Báo cáo Y học: The trans-sialidase from the African trypanosome Trypanosoma brucei potx

The recombinant TbTS protein has both sialidase and trans-sialidase activity, but it is about 10 times more efficient in transferring than in hydro-lysing sialic acid.. brucei trans-sialid

The trans -sialidase from the African trypanosome

Trypanosoma brucei

Georgina Montagna1, M Laura Cremona1, Gasto´n Paris1, M Fernanda Amaya2, Alejandro Buschiazzo2, Pedro M Alzari2and Alberto C C Frasch1

1

Instituto de Investigaciones Biotecnolo´gicas – Instituto Tecnolo´gico de Chascomu´s, Consejo Nacional de Investigaciones

Cientı´ficas y Te´cnicas, Universidad Nacional de General San Martı´n, Provincia de Buenos Aires, Argentina;

2

Unite´ de Biochimie Structurale, CNRS URA 2185, Institut Pasteur, Paris, France

Trypanosoma bruceiis the cause of the diseases known as

sleeping sickness in humans (T brucei ssp gambiense and

ssp rhodesiense) and ngana in domestic animals (T brucei

brucei) in Africa Procyclic trypomastigotes, the tsetse vector

stage, express a surface-bound trans-sialidase that transfers

sialic acid to the glycosylphosphatidylinositol anchor of

procyclin, a surface glycoprotein covering the parasite

sur-face Trans-sialidase is a unique enzyme expressed by a few

trypanosomatids that allows them to scavenge sialic acid

from sialylated compounds present in the infected host The

only enzyme extensively characterized is that of the

Ameri-can trypanosome T cruzi (TcTS) In this work we identified

and characterized the gene encoding the trans-sialidase from

T brucei brucei(TbTS) TbTS genes are present at a small

copy number, at variance with American trypanosomes

where a large gene family is present The recombinant TbTS

protein has both sialidase and trans-sialidase activity, but it is about 10 times more efficient in transferring than in hydro-lysing sialic acid Its N-terminus contains a region of 372 amino acids that is 45% identical to the catalytic domain of TcTS and contains the relevant residues required for cata-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 bruceissp 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

ngana disease 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 acceptor molecules present on the surface of parasite plasma membrane [5] Indeed, the presence of trans-sialidase activity is unique to a few trypanosomes, being absent in all other cell types tested so far Trans-sialidase is a modified sialidase that instead of hydrolysing sialic acid, transfers the monosaccharide to another sugar moiety The only trans-sialidase extensively studied is the one from Trypanosoma cruzi(TcTS) The enzyme is involved in sequestering sialic acid from sialoglycoconjugates present in the blood and other tissues in the infected vertebrate host The sialic acid is transferred to terminal galactoses present in mucins, highly O-glycosylated proteins that cover the parasite surface [5] Sialylated mucins have been suggested to be involved in invasion of the mammalian host cells and in protection against complement lysis [6–8]

In T cruzi and T rangeli (a related American parasite which only displays sialidase activity), trypanosomal sialidases are encoded by a multigenic family [9,10] In

T cruzi, there are about 140 genes, half of them encoding proteins that display enzymatic activity The other mem-bers code for proteins lacking activity due to a mutation

Correspondence to Instituto de Investigaciones Biotecnolo´gicas,

Universidad Nacional de General San Martı´n, INTI,

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

1650 San Martı´n, Pcia de Buenos Aires, Argentina.

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

E-mail: cfrasch@iib.unsam.edu.ar

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)

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%

identical to TcTS [13], and some of them also lack

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

one or more noncatalytic domains, which may be involved

in carbohydrate recognition, as for the enzymes from

Vibrio cholerae[15] and Micromonospora viridifaciens [16]

The three-dimensional structure of TrSA [17] showed that

trypanosomal enzymes fold into two distinct structural

domains: the b propeller catalytic domain and a tightly

associated C-terminal domain with the characteristic

b barrel topology of plant lectins These crystallographic

studies also showed that they share a similar active site

architecture, where several amino-acid residues critical for

enzyme function, are strictly conserved In T cruzi and

T rangeli, a conserved tryptophan residue (W313) was

recently shown to be implicated in the binding of the

substrate and to be determinant for the specificity for

a-(2,3) linkages [18] Other residues in the surrounding of

the active site differ when the structures of sialidase and

trans-sialidase are compared In particular, two residues

from TcTS, Y119 and P284, were found to be critical for

the transfer reaction and were proposed to modulate the

substrate-binding cleft, providing trans-sialidase with the

capacity for transferring the monosaccharide

We report here the first gene coding for a trans-sialidase

belonging to African trypanosomes The deduced

sialidase protein sequence is only 38% similar to the

trans-sialidase of T cruzi, but conserves all the amino-acid

residues that are relevant for the enzymatic activity Single

point mutation at critical positions, revealed distinct

features between trans-sialidase active sites in American

and 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 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-32P]dCTP

in a 30-cycle primer extension reaction after optimization of the template and MgCl2 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 GCTAGCCTCTGCAGCCGACAT-3¢) The filters were hybridized with the probes described using hybridization solution containing 0.5M NaH2PO4, 7% SDS, 1 mM

EDTA and 1% BSA, at 62C

Cloning oftrans-sialidase genes PCR was carried out using Vent DNA Polymerase (New England Biolabs) on 100 ng of parasite DNA PCR primers contained restriction enzymes sites to facilitate the subsequent cloning steps in the expression vector For TbTS the primers were as follows: AminoMet (5¢-AT GGAGGAACTCCACCAACAAAT-3¢, forward) and STOP (5¢-TATAGATCTTCAAATCGCCAACACATA CAT-3¢, reverse, underlined is the BglII restriction site) For TbTS-like: TbTSIIamino (5¢-CTTGCTAGCATG CGCGTTGTATACCAG-3¢, forward, underlined is the NheI restriction site) and TbTSIIStop (5¢-AGAGATCT AGAACGCGTGGTCTGC-3¢, reverse, underlined is the BglII restriction site) Primer sequences for TbTS were 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),

according to the manufacturer’s instructions All clones

were sequenced to confirm mutation of target sites

Expression oftrans-sialidase genes in bacteria

and protein purification

The plasmid containing the complete ORF of the TbTS

(pGEM-TbTS) was cut with EcoRI and the DNA fragment

corresponding to TbTS gene was ligated into the expression

vector pTrcHisC (Invitrogen) The His-tag encoded in the

plasmid vector was used to purify the recombinant protein

A TbTS construct starting at the codon for leucine 28 was

obtained by PCR on the pGEM-TbTS plasmid using the

followings primers: LTSK (5¢-TATGCTAGCTTGACT

TCCAAGGCTGCGG-3¢, forward, underlined is the NheI

restriction site) and STOP (see above) After digestion with

the corresponding restriction enzymes, the fragment was

ligated to pTrcHisC vector A similar procedure was carried

out for TbTS-like gene, but the PCR reaction was

performed using pGEM-TbTS-like as template and the

followings primers: TbTSIILCS (5¢-CTTGCTAGCCTC

TGCAGCCGACAT-3¢, forward, underlined is the NheI

restriction site) and TbTSIIcarboxi (5¢-TAGAGATCTTA

CATAAATAGGGAATA-3¢, reverse, underlined is the

BglII restriction site) The constructs were introduced in

E coli BL21 (DE3) pLysS cells by the calcium chloride

method Overnight cultures were diluted 1 : 50 in Terrific

Broth and grown at 37C up to D6000.8–1.0, with constant

agitation at 250 r.p.m Bacteria were induced to over

express recombinant protein by adding 0.5 mM isopropyl

thio-b-D-galactoside (Sigma) and induction was maintained

at 18C for 12–16 h Cells were harvested, washed with

NaCl/Tris (20 mMTris/HCl pH 7.6 and 50 mMNaCl) and

frozen ()80 C) until needed After thawing, lysis was

carried 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)1DNAse 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) Ni2+-charged equilibrated in 20 mMPipes pH 6.9 and

0.5MNaCl (buffer IMAC) The column was washed with

30 mM imidazole in buffer IMAC Elution was achieved

using a linear gradient 30–250 mM imidazole in buffer

IMAC The activity peak was pooled, dialyzed against

20 mMBistris pH 7.4 and further purified by FPLC anion

exchange (Mono Q) equilibrated with the same buffer The

protein was eluted by applying a linear gradient of

0–250 mMtrisodium citrate Purified proteins were analysed

by SDS/PAGE under reducing conditions, stained with

Coomasie Blue R250, and quantitated with Kodak 1D3.0

software using purified BSA as standard

Enzyme activity assays

Enzyme activity assays were carried out using the purified

proteins as described in the previous section Neuraminidase

activity was determined by measuring the fluorescence of

4-methylumbelliferone released by the hydrolysis of

0.2 mM 2¢-(4-methylumbelliferyl)-a-D-N-acetylneuraminic

acid (MUNen5Ac, Sigma) The assay was performed in

50 lL in 20 mM Pipes pH 6.9 After incubation at 35C,

the reaction was stopped by dilution in 0.2 sodium

carbonate pH 10, and fluorescence was measured with a DYNAQuantTM200 fluorometer (Hoefer Pharmacia Inc) Trans-sialidase activity was measured in 20 mM Pipes

pH 6.9, using 1 mM Neu5Ac-a-(2–3) lactose as sialic acid donor and 12 lM[D-glucose-1-14C]lactose (55 mCiÆmmol)1) (Amersham) as acceptor, in 30 lL final volume at 35C The reaction was stopped by dilution, and sialyl-[14C]lactose was quantitated with a b-scintillation counter as described previously [21] Suitable modifications were made to the standard reaction to obtain the kinetic constants MUNen5Ac is an unspecific substrate and it does not allow

a distinction between hydrolysis of a-(2,3)- and a-(2,6)-linked sialic acid Therefore, in order to determine the substrate 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 the thiobarbituric method [22] Predefined quantities of wild-type or mutant proteins were incubated with 0.5 mM of either 3¢SL or 6¢SL and 50 mM Hepes pH 7.0, in a final volume of 20 lL for 30 min at 35C The enzymatic reactions were stopped by adding 15 lL of 25 mMNaIO4 solution prepared in 125 mM sulfuric acid solution The mixtures were vortexed and allowed to react in a water bath

at 37C 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 mM, 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 lL were separated by high-performance liquid chromatography through a C18reverse phase column (Pharmacia Biotech) using 2 : 3 : 5 water/methanol/buffer (buffer: 0.2% phos-phoric acid; 0.23M 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

R E S U L T S

TheT brucei trans-sialidase primary sequence conserves most of the structurally relevant amino-acid residues of bacterial and protozoan sialidases

corres-ponding to the catalytic domain of TcTS (L26499, a member of family I of T cruzi trans-sialidase/sialidase superfamily [23]) on the T brucei Genome Project Database (Sanger Centre) The search identified six GSSs with aBLAST

Evalue 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 FRIP and Asp box motifs were conserved in the deduced sequence, this open reading frame might code for a T brucei sialidase-related protein These data were used to design oligonucleotides for the amplification by PCR on genomic DNA to clone the gene coding for the complete TbTS Eleven genes from independent PCRs were sequenced and organized into eight different groups according to their nucleotide sequence (Fig 1) The differences among genes

seem not to be randomly distributed, but rather, localized at

nine positions Combinations of mutations at these nine

positions generated eight genes having from one to five

differences Five out of the nine differences are in the first

and second codon positions, giving rise to a high proportion

of nonconservative mutations Most of the differences

(seven out of nine) are located in the catalytic domain (see

below), but they are placed at positions irrelevant for the

enzymatic activity because the corresponding recombinant

proteins displayed both sialidase and trans-sialidase activity

(see next section) The deduced primary structure of the

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 critical substitutions observed at the periphery of the cleft in TcTS are conserved in the deduced primary sequence of TbTS, including an aromatic residue (Y120 in TcTS) that was 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 the subterminal VTVxNVfLYNR motif (VIVRNVLLYHR

in T brucei) that in the case of T cruzi, defines the trypanosome trans-sialidase/sialidase superfamily of surface proteins [26] It has been recently shown that this sequence is involved in host cell binding during T cruzi infection process [27]

Expression and properties ofT brucei recombinant trans-sialidase

The entire ORF starting at the codon for the first methionine was identified by sequencing the 5¢ UTR of TbTS mRNA A construct expressed from the codon for this first methionine produced a protein of approximately 84.4 kDa that lacked sialidase and trans-sialidase activities (data not shown) An analysis of the putative start of the mature protein N-terminus using the IPSORT program (Human Genome Center, Institute of Medical Sciences, University of Tokio), predicted the existence of a signal peptide that ends just before leucine 28 The insert was then 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-acid protein with a predicted molecular mass of 81.4 kDa 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.

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 mM) and TcTS (4.3 mM) were similar, as it was the turnover of both enzymes (apparent Vmax for sialyl-lactose is 51 161 nmolÆmin)1Æmg)1 for TbTS and

32 692 nmolÆmin)1Æmg)1) for TcTS (Fig 3; [18]) As in the case of T cruzi trans-sialidase [18], TbTS behaves as a very efficient sialyl-transferase: in excess of both the donor and acceptor substrates, the enzyme is 11.1 times more efficient

in transferring than hydrolysing donor sialic acid, as can be concluded by comparing the Vmax of the hydrolysis and transference activities (Fig 3) We have also measured the trans-sialidase-sialidase activity ratio in the native T brucei brucei enzyme from procyclic forms, as described under Experimental procedures This ratio was 8.9 Thus, there is a good agreement between values obtained with the recom-binant and native enzymes

Point mutations at critical amino-acid residues revealed features of the catalytic site of African trypanosomestrans-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

I

-1 min.mg) x 10

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 min.mg)

1/S (mM -1 )

Km: 2.27 mM Vmax: 51161 nmol min -1 mg -1

7 8

Elution (mL)

250

0

kDa

66 97.4

45

9 10 11 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

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

stants were obtained using lactose fixed

con-centration of 2 m M and varying the

concentration of the sialyl lactose according to

the experiment Data are the mean of three

independent experiments.

Catalytic domain Lectin-like

domain

TcTS

TbTS

372

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 0 104.29 ± 8.3 (11.2) 0

trans-sialidase activity TcTSa sialidase activity TcTSb

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

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.

trans-sialidase activities, as in TcTS The change of the

aromatic side chain (W400 A) that in the case of TrSA and

TcTS lost the capability of hydrolysing MUNen5Ac [18],

retained 11.6% of the sialidase activity when MUNen5Ac is

used as substrate (Fig 4B) The mutation at Y191S

suppressed both activities, at variance with the American

trypanosome trans-sialidase, where the substitution of Y120

practically abolishes the sialyltransferase activity while

preserving some of the sialidase activity [17,18] The

differences observed in the effect of the mutations at these

positions could arise from distinct organizations of the

catalytic sites of both trans-sialidases

The trans-sialylation activity of TbTS W400A was lost as

in TcTS W312A mutant (Fig 4 and [18]), thus indicating

that the transfer, but not the hydrolysis reaction requires a

precise orientation of the bound substrate in both enzymes

The exposed tryptophan residue in TcTS and TrSA

determined the high specificity of these enzymes towards

sialyl-a-(2,3) substrates [18], which could be explained by

unfavourable interactions of this aromatic side-chain with

sialyl-a-(2,6)-linked oligosaccharides To test if this is also

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 theT brucei

andT 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 sialidases, are also conserved in the three trypanosomal enzymes (shown in b lue 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) that could serve as the proton donor in the reaction; and two residues (E231, Y343) that probably serve to stabilize the transition state intermediate Other amino-acid residues conserved in the active site of the three trypanosomal enzymes (but not necessarily in other sialidases) include R54 and D97, whose side chains make hydrogen bonding interactions with the bound inhibitor; W121, L(I)177 and Q196, all of which are part of the pocket that binds the N-acetyl group of sialic acid; D248 and E358, whose carboxylate groups make hydrogen bonds with two arginine side-chains of the triad; and W313 and Y365, are both favourably 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 that they could be important for transglycosylation activity Two

of 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 two trans-sialidases [17,18,28] The presence of a tyrosine residue

at position 120 was shown to be critical for TcTS activity [17], probably because this aromatic side-chain residue is involved in substrate binding Also, the conservation of a sequence 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], who 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 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).

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

that TbTS genes are present in a small copy number (Fig 7),

a situation that is different from American trypanosomes

where the trans-sialidase family genes comprises at least 140

members Regarding the results obtained with enzymes that

cut at least once on each gene unit (BssHII, EcoRV and

HindIII, Fig 7A, panel I), a minimum of two

trans-sialidase-related genes can be estimated from the Southern

blot analysis It is likely that the TbTS genes are organized in

tandem, as previous evidence from cloning and sequencing

(see Fig 1) suggested that several copies may exist

Database using the fragment corresponding to the putative

catalytic domain of TbTS identified a BAC clone with a

TbTS We decided then to analyse the presence of

TS-related genes on the genome of T brucei, b ecause in

American trypanosomes these genes are abundantly

repre-sented in the parasite genome [5] We designed primers

based on the sequence of the BAC clone and performed a

PCR on genomic DNA These PCR resulted in a gene of

2109 bp that was called TbTS-like The deduced primary

sequence showed a partial conservation of the typical

sialidase motifs (FRIP and Asp box), and the absence of the

residues shown to be important for activity in the

three-dimensional structure of bacterial and protozoan sialidases

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 theIPSORTprogram 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)

D I S C U S S I O N

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] The function of procyclins is unknown, although they contribute to the establishment of strong infections in the fly vector Parasites that have no surface procyclin because of a defect in GPI synthesis are less efficient at establishing infection in flies [32] Impairment of this process offers a possibility for controlling vector parasitemia (see below) Extensive work has been carried out on the molecu-larbiology, biochemistry and structure of the surface

9.4 23.1

6.6

4.4

kpb

A

2.3

2.0

SxDxGxTW

TbTS

LTIxNAMLYNR

YRSP

683 1

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 cruzi DNA 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.

trans-sialidase of American trypanosome T cruzi (reviewed

in [5]), the agent of Chagas’ disease Both American and

African trans-sialidases are developmentally regulated

sur-face glycoproteins [24,34] They share a number of features

that are unusual for the rest of microbial sialidases, such as a

neutral optimum pH (6.9 for T brucei, 7.2 for T cruzi), the

independence of divalent cations, a relative resistance

towards the natural sialidase inhibitor Neu2en5Ac and the

same substrate specificity [24,33] In spite of not being

closely related in their overall primary structure, TbTS

conserves most of the amino acids relevant for the catalytic

site of American trans-sialidase The identity increases up to

45% in the region corresponding to the catalytic domain,

but TbTS contains an extra region of 100 amino acids

towards its N-terminal end In its C-terminal region, the

identity falls to 30% relative to the lectin-like domain of

American trans-sialidase The trans-sialidase gene products

of T cruzi and T brucei have a significant degree of

structural and biochemical similarity to the sialidases found

in bacteria and viruses (Fig 8) The comparison of inferred

gene trees with species trees made by alignment of the

nucleotide and predicted amino-acid sequences of sialidases

and trans-sialidase suggested that the genes encoding the

T cruzi trans-sialidase of mammalian forms might be

derived from genes expressed in the insect forms of the

genus Trypanosoma [35] It was recently demonstrated by

analysis of DNA sequences from 62 different species of this

genus that there is evidence for a common ancestor for

T cruziand T brucei around 100 million years ago [36], an

ancestor that could have carried the primitive trans-sialidase

gene

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 choleraeand 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 [38] sialidases, display a high specificity for a-(2,3)-linked sialic acid conjugates We have demonstrated that a conserved tryptophan residue in American trypanosome sialidase and trans-sialidase is directly involved in the binding of sialic acid donor substrates, as the single point mutant Wfi A allowed a looser accommodation of the donor substrate, broadening their substrate specificity [18]

On the other hand, a significant decrease of hydrolytic activity against the fluorogenic substrate MUNen5Ac was shown in the case of T cruzi: hydrolysis was undetectable in the TcTS mutant In TbTS mutant, the activity falls 10-fold relative to the activity of the wild-type towards this substrate

It has been shown recently that the lectin-like domain of a trans-sialidase-related protein is involved in host cell binding activity during the T cruzi cell invasion process [27,39] The binding site to cytokeratin 18 colocalizes with the trans-sialidase/sialidase superfamily motif (VTVxNVfLYNR) [27] Because this motif is conserved in TbTS, it is possible that a cell binding activity in the lectin-like domain of TbTS could 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

trypomastigote TcTS

procyclic form TbTS

StSA

SxDxGxTW FRIP

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.

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

livestock There has been extensive international

collabor-ation and considerable expenditure on mechanisms to

control the disease and its vector [42] Given the limited

range and effectiveness of the drugs available as resistance

has emerged, modulating tsetse vector infection appears to

be an important strategy in reducing the incidence of this

disease Major advances being made by molecular

biologi-cal and genomic research will eventually lead to the

development of new approaches to control disease

trans-mission by insect vectors Although not demonstrated here,

trans-sialidase might have a relevant function for the

survival of T brucei in the tsetse vector In fact, the same

enzymatic activity has a relevant function for the survival of

T cruzi Furthermore, the gene encoding this enzyme might

have been generated millions of years ago and have been

conserved, probably as a result of its important function

Further work will demonstrate if TbTS is indeed an

essential enzyme for the parasite If so, treatment of cows

with a putative inhibitor could be used to prevent infection

in the tsetse fly and its dissemination A similar approach to

that proposed by vaccination in Plasmodium infections, the

so-called transmission blocking malaria vaccines [43]

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

We would like to thank Graciela 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

Promocio´n Cientı´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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