incubated with 0.5 mM ofeither 3¢SL or 6¢SL and 50 mM Hepes pH 7.0, in a finalvolume of 20 lL for 30 min at 35 �C. The enzymaticreactions were stopped by adding 15 lL of 25 mM NaIO4

solution prepared in 125 mM sulfuric acid solution. Themixtures were vortexed and allowed to react in a water bathat 37 �C for 30 min. Samples were then neutralized with13 lL of sodium arsenite 2% w/v in HCl (0.5 N) by slowaddition of the reactive. Tubes were gently vortexed tocomplete the reduction reaction. After the total disappear-ance of yellow colour (5 min) 152 lL of thiobarbituric acid(36 mM, pH 9.0) were added and then incubated in a boilingwater bath for 15 min Samples were then cooled in an ice-water bath for 5 min, followed by room-temperature colourstabilization. The samples were centrifuged, and 20 lLwereseparated by high-performance liquid chromatographythrough a C18 reverse phase column (Pharmacia Biotech)using 2 : 3 : 5 water/methanol/buffer (buffer: 0.2% phos-phoric acid; 0.23 M sodium perchlorate). Absorbance wasmeasured at 549 nm. A sialic acid calibration curve waspreviously set, and absorbance values were always read inthe linear range.

R E S U L T S

The T. brucei trans-sialidase primary sequenceconserves most of the structurally relevant amino-acidresidues of bacterial and protozoan sialidases

BLAST searches were performed using sequences corres-ponding to the catalytic domain of TcTS (L26499, amember of family I of T. cruzi trans-sialidase/sialidasesuperfamily [23]) on theT. bruceiGenome Project Database(Sanger Centre). The search identified sixGSSs with a BLAST

E value between 2.6 · 10)36 and 0.73. When assembled,these fragments built up an open reading frame of 2316 bp.Because various sialidase amino-acid motifs such as FRIPand Asp box motifs were conserved in the deducedsequence, this open reading framemight code for aT. bruceisialidase-related protein. These data were used to designoligonucleotides for the amplification by PCR on genomicDNA to clone the gene coding for the complete TbTS.Eleven genes from independent PCRs were sequenced andorganized into eight different groups according to theirnucleotide sequence (Fig. 1). The differences among genes

� FEBS 2002 The trans-sialidase of African trypanosomes (Eur. J. Biochem. 269) 2943

seem not to be randomly distributed, but rather, localized atnine positions. Combinations of mutations at these ninepositions generated eight genes having from one to fivedifferences. Five out of the nine differences are in the firstand second codon positions, giving rise to a high proportionof nonconservative mutations. Most of the differences(seven out of nine) are located in the catalytic domain (seebelow), but they are placed at positions irrelevant for theenzymatic activity because the corresponding recombinantproteins displayed both sialidase and trans-sialidase activity(see next section). The deduced primary structure of theprotein coded by these genes showed that TbTS is organizedinto three putative regions (Fig. 2). AnN-terminal region of

100 amino acids, which is absent in TcTS, amiddle region of372 amino acids, which is 45% identical to the catalyticdomain of the T. cruzi enzyme and a C-terminal region of298 amino acids followed by an hydrophobic region likelyto correspond to a GPI-anchor signal. TbTS is probablyanchored by GPI to the surface membrane since nativeprocyclic trans-sialidase can be released from the parasite bytreatment with phospholipase D [24]. The 298 amino acidsin the C-terminal domain are 30% identical to the TcTSlectin-like domain. TbTS does not have a repetitive domainat the C-terminus that is homologous to the T. cruzi SAPAdomain.The catalytic region revealed the conservation of most of

the structurally relevant residues displayed in bacterial andprotozoan sialidases and trans-sialidases (Fig. 2), such as anarginine triad that binds to the carboxylate group commonto all the sialic acid derivatives (R133, R346, R431), aglutamic acid (E473) that stabilizes one of the arginine sidechains, a negatively charged group (D157) proposed as apossible proton donor in the hydrolytic reaction and twoessential residues at the bottom of the site (E331, Y457),which are well positioned to stabilize a putative sialosylcation intermediate [25]. This tyrosine residue was found tobe 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 acidchanges close to the substrate-binding cleft that mightmodulate the sialyltransferase activity [17]. Most of thesecritical substitutions observed at the periphery of the cleft inTcTS are conserved in the deduced primary sequence ofTbTS, including an aromatic residue (Y120 in TcTS) thatwas found to have a crucial role in the transfer reaction [17].TbTS also conserves an exposed aromatic side chain (W400)that favours, in the case of microbial sialidases and trans-sialidases, the high specificity for sialyl-a-(2,3) substrates[18]. The TbTS genes present partially conserved thesubterminal VTVxNVfLYNR motif (VIVRNVLLYHRin T. brucei) that in the case of T. cruzi, defines thetrypanosome trans-sialidase/sialidase superfamily of surfaceproteins [26]. It has been recently shown that this sequence isinvolved in host cell binding during T. cruzi infectionprocess [27].

Expression and properties of T. brucei recombinanttrans-sialidase

The entire ORF starting at the codon for the firstmethionine was identified by sequencing the 5¢ UTR ofTbTS mRNA. A construct expressed from the codon forthis first methionine produced a protein of approximately84.4 kDa that lacked sialidase and trans-sialidase activities(data not shown). An analysis of the putative start of themature protein N-terminus using the IPSORT program(Human Genome Center, Institute of Medical Sciences,University of Tokio), predicted the existence of a signalpeptide that ends just before leucine 28. The insert was thendesigned to have this amino acid at position +1. The newconstruct, which includes an N-terminal extension of 10amino acids expressing a His-tag, codes for a 745 amino-acid protein with a predicted molecular mass of 81.4 kDaand displaying both sialidase and trans-sialidase activity(data not shown). All further work was performed with thisprotein. 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

indicatedwithverticalbars and theboxeshighlight 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) � FEBS 2002

through passage on a iminodiacetic acid metal affinitycolumn followed by FPLC anionic exchange (see Experi-mental procedures for details). After the anionic exchangecolumn, the proteinwas > 95%pure (Fig. 3).MUNen5Acwas used as substrate to assay for sialidase activity, and amix of Neu5Ac-a-(2,3) and Neu5Ac-a-(2,6)-lactose as sialicacid donor and lactose as acceptor for the trans-sialidase

activity (Fig. 3). The affinity for sialyl-lactose as substrate ofTbTS (2.27 mM) and TcTS (4.3 mM) were similar, as itwas the turnover of both enzymes (apparent Vmax forsialyl-lactose is 51 161 nmolÆmin)1Æmg)1 for TbTS and32 692 nmolÆmin)1Æmg)1) for TcTS (Fig. 3; [18]). As in thecase of T. cruzi trans-sialidase [18], TbTS behaves as a veryefficient sialyl-transferase: in excess of both the donor andacceptor substrates, the enzyme is 11.1 times more efficientin transferring than hydrolysing donor sialic acid, as can beconcluded by comparing the Vmax of the hydrolysis andtransference activities (Fig. 3). We have also measured thetrans-sialidase-sialidase activity ratio in the native T. bruceibrucei enzyme from procyclic forms, as described underExperimental procedures. This ratiowas 8.9. Thus, there is agood agreement between values obtained with the recom-binant and native enzymes.

Point mutations at critical amino-acid residuesrevealed features of the catalytic site of Africantrypanosomes trans-sialidase

Based on the crystal structure of TrSA [17], mutants ofTbTS at key positions involved in substrate binding andspecificity were constructed and characterized. Thesemutants include (see Fig. 4A) the exposed aromatic sidechain 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 thecatalytic cleft (Y191 in TbTS), a proline residue that wasfound to increase the sialidase activity in TrSA (P371 inTbTS) and a tyrosine residue that is well positioned tostabilize a putative sialosyl cation intermediate (Y430 inTbTS) [17].The mutant proteins were produced and purified with the

same criteria described for the wild-type in the previoussection. As shown in Fig. 4B, mutations at positions 371and 430 of TbTS completely abolished both sialidase and

II

A B

I

1/v

(nm

ol s

ialic

aci

d-1.m

in.m

g) x

10-3

1/S (mM-1)

5.0

4.0

3.0

2.0

1.0

0 5 10 15 20 25

Km: 1.2mM

Vmax: 4582 nmol.min-1.mg-1

2.5

2.0

1.5

1.0

0.5

0 2.0 4.0 6.0

1/v

(nm

ol s

ialic

aci

d-1.m

in.m

g) ×

10-

3

1/S (mM-1)

Km: 2.27 mMVmax: 51161 nmol.min-1.mg-1

7 8

0 10 20Elution (mL)

250

0

Citrate (m

M)

kDa7 8 9 10 11

66

97.4

45

Abs

orba

nce

280

nm

9 10 11Fractions

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

varying the concentrations of MUNen5Ac as

sialic acid donor (see Experimental proce-

dures). II, the trans-sialidase activity was

measured using sialyl-a-(2,3)-lactose and lac-

tose as the sialic acid donor and acceptor

substrates, respectively. The apparent con-

stants were obtained using lactose fixed con-

centration of 2 mM and varying the

concentration of the sialyl lactose according to

the experiment. Data are the mean of three

independent experiments.

Catalytic domain Lectin-likedomain

TcTS

TbTS

1 642119 283 312 342

Y T W Y

372

1 73 743191

Y

371 400 430

T W Y

461

TbTS wild type 8074.33 ± 691.52 (100) 100 933.8 ± 60.78 (100) 100

TbTS Y430-H 0 0 0 0

TbTS T371-Q 0 0 0 0

TbTS Y191-S 0 0.6 0 12.8

TbTS W400-A 0 0 0104.29 ± 8.3 (11.2)

trans-sialidase activity TcTSa bTcTSsialidase activity

A

B

Fig. 4. Site-directed mutagenesis on TbTS. (A) Relative positions of

the site-directedmutagenesis 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

controls is indicated in parenthesis. The values are the mean and

standard deviation of three independent determinations. The per-

centage of trans-sialidase (a) and sialidase (b) activity of TcTS referred

to wild-type controls.

� FEBS 2002 The trans-sialidase of African trypanosomes (Eur. J. Biochem. 269) 2945

trans-sialidase activities, as in TcTS. The change of thearomatic side chain (W400 A) that in the case of TrSA andTcTS lost the capability of hydrolysing MUNen5Ac [18],retained 11.6% of the sialidase activity whenMUNen5Ac isused as substrate (Fig. 4B). The mutation at Y191Ssuppressed both activities, at variance with the Americantrypanosome trans-sialidase, where the substitution of Y120practically abolishes the sialyltransferase activity whilepreserving some of the sialidase activity [17,18]. Thedifferences observed in the effect of the mutations at thesepositions could arise from distinct organizations of thecatalytic sites of both trans-sialidases.The trans-sialylation activity of TbTSW400A was lost as

in TcTS W312A mutant (Fig. 4 and [18]), thus indicatingthat the transfer, but not the hydrolysis reaction requires aprecise orientation of the bound substrate in both enzymes.The exposed tryptophan residue in TcTS and TrSAdetermined the high specificity of these enzymes towardssialyl-a-(2,3) substrates [18], which could be explained byunfavourable interactions of this aromatic side-chain withsialyl-a-(2,6)-linked oligosaccharides. To test if this is alsothe case of TbTS, the mutant protein W400A was obtainedand assayed for activity using sialyl-a-(2,3)-lactose (3¢SL)and sialyl-a-(2,6)-lactose (6¢SL). The mutated enzyme wasnow capable of hydrolysing the a-(2,6) regioisomer, losingthe strict specificity of the wild-type enzyme for the sialyl-a-(2,3) substrate (Fig. 5).

The active sites of the T. bruceiand T. cruzi trans-sialidases are highly conserved

As expected from their similar function and commonevolutionary origin, critical active site residues are largelyconserved in all trypanosomal sialidases and trans-siali-dases. The 3D structure of the T. rangeli sialidase bound to2,3-didehydro-2-deoxy-N-acetylneuraminic acid (Neu2-en5Ac, a sialidase inhibitor) [17] showed 33 amino acidsthat are positioned close to the inhibitor. They have at leastone atom at less than 7 A from Neu2en5Ac. Among thesepositions, 26 amino acids (79%) are conserved betweenTcTS and TbTS, 24 (73%) are conserved between TbTSand TrSA, and 22 (67%) are conserved between TcTS andTrSA. These relative similarities differ significantly from

those found when comparing the entire catalytic domains(Fig. 4), thus revealing a functional constraint on theevolution of trans-sialidases.All amino-acid residues that have been found to be

important for the function in other viral and bacterialsialidases, are also conserved in the three trypanosomalenzymes (shown in blue in Fig. 6): the arginine triad (R36,R246 and R315, TrSA numbering) that binds the carboxy-late group of sialic acid; the aspartic acid residue (D60) thatcould serve as the proton donor in the reaction; and tworesidues (E231, Y343) that probably serve to stabilize thetransition state intermediate. Other amino-acid residuesconserved in the active site of the three trypanosomalenzymes (but not necessarily in other sialidases) include R54and D97, whose side chains make hydrogen bondinginteractions with the bound inhibitor; W121, L(I)177 andQ196, all of which are part of the pocket that binds theN-acetyl group of sialic acid; D248 and E358, whosecarboxylate groupsmake hydrogen bonds with two arginineside-chains of the triad; and W313 and Y365, are bothfavourably positioned to interact with the substrate.Of particular interest are seven positions that are

invariant in the two trans-sialidases (T. brucei and T. cruzi),but differ in TrSA (shown in red in Fig. 6), suggesting thatthey could be important for transglycosylation activity. Twoof these have been previously shown to be critical for trans-sialidase activity, namely TrSA S120 and Q284, substituted,respectively, by tyrosine and proline residues in the twotrans-sialidases [17,18,28]. The presence of a tyrosine residueat position 120 was shown to be critical for TcTS activity[17], probably because this aromatic side-chain residue isinvolved in substrate binding. Also, the conservation of asequence PGS at positions 284–286 of both trans-sialidases(substituted by the sequence QDC in TrSA, see Fig. 6)confirm previous findings of Smith & Eichinger [28], whostudied 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

red are conserved in the two trypanosomal trans-sialidases, but differ in

TrSA, and could be important for transglycosylation (see text).

2946 G. Montagna et al. (Eur. J. Biochem. 269) � FEBS 2002

esis between TrSA and TcTS. Along similar lines, Pariset al. [18] demonstrated that the substitution Q284-P inTrSA increased significantly the hydrolytic activity of theenzyme. The three other positions in the neighbourhood ofthe active site that differ between trypanosomal sialidaseand trans-sialidase are M96, F114 and V180 in TrSA,substituted by valine, tyrosine and alanine residues in thetrans-sialidases, respectively (Fig. 6). Although it is difficultto assess the functional role of these substitutions in theabsence of a crystal structure for trans-sialidase, they couldcontribute to modulation of specific protein–sialic acidinteractions, which are important for the transfer reaction tooccur.

Genomic organization of TbTS genes

Southern blot analysis of total DNA from T. brucei bruceistrain probed with the catalytic region of the genes showedthat TbTS genes are present in a small copy number (Fig. 7),a situation that is different from American trypanosomeswhere the trans-sialidase family genes comprises at least 140members. Regarding the results obtained with enzymes thatcut at least once on each gene unit (BssHII, EcoRV andHindIII, Fig. 7A, panel I), a minimum of two trans-sialidase-related genes can be estimated from the Southernblot analysis. It is likely that the TbTS genes are organized intandem, as previous evidence from cloning and sequencing(see Fig. 1) suggested that several copies may exist.A BLAST search on the T. brucei Genome Project

Database using the fragment corresponding to the putativecatalytic domain of TbTS identified a BAC clone with aBLAST E value of 6 · 10)45 that showed 30% similarity withTbTS. We decided then to analyse the presence ofTS-related genes on the genome of T. brucei, because inAmerican trypanosomes these genes are abundantly repre-sented in the parasite genome [5]. We designed primersbased on the sequence of the BAC clone and performed aPCR on genomic DNA. These PCR resulted in a gene of2109 bp that was called TbTS-like. The deduced primarysequence showed a partial conservation of the typicalsialidase motifs (FRIP andAsp box), and the absence of theresidues shown to be important for activity in the three-dimensional structure of bacterial and protozoan sialidasesand trans-sialidases (data not shown). Southern blotanalysis with a probe corresponding to the central part ofthis gene (Fig. 7B) demonstrated that it is present inone copy in T. brucei (Fig. 7A, panel II). We analysedTbTS-like gene with the IPSORT program to subcloning andtested its product for enzymatic activity. As expected, thenew construct coded for a protein of 703 amino acidsthat displayed no sialidase/trans-sialidase activity whenexpressed in bacteria (Fig. 7B).

D I S C U S S I O N

We are describing for the first time the gene coding for anactive trans-sialidase of the African trypanosome Trypan-osoma brucei brucei. Both sialidase and trans-sialidaseactivities are mediated by the same protein, encoded bythe gene identified here. The trans-sialidase in Africantrypanosomes is expressed in the procyclic form, the stage ofthe parasite that replicates in the tsetse fly midgut. Procyclicforms are characterized by the synthesis of a surface coat

composed of procyclins (otherwise known as procyclic acidrepetitive protein). Each cell is covered by approximately sixmillion procyclin molecules [29] that are attached to thesurface membrane by GPI anchors [4]. It has been shownthat isolated de-sialylated procyclin can be sialylated byculture-purified trans-sialidase [30]. The unusual GPIanchor of procyclin was known to contain five sialic acidmolecules on its structure, but it might be sialylated inregions other than the GPI anchor, because the number ofsialic acid residues is about 10 per procyclin molecule [31].The function of procyclins is unknown, although theycontribute to the establishment of strong infections in the flyvector. Parasites that have no surface procyclin because of adefect in GPI synthesis are less efficient at establishinginfection in flies [32]. Impairment of this process offers apossibility for controlling vector parasitemia (see below).Extensive work has been carried out on the molecu-

larbiology, biochemistry and structure of the surface

Eco

RI

Kpn

I

SacI

I

Bss

HII

Eco

RV

Hin

dIII

Eco

RI

Kpn

I

SacI

I

Bss

HII

Eco

RV

9.4

23.1

6.6

4.4

kpb

A

I II

2.3

2.0

SxDxGxTWFRIP VIVxNVLLYNR

1 100 472 770

TbTS

LTIxNAMLYNR

5/5 4/5 4/5

2/5 2/5 3/5

YRSP

6831

TbTS like

B

30% similarity

TbTs probe

TbTs like probe

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 �C in 0.1 · NaCl/Cit, 0.1% SDS. As controls, maize

DNA digested with EcoR1 and T. cruziDNA digested with PstI were

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.

� FEBS 2002 The trans-sialidase of African trypanosomes (Eur. J. Biochem. 269) 2947

trans-sialidase of American trypanosome T. cruzi (reviewedin [5]), the agent of Chagas’ disease. Both American andAfrican trans-sialidases are developmentally regulated sur-face glycoproteins [24,34]. They share a number of featuresthat are unusual for the rest ofmicrobial sialidases, such as aneutral optimum pH (6.9 for T. brucei, 7.2 for T. cruzi), theindependence of divalent cations, a relative resistancetowards the natural sialidase inhibitor Neu2en5Ac and thesame substrate specificity [24,33]. In spite of not beingclosely related in their overall primary structure, TbTSconserves most of the amino acids relevant for the catalyticsite of American trans-sialidase. The identity increases up to45% in the region corresponding to the catalytic domain,but TbTS contains an extra region of 100 amino acidstowards its N-terminal end. In its C-terminal region, theidentity falls to 30% relative to the lectin-like domain ofAmerican trans-sialidase. The trans-sialidase gene productsof T. cruzi and T. brucei have a significant degree ofstructural and biochemical similarity to the sialidases foundin bacteria and viruses (Fig. 8). The comparison of inferredgene trees with species trees made by alignment of thenucleotide and predicted amino-acid sequences of sialidasesand trans-sialidase suggested that the genes encoding theT. cruzi trans-sialidase of mammalian forms might bederived from genes expressed in the insect forms of thegenus Trypanosoma [35]. It was recently demonstrated byanalysis of DNA sequences from 62 different species of thisgenus that there is evidence for a common ancestor forT. cruzi and T. brucei around 100 million years ago [36], anancestor that could have carried the primitive trans-sialidasegene.The identity in the catalytic region of the two enzymes led

us to investigate whether the same architecture of the activesite is likely to be shared by both enzymes. There is growingevidence suggesting the existence of distinct donor- andacceptor-binding sites to account for the sialyl-transferaseactivity of T. cruzi enzyme, supported by recent crystallo-graphic data of enzyme–substrate analog complexes. Aninhibitor contacting residue (Y119) and a shallow depres-sion (formed by P283, Y248 and W312) are favourablypositioned in the T. cruzi enzyme to be involved in bindingthe acceptormolecule. P284 has been shown to be one of the

essential amino-acid residues for trans-sialylation, as aTrSA-TcTS chimerical molecule displaying only sialidaseactivity was able to trans-sialylate after mutation of Q284 toa proline residue [28]. The mutation of the homologousresidue, P371Q, seems to induce the same effect on thestructure 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 thetransfer/hydrolysis ratio towards a more efficient hydrolase,while the inverse substitution in TrSA retains a significantsialidase activity [17]. The substitution of the homologousresidue in TbTS, Y191, causes a dramatic effect on thisenzyme, abolishing both sialidase and trans-sialidase activ-ities. Many microbial sialidases, such as the enzymes fromVibrio cholerae and influenza virus can cleave a-(2,3), a-(2,6)and even a-(2,8)-linked sialic acid conjugates [14,37]. Bothtrypanosome sialidase and trans-sialidases, as well asSalmonella typhimurium (StSA) [25] andMacrobdella decora[38] sialidases, display a high specificity for a-(2,3)-linkedsialic acid conjugates. We have demonstrated that aconserved tryptophan residue in American trypanosomesialidase and trans-sialidase is directly involved in thebinding of sialic acid donor substrates, as the single pointmutant W fi A allowed a looser accommodation of thedonor substrate, broadening their substrate specificity [18].On the other hand, a significant decrease of hydrolyticactivity against the fluorogenic substrate MUNen5Ac wasshown in the case ofT. cruzi: hydrolysis was undetectable inthe TcTS mutant. In TbTS mutant, the activity falls 10-foldrelative to the activity of the wild-type towards thissubstrate.It has been shown recently that the lectin-like domain of a

trans-sialidase-related protein is involved in host cell bindingactivity during the T. cruzi cell invasion process [27,39]. Thebinding site to cytokeratin 18 colocalizes with the trans-sialidase/sialidase superfamily motif (VTVxNVfLYNR)[27]. Because this motif is conserved in TbTS, it is possiblethat a cell binding activity in the lectin-like domain of TbTScould play a role in T. brucei infection in tsetse flies.Efforts to develop inhibitors based on the structure are

currently being made for the trans-sialidase of American

trypomastigoteTcTS

procyclic form TbTS

StSA

SxDxGxTWFRIP

Try

pano

som

a

epimastigoteTrSA

bact

eria

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) � FEBS 2002

trypanosomes as new alternatives for chemotherapy. Thesecompounds are needed urgently, because the available drugsare only effective in 50% of the acute infections and theirusefulness for parasitological cure in chronic infections iscontroversial [40,41]. Since the first years of the 20thcentury, human and animal trypanosomiasis have beenrecognized as a cause of morbidity and mortality through-out sub-SaharanAfrica and amajor constraint on the use oflivestock. There has been extensive international collabor-ation and considerable expenditure on mechanisms tocontrol the disease and its vector [42]. Given the limitedrange and effectiveness of the drugs available as resistancehas emerged, modulating tsetse vector infection appears tobe an important strategy in reducing the incidence of thisdisease. Major advances being made by molecular biologi-cal and genomic research will eventually lead to thedevelopment of new approaches to control disease trans-mission by insect vectors. Although not demonstrated here,trans-sialidase might have a relevant function for thesurvival of T. brucei in the tsetse vector. In fact, the sameenzymatic activity has a relevant function for the survival ofT. cruzi. Furthermore, the gene encoding this enzymemighthave been generated millions of years ago and have beenconserved, probably as a result of its important function.Further work will demonstrate if TbTS is indeed anessential enzyme for the parasite. If so, treatment of cowswith a putative inhibitor could be used to prevent infectionin the tsetse fly and its dissemination. A similar approach tothat proposed by vaccination in Plasmodium infections, theso-called transmission blocking malaria vaccines [43].

A C K N O W L E D G E M E N T S

Wewould like to thankGraciela Gotz for revising the manuscript. This

work was supported by grants from the World Bank/UNDP/WHO

Special Program for Research and Training in Tropical Diseases

(TDR), ECOS-SeCyT (France-Argentina), the Human Frontiers

Science Program, the Institut Pasteur and the Agencia Nacional de

Promocion Cientıfica y Tecnologica, 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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