DOI: 10.1002/cbic.201100421 The Chemistry and Biology of … · 2012-01-11 · and in human serum....

DOI: 10.1002/cbic.201100421

The Chemistry and Biology of Trypanosomal trans-Sialidases: Virulence Factors in Chagas Disease andSleeping SicknessRoland Schauer*[a] and Johannis P. Kamerling[b]

2246 � 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2011, 12, 2246 – 2264

Introduction

Sialidases are glycosidases that occur in viruses, in pro- and eu-karyotic microorganisms and in animals, especially of the deu-terostome lineage. Most of them are exo-glycosidases that re-lease terminal sialic acid residues from their (a2–3), (a2–6),(a2–8) and/or (a2–9) glycosidic linkages.[1–3] In vertebrate cells,sialidases have a catabolic role, and they are engaged in specif-ic physiological and pathological cellular events.[2, 4–6] In micro-organisms, these enzymes often are virulence factors thatenable spreading and infection of host cells.[7–9] They also canserve nutritional purposes in bacteria, by consuming sialic acidfrom the host or from food matter in the intestine.

As with most enzymatic reactions, the sialidase reaction istheoretically reversible. This has been exploited by bacterialand viral sialidases by choosing suitable conditions for the(a2–3)- or (a2–6)-sialylation of oligosaccharides in the finalstep of chemical synthesis.[2, 5, 9] Typical examples comprise thesialidases from Arthrobacter ureafaciens,[10, 11] Clostridium perfrin-gens,[11, 12] Vibrio cholerae,[11–14] Salmonella typhimurium,[12, 14, 15]

Corynebacterium diphtheriae,[16] (here expressed on the surfaceof Saccharomyces cerevisiae[17]) and Newcastle diseasevirus.[11, 12] Also, a human plasma sialidase has been isolatedwith activities for cleaving and synthesising (a2–3), (a2–6) and(a2–8) linkages.[18–20] In a number of cases bacterial sialyltrans-ferases have also been discovered that have sialidase/trans-sial-idase activities, for example, the recombinant sialyltransferasesfrom Pasteurella multocida (transfer of (a2–3) linkages),[21] Cam-pylobacter jejuni (transfer of (a2–8) linkages)[22] andPhotobacterium damsela (transfer of (a2–6) linkages).[23]

Besides the sialidases and sialyltransferases mentionedabove, in some trypanosomal species a particular class of siali-dases has been discovered; these behave like normal sialidasesif only water is present, but preferentially transfer sialyl resi-dues from one glycan chain to the terminal galactose residueof another nonsialylated oligosaccharide or glycoconjugate.Thus, they are well suited for glycan sialylation. These so-calledtrans-sialidases (EC 3.2.1.18) gained great attention becausethey play crucial roles in the pathogenicity of some trypano-some species, such as Trypanosoma cruzi in South America andthe Trypanosoma brucei group in Africa. They are virulence fac-tors in widespread and devastating diseases like the South

American Chagas disease and the African sleeping sickness.Trypanosomes acquire sialic acids from their host with the aidof trans-sialidases, and incorporate them into their cell-surfaceglycoproteins. This, however, occurs at different developmentalstages of the parasites and therefore contributes to the viru-lence of the South American and African trypanosomes in dif-ferent ways.

In this review, the occurrence and isolation of trans-sialid-ases, their assay systems, enzyme properties, substrate specific-ity, inhibitors, reaction mechanism and protein structures willbe described. Furthermore, the different mechanisms by whichtrans-sialidases contribute to the pathogenicity of the parasiteswill be discussed, as well as the epidemiology and some clini-cal features of Chagas disease and sleeping sickness, which arestill considered as “neglected diseases”.

Occurrence and Isolation of trans-Sialidases

The first hint of the existence of an unusual sialic acid transferreaction was given in 1983 when the sialic acids N-acetylneura-minic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc)were detected in T. cruzi,[24, 25] and it was found that these werenot synthesised by the parasites themselves. As the molar ratioof these trypanosomal sialic acids corresponded to that in theincubation medium, their acquisition from this source (or inthe case of an infection, from host glycoconjugates) was as-sumed. Furthermore, the involvement of a sialidase activitywas suspected in these trypanosomes,[26] and this was later lo-calised on their cell surfaces. These observations led to theidentification of the trans-sialidase enzymes, first in the Ameri-can species T. cruzi,[27–29] and later in the African speciesT. brucei[30, 31] and Trypanosoma congolense.[32, 33] Closer investi-gation revealed the presence of this enzyme in the wholeT. brucei group, that is, in several strains of T. brucei brucei,T. brucei rhodesiense and T. brucei gambiense.[32] The trans-siali-

[a] Prof. Dr. R. SchauerBiochemisches Institut, Christian-Albrechts-Universit�t KielOlshausenstrasse 40, 24098 Kiel (Germany)E-mail : [email protected]

[b] Prof. Dr. J. P. KamerlingBijvoet Center for Biomolecular Research, Utrecht UniversityPadualaan 8, 3584 CH Utrecht (The Netherlands)

trans-Sialidases constitute a special group of the sialidasefamily. They occur in some trypanosome species and, in aunique reversible reaction, transfer sialic acids from one glyco-sidic linkage with galactose (donor) to another galactose (ac-ceptor), to form (a2–3)-sialyl linkages. Trypanosomes causesuch devastating human diseases as Chagas disease in SouthAmerica (Trypanosoma cruzi) or sleeping sickness in Africa (Try-panosoma brucei). The trans-sialidases strongly contribute tothe pathogenicity of the trypanosomes by scavenging sialicacids from the host or blood meal to coat the parasite surface;this aids their survival strategy in the insect’s intestine, and inthe blood circulation or cells of the host, and serves to com-

promise the immune system of the human or animal host.American and African trypanosomes express trans-sialidases atdifferent stages of their vector/host development. They aretransmitted to humans by insect vectors (tsetse fly or otherinsect “bug” species). trans-Sialidase activity with varying link-age specificity has also been found in a few bacteria speciesand in human serum. trans-Sialidases are of increasing practicalimportance for the chemo-enzymatic synthesis of sialylatedglycans. The search for appropriate inhibitors of trans-sialidasesand vaccination strategies is intensifying, as less toxic medica-ments for the treatment of these widespread and often chron-ic tropical diseases are required.

ChemBioChem 2011, 12, 2246 – 2264 � 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chembiochem.org 2247

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dases are glycosylphosphatidylinositol (GPI)-anchored on thecell surface of the parasites. In contrast to “classical” sialidases,trypanosomal trans-sialidases catalyse the reversible transfer ofpreferentially (a2–3)-linked sialic acids from donor glycans di-rectly to terminal bGal-containing acceptor molecules, therebygiving rise to new (a2–3) glycosidic linkages (Figure 1).[34, 35]

Thus, like classical sialidases, trans-sialidases are “retaining” sia-lidases (note that endo-sialidases are “inverting” sialidases[36]).In the absence of an appropriate acceptor, these enzymes actas sialidases that transfer the glycosidically linked sialic acid toa water molecule instead of to a terminal bGal residue; howev-er, this activity is slower than that of the transfer reaction. Evi-dently, the trans-sialidases represent a kind of sialyltransferasethat can form new sialic acid–glycan linkages without the needfor prior energy-consuming sialic acid activation by cytidine tri-phosphate (CTP).

The activity of trans-sialidases in Endotrypanum species hasalso been described.[37] These protozoic microorganisms aredistantly related to the trypanosomes. Many other members ofthe kinetoplastida lack both sialidase and trans-sialidase activi-ties, or express only sialidase, such as Trypanosoma vivax[38]

and Trypanosoma rangeli[39] (for reviews, see refs. [40–42]). It ispossible to discriminate between morphologically indistin-guishable trypanosomatids by measurement of trans-sialidase

and sialidase activities.[43] Furthermore, the occurrence of sialicacids on major cell-surface epitopes correlates with the expres-sion of trans-sialidase.[44]

In contrast to these intermolecular trans-sialidases, the exis-tence of an intramolecular trans-sialidase that forms 2,7-anhy-dro-Neu5Ac (Neu2,7an5Ac) upon release of sialic acid from theglycosidic linkage has been described in the leech.[45] Thisenzyme (neuraminidase B) has also been found in Streptococ-cus pneumoniae,[46] and has a strict specificity for (a2–3)-linkedsialic acid substrates. The formation of Neu2,7an5Ac in addi-tion to Neu5Ac was also observed by the action of the siali-

Figure 1. A) Reversible trans-glycosylation of (a2–3)-linked N-acetylneura-minic acid between Neu5Ac(a2–3)Gal-OR1 and Neu5Ac(a2–3)Gal-OR2, cata-lysed by trypanosomal trans-sialidases. The 4-methylumbelliferyl and p-nitro-phenyl a-glycosides of N-acetylneuraminic acid are frequently used in an ir-reversible reaction. B) Trypanosomes are unable to synthesise sialic acids.However, some species express trans-sialidases that are used to transfersialic acids from host cell glycoconjugates to terminal b-galactose residuesof GPI-anchored glycoproteins on the pathogen surface, as shown here formucins of T. cruzi (adapted from ref. [104], and with thanks to T. Jacobs formodifications).

Hans Kamerling studied chemistry and

obtained his PhD degree (1972) at

Utrecht University. At this university,

he has been active in the glycoscience

field since 1969 (structural analysis and

synthesis of carbohydrates), became

University Fund Professor of Organic

Chemistry of Natural Products in 1990,

Professor of Bio-Organic Chemistry of

Carbohydrates in 2000, and retired in

2009. Since 2008, he has been Honora-

ry Professor of Chemical Glycobiology

at the University of Groningen, and was honoured with the Dutch

Royal Distinction “Officer in the Order of Orange-Nassau” in 2009.

He is the author of over 420 scientific publications.

Roland Schauer studied medicine and

biochemistry at the University of T�-

bingen and obtained his M.D. in 1962

and a Biochemistry Diploma in 1966.

In 1967 he started his work on sialic

acids at the Ruhr University in

Bochum, where he was appointed As-

sociate Professor in 1973. In 1976 he

became a Full Professor and Director

of the Institute of Biochemistry at Kiel

University and became Emeritus Pro-

fessor in 2001. He has organised a

number of scientific conferences, is author or co-author of 400

publications, and received the 2009 Rosalind Kornfeld Award for

Lifetime Achievement in Glycobiology from the Society for Glycobi-

ology.

2248 www.chembiochem.org � 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2011, 12, 2246 – 2264

R. Schauer and J. P. Kamerling

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dase from Clostridium tertium (unpublished results from R.S.’slaboratory).

The first isolation of trans-sialidase was reported fromT. cruzi,[29] followed by a corresponding enzyme from culturedprocyclic forms of the African trypanosome species T. brucei[30]

and T. congolense.[33] The enzymes are bound to cell mem-branes of the trypanosomes by glycolipid anchors, thereforedetergents are required for their solubilisation. When grown incell culture, some of the enzyme is released and can be foundin the medium. The molecular masses of the trans-sialidaseswere estimated to be 160–200, 67, and 90 kDa (monomericform) for the enzymes from T. cruzi, T. brucei and T. congolense,respectively. Regarding kinetic, structural and other properties,the trans-sialidases from the two continents show striking simi-larities (see below). T. congolense, however, expresses varioustrans-sialidase forms that exhibit either high or low trans-siali-dase activities when compared with their sialidase activities.[33]

Many forms were also found in T. cruzi,[40, 47–50] translated frommultiple trans-sialidase genes. The various trans-sialidase pro-teins expressed by T. cruzi also show variable enzyme activitiesand trans-sialidase/sialidase activity ratios. Some of them evenare inactive. Expression of this enzyme also depends on thetrypanosome strain, and this much influences its pathogenici-ty.[49] The expression of trans-sialidases also differs betweenparasites living in mammalian (trypomastigotes) and insect(epimastigotes) hosts. This results from the expression of twogroups of non-overlapping sets of proteins translated from thecomplex gene family of T. cruzi, depending on the host. At themolecular level, underlying this differential expression, post-transcriptional events mainly driven by specific, highly con-served 3’ untranslated regions (3’ UTRs) were elucidated to beinvolved in gene expression.[50] In contrast, only a smallnumber of trans-sialidase genes were discovered in T. brucei.[51]

Trypanosomal trans-sialidases also exhibit pronounced homol-ogy with classical sialidases; some of these aspects are furtherdiscussed below.

trans-Sialidase Reaction Mechanism andProtein Structures

These trans-sialidases share protein structures and conservedamino acids (involved in enzyme catalysis) with microbial andanimal sialidases. An early observation was that the T. cruzitrans-sialidase, also called “shed acute-phase antigen” (SAPA),consists of an N-terminal half that contains the enzymatic func-tion, and a highly antigenic C-terminal portion, mainly com-posed of a tandem series of twelve amino acid repeats, calledSAPA repeats.[52] A recombinant T. cruzi trans-sialidase lackingthe repeats was shown to retain enzymatic activity.[53] Four Aspboxes (Ser-X-Asp-X-Gly-X-Thr-Trp) are located near the N termi-nus of the enzyme; these seem not to be directly involved inenzyme catalysis but might preserve the protein structure, sim-ilar to the function of Asp boxes in bacterial and human siali-dases.[5, 54] The American parasite T. rangeli secretes a sialidasethat has no trans-sialidase activity or SAPA repeats, but has70 % amino acid identity with the T. cruzi trans-sialidase.[55] X-ray crystallographic studies of the T. rangeli sialidase revealed a

canonical b-propeller topology of the active site centre thatwas similar to that of the classical sialidases, and enabled mod-elling of the structure of T. cruzi trans-sialidase. This, togetherwith mutagenesis experiments, allowed the identification ofthe amino acids at the active site, changes to which trans-formed this sialidase into an efficient sialyltransferase.[55, 56] Itwas found that the binding site for the acceptor carbohydrateis distinct from the donor ((a2–3)-linked sialic acid) bindingsite; this was in contrast to previously known sialidases. A tyro-sine residue (Tyr120) was found by mutagenesis to be crucialfor binding of the acceptor substrate, and a model was provid-ed for both the hydrolysis and transfer reactions catalysed byT. cruzi trans-sialidase; this contributed to the understanding ofhow the glycosidase structure achieves glycosyltransferase ac-tivity. According to the model (Figure 2), the carboxylate groupof sialic acid interacts with an arginine triad (Arg35, Arg245and Arg314), a glutamic acid residue (Glu230) stabilises one ofthese arginines, an aspartic acid (Asp59) is essential for cataly-sis by proton transfer, and a tyrosine (Tyr342) is in contact withthe transient oxocarbonium ion at the C2 carbon of sialic acid,as is the case in the active clefts of all sialidases.[55, 57] This is fol-lowed by nucleophilic attack of either water or the hydroxylgroup at C3 of galactose or a galactoside (Scheme 1).

Energy analysis of the catalytic mechanism of T. cruzi trans-sialidase revealed that ligand binding facilitates proton trans-fer.[58] Computational experiments suggest a long-lived cova-lent intermediate in the catalytic mechanism, and identifiedthe Tyr342/Glu230 pair as an unusual catalytic couple

Figure 2. The active site of T. cruzi trans-sialidase with the catalytic aminoacid residues Tyr342, Glu230, and Asp59 (compare Scheme 1). The noncata-lytic Asp96 residue is important for stabilisation of the proton transfer fromTyr342 to Glu230. (Note that Asp96 interacts with the acetamido group ofNeu5Ac.) The relevant arginine residues Arg35, Arg245, Arg314, and the sub-strate N-acetylneuraminyllactose are also included. Hydrogen atoms are notshown for these residues. By courtesy of A. Roitberg; see also ref. [58] .


Trypanosomal trans-Sialidases

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(Figure 2). The tyrosine hydroxyl proton is transferred to thecarboxylate group of glutamate before the nucleophilic attack.This means that binding of the substrate (i.e. , formation of theenzyme’s holo form) is necessary before the transfer reactionbecomes energetically possible.

Comparison of the crystal-derived structure of the T. cruzitrans-sialidase with that of T. rangeli sialidase revealed structur-al differences in the active-site cleft, which could be responsi-ble for the different types of enzyme reaction. The substrate-binding pocket of T. cruzi trans-sialidase appears narrower andmore hydrophobic, and might favour trans-sialylation by theexclusion of water.[57] This was confirmed by molecular dynam-ics calculations that showed that T. cruzi trans-sialidase has avery flexible, widely open catalytic cleft, mostly due to Trp312loop motion in the apo form (Figure 3 A).[59] After ligand bind-ing, this flexibility and solvent exposure is much reduced. Thisis in contrast to the T. rangeli sialidase, which maintains a moreopen catalytic cleft in both apo and holo forms, required forhydrolytic activity. A proline residue (Pro283), influencing theposition of a conserved tryptophan near the centre active site,is also necessary for T. cruzi trans-sialidase activity. Remarkabledifferences between classical sialidases and trans-sialidases arethe structural changes that occur upon absorption of sub-strates or Neu5Ac2en into the crystals ; these become unstablein the case of trans-sialidase due to disturbance of the molecu-lar packing. The reason for this phenomenon is conformationalchanges caused by the sialic acid of the donor substrates.Tyr342 is sterically displaced by about 2 � upon substrate bind-

ing, thereby leading to a shortening of the distance betweenthe tyrosine hydroxyl group and the C2 atom of the sialicacid.[57, 60] The plasticity of the catalytic pocket induced by bind-ing of sialic acid is essential for catalysis by trans-sialidase, andwas not observed in all the known 3D structures of classicalprokaryotic and eukaryotic sialidases, including that of theenzyme from T. rangeli. The latter sialidase and T. cruzi trans-sialidase also interact in different ways with the sialic acid glyc-erol side-chain. Only a single interaction was seen in the T. ran-geli sialidase, but multiple ones occurred with the T. cruzi trans-sialidase.[61]

By using surface plasmon resonance and NMR spectroscopy,highly valuable information on how the trans-sialidase reactionproceeds was obtained from binding studies with either activeT. cruzi trans-sialidase[57, 62, 63] or with inactive natural mutants.[64]

While, for example, 3’-sialyllactose readily bound to theenzyme protein, the acceptor substrate, lactose or other asialoglycoconjugates did not interact with the enzyme at all, unlesspre-incubated with the sialylated donor substrate. These stud-ies show that sialic acid modulates the affinity for the asialo ac-ceptor substrate and that the association of the asialo receptorwith the active site is an absolute requirement for the transferreaction. Investigation of crystals soaked with donor or accept-or substrates confirmed that this occurs by a structural changein the sequential binding of the enzyme (Figure 3 B). Duringthis interaction the amino acids Trp312 and Tyr119 make stack-ing interactions with lactose, and thus enable positioning ofthe HO3 group of the galactose moiety in the ternary enzyme

Scheme 1. Mechanism of action of trypanosomal trans-sialidases, as reported for T. cruzi trans-sialidase, based on refs. [58] and [117]. For clarity, in the inter-mediate structures the d+ and d� charges have been omitted.



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complex, thereby allowing nucleophilic attack of the anomericcarbon in the sialylated transition species.[57]

Based on molecular dynamics investigations into the activesite plasticity of T. cruzi trans-sialidase, a dual role for Trp312 isassumed.[65] According to this, the tryptophan residue assistsTyr119 in orienting the substrate for sialylation by the trans-sialidase (Figure 3). In addition, Trp312 behaves like a molecu-lar shovel because of its lever-like motion. This releases thedonor product from the active site following delivery of sialicacid, and loads the acceptor substrate into position for subse-quent molecule binding.

Point mutations of these amino acids confirmed their rolesin catalysis and that Tyr119 is part of the second binding-site.[66] In spite of the ablation of trans-sialidase activity, theprotein hydrolysed both (a2–3)- and (a2–6)-linked sialic acid.This demonstrates the requirement for precise orientation ofthe substrates within the catalytic centre for the transfer reac-tion (in contrast to hydrolysis), and the presence of distinctbinding-sites for acceptor and donor substrates. These struc-tural features are unique to trans-sialidases. Interestingly, sialic

acid (a2–6)-linked to lactose cannot trigger the conformationalswitch required for the trans-glycosylation reaction, in contrastto (a2–3)-linked sialic acid.[64] The sequential binding of donor(a2–3)-linked sialic acid and acceptor galactose and thechange in protein conformation involved in the T. cruzi trans-sialidase reaction is depicted in Figure 4.

The binding of donor substrate to T. cruzi trans-sialidase wasalso demonstrated by saturation transfer difference (STD) NMRexperiments with the p-nitrophenyl glycoside of aNeu5Ac.[63]

This technique allowed comparison of the rate of substrate hy-drolysis with that of sialic acid transfer. Interestingly, shorteningof the Neu5Ac glycerol side-chain favoured hydrolysis overtransfer, whereas 9-O-acetylation of Neu5Ac had the oppositeeffect.

Mutants of T. rangeli sialidase have been obtained that ex-hibit some trans-sialidase activity that arises from the forma-tion of a trans-sialidase-like binding-site for the acceptorsugar.[67] This interaction is a prerequisite for trans-sialidase ac-tivity, together with fine-tuning of protein–substrate interac-tions and the flexibility of crucial active-site residues.

Figure 3. Molecular dynamics (MD) studies of the active site plasticity of the T. cruzi trans-sialidase. A) Visualisation of the flexibility of the Tyr119 and Trp312residues in the sialylated enzyme/lactose complex, demonstrating that Tyr119 switches from the stacked to a rotated position and Trp312 moves betweenstacked and open conformations. B) Selected steps from the MD trajectory of the sialylated-enzyme/lactose complex. (I) Initially Tyr119 is in the stacking posi-tion and the Trp312 loop is stacked; (II) at 22 ns, Tyr 119 rotates away from the stack, followed by a Trp312 motion to shift the Trp312/lactose complex of thestack; and (III) full opening of the Trp312 loop enables lactose to vacate the active site at 32 ns. Reproduced from ref. [65], with permission of Elsevier/RightsLink.



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The catalytic mechanisms of the trans-sialidase re-action of both the American and African trypano-somes (T. cruzi and the T. brucei group, respectively)presumably are similar, and a common ancestor ofboth trypanosome types has been predicted to haveexisted around 100 million years ago, and to havecarried a primitive trans-sialidase gene.[68] When theAfrican and American continents separated, the samehappened with the protozoan species. Thereafter, aremarkable subspecies diversity of T. cruzi developedin South America, as was concluded on the basis ofproteomic diversity.[69]

A crystal structure of the T. brucei trans-sialidase isyet to be resolved, but its protein sequence fromgene sequencing experiments is available, and itscatalytic domain shows 45 % identity with that of thecorresponding region of the T. cruzi trans-sialidase;most of the amino acids essential for the catalyticsite of the American trans-sialidase are also con-served.[51] Seven positions are invariant in the twotrans-sialidases. Exchange of tryptophan (Trp400) byalanine in the trans-sialidase of T. brucei abolishedtrans-sialylation activity but enabled the mutant toadditionally hydrolyse (a2–6) sialic acid linkages. Thisis remarkably similar to the behaviour of a T. cruzitrans-sialidase W312A mutant.[66]

Most of the critical active-site residues common toother trypanosomal sialidases and trans-sialidases arealso conserved in the two trans-sialidases isolatedfrom the animal-pathogenic African trypanosome T. congo-lense ; these have pronounced differences in their capacity forsialic acid transfer as compared with hydrolytic activity.[33] Thepartial sequences obtained for these two enzymes by a PCR-based approach showed 50 % identity, but they are also similarto those of viral, bacterial and animal sialidases, as well as

other trypanosomal trans-sialidases.[70] The similarities and dif-ferences at the active site between American and African(trans-)sialidases are depicted in Figure 5.[55, 70]

Structural, kinetic, and mechanistic analyses of T. cruzi trans-sialidase revealed the transfer reaction to proceed by a classi-cal ping-pong bi-bi kinetic mechanism, in which the donor andacceptor substrates must separately bind at the same site. Inthe first step, an intermediate with sialic acid covalently linkedto Tyr342 as the active-site nucleophile is formed, as shownwith fluorinated Neu5Ac derivatives[60, 65, 71, 72] (and unpublishedresults from R.S.’s laboratory). Asp59 serves as the acid–basecatalyst in this reaction. Studies with the T. rangeli sialidase,which is a “true” sialidase, have shown a corresponding mech-anism,[61] thus leading to the assumption that probably all exo-sialidases (as retaining glycosidases), operate by a similarmechanism, with the transient formation of a sialylatedenzyme conjugate. For the trans-sialidases, however, sialic acidis transferred in a second step to galactose by essentially a re-verse of the preceding sialylation process, again by involvingelectrophilic migration of the anomeric centre onto the HO3group of galactose.[60] This trans-glycosylation process might

be facilitated by the longer lifetime of the sialylated tyrosineintermediate observed in T. cruzi trans-sialidase, together withother intriguing differences in the reaction mechanism thatmake trans-sialidases unique.

Figure 4. Proposed events in the transfer reaction of T. cruzi trans-sialidase,to show that correct positioning of the correct sialoside donor ((a2–3) link-age, not (a2–6)) in the inactive binding site of the enzyme is necessary forthe protein to undergo the conformational change that allows the b-galac-toside acceptor to bind. Reproduced from ref. [64] with permission. Copy-right: the American Society for Biochemistry and Molecular Biology, 2004.

Figure 5. Model of the N-terminal domain of T. congolense trans-sialidase 1, according tothe crystal structure of the T. rangeli sialidase/Neu2en5Ac complex.[55] A) Conservedmotifs in the N-terminal domain showing Asp boxes (orange) conserved in bacterial andviral sialidases, the motif LYCHLE (purple) common to all known trypanosomal trans-siali-dases, and the motifs ISRVIGNS and VPVMLITHP (green), which have been found in allstudied African trans-sialidase genes. B) Putative active site of T. congolense trans-sialidase1 with the inhibitor Neu2en5Ac (yellow). Residues shown in red are conserved in T. ran-geli sialidase, T. cruzi trans-sialidase, T. brucei trans-sialidase and T. congolense trans-siali-dase 1; residues shown in blue are conserved in the three trans-sialidases, but are differ-ent in the sialidase. The tyrosine residue (green) is unique to T. congolense trans-sialidase1; at this position, T. rangeli sialidase and the trans-sialidases of T. cruzi and T. brucei con-tain tryptophan residues. The alanine residue (light blue) differs between the Americanand African trypanosomal enzymes. Residues are numbered according to the T. congo-lense trans-sialidase 1 sequence,[70] except where sequence information is incomplete,the T. rangeli sialidase sequence residues R, R and D were used.[55] Reproduced fromref. [70] with permission. Copyright : Walter de Gruyter, 2003.



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trans-Sialidase Assays

Various radioactive and non-radioactive assays have been re-ported for the detection of trans-sialidase activity.[35, 73] A fluori-metric 96-well plate assay, in which 4-methylumbelliferyl b-d-galactopyranoside (MU-bGal) was used as the acceptor sub-strate and 3’-sialyllactose as the sialic acid donor, enabled thespecific, sensitive and relatively rapid detection of enzyme ac-tivity on a larger scale. The resulting Neu5Ac-(MU-bGal) wasseparated from the substrate MU-bGal by ion-exchange chro-matography in 96-well filter plates, hydrolysed, and the liberat-ed MU was measured in a fluorimeter, thus giving the trans-sialidase activity.

In a recently described assay, synthetic benzyl b-d-Fucp-(1!6)-a-d-GlcpNAc (d-Fucp is 6-deoxy-d-Galp) was used as the ac-ceptor substrate and 3’-sialyllactose or fetuin as donor. In thetransfer reaction terminal b-galactose was created from thedonor, and this was quantified spectrophotometrically in a gal-actose oxidase assay (terminal b-fucose is not a substrate forgalactose oxidase).[74] This assay also allowed discrimination be-tween trans-sialidase and sialidase activity by omitting the di-saccharide benzyl glycoside in the latter.

Donor and Acceptor Specificities oftrans-Sialidases

Trypanosomal trans-sialidases are similar with regard to theirsubstrate specificity. Neu5Ac is hydrolysed or transferred mostefficiently, followed by Neu5Gc and, to much lesser degree, byO-acetylated sialic acid.[2, 75] Trypanosomal trans-sialidases havebroad sialyl donor and acceptor specificities, and favour oligo-saccharides and glycoproteins, as has been demonstrated withT. cruzi,[76–78] T. brucei [30, 31, 44] and T. congolense.[32, 33]

Trypanosoma cruzi trans-sialidase: One of the first papersfocusing on detailed studies on the substrate specificity of thenative T. cruzi trans-sialidase dates from 1992. The plasmamembrane associated enzyme was isolated from supernatantsof trypomastigote cultures.[34] A wide range of potential sialicacid donors (gangliosides, oligosaccharides) was tested with[14C]lactose as acceptor, usually at pH 7.2 and 37 8C. Whencomparing (a2–3)-, (a2–6)-, and (a2–9)-linked Neu5Ac, it wasfound that only terminal (a2–3)-linked Neu5Ac was transferredin high yield to the nonreducing bGal residue to yield Neu5Ac-(a2–3)14C-Gal(b1–4)Glc only, thereby demonstrating the regio-specificity of the trans-sialidase. Donors containing theNeu5Ac(a2–3)Gal(b1–4)R sequence were better than donorscontaining the Neu5Ac(a2–3)Gal(b1–3)R sequence. The nearbypresence of a Fuc residue hampered donation by Neu5Ac.Compounds with internal Neu5Ac(a2–3)Gal(b1- units did notfunction as donors. Modifications at C9 (9d-Neu5Ac or Neu5-Ac9Me) did not alter the transfer reaction ability, but modifica-tions at C4 (4d-Neu5Ac or Neu5Ac4Me), C7 (7d-Neu5Ac) andC8 (8d-Neu5Ac or Neu5Ac8Me) led to inactive donors. Finally,4-methylumbelliferyl a-N-acetylneuraminic acid (MU-aNeu5Ac)also turned out to be a donor, to create Neu5Ac(a2–3)Gal(b1-units. By using the T. cruzi trans-sialidase-mediated formationof Neu5Ac(a2–3)14C-Gal(b1–4)Glc from Neu5Ac(a2–3)Gal(b1–

4)Glc as a donor and 14C-lactose as an acceptor, the effects of awide range of potential acceptors were tested. Compoundscontaining bGalNAc, bGlc, bGlcNAc, aGal, aGlc or aMan at thenonreducing end were inactive, whereas the type of linkage(Gal(b1–3), Gal(b1–4) or Gal(b1–6)) influenced the efficiency ofthe transfer reaction. Tests with milk oligosaccharides showedhigher activity for (b1–4)-linked Gal than for (b1–3)-linked Gal.It should be noted that in the presence of poor acceptors, theT. cruzi trans-sialidase functioned more as a sialidase than as atrans-sialidase, and free Neu5Ac was generated.

In a subsequent evaluation of T. cruzi trans-sialidase isolatedfrom trypomastigotes with a wide range of oligosaccharide,glycolipid, and glycoprotein acceptors, most earlier findingswere confirmed.[76] Oligosaccharides with terminal Lewisx orLewisa epitopes were not acceptor substrates. Compared withNeu5Ac(a2–3)Gal(b1–4)Glc, MU-aNeu5Ac and pNP-aNeu5Ac(pNP = p-nitrophenyl) were extremely poor sialic acid donors.The T. cruzi trans-sialidase has an apparent pH optimum of 7.9and a optimal temperature of 13 8C. The kinetic properties ofthe enzyme suggested that the trans-sialylation reaction mightoccur by a rapid-equilibrium random or steady-state orderedmechanism. The effectiveness of immobilised T. cruzi trans-siali-dase as a synthetic reagent was evaluated by using Neu5Ac-(a2–3)Gal(b1–4)Glc as donor and lactose–bovine serum albu-min (BSA) and Gal(b1–3)GlcNAc(b1–3)Gal(b1–4)Glc as acceptor.

In order to synthesise valuable sialic acid related glycopep-tide and oligosaccharide probes that might be useful in fluo-rescence energy transfer measurements and photoaffinity la-belling experiments, periodate-oxidised MU-aNeu5Ac was cou-pled with different primary amines by reductive amination,and the obtained products were incubated with T. cruzi trans-sialidase and lactose as the model acceptor.[79] The chemo-en-zymatic preparation of a water-soluble polyacrylamide polymerdecorated with Neu5Ac(a2–3)Gal(b1–4)GlcNAc is another niceapplication of the useful T. cruzi trans-sialidase.[80] A further in-teresting application is the T. cruzi trans-sialidase-catalysed(a2–3)-sialylation of pyridyl-2-amino-oligosaccharides with ter-minal bGal units (related to N-glycoprotein glycans) whereused as model compounds in N-glycan profiling studies.[81]

Another report from the same period describes the proper-ties of SAPA as being not only the major sialidase but also themajor trans-sialidase of T. cruzi.[82] In these studies it was shownthat 3’-sialyllactose, MU-aNeu5Ac, and foetal calf serum fetuinwere effective donors for the (a2–3)-sialylation of [Gal-14C]-N-acetyllactosamine. Colominic acid, an (a2–8)-linked polymer ofNeu5Ac, was not effective. Interestingly, C7-Neu5Ac-fetuin wasalso able to act as sialic acid donor. Also, fetuin-derived O-gly-cans (alditols) and N-glycopeptides turned out to be suitabledonors.[83] In the case of a series of gangliosides tested as po-tential donors, it was found that compounds with only internalNeu5Ac(a2–3)Gal(b1- or Neu5Ac(a2–8)Neu5Ac(a2–3)Gal(b1-units do not function as donors. However, the conversion ofasialo-GM1a into GD1a with a suitable sialic acid donorshowed that internal Gal(b1-residues can be (a2–3)-sialylated[83]

(see also ref. [78]). Besides N-acetyllactosamine, the N- and O-glycans in asialofetuin and asialo bovine submaxillary glandmucin also served as acceptors for Neu5Ac transfer.



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A real breakthrough for the application of T. cruzi trans-siali-dase in the synthesis of (a2–3)-sialylated compounds was thegeneration of the enzyme in its recombinant form.[53, 57, 66, 84] Asmentioned above, the recombinant construct, expressed inE. coli, lacks the tandem 12 amino acid repeats that are presentin the SAPA antigen but not important for enzymatic activity.One study focused on some kinetic properties of a recombi-nant His-tagged T. cruzi trans-sialidase expressed in E. coli withNeu5Ac(a2–3)Gal(b1–4)Glc and MU-aNeu5Ac as donors; itturned out that the rates of sialic acid transfer to water (hydro-lysis) and to terminal bGal residues have a unique behaviourwith respect to the reaction temperature and the acceptorconcentration.[85] A study of the transfer of Neu5Gc was carriedout with the donors Neu5Gc(a2–3)Gal(b1–4)Glc(b1–O)CH2CH2N3 and Neu5Gc(a2–3)Gal(b1–3)GlcNAc(b1–O)CH2CH2N3, and with lactose, lactitol and N-acetyllactosamineas acceptors, and different yields were obtained.[75] Applica-tions of T. cruzi trans-sialidase for the synthesis of relevantsialyl-oligosaccharides with Neu5Ac(a2–3)Gal(b1–4)Glc and/orMU-aNeu5Ac as donors have been demonstrated for Neu5Ac-(a2–3)Gal(b1–4)GlcNAc (3’-sialyl-N-acetyllactosamine),[86]

Neu5Ac(a2–3)Gal(b1–3)GlcNAc (3’-sialyl-lacto-N-biose I)[87] andNeu5Ac(a2–3)Gal(b1–4)Xyl-pNP.[88] In a report dealing with thesynthesis of Neu5Ac(a2–3)Gal(b1–4)Glc(b1–O)(CH2)7CH3 frompNP-aNeu5Ac as donor and octyl b-lactoside as acceptor andrecombinant T. cruzi trans-sialidase, it was stated that theenzyme proved to be labile in both partially and fully purifiedforms. Therefore, for routine use, crude E. coli lysate containingtrans-sialidase activity was preferred.[89] To monitor the hydroly-sis of sialylated donors and the transfer of sialic acid fromdonor to acceptor molecules when using recombinant T. cruzitrans-sialidase, an extensive NMR study with a variety ofdonors and acceptors was carried out.[62, 90] As confirmed byNMR spectroscopy, T. cruzi trans-sialidase catalyses the hydroly-sis of the sialyl glycosidic linkage with retention of configura-tion.[62, 90, 91]

The acceptor substrate specificity of T. cruzi trans-sialidasefor lactose variants was evaluated for, among others, lactitoland lactobionic acid.[92] With Neu5Ac(a2–3)Gal(b1–4)Glc asdonor, both turned out to be good acceptors for sialic acid.Lactitol effectively inhibited the transfer of Neu5Ac to N-acetyl-lactosamine; when incubated with live trypanosomes andT. cruzi trans-sialidase, it also inhibited the resialylation of theparasite mucins. Also Gal(b1–3)Ara and Gal(b1–3)Ara-ol wereshown to be good acceptors. Remarkably, when Neu5Ac(a2–3)lactitol, Neu5Ac(a2–3)lactobionic acid and Neu5Ac(a2–3)-N-acetyllactosamine were tested as donor substrates with lactoseas acceptor, the lactitol variant was not active at all. This con-trasted with a study that demonstrates that Neu5Ac(a2–3)lacti-tol can act as donor in the sialylation of lactose.[27, 93] A repeti-tion of the experiments with Neu5Ac(a2-3)lactitol by the au-thors that found no activity showed some transfer of Neu5Acto lactose.[94]

Series of (a2–3)-sialylated glycans were prepared with re-combinant T. cruzi trans-sialidase by using Gal(b1–3)GalNAc-(a1–O)Ser/Thr, lactosides and lactosamide derivatives as ac-ceptors, and pNP-aNeu5Ac or MU-aNeu5Ac as donors.[95] At-

tention was paid to pH, temperature, and incubation times,and, under the conditions used, no hydrolysis of the donorsubstrates was observed. In the context of investigations di-rected at the binding of (a2–3)-sialylated oligosaccharides tomyelin-associated glycoprotein (MAG), C7 and C8 analogues ofpNP-aNeu5Ac were prepared, and their suitability for transferby recombinant T. cruzi trans-sialidase were proven.[96] In fur-ther studies, the efficiency of pNP-aNeu5Ac, pNP-aNeu5Prop(Prop = propanoyl), pNP-aNeu5Ac9But (But = butanoyl), pNP-aNeu5But, pNP-aNeu5iBut (iBut = isobutanoyl) and pNP-aNeu5Gc as donors was reported in a T. cruzi trans-sialidase-catalysed reaction with Gal(b1–6)Glc(a1–O)Me as acceptor.[97]

The artificial donors pNP-aNeu5But and pNP-aNeu5iBut turnedout to be completely inactive. When comparing the varioustested substrates, including pNP-aC7Neu5Ac and pNP-aC8-Neu5Ac, it is interesting to note that the C7–C9 glycerol sidechain of Neu5Ac is located outside the binding pocket and theC5 N-acetyl group is located deep within the pocket. Addition-ally, NMR studies have been carried out on the binding ofT. cruzi trans-sialidase to pNP-aNeu5Ac, pNP-aC8Neu5Ac, pNP-aC7Neu5Ac and pNP-aNeu5,9Ac2, and information was collect-ed on the rate of substrate hydrolysis versus the rate of sialicacid transfer.[63]

In the context of studying the transfer of sialic acid fromhost glycoconjugates to the surface GPI-anchored glycopro-teins of T. cruzi, in earlier investigations attention was paid tothe aGlcNAc-bound O-glycans ((a2–3)-sialylated branched gal-acto-oligomers) of the mucins from the parasitic G, Y, CL-Brener, Dm28c and Tulahuen strains.[98–103] The O-glycans onthe cell surface were excellent sialic acid acceptors for trans-sialidase.[77, 78, 104, 105] This was shown with, among others, azido-modified Neu5Ac, which was readily transferred to galactoseresidues of surface mucins of living parasites.[106] This sialic acidderivative was found to be also transferred to a complex pat-tern of glycoproteins on mouse thymocytes, lymphocytes andspleen cells by parasite trans-sialidase shed into the bloodstream; CD45 was the main acceptor, followed by integrins.This sialylation of both parasite and host membrane glycopro-teins by trans-sialidase has great biological implications, espe-cially on the immune system, as will be discussed below. Inthis context, it is of interest to mention that sialidase-treatedsheep and human erythrocytes could be resialylated up to50 % using T. cruzi trans-sialidase and 3’-sialyllactose, and thatresialylation of sheep erythrocytes restored their resistance tolysis by human complement.[107]

To get a detailed insight into the terminal Gal residues thatcan be sialylated, the established major O-glycan structure ofthe G strain, Galp(b1–2)[Galp(b1–3)]Galp(b1–6)[Galf(b1–4)]GlcpNAc, and the corresponding alditol and benzyl a-glyco-side were synthesised and subjected to sialic acid transfer byusing recombinant T. cruzi trans-sialidase with Neu5Ac(a2–3)Gal(b1–4)Glc as the donor.[108] It was found that selective(a2–3)-sialylation occurred only at the less hindered terminalGalp(b1–3) unit. However, when using the terminal Galp(b1–2)[Galp(b1–3)]Gal fragment in its free, alditol, or benzyl a-gly-coside form, only the flexible alditol form was sialylated, ateither one of the two terminal Gal units (disialylation, if pres-



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ent, was negligible).[109] All three compounds were effective in-hibitors of the T. cruzi trans-sialidase-mediated (a2–3)-sialyla-tion of N-acetyllactosamine. Additionally, in these studies therates of sialylation of the major synthetic product, and a seriesof related fragments (as free oligosaccharide, alditol, andbenzyl a-glycoside) were compared, with the aim of relatingthe presence of Galf with the ability of these compounds toact as substrates in the T. cruzi trans-sialidase-catalysed transferreaction. Although there were differences, all fragments wereshown to be good acceptor molecules.[105, 108, 110–113] In the re-combinant T. cruzi trans-sialidase-mediated (a2–3) sialylation ofa synthetic T. cruzi-related glycopeptide fragment, Thr-Thr-(LacNAc-Thr)-Thr-Thr-Gly with Gal(b1–4)GlcNAc (= LacNAc) cou-pled a-glycosidically (natural linkage) and b-glycosidically (non-natural substrate), with calf serum fetuin as a sialic acid donor,it turned out that the linkage between GlcNAc and Thr doesnot present a problem for good yield.[114]

Trypanosoma brucei trans-sialidase: The native T. bruceitrans-sialidase from procyclic trypomastigotes has been shownto transfer sialic acids from a variety of sialoglycoconjugates(i.e. , serum glycoconjugates, human and bovine erythrocytes)to terminal bGal residues of oligosaccharides and glycoconju-gates (i.e. , sialidase-treated erythrocytes) by (a2–3) linkages.[31]

With [Glc-1-14C]lactose as acceptor, of the range of testeddonors, Neu5Ac(a2–3)Gal(b1–4)Glc and Neu5Ac(a2–3)Gal(b1–4)Glc-ol, followed by fetuin (all of which (a2–3) linkages) andMU-aNeu5Ac, were the best donors. Compounds with (a2–6)linkages, such as Neu5Ac(a2–6)Gal(b1–4)Glc, a1-acid glycopro-tein, and bovine submandibular gland mucin, or with (a2–8)linkages, such as in colominic acid, and bovine brain ganglio-sides with (a2–3) and (a2–8) linkages, showed only minimaltransfer of sialic acid. Free lactose, lactitol, bGal1Me, a dianten-nary asialo N-glycan, and asialo-fetuin were found to be goodacceptors, whereas the Lewisx determinant was a poor sialicacid acceptor.

Trypanosoma congolense trans-sialidase: T. congolensetrans-sialidase from procyclic trypomastigotes also uses prefer-entially (a2–3)-linked sialic acid (both Neu5Ac and Neu5Gc) fortransfer to terminal bGal residues, thus generating Sia(a2–3)Gal(b1- elements.[32, 73] Of all tested donors with lactose as ac-ceptor, Neu5Ac(a2–3)Gal(b1–4)Glc and Neu5Gc(a2–3)Gal(b1–4)Glc turned out to be the best, with fetuin and MU-aNeu5Acsecond. Neu5Ac(a2–6)Gal(b1–4)Glc and Neu5Ac(a2–8)Neu5Acshowed only minimal activity, whereas bovine brain ganglio-sides, collocalia mucin and bovine submandibular gland mucindid not lead to Neu5Ac transfer. At the acceptor side, lactose,4MU-bGal, pNP-bGal, a diantennary asialo N-glycan and asialo-fetuin were good acceptors, whereas bGal1Me and the Lewisx

structure were moderate acceptors. Surprisingly, a lactose–BSAconjugate was a very moderate acceptor. Similar experimentswere repeated with two subforms of the T. congolense trans-sialidase, which markedly differed in catalytic efficiency.[33] Inthis study, it was found for both T. congolense trans-sialidasesubforms that sialic acids in (a2–6) linkage also served as rea-sonable donors (e.g. , Neu5Ac(a2–6)Gal(b1–4)Glc), althoughonly (a2–3) linkages were created in the transfer reaction. Be-sides the usual substrates (Neu5Ac(a2–3)Gal(b1–4)Glc, Neu5Ac-

(a2–3)Gal(b1–4)GlcNAc and fetuin), sialylated milk oligosac-charide mixtures and k-caseine-derived glycomacropeptidealso proved to be good donors. A series of neutral milk oligo-saccharides were good acceptors, especially when the tetra-meric T. congolense trans-sialidase subform was used, but inagreement with the findings for T. cruzi trans-sialidase, T. con-golense trans-sialidase preferred the terminal Gal(b1–4)GlcNAcsequence over the terminal Gal(b1–3)GlcNAc sequence.

trans-Sialidase Inhibitors

Inhibitors of trans-sialidases are gaining great significance aspossible drugs, for example, against Chagas disease. trans-Siali-dases are only weakly inhibited by substances that are knownas potential inhibitors of classical sialidases,[2, 5, 115, 116] such as 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu2en5Ac), 4-amino-Neu2en5Ac, 4-guanidino-Neu2en5Ac and N-(4-nitrophe-nyl)oxamic acid.[2, 31, 33] These molecules can inhibit trans-siali-dases from both the African and American pathogenic trypa-nosomes by 50 %, but only in the higher millimolar range. Inparticular, Neu2en5Ac, which is more widely used as a sialidaseinhibitor than the oxamic acid derivative, can inhibit verte-brate, bacterial and viral sialidases at micromolar concentra-tions to various extents. Free Neu5Ac is a weak inhibitor ofclassical sialidases,[2] but does not act at all on the trans-siali-dases. Correspondingly, the 2,3-difluoro derivative of Neu5Ac,by forming covalent intermediates with sialidases and trans-sialidases, was required at high concentrations (20 mm) to in-activate the enzyme completely.[71] It turned out to be possibleto make 3-fluorosialyl fluoride more specific as an inhibitor forT. cruzi trans-sialidase over the human sialidase Neu2 by incor-poration of an aromatic group at C9.[117] These substances in-teract hydrophobically with the phenyl side chain of Tyr119,and thus impair the binding of the lactoside acceptors.

Flavonoid and anthraquinone derivatives, found in a naturalproduct library, represent another new class of relativelystrong trans-sialidase inhibitors. 6-Chloro-9,10-dihydro-4,5,7-tri-hydroxy-9,10-dioxo-2-anthracenecarboxylic acid was revealedto be an excellent and quite specific trans-sialidase inhibitor at0.58 mm.[118] Sulfonamide-containing hydroxylated chalcone andquinolinone derivatives are also strong T. cruzi trans-sialidase-specific inhibitors with Ki values between 2.0 and 0.2 mm.[119]

The tested compounds did not show any significant inhibitionof human sialidase Neu2. It was suggested that the anionicform of these substances binds to the enzyme’s site that isnormally occupied by the carboxylate group of sialic acids. The2-difluoromethyl-4-nitrophenyl glycoside of aNeu5Ac is amechanism-based irreversible inhibitor of trans-sialidase withan IC50 of 0.6 mm.[120] This substance inhibits infection of mam-malian cells by T. cruzi and thus might be suitable for chemo-therapy against Chagas disease. When testing a library of 1,2,3-triazole-substituted galactose derivatives synthesised by “clickchemistry”, substances were found that inhibited T. cruzi trans-sialidase modestly, but had trypanocidal activity in cell cul-tures.[121] Chiral heterocyclic systems (2,3-dihydro-1,4-benzodi-thiine and methyl-2,3-dihydro-1,4-benzodithiine) on carbohy-drate templates were found to be strongly toxic for the blood-



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stream form of T. brucei with an IC50 value of 11 mm.[122] C-Sialo-sides, which have aromatic residues in an a-configuration,were also revealed to be promising T. cruzi trans-sialidase in-hibitors, by targeting Tyr119 and Trp312 of the acceptor bind-ing region.[123]

As trans-sialidases possess a lactose-binding site in theiractive centre, lactose and its derivatives, especially lactitol, in-hibit sialic acid transfer by the T. cruzi enzyme.[124–128] In orderto construct inhibitors of T. cruzi trans-sialidase with a longerlife-time and better bioavailability, poly(ethylene glycol) (PEG)was conjugated with lactose (reductive amination by PEGami-no), lactobionolactone (amide formation from a carboxyl func-tion and PEGamino), and Gal(b1–6)GlcN(a1–O)Bn (amide for-mation from an amino function and PEG-NHS ester).[125] Inhibi-tion by these compounds was tested against the recombinantT. cruzi trans-sialidase-mediated transfer reaction of Neu5Acfrom 3’-sialyllactose to [Glc-1-14C]lactose. Although the Gal(b1–6)GlcN(a1–O)Bn-PEG conjugate gave good results, comparablewith the free saccharide, there was rapid clearance from theblood.

Oligosaccharides from the mucins of T. cruzi can also inhibitthe transfer of sialic acid to substrates such as N-acetyllactos-amine.[105] It should be noted that heavy metal ions, especiallymercury, are potent inhibitors of trans-sialidases, which do notrequire calcium ions, unlike some sialidases.[2] Furthermore,potent inhibitors of T. cruzi propagation in vitro and in vivo areantibodies directed against trans-sialidase or the a-galactosylresidues of trypanosomal mucins. Inhibition of sialylation byanti-trans-sialidase antibodies increases the killing of the para-sites by anti-a-galactosyl antibodies, because normally themucin is heavily sialylated (ca. 107 sialic acid residues per cell),which has a strong protective effect for the trypanosome.[126]

Incubation of human myoblastoma cells (86-HG-39) with N-propionylmannosamine or other N-acyl-mannosamines wasfound to appreciably attenuate sialylation of T. cruzi parasitesand correspondingly the infection of the myoblastoma cells,because the N-acyl-modified sialic acids were transferred bytrans-sialidase at reduced rates.[129] With a newly developed a-galactose-based vaccine, complete protection of mice fromT. cruzi infection was achieved.[127] It was also possible to pro-tect mice from a lethal challenge of T. cruzi by genetic vaccina-tion with a pool of trans-sialidase genes.[128] In contrast, vacci-nation with trypanosomal mucin genes was not successful.

Biology and Pathology of trans-Sialidases

The trans-sialidases of African and American trypanosomes arestrong virulence factors involved in both Chagas disease andsleeping sickness and in a variety of animal diseases. Here, theepidemiology, vectors and medical aspects of these diseaseswill be summarised, followed by some cell biological mecha-nisms of the pathogenesis.

Chagas disease

Chagas disease, caused by T. cruzi, is endemic to Latin America:10–12 million people are affected, and about 15 000 die each

year.[130] It is an old human disease, as T. cruzi DNA was foundin ~9000-year-old mummies in Chile and Peru.[131] Recently,Chagas disease has been spread by travellers to other coun-tries, such as North America, Europe, Australia and Japan.[131] InEurope, for example, about 80 000 people are reported to carrythe parasite. T. cruzi was discovered to be the causative agentby Carlos Chagas in Brasil 102 years ago.[132] These parasitesenter the body through skin lesions and mucous membranes,and cause sometimes acute but mostly mild fever, swelling oflymph nodes and other tissues and skin lesions.[130] In many pa-tients, the parasites persist mainly in cells of the host, andcause a chronic phase, which often remains undetected forabout 30 years, until severe complications arise that can leadto sudden heart failure and death. Hypertrophy of the heartand colon (“megacolon”) are the predominant final symptomsof this disease.

The parasites are transmitted through skin lesions fromfaeces-contaminated blood-sucking insects, mainly Triatoma in-festans, T. dimidiada, and Rhodnius prolixus (the “kissing bug”).These species can transfer the protozoa between humans andfrom animals to man. Thus, Chagas disease can be consideredas a zoonosis. The animal hosts are various mammals, includ-ing rats and dogs. However, as trypanosomes can also pene-trate mucous epithelia, for example, eyes and mouth, infectionis also possible by food or, in the case of babies, at birth.

In the mammalian stage, T. cruzi mainly lives and multipliesinside the cells of many tissues. The life cycle is shown inFigure 6.[130, 133, 134] The parasites are acquired as trypomasti-gotes from the insect and penetrate into the host’s cells,where they multiply as amastygotes. After transformation intotrypomastigotes and cell lysis, they enter the blood stream andare taken up by blood-sucking insects. In the insect’s gut theybecome epimastigotes and multiply in the midgut. trans-Siali-dase is expressed in all stages, but with different enzymaticand biological activities.

Six separate groups of T. cruzi are known, and these occur indistinct areas of South and North America. The phylogeneticmapping of proteomic diversity has been mentioned above.[69]

The diagnosis of Chagas disease, including in the silentphase, is possible by using serum antibodies and PCR reac-tions. Although drugs are available for the treatment ofChagas disease, new drugs with less toxicity are under investi-gation. One target of such medicaments is believed to be thetrans-sialidase because it has been revealed to be a crucialpathogenic factor involved in spreading and survival of trypa-nosomes in the host. This research is being pursued in severallaboratories (see “trans-Sialidase Inhibitors” above).

In order to understand the biochemical role of trans-sialidasein the life-cycle of T. cruzi, the expression of this enzyme wasstudied in the insect vector and during the blood stage and in-tracellular life in the mammalian host. It was found that trans-sialidase seems to play a minor role in the insect’s gut but isvery important for replication and persistence in the host. Thisis quite in contrast to the behaviour of T. brucei, which doesnot invade cells (see below). After the blood meal, T. cruzi epi-mastigotes adhere to the endothelial cells of the insect’s pos-terior midgut, as investigated with Rhodnius prolixus.[135] Perimi-



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crovillar membrane glycoproteins of the insect’s intestine andhydrophobic proteins of the trypanosome surface seem toplay roles in this interaction. The binding can be inhibited byvarious N-acetylhexosamines, galactose, mannose and sialicacid. Neu5Ac exhibited the strongest inhibitory potency. Ac-cordingly, sialylated glycans seem to be involved in the bind-ing of trypanosomes before leaving with the insect faeces.

Studies have been reported that have questioned the role oftrans-sialidase and sialic acids for the survival of T. cruzi in theinsect.[136] The T. cruzi Silvio strain has two subpopulations, asmaller one that expresses trans-sialidase (TS+) and a largerone without enzyme activity (TS�). Surprisingly, when infectingR. prolixus with these subpopulations, the absence of trans-sial-idase activity led to a higher parasite load in the insect. Theaddition of monoclonal anti-trans-sialidase antibody to TS+

cells stimulated their multiplication, while the addition oftrans-sialidase to TS� trypanosomes reduced parasite numbersin the insect. These studies suggest a different biological rolefor TS+ and TS� subpopulations in the insect vector and verte-brate host. TS� parasites might more easily reproduce in theinsect, whereas TS+ trypanosomes are better suited to invasionof vertebrate cells.[136, 137] Thus, the polymorphism of T. cruzimight increase their fitness in different host environments. Fur-ther evidence that sialic acid is not essential for T. cruzi survival

and development in the vector is suggested by the observa-tion that the parasites grew well when feeding various insectspecies with a blood-free diet.[138]

It should be noted that during the insect stage (epimasti-gotes) the trans-sialidase of TS+ parasites lacks the carboxy ter-minus tandem repeats of the enzyme found in the vertebratestage.[136] This protein motif is expressed in the trans-sialidaseof T. cruzi trypomastigotes and influences their immunologicalproperties.

In the last 20 years, many studies have examined the role oftrans-sialidase as a strong virulence factor during the life andmultiplication of T. cruzi in the mammalian host.[49, 136] Theseshowed that sialylation events catalysed by trans-sialidase oneither parasite or host cells have many effects and finallyenable T. cruzi to survive for a long time—often several de-cades—in their hosts. Chagas disease can have a rather chroniccourse.

The trans-sialidase activity helps the parasites to better resistthe host’s innate and acquired immune systems, to enter cells,and even to manipulate the host’s immune system and thebiology of various other cell types; this enables the parasites’long residency in their hosts, and thus supplies a constant try-panosome reservoir for further distribution.[139] Some character-

Figure 6. Life cycle of the South American trypanosome T. cruzi. Trypomastigotes enter the human body through skin lesions or mucous membranes. Aftercell invasion, amastigotes multiply by binary fission, and trypomastigotes are released into the blood stream, and thereby infect other tissues; they can betaken up by Triatomine insects, which serve as vectors. Inside the vector, trypomastigotes transform into multiplicative epimastigotes and further into infec-tious trypomastigotes. Parasites are released in insect faeces, and thereby infect other mammals. Obtained from ref. [133] , Bentham Science Publishers.



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istic examples from the wealth of publications will be present-ed in the following.

The importance of trans-sialidases during the time of infec-tion of the host by T. cruzi is mirrored by a ten- to 20-foldhigher expression of this enzyme in trypomastigotes relative tothat in the epimastigotes of the vector stage.[25, 140] Intracellularamastigotes were found not to express significant trans-siali-dase activity. At the site of the insect’s bite, metacyclic trypa-nosomes (Figure 6) begin to invade many kinds of cells by, forexample, activating signal transduction pathways, hinderingapoptosis, the mobilisation of Ca2 + , and the engagement of ly-sosomes.[141–144] It was found that this process involves activeand inactive members of the trans-sialidase superfamily (GP85/TS) and sialylated members of the mucin superfamily on thetrypanosome surface, as well as different components of theextracellular matrix of the host tissue, such as fibronectin, lami-nin, collagen, galectin-3, and other substances.[142, 145, 146] Allmembers of the GP85/TS glycoprotein superfamily contain theconserved sequence VTVXNVFLYNR, of which the FLY motifwas found to strongly enhance parasite entry into mammaliancells by stimulating a signalling cascade.[147] For example, trans-sialidase triggers NF-kB activation, thereby leading to the ex-pression of adhesion molecules and thus trypanosome entryinto host cells.[148] After endocytosis, the parasitophorous va-cuole is degraded with the aid of trypanosomal proteases. Ithas been found that expression of trans-sialidase facilitatesparasite escape from its vacuole.[140] In the cytosol the trypano-somes differentiate into amastigotes and start to divide. In thiscompartment they can again differentiate into trypomastigotesbefore they are liberated into the intercellular space or theblood stream, where they infect other cells or are ingested byan insect with a blood meal,[142] thus starting a new trypano-some lifecycle.

The crucial role of trans-sialidase in the infection mechanismof T. cruzi has been well established since the finding that sialicacid deficient CHO(Lcc2) cells are relatively resistant to trypa-nosome invasion.[149] However, there are still many open ques-tions concerning how this enzyme and sialylated structures onthe parasite and host cells manage to exploit important cellu-lar processes in target tissues, and to compromise the immunesystem to favour long survival. As well as the trans-sialidase onthe parasite’s surface, the soluble enzyme shed into the bloodserum has to be considered.[141] Its peptide repeats, which arenot involved in catalysis, are players in this complex infectionmechanism too, because they generate a strong immune re-sponse.[144, 150] Stimulation of interleukin 6 (IL-6) by this proteinmotif was observed in human intestinal microvascular endo-thelial cells and in peripheral blood mononuclear cells.[151] Fur-thermore, the peptide repeats raise the half-life of trans-siali-dase activity in the blood;[152] such stabilising effects can alsobe achieved by genetic fusion of simple amino acid sequencesfrom other proteins.[152]

The following will summarise typical pathologies (mostly ofthe shed, soluble trans-sialidase), which might be involved inthe complex pathogenesis of Chagas disease. Pronouncedthrombocytopenia is observed during the acute phase of thedisease, because trans-sialidase activity reduces sialylation of

thrombocytes,[153] and this is known to lead to their rapid elimi-nation by phagocytosis.[154] Active or inactive trans-sialidasecan bind to mammalian cells, for example, heart cells or T lym-phocytes, in a lectin-like manner.[155, 156] Of special importancein the biology of Chagas disease is the binding of trypano-somes by their mucin-bound sialic acid coat to siglecs (sialicacid-binding immunoglobulin-like lectins). In mice, the bindingof sialoadhesin (siglec-1)-positive macrophages to sialylated li-gands on T. cruzi cells might be important in the initial trypo-mastigote infection.[104, 157] Siglec-E of mouse phagocytic anddendritic cells was found to be the target of sialic acid bindingfor the T. cruzi Tulahuen strain that expresses high trans-siali-dase activity (Figure 7).[104, 158] This event suppresses secretionof the pro-inflammatory cytokine IL-12 and consequent T-cellactivation. The molecular mechanism behind this phenomenonmight be disruption of cis interactions between siglecs andsialic acids on immune-competent host cells. T. cruzi trans-siali-dase activity mediates the transfer of the host cell’s sialic acidto the parasite cell by its trans-sialidase activity. The resultingtrans interaction with the parasite can impair the immune re-sponse, similarly to the inhibition of the antibacterial functionof neutrophils after the ligation to siglec-9 of sialylated glycansby group B Streptococcus.[159] Such sialic acid-mediated molecu-lar mimicry seems to be the basic mechanism by which para-sites sabotage host cytokine-secretion.[160] The outstanding roleof trans-sialidase in these events, aimed at weakening thehost’s defence system and improving the parasite’s chances ofsurvival, is mirrored by an increase in virulence of Leishmaniamajor into which trans-sialidase had been transfected.[160, 161]

Correspondingly, trans-sialidase is a kind of cytokine mimetic, a“parasitokine”.[160] Sensitising mice with small doses of trans-sialidase turned them into highly susceptible hosts for T. cruziinfection.[161] This virulence-enhancing activity can in part beexplained by polyclonal lymphocyte activation and hypergam-maglobulinemia, as well as the loss of self-tolerance of lym-phocytes observed in the acute phase of Chagas disease. Thus,trans-sialidase is a T-cell-independent B-cell mitogen and indu-ces nonspecific Ig secretion.

Figure 7. Representation of the interaction of siglecs with T. cruzi cells. Mostsiglecs are involved in cis interactions by binding to sialylated glycans oncell surfaces. Removal of sialic acid from the cell surface glycans by trypano-somal trans-sialidase leads to release of siglecs from their cis interactions.The trans-sialidase-mediated sialylation by T. cruzi parasites generates li-gands for trans interactions. Reproduced from ref. [104] with permission.Copyright : Elsevier, 2010.



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Cross-reactive epitopes located in the catalytic region oftrans-sialidase were reported to diminish the elicitation oftrans-sialidase-neutralising antibodies.[162] This cross-reactivity isbelieved to delay the humoral response that would otherwiseresult from the highly immunogenic SAPA repeats located atthe trans-sialidase C terminus.

The biology of T-cells is also strongly disturbed in T. cruzi in-fection. trans-Sialidase has been shown to lead to depletion byapoptosis of mouse thymocytes in a nurse cell complex.[163]

Supposedly, the enzyme influences sialylation of the maturingthymocytes, thus mimicking the expression of endogenoussialyltransferase. These are most important during develop-ment of the cells, although at the wrong place and wrongtime this might lead to a decrease in the supply of matureT lymphocytes. trans-Sialidase can also subvert T-cell sialylationand might compromise antigen-specific CD8+ T-cell respons-es.[164] Normally, these cytotoxic lymphocytes are desialylatedupon activation, thereby increasing their effector activity. trans-Sialidase can resialylate CD8+ T-cells and thus reduce the anti-gen-specific response of these cells, and consequently mightfavour persistence of the parasites in the host. CD43, which isinvolved in lymphocyte signalling, is a candidate target of thisdistorted sialylation. In patients with mild Chagas symptoms ahigher frequency of interferon-gamma (IFN-g)-producing T-cellsspecific for T. cruzi was identified than in individuals withsevere symptoms.[165] A multitude of HLA supertype-binder epi-topes encoded within multiple trans-sialidase genes weretested for their ability to stimulate a recall CD8+ T cell re-sponse in peripheral blood, irrespective of the HLA haplotypeof the patients. It could be shown with some of these HLA-re-stricted trans-sialidase peptides that the CD8+ T-cell compart-ment specific for T. cruzi exhibits a very low level of polyfunc-tional cytokine response characteristic for chronic T. cruzi infec-tion. These peptides might become useful for monitoringimmune competence and other parameters of disease statusin this illness.

T. cruzi follows another strategy to optimize the host–para-site equilibrium by reducing the damage of nerve cells. It hadattracted attention that in the long intermittent, often symp-tom-less phase of Chagas disease, the average numbers ofneurons in both cardiac and gastrointestinal ganglia increasewith the age of the patients, compared to those in non-Chagas individuals.[166] Also, signs of neurite development,axon regeneration and sprouting of sympathetic and parasym-pathetic nerve fibres of the heart and colon were observed inrodents. Furthermore, it was found that T. cruzi trans-sialidaseinduces neurite outgrowth of pheochromocytoma PC12 cellsand reduces apoptosis caused by growth factor deprivation.[166]

The enzyme interacts especially with ciliary neurotrophic factor(CNTF) and the leukaemia inhibitory factor (LIF), neurocyto-kines of the IL-6 family produced by Schwann cells, to rescueneurons from death. Extension of these studies into the neuro-protective effects of trans-sialidase[167, 168] confirmed that trans-sialidase can mimic neurotrophic factors such as nerve growthfactor (NGF) and brain-derived neurotrophic factor (BDNF) ; thiswas evident in cell survival responses against oxidative stress,hypoxia-reduced neurite retraction and serum/glucose depriva-

tion in various in vitro neuronal insult models. At the molecularlevel, trans-sialidase binds to the NGF tyrosine kinase receptor-A (TrkA), hydrolyses (a2–3)-linked sialyl residues of the recep-tor, and thereby leads to receptor internalisation and activa-tion, and to neuronal differentiation.[167] The interaction oftrans-sialidase with the b1-adrenergic receptor (involved inneurotransmission) is assumed to also influence the pathologyof Chagas disease.[169]

It should finally be mentioned that trans-sialidase mightgain clinical significance. The addition of trans-sialidase to cul-tured human monocytes from the peripheral blood of healthydonors or from mice inhibited proliferation of these cells, simi-lar to that observed with lymphoid organs.[170] The exploitationof trans-sialidases for immunosuppression in organ transplan-tation medicine was discussed. trans-Sialidase is also beingconsidered as a therapeutic tool for the treatment of chronicinflammatory diseases with fibrosis, for example atherosclero-sis.[171] The enzyme is possibly active against mycoplasma andchlamydia, often found as co-infecting agents in affectedtissue. This was deduced from the observation that chagasicpatients usually do not show severe atherosclerosis and pres-ent less mycoplasma in the heart than persons not infected byT. cruzi.

After the injection of T. cruzi trans-sialidase into rats infectedwith Mycoplasma pulmonis and Chlamydia pneumoniae, clinicalsymptoms such as pneumonia improved.[171] Also, in tumourcell culture trans-sialidase reduced the infection rate of M. pul-monis ; in atherosclerotic rabbits trans-sialidase together withanti-oxidants diminished intimal atherosclerotic thickness andincreased the medial layer thickness. The reason for this reduc-tion of the infectivity of M. pulmonis may be explained by a re-duction of the sialylation by trans-sialidase of cell membranesites required for Chlamydia binding and infection.

Sleeping sickness

The main aspects of this disease, caused by various subspeciesof T. brucei in Africa, affects both humans and domesticatedanimals. The animal form is called Nagana. The following infor-mation is mostly taken from very informative reviews.[133, 172, 173]

Trypanosomiasis, caused by the trypanosomes mentioned, isendemic in sub-Saharan Africa. It mainly affects remote andundeveloped rural regions, and influences the socioeconomyin villages. Nagana causes the loss of livestock. Sick people arenot often found in modern towns. Travellers can also carry thedisease to other continents, although there it cannot spreadfurther.

The prevalence of African trypanosomiasis declined duringthe last 100 years due to control, intervention programmesand medical treatment. The disease was almost eliminated inthe mid-1960s, but surged in the late 1990s. It is on the declineagain and presently about 70 000 sick people are known to beinfected, although it is difficult to identify all patients inremote areas. Sleeping sickness is a chronic, severe disease,which leads to death within three years if untreated. Varioustypes exist in different African regions. The main form occursin the central and western parts of Africa and is caused by



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T. brucei gambiense (West African trypanosomiasis). In the moresouth-eastern regions T. brucei rhodesiense prevails. It causes amore acute illness, which is fatal within weeks or months (EastAfrican trypanosomiasis).

Two stages are observed in sleeping sickness. After the biteby a tsetse fly, an inflamed area of the skin (a “chancre”) isformed, from where the trypanosomes enter the blood andlymph system and initiate the haemolymphatic stage, whichhas intermittent fever and a variety of symptoms. Later theparasites cross the blood–brain barrier and in this second,meningoencephalitic, stage can be found in the cerebrospinalfluid. In contrast to T. cruzi, these parasites usually do not entercells. Sleep disturbances and neuropsychiatric disorders pre-dominate in this phase, and the disease concludes with a ter-minal coma.

Trypanosomiasis can be diagnosed, even if asymptomatic,by immunological means, PCR, proteomic fingerprinting and,best, by microscopy search for parasites in body fluids. Variousmedicaments are available for effective treatment, althoughvaccines do not exist. The best preventive method is the re-duction of the tsetse fly bites, by control of bush growth in ag-ricultural areas, by using fly traps, insecticide spraying, and thesterile insect technique.[133, 172]

T. brucei gambiense, the main causative agent of humansleeping sickness, is transmitted mainly from human to human,while for T. brucei rhodesiense, cattle, which themselves do notfall sick, are an important reservoir. T. brucei brucei, T. congo-lense, and T. evansi are animal-only pathogenic.

In contrast to T. cruzi, the trans-sialidases of some African try-panosome species are expressed only in the insectstage.[31, 32, 133, 172] Trypanosomes from the blood meal develop inthe midgut of the tsetse fly to the procyclic form (Figure 8),which multiplies and expresses trans-sialidase. It is interestingto note (and this points to its function) that this enzyme isonly expressed in those trypanosomes that exhibit this type ofdevelopmental cycle in the vector, such as T. brucei group andT. congolense.[32] The other pathogenic (for animals only) Afri-can trypanosomes T. equiperdum and T. evansi do not expresstrans-sialidase activity. They are transferred between mammali-an hosts and lack the intestinal stage in the tsetse fly. Also,T. vivax lacks transformation in the fly’s gut and corresponding-ly trans-sialidase activity. It expresses classical sialidase activityin the host’s blood stream.[32, 38] This distribution of trans-siali-dase activity leads to the assumption of a crucial role of thisenzyme and of sialic acids for its life in the insect. Culturingtrypanosomes in sialic acid containing medium revealed sialy-lation of their cell surface, but only for (sub-)species with aninsect stage.[31, 32] The site of sialylation was found to be theglycolipid anchor of the developmentally regulated PARP (aprocyclin, “procyclic acidic repetitive protein”) in T. brucei.[31] Itwas estimated that there are five sialic acid residues in thismembrane anchor, with a polylactosamine structure.[174] Al-though trypanosomal trans-sialidases have a narrow specificityregarding the sialic acid accepting sugar and the linkageformed, they exhibit broad specificity concerning the sialicacid donor and acceptor glycans. Transfer of sialic acids fromvarious oligosaccharides, glycoconjugates, for example, serum

glycoproteins or PARP and, from cells like erythrocytes andeven the trypanosomes themselves was demonstrated notonly with solubilised enzyme but also with trans-sialidase stillbound to procyclic trypanosomes in culture.[31, 32]

It is not known whether trans-sialidase is additionally re-quired to sialylate molecules of the insect’s gut, or whethersialic acids help the trypanosomes to attach to the fly’s epithe-lia by lectins. It is more feasible, based on knowledge aboutthe manyfold functions of sialic acids,[2, 175–178] that coating ofthe trypanosomes with sialic acids might protect them duringtheir intestinal life from destruction by proteases and other di-gestive enzymes, by complement factors, immunoglobulins,trypanocidal lectins, and antimicrobial peptides.[31, 32, 179] Someof these agents might be derived from the blood meal. Fur-thermore, it is conceivable that sialic acids are required fortransformation of the procyclic trypanosomes into the epimas-tigotic and metacyclic forms in the fly’s salivarygland.[29, 31, 133, 172] The latter form is infective and is transferredinto the host’s skin during the insect’s bite. From the develop-ing shancre the trypanosomes enter the blood stream andloose trans-sialidase activity. It is, however, not known whethertrans-sialidase activity persists in the salivary gland or is re-quired during the short stay of the trypanosome in inflamedskin after the bite.

Strong evidence exists to support the theory that sialic acidsare required for trypanosome survival in fly vectors,[179] and it

Figure 8. Life cycle of the African trypanosome, T. brucei. In man the blood-stream forms show a polymorphism with A) dividing (bent arrows) slenderforms, B) intermediate forms, and C) “stumpy” forms. In the tsetse fly vector,bloodstream forms transform to D) dividing midgut forms, then to E) the mi-grating epimastigote forms, which develop in the salivary glands to F) theinfective metacyclic forms, which are injected during the next blood mealinto the mammalian host. Reproduced from refs. [172], and [181] with per-mission. Copyright : Karger Verlag.



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has been shown that disruption of the linkage of procyclin ortrans-sialidase to the glycolipid anchor, or disturbance of GPIsynthesis by gene knockout (KO) with cultured procyclic trypa-nosomes, resulted in less procyclin or trans-sialidase activity.These strongly reduced their survival in the fly’s midgut. Aftertransfection of GPI8KO cells, which lack GPI transamidase activ-ity, with a truncated gene that expressed a soluble form ofT. brucei trans-sialidase, partial restoration of the viability of theparasites was obtained, possibly a consequence of acquisitionof sialic acids by the secreted trans-sialidase from the incuba-tion medium. This confirms the hypothesis that an intact sialo-glycocalyx coat is essential for the life cycle of African trypano-somes that require a procyclic insect stage.

Therefore, inhibitors of GPI synthesis or trans-sialidase activi-ty could prevent parasite transmission, although this is ratherhypothetical as the chemotherapeutic targets are expressedonly in the tsetse fly and therefore not easily accessible.

In the host, the trypanosome starts to express variable sur-face glycoproteins (VSG), for which they possess a large genenumber.[32, 133, 172, 180] These variable antigens, which are not sialy-lated represent a potent defence mechanism that enablesthese African trypanosomes to survive in the mammalian hostand to cause the symptoms of chronic and very severe illness.

Conclusion

trans-Sialidases are rare and unique enzymes. They were dis-covered first in trypanosomes about 20 years ago, and werefound to be expressed by only a few microorganisms. Trypano-somal trans-sialidases have attracted great attention, and haveled to a wealth of literature within a short time, especially fromSouth American laboratories. They have been revealed to bestrong virulence factors in serious and wide-spread tropical dis-eases (Chagas disease and sleeping sickness), especially inmore remote and poorly developed areas of tropical SouthAmerica and Africa. These severe, often deadly diseases needmore attention, better prevention strategies, and treatmentwith less toxic medicaments. Millions of people are affected,and the diseases are, due to globalisation, slowly spreading tonon-tropical countries. In order to find trans-sialidase inhibitorsand vaccines, more basic research is required to increase in-sight into the molecular strategies by which the parasites ach-ieve long-lasting infection. The hosts serve as persistent reser-voirs for the parasite, while spreading occurs with the aid of in-sects. While the T. brucei group require trans-sialidases for para-site growth in the insect stage, the corresponding enzyme ofT. cruzi exerts its function mainly in the vertebrate host: foruptake into cells, exploitation of cellular processes, controlledweakening of the host’s immune system, and prevention ofsignificant damage of the nerve system. This in vivo propertyof T. cruzi trans-sialidases might also be considered for use inthe treatment of immunological, inflammatory or neurologicaldiseases.

trans-Sialidases also represent a new type of sialyltransfer-ases and thus enrich the sialylation strategies of microorgan-isms. This property can be exploited for the (a2–3) sialylation

of many glycan structures—a promising biotechnologicalapplication.

Acknowledgements

We thank Ralf Schwanbeck, Biochemisches Institut, Christian-Al-brechts-Universit�t Kiel, for help in preparing the figures.

Keywords: donor–acceptor systems · Chagas disease ·inhibitors · reaction mechanisms · sialic acids · sleepingsickness · trans-sialidases · trypanosomes

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DOI: 10.1002/cbic.201100421 The Chemistry and Biology of … · 2012-01-11 · and in human serum....

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