Virulence Factors in Chagas Disease and Sleeping Sickness Roland Schauer*[A] and Johannis P

DOI: 10.1002/cbic.201100421 The Chemistry and Biology of Trypanosomal trans- Sialidases: Virulence Factors in Chagas Disease and Sleeping Sickness Roland Schauer*[a] and Johannis P. Kamerling[b]

2246 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2011, 12, 2246 – 2264 trans-Sialidases constitute a special group of the sialidase promise the immune system of the human or animal host. family. They occur in some trypanosome species and, in a American and African trypanosomes express trans-sialidases at unique reversible reaction, transfer sialic acids from one glyco- different stages of their vector/host development. They are sidic linkage with galactose (donor) to another galactose (ac- transmitted to humans by insect vectors (tsetse fly or other ceptor), to form (a2–3)-sialyl linkages. Trypanosomes cause insect “bug” species). trans-Sialidase activity with varying link- such devastating human diseases as Chagas disease in South age specificity has also been found in a few bacteria species America (Trypanosoma cruzi) or sleeping sickness in Africa (Try- and in human serum. trans-Sialidases are of increasing practical panosoma brucei). The trans-sialidases strongly contribute to importance for the chemo-enzymatic synthesis of sialylated the pathogenicity of the trypanosomes by scavenging sialic glycans. The search for appropriate inhibitors of trans-sialidases acids from the host or blood meal to coat the parasite surface; and vaccination strategies is intensifying, as less toxic medica- this aids their survival strategy in the insect’s intestine, and in ments for the treatment of these widespread and often chron- the blood circulation or cells of the host, and serves to com- ic tropical diseases are required.

Introduction American Chagas disease and the African sleeping sickness. Trypanosomes acquire sialic acids from their host with the aid Sialidases are glycosidases that occur in viruses, in pro- and eu- of trans-sialidases, and incorporate them into their cell-surface karyotic microorganisms and in animals, especially of the deu- glycoproteins. This, however, occurs at different developmental terostome lineage. Most of them are exo-glycosidases that re- stages of the parasites and therefore contributes to the viru- lease terminal sialic acid residues from their (a2–3), (a2–6), lence of the South American and African trypanosomes in dif- (a2–8) and/or (a2–9) glycosidic linkages.[1–3] In vertebrate cells, ferent ways. sialidases have a catabolic role, and they are engaged in specif- In this review, the occurrence and isolation of trans-sialid- ic physiological and pathological cellular events.[2,4–6] In micro- ases, their assay systems, enzyme properties, substrate specific- organisms, these enzymes often are virulence factors that ity, inhibitors, reaction mechanism and protein structures will enable spreading and infection of host cells.[7–9] They also can be described. Furthermore, the different mechanisms by which serve nutritional purposes in bacteria, by consuming sialic acid trans-sialidases contribute to the pathogenicity of the parasites from the host or from food matter in the intestine. will be discussed, as well as the epidemiology and some clini- As with most enzymatic reactions, the sialidase reaction is cal features of Chagas disease and sleeping sickness, which are theoretically reversible. This has been exploited by bacterial still considered as “neglected diseases”. and viral sialidases by choosing suitable conditions for the (a2–3)- or (a2–6)-sialylation of oligosaccharides in the final step of chemical synthesis.[2,5,9] Typical examples comprise the Occurrence and Isolation of trans-Sialidases [10,11] sialidases from Arthrobacter ureafaciens, Clostridium perfrin- The first hint of the existence of an unusual sialic acid transfer [11,12] [11–14] [12,14,15] gens, Vibrio cholerae, Salmonella typhimurium, reaction was given in 1983 when the sialic acids N-acetylneura- [16] Corynebacterium diphtheriae, (here expressed on the surface minic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) [17] of Saccharomyces cerevisiae ) and Newcastle disease were detected in T. cruzi,[24,25] and it was found that these were [11,12] virus. Also, a human plasma sialidase has been isolated not synthesised by the parasites themselves. As the molar ratio with activities for cleaving and synthesising (a2–3), (a2–6) and of these trypanosomal sialic acids corresponded to that in the [18–20] (a2–8) linkages. In a number of cases bacterial sialyltrans- incubation medium, their acquisition from this source (or in ferases have also been discovered that have sialidase/trans-sial- the case of an infection, from host glycoconjugates) was as- idase activities, for example, the recombinant sialyltransferases sumed. Furthermore, the involvement of a sialidase activity [21] from Pasteurella multocida (transfer of (a2–3) linkages), Cam- was suspected in these trypanosomes,[26] and this was later lo- [22] pylobacter jejuni (transfer of (a2–8) linkages) and calised on their cell surfaces. These observations led to the [23] Photobacterium damsela (transfer of (a2–6) linkages). identification of the trans-sialidase enzymes, first in the Ameri- Besides the sialidases and sialyltransferases mentioned can species T. cruzi,[27–29] and later in the African species above, in some trypanosomal species a particular class of siali- T. brucei[30,31] and Trypanosoma congolense.[32,33] Closer investi- dases has been discovered; these behave like normal sialidases gation revealed the presence of this enzyme in the whole if only water is present, but preferentially transfer sialyl resi- T. brucei group, that is, in several strains of T. brucei brucei, dues from one glycan chain to the terminal galactose residue T. brucei rhodesiense and T. brucei gambiense.[32] The trans-siali- of another nonsialylated oligosaccharide or glycoconjugate. Thus, they are well suited for glycan sialylation. These so-called [a] Prof. Dr. R. Schauer trans-sialidases (EC 3.2.1.18) gained great attention because Biochemisches Institut, Christian-Albrechts-Universitt Kiel they play crucial roles in the pathogenicity of some trypano- Olshausenstrasse 40, 24098 Kiel (Germany) E-mail: [email protected] some species, such as Trypanosoma cruzi in South America and [b] Prof. Dr. J. P. Kamerling the Trypanosoma brucei group in Africa. They are virulence fac- Bijvoet Center for Biomolecular Research, Utrecht University tors in widespread and devastating diseases like the South Padualaan 8, 3584 CH Utrecht (The Netherlands)

ChemBioChem 2011, 12, 2246 – 2264 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chembiochem.org 2247 R. Schauer and J. P. Kamerling dases are glycosylphosphatidylinositol (GPI)-anchored on the cell surface of the parasites. In contrast to “classical” sialidases, trypanosomal trans-sialidases catalyse the reversible transfer of preferentially (a2–3)-linked sialic acids from donor glycans directly to terminal bGal-containing acceptor molecules, thereby giving rise to new (a2–3) glycosidic linkages (Figure 1).[34,35] Thus, like classical sialidases, trans-sialidases are “retaining” sialidases (note that endo-sialidases are “inverting” sialidases[36]). In the absence of an appropriate acceptor, these enzymes act as sialidases that transfer the glycosidically linked sialic acid to a water molecule instead of to a terminal bGal residue; however, this activity is slower than that of the transfer reaction. Evi- dently, the trans-sialidases represent a kind of sialyltransferase that can form new sialic acid–glycan linkages without the need for prior energy-consuming sialic acid activation by cytidine tri- phosphate (CTP). The activity of trans-sialidases in Endotrypanum species has also been described.[37] These protozoic microorganisms are distantly related to the trypanosomes. Many other members of the kinetoplastida lack both sialidase and trans-sialidase activities, or express only sialidase, such as Trypanosoma vivax[38] and Trypanosoma rangeli[39] (for reviews, see refs. [40–42]). It is possible to discriminate between morphologically indistin- guishable trypanosomatids by measurement of trans-sialidase

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 Figure 1. A) Reversible trans-glycosylation of (a2–3)-linked N-acetylneura- University and became Emeritus Pro- minic acid between Neu5Ac(a2–3)Gal-OR1 and Neu5Ac(a2–3)Gal-OR2, cata- fessor in 2001. He has organised a lysed by trypanosomal trans-sialidases. The 4-methylumbelliferyl and p-nitro- number of scientific conferences, is author or co-author of 400 phenyl a-glycosides of N-acetylneuraminic acid are frequently used in an ir- publications, and received the 2009 Rosalind Kornfeld Award for reversible reaction. B) Trypanosomes are unable to synthesise sialic acids. However, some species express trans-sialidases that are used to transfer Lifetime Achievement in Glycobiology from the Society for Glycobi- sialic acids from host cell glycoconjugates to terminal b-galactose residues ology. of GPI-anchored glycoproteins on the pathogen surface, as shown here for mucins of T. cruzi (adapted from ref. [104], and with thanks to T. Jacobs for Hans Kamerling studied chemistry and modifications). obtained his PhD degree (1972) at Utrecht University. At this university, he has been active in the glycoscience field since 1969 (structural analysis and and sialidase activities.[43] Furthermore, the occurrence of sialic synthesis of carbohydrates), became acids on major cell-surface epitopes correlates with the expres- University Fund Professor of Organic sion of trans-sialidase.[44] Chemistry of Natural Products in 1990, In contrast to these intermolecular trans-sialidases, the exis- Professor of Bio-Organic Chemistry of tence of an intramolecular trans-sialidase that forms 2,7-anhy- Carbohydrates in 2000, and retired in dro-Neu5Ac (Neu2,7an5Ac) upon release of sialic acid from the 2009. Since 2008, he has been Honora- glycosidic linkage has been described in the leech.[45] This ry Professor of Chemical Glycobiology enzyme (neuraminidase B) has also been found in Streptococ- at the University of Groningen, and was honoured with the Dutch cus pneumoniae,[46] and has a strict specificity for (a2–3)-linked Royal Distinction “Officer in the Order of Orange-Nassau” in 2009. sialic acid substrates. The formation of Neu2,7an5Ac in addi- He is the author of over 420 scientific publications. tion to Neu5Ac was also observed by the action of the siali-

2248 www.chembiochem.org 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2011, 12, 2246 – 2264 Trypanosomal trans-Sialidases dase from Clostridium tertium (unpublished results from R.S.’s canonical b-propeller topology of the active site centre that laboratory). was similar to that of the classical sialidases, and enabled mod- The first isolation of trans-sialidase was reported from elling of the structure of T. cruzi trans-sialidase. This, together T. cruzi,[29] followed by a corresponding enzyme from cultured with mutagenesis experiments, allowed the identification of procyclic forms of the African trypanosome species T. brucei[30] the amino acids at the active site, changes to which trans- and T. congolense.[33] The enzymes are bound to cell mem- formed this sialidase into an efficient sialyltransferase.[55,56] It branes of the trypanosomes by glycolipid anchors, therefore was found that the binding site for the acceptor carbohydrate detergents are required for their solubilisation. When grown in is distinct from the donor ((a2–3)-linked sialic acid) binding cell culture, some of the enzyme is released and can be found site; this was in contrast to previously known sialidases. A tyro- in the medium. The molecular masses of the trans-sialidases sine residue (Tyr120) was found by mutagenesis to be crucial were estimated to be 160–200, 67, and 90 kDa (monomeric for binding of the acceptor substrate, and a model was provid- form) for the enzymes from T. cruzi, T. brucei and T. congolense, ed for both the hydrolysis and transfer reactions catalysed by respectively. Regarding kinetic, structural and other properties, T. cruzi trans-sialidase; this contributed to the understanding of the trans-sialidases from the two continents show striking simi- how the glycosidase structure achieves glycosyltransferase ac- larities (see below). T. congolense, however, expresses various tivity. According to the model (Figure 2), the carboxylate group trans-sialidase forms that exhibit either high or low trans-siali- of sialic acid interacts with an arginine triad (Arg35, Arg245 dase activities when compared with their sialidase activities.[33] and Arg314), a glutamic acid residue (Glu230) stabilises one of Many forms were also found in T. cruzi,[40, 47–50] translated from these arginines, an aspartic acid (Asp59) is essential for cataly- multiple trans-sialidase genes. The various trans-sialidase pro- sis by proton transfer, and a tyrosine (Tyr342) is in contact with teins expressed by T. cruzi also show variable enzyme activities the transient oxocarbonium ion at the C2 carbon of sialic acid, and trans-sialidase/sialidase activity ratios. Some of them even as is the case in the active clefts of all sialidases.[55,57] This is fol- are inactive. Expression of this enzyme also depends on the lowed by nucleophilic attack of either water or the hydroxyl trypanosome strain, and this much influences its pathogenici- group at C3 of galactose or a galactoside (Scheme 1). ty.[49] The expression of trans-sialidases also differs between parasites living in mammalian (trypomastigotes) and insect (epimastigotes) hosts. This results from the expression of two groups of non-overlapping sets of proteins translated from the complex gene family of T. cruzi, depending on the host. At the molecular level, underlying this differential expression, post- transcriptional events mainly driven by specific, highly conserved 3’ untranslated regions (3’ UTRs) were elucidated to be involved in gene expression.[50] In contrast, only a small number 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 further discussed below. trans-Sialidase Reaction Mechanism and Protein Structures These trans-sialidases share protein structures and conserved amino acids (involved in enzyme catalysis) with microbial and animal sialidases. An early observation was that the T. cruzi trans-sialidase, also called “shed acute-phase antigen” (SAPA), consists of an N-terminal half that contains the enzymatic func- Figure 2. The active site of T. cruzi trans-sialidase with the catalytic amino acid residues Tyr342, Glu230, and Asp59 (compare Scheme 1). The noncata- tion, and a highly antigenic C-terminal portion, mainly com- lytic Asp96 residue is important for stabilisation of the proton transfer from posed of a tandem series of twelve amino acid repeats, called Tyr342 to Glu230. (Note that Asp96 interacts with the acetamido group of SAPA repeats.[52] A recombinant T. cruzi trans-sialidase lacking Neu5Ac.) The relevant arginine residues Arg35, Arg245, Arg314, and the sub- the repeats was shown to retain enzymatic activity.[53] Four Asp strate N-acetylneuraminyllactose are also included. Hydrogen atoms are not shown for these residues. By courtesy of A. Roitberg; see also ref. [58]. boxes (Ser-X-Asp-X-Gly-X-Thr-Trp) are located near the N terminus of the enzyme; these seem not to be directly involved in enzyme catalysis but might preserve the protein structure, similar to the function of Asp boxes in bacterial and human siali- Energy analysis of the catalytic mechanism of T. cruzi trans- dases.[5,54] The American parasite T. rangeli secretes a sialidase sialidase revealed that ligand binding facilitates proton trans- that has no trans-sialidase activity or SAPA repeats, but has fer.[58] Computational experiments suggest a long-lived cova- 70% amino acid identity with the T. cruzi trans-sialidase.[55] X- lent intermediate in the catalytic mechanism, and identified ray crystallographic studies of the T. rangeli sialidase revealed a the Tyr342/Glu230 pair as an unusual catalytic couple

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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 intermediate structures the d+ and d charges have been omitted.

(Figure 2). The tyrosine hydroxyl proton is transferred to the ing, thereby leading to a shortening of the distance between carboxylate group of glutamate before the nucleophilic attack. the tyrosine hydroxyl group and the C2 atom of the sialic This means that binding of the substrate (i.e., formation of the acid.[57, 60] The plasticity of the catalytic pocket induced by bind- enzyme’s holo form) is necessary before the transfer reaction ing of sialic acid is essential for catalysis by trans-sialidase, and becomes energetically possible. was not observed in all the known 3D structures of classical Comparison of the crystal-derived structure of the T. cruzi prokaryotic and eukaryotic sialidases, including that of the trans-sialidase with that of T. rangeli sialidase revealed structur- enzyme from T. rangeli. The latter sialidase and T. cruzi trans- al differences in the active-site cleft, which could be responsi- sialidase also interact in different ways with the sialic acid glyc- ble for the different types of enzyme reaction. The substrate- erol side-chain. Only a single interaction was seen in the T. ran- binding pocket of T. cruzi trans-sialidase appears narrower and geli sialidase, but multiple ones occurred with the T. cruzi trans- more hydrophobic, and might favour trans-sialylation by the sialidase.[61] exclusion of water.[57] This was confirmed by molecular dynam- By using surface plasmon resonance and NMR spectroscopy, ics calculations that showed that T. cruzi trans-sialidase has a highly valuable information on how the trans-sialidase reaction very flexible, widely open catalytic cleft, mostly due to Trp312 proceeds was obtained from binding studies with either active loop motion in the apo form (Figure 3A).[59] After ligand bind- T. cruzi trans-sialidase[57, 62,63] or with inactive natural mutants.[64] ing, this flexibility and solvent exposure is much reduced. This While, for example, 3’-sialyllactose readily bound to the is in contrast to the T. rangeli sialidase, which maintains a more enzyme protein, the acceptor substrate, lactose or other asialo open catalytic cleft in both apo and holo forms, required for glycoconjugates did not interact with the enzyme at all, unless hydrolytic activity. A proline residue (Pro283), influencing the pre-incubated with the sialylated donor substrate. These stud- position of a conserved tryptophan near the centre active site, ies show that sialic acid modulates the affinity for the asialo ac- is also necessary for T. cruzi trans-sialidase activity. Remarkable ceptor substrate and that the association of the asialo receptor differences between classical sialidases and trans-sialidases are with the active site is an absolute requirement for the transfer the structural changes that occur upon absorption of sub- reaction. Investigation of crystals soaked with donor or accept- strates or Neu5Ac2en into the crystals; these become unstable or substrates confirmed that this occurs by a structural change in the case of trans-sialidase due to disturbance of the molecu- in the sequential binding of the enzyme (Figure 3B). During lar packing. The reason for this phenomenon is conformational this interaction the amino acids Trp312 and Tyr119 make stack- changes caused by the sialic acid of the donor substrates. ing interactions with lactose, and thus enable positioning of Tyr342 is sterically displaced by about 2 upon substrate bind- the HO3 group of the galactose moiety in the ternary enzyme

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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 Trp312 residues in the sialylated enzyme/lactose complex, demonstrating that Tyr119 switches from the stacked to a rotated position and Trp312 moves between stacked and open conformations. B) Selected steps from the MD trajectory of the sialylated-enzyme/lactose complex. (I) Initially Tyr119 is in the stacking position 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 the stack; 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/Rights Link.

complex, thereby allowing nucleophilic attack of the anomeric acid (a2–6)-linked to lactose cannot trigger the conformational carbon in the sialylated transition species.[57] switch required for the trans-glycosylation reaction, in contrast Based on molecular dynamics investigations into the active to (a2–3)-linked sialic acid.[64] The sequential binding of donor site plasticity of T. cruzi trans-sialidase, a dual role for Trp312 is (a2–3)-linked sialic acid and acceptor galactose and the assumed.[65] According to this, the tryptophan residue assists change in protein conformation involved in the T. cruzi trans- Tyr119 in orienting the substrate for sialylation by the trans- sialidase reaction is depicted in Figure 4. sialidase (Figure 3). In addition, Trp312 behaves like a molecu- The binding of donor substrate to T. cruzi trans-sialidase was lar shovel because of its lever-like motion. This releases the also demonstrated by saturation transfer difference (STD) NMR donor product from the active site following delivery of sialic experiments with the p-nitrophenyl glycoside of aNeu5Ac.[63] acid, and loads the acceptor substrate into position for subse- This technique allowed comparison of the rate of substrate hy- quent molecule binding. drolysis with that of sialic acid transfer. Interestingly, shortening Point mutations of these amino acids confirmed their roles of the Neu5Ac glycerol side-chain favoured hydrolysis over in catalysis and that Tyr119 is part of the second binding- transfer, whereas 9-O-acetylation of Neu5Ac had the opposite site.[66] In spite of the ablation of trans-sialidase activity, the effect. protein hydrolysed both (a2–3)- and (a2–6)-linked sialic acid. Mutants of T. rangeli sialidase have been obtained that ex- This demonstrates the requirement for precise orientation of hibit some trans-sialidase activity that arises from the forma- the substrates within the catalytic centre for the transfer reaction of a trans-sialidase-like binding-site for the acceptor tion (in contrast to hydrolysis), and the presence of distinct sugar.[67] This interaction is a prerequisite for trans-sialidase ac- binding-sites for acceptor and donor substrates. These struc- tivity, together with fine-tuning of protein–substrate interac- tural features are unique to trans-sialidases. Interestingly, sialic tions and the flexibility of crucial active-site residues.

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other trypanosomal trans-sialidases.[70] The similarities and differences 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 classical ping-pong bi-bi kinetic mechanism, in which the donor and acceptor substrates must separately bind at the same site. In the first step, an intermediate with sialic acid covalently linked to Tyr342 as the active-site nucleophile is formed, as shown with fluorinated Neu5Ac derivatives[60,65, 71,72] (and unpublished results from R.S.’s laboratory). Asp59 serves as the acid–base catalyst in this reaction. Studies with the T. rangeli sialidase, which is a “true” sialidase, have shown a corresponding mechanism,[61] thus leading to the assumption that probably all exo- sialidases (as retaining glycosidases), operate by a similar Figure 4. Proposed events in the transfer reaction of T. cruzi trans-sialidase, mechanism, with the transient formation of a sialylated to show that correct positioning of the correct sialoside donor ((a2–3) linkage, not (a2–6)) in the inactive binding site of the enzyme is necessary for enzyme conjugate. For the trans-sialidases, however, sialic acid the protein to undergo the conformational change that allows the b-galac- is transferred in a second step to galactose by essentially a re- toside acceptor to bind. Reproduced from ref. [64] with permission. Copy- verse of the preceding sialylation process, again by involving right: the American Society for Biochemistry and Molecular Biology, 2004. electrophilic migration of the anomeric centre onto the HO3 group of galactose.[60] This trans-glycosylation process might

The catalytic mechanisms of the trans-sialidase reaction of both the American and African trypanosomes (T. cruzi and the T. brucei group, respectively) presumably are similar, and a common ancestor of both trypanosome types has been predicted to have existed around 100 million years ago, and to have carried a primitive trans-sialidase gene.[68] When the African and American continents separated, the same happened with the protozoan species. Thereafter, a remarkable subspecies diversity of T. cruzi developed in South America, as was concluded on the basis of proteomic diversity.[69] A crystal structure of the T. brucei trans-sialidase is yet to be resolved, but its protein sequence from gene sequencing experiments is available, and its Figure 5. Model of the N-terminal domain of T. congolense trans-sialidase 1, according to catalytic domain shows 45 % identity with that of the the crystal structure of the T. rangeli sialidase/Neu2en5Ac complex.[55] A) Conserved corresponding region of the T. cruzi trans-sialidase; motifs in the N-terminal domain showing Asp boxes (orange) conserved in bacterial and viral sialidases, the motif LYCHLE (purple) common to all known trypanosomal trans-siali- most of the amino acids essential for the catalytic dases, and the motifs ISRVIGNS and VPVMLITHP (green), which have been found in all site of the American trans-sialidase are also con- studied African trans-sialidase genes. B) Putative active site of T. congolense trans-sialidase served.[51] Seven positions are invariant in the two 1 with the inhibitor Neu2en5Ac (yellow). Residues shown in red are conserved in T. ran- trans-sialidases. Exchange of tryptophan (Trp400) by geli sialidase, T. cruzi trans-sialidase, T. brucei trans-sialidase and T. congolense trans-sialidase 1; residues shown in blue are conserved in the three trans-sialidases, but are differ- alanine in the trans-sialidase of T. brucei abolished ent in the sialidase. The tyrosine residue (green) is unique to T. congolense trans-sialidase trans-sialylation activity but enabled the mutant to 1; at this position, T. rangeli sialidase and the trans-sialidases of T. cruzi and T. brucei con- additionally hydrolyse (a2–6) sialic acid linkages. This tain tryptophan residues. The alanine residue (light blue) differs between the American is remarkably similar to the behaviour of a T. cruzi and African trypanosomal enzymes. Residues are numbered according to the T. congolense trans-sialidase 1 sequence,[70] except where sequence information is incomplete, [66] trans-sialidase W312A mutant. the T. rangeli sialidase sequence residues R, R and D were used.[55] Reproduced from Most of the critical active-site residues common to ref. [70] with permission. Copyright: Walter de Gruyter, 2003. other trypanosomal sialidases and trans-sialidases are also conserved in the two trans-sialidases isolated from the animal-pathogenic African trypanosome T. congo- be facilitated by the longer lifetime of the sialylated tyrosine lense; these have pronounced differences in their capacity for intermediate observed in T. cruzi trans-sialidase, together with sialic acid transfer as compared with hydrolytic activity.[33] The other intriguing differences in the reaction mechanism that partial sequences obtained for these two enzymes by a PCR- make trans-sialidases unique. based approach showed 50% identity, but they are also similar to those of viral, bacterial and animal sialidases, as well as

2252 www.chembiochem.org 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2011, 12, 2246 – 2264 Trypanosomal trans-Sialidases trans-Sialidase Assays 4)Glc as a donor and 14C-lactose as an acceptor, the effects of a wide range of potential acceptors were tested. Compounds Various radioactive and non-radioactive assays have been re- containing bGalNAc, bGlc, bGlcNAc, aGal, aGlc or aMan at the ported for the detection of trans-sialidase activity.[35,73] A fluori- nonreducing end were inactive, whereas the type of linkage metric 96-well plate assay, in which 4-methylumbelliferyl b-d- (Gal(b1–3), Gal(b1–4) or Gal(b1–6)) influenced the efficiency of galactopyranoside (MU-bGal) was used as the acceptor sub- the transfer reaction. Tests with milk oligosaccharides showed strate and 3’-sialyllactose as the sialic acid donor, enabled the higher activity for (b1–4)-linked Gal than for (b1–3)-linked Gal. specific, sensitive and relatively rapid detection of enzyme ac- It should be noted that in the presence of poor acceptors, the tivity on a larger scale. The resulting Neu5Ac-(MU-bGal) was T. cruzi trans-sialidase functioned more as a sialidase than as a separated from the substrate MU-bGal by ion-exchange chro- trans-sialidase, and free Neu5Ac was generated. matography in 96-well filter plates, hydrolysed, and the liberat- In a subsequent evaluation of T. cruzi trans-sialidase isolated ed MU was measured in a fluorimeter, thus giving the trans- from trypomastigotes with a wide range of oligosaccharide, sialidase activity. glycolipid, and glycoprotein acceptors, most earlier findings In a recently described assay, synthetic benzyl b-d-Fucp-(1! were confirmed.[76] Oligosaccharides with terminal Lewisx or 6)-a-d-GlcpNAc (d-Fucp is 6-deoxy-d-Galp) was used as the ac- Lewisa epitopes were not acceptor substrates. Compared with ceptor substrate and 3’-sialyllactose or fetuin as donor. In the Neu5Ac(a2–3)Gal(b1–4)Glc, MU-aNeu5Ac and pNP-aNeu5Ac transfer reaction terminal b-galactose was created from the (pNP =p-nitrophenyl) were extremely poor sialic acid donors. donor, and this was quantified spectrophotometrically in a gal- The T. cruzi trans-sialidase has an apparent pH optimum of 7.9 actose oxidase assay (terminal b-fucose is not a substrate for and a optimal temperature of 138C. The kinetic properties of galactose oxidase).[74] This assay also allowed discrimination be- the enzyme suggested that the trans-sialylation reaction might tween trans-sialidase and sialidase activity by omitting the di- occur by a rapid-equilibrium random or steady-state ordered saccharide benzyl glycoside in the latter. mechanism. The effectiveness of immobilised T. cruzi trans-sialidase as a synthetic reagent was evaluated by using Neu5Ac- Donor and Acceptor Specificities of (a2–3)Gal(b1–4)Glc as donor and lactose–bovine serum albu- trans-Sialidases min (BSA) and Gal(b1–3)GlcNAc(b1–3)Gal(b1–4)Glc as acceptor. In order to synthesise valuable sialic acid related glycopep- Trypanosomal trans-sialidases are similar with regard to their tide and oligosaccharide probes that might be useful in fluo- substrate specificity. Neu5Ac is hydrolysed or transferred most rescence energy transfer measurements and photoaffinity la- efficiently, followed by Neu5Gc and, to much lesser degree, by belling experiments, periodate-oxidised MU-aNeu5Ac was cou- O-acetylated sialic acid.[2,75] Trypanosomal trans-sialidases have pled with different primary amines by reductive amination, broad sialyl donor and acceptor specificities, and favour oligo- and the obtained products were incubated with T. cruzi trans- saccharides and glycoproteins, as has been demonstrated with sialidase and lactose as the model acceptor.[79] The chemo-en- T. cruzi,[76–78] T. brucei [30,31, 44] and T. congolense.[32, 33] zymatic preparation of a water-soluble polyacrylamide polymer Trypanosoma cruzi trans-sialidase: One of the first papers decorated with Neu5Ac(a2–3)Gal(b1–4)GlcNAc is another nice focusing on detailed studies on the substrate specificity of the application of the useful T. cruzi trans-sialidase.[80] A further in- native T. cruzi trans-sialidase dates from 1992. The plasma teresting application is the T. cruzi trans-sialidase-catalysed membrane associated enzyme was isolated from supernatants (a2–3)-sialylation of pyridyl-2-amino-oligosaccharides with ter- of trypomastigote cultures.[34] A wide range of potential sialic minal bGal units (related to N-glycoprotein glycans) where acid donors (gangliosides, oligosaccharides) was tested with used as model compounds in N-glycan profiling studies.[81] [14C]lactose as acceptor, usually at pH 7.2 and 378C. When Another report from the same period describes the proper- comparing (a2–3)-, (a2–6)-, and (a2–9)-linked Neu5Ac, it was ties of SAPA as being not only the major sialidase but also the found that only terminal (a2–3)-linked Neu5Ac was transferred major trans-sialidase of T. cruzi.[82] In these studies it was shown in high yield to the nonreducing bGal residue to yield Neu5Ac- that 3’-sialyllactose, MU-aNeu5Ac, and foetal calf serum fetuin (a2–3)14C-Gal(b1–4)Glc only, thereby demonstrating the regio- were effective donors for the (a2–3)-sialylation of [Gal-14C]-N- specificity of the trans-sialidase. Donors containing the acetyllactosamine. Colominic acid, an (a2–8)-linked polymer of Neu5Ac(a2–3)Gal(b1–4)R sequence were better than donors Neu5Ac, was not effective. Interestingly, C7-Neu5Ac-fetuin was containing the Neu5Ac(a2–3)Gal(b1–3)R sequence. The nearby also able to act as sialic acid donor. Also, fetuin-derived O-gly- presence of a Fuc residue hampered donation by Neu5Ac. cans (alditols) and N-glycopeptides turned out to be suitable Compounds with internal Neu5Ac(a2–3)Gal(b1- units did not donors.[83] In the case of a series of gangliosides tested as po- function as donors. Modifications at C9 (9d-Neu5Ac or Neu5- tential donors, it was found that compounds with only internal Ac9Me) did not alter the transfer reaction ability, but modifica- Neu5Ac(a2–3)Gal(b1- or Neu5Ac(a2–8)Neu5Ac(a2–3)Gal(b1- tions at C4 (4d-Neu5Ac or Neu5Ac4Me), C7 (7d-Neu5Ac) and units do not function as donors. However, the conversion of C8 (8d-Neu5Ac or Neu5Ac8Me) led to inactive donors. Finally, asialo-GM1a into GD1a with a suitable sialic acid donor 4-methylumbelliferyl a-N-acetylneuraminic acid (MU-aNeu5Ac) showed that internal Gal(b1-residues can be (a2–3)-sialylated[83] also turned out to be a donor, to create Neu5Ac(a2–3)Gal(b1- (see also ref. [78]). Besides N-acetyllactosamine, the N- and O- units. By using the T. cruzi trans-sialidase-mediated formation glycans in asialofetuin and asialo bovine submaxillary gland of Neu5Ac(a2–3)14C-Gal(b1–4)Glc from Neu5Ac(a2–3)Gal(b1– mucin also served as acceptors for Neu5Ac transfer.

ChemBioChem 2011, 12, 2246 – 2264 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chembiochem.org 2253 R. Schauer and J. P. Kamerling

A real breakthrough for the application of T. cruzi trans-siali- tention was paid to pH, temperature, and incubation times, dase in the synthesis of (a2–3)-sialylated compounds was the and, under the conditions used, no hydrolysis of the donor generation of the enzyme in its recombinant form.[53,57,66, 84] As substrates was observed. In the context of investigations di- mentioned above, the recombinant construct, expressed in rected at the binding of (a2–3)-sialylated oligosaccharides to E. coli, lacks the tandem 12 amino acid repeats that are present myelin-associated glycoprotein (MAG), C7 and C8 analogues of in the SAPA antigen but not important for enzymatic activity. pNP-aNeu5Ac were prepared, and their suitability for transfer One study focused on some kinetic properties of a recombi- by recombinant T. cruzi trans-sialidase were proven.[96] In fur- nant His-tagged T. cruzi trans-sialidase expressed in E. coli with ther studies, the efficiency of pNP-aNeu5Ac, pNP-aNeu5Prop Neu5Ac(a2–3)Gal(b1–4)Glc and MU-aNeu5Ac as donors; it (Prop =propanoyl), pNP-aNeu5Ac9But (But= butanoyl), pNP- turned out that the rates of sialic acid transfer to water (hydro- aNeu5But, pNP-aNeu5iBut (iBut =isobutanoyl) and pNP- lysis) and to terminal bGal residues have a unique behaviour aNeu5Gc as donors was reported in a T. cruzi trans-sialidase- with respect to the reaction temperature and the acceptor catalysed reaction with Gal(b1–6)Glc(a1–O)Me as acceptor.[97] concentration.[85] A study of the transfer of Neu5Gc was carried The artificial donors pNP-aNeu5But and pNP-aNeu5iBut turned out with the donors Neu5Gc(a2–3)Gal(b1–4)Glc(b1– out to be completely inactive. When comparing the various

O)CH2CH2N3 and Neu5Gc(a2–3)Gal(b1–3)GlcNAc(b1– tested substrates, including pNP-aC7Neu5Ac and pNP-aC8-

O)CH2CH2N3, and with lactose, lactitol and N-acetyllactosamine Neu5Ac, it is interesting to note that the C7–C9 glycerol side as acceptors, and different yields were obtained.[75] Applica- chain of Neu5Ac is located outside the binding pocket and the tions of T. cruzi trans-sialidase for the synthesis of relevant C5 N-acetyl group is located deep within the pocket. Addition- sialyl-oligosaccharides with Neu5Ac(a2–3)Gal(b1–4)Glc and/or ally, NMR studies have been carried out on the binding of MU-aNeu5Ac as donors have been demonstrated for Neu5Ac- T. cruzi trans-sialidase to pNP-aNeu5Ac, pNP-aC8Neu5Ac, pNP- [86] (a2–3)Gal(b1–4)GlcNAc (3’-sialyl-N-acetyllactosamine), aC7Neu5Ac and pNP-aNeu5,9Ac2, and information was collect- Neu5Ac(a2–3)Gal(b1–3)GlcNAc (3’-sialyl-lacto-N-biose I)[87] and ed on the rate of substrate hydrolysis versus the rate of sialic Neu5Ac(a2–3)Gal(b1–4)Xyl-pNP.[88] In a report dealing with the acid transfer.[63] synthesis of Neu5Ac(a2–3)Gal(b1–4)Glc(b1–O)(CH2)7CH3 from In the context of studying the transfer of sialic acid from pNP-aNeu5Ac as donor and octyl b-lactoside as acceptor and host glycoconjugates to the surface GPI-anchored glycopro- recombinant T. cruzi trans-sialidase, it was stated that the teins of T. cruzi, in earlier investigations attention was paid to enzyme proved to be labile in both partially and fully purified the aGlcNAc-bound O-glycans ((a2–3)-sialylated branched gal- forms. Therefore, for routine use, crude E. coli lysate containing acto-oligomers) of the mucins from the parasitic G, Y, CL- trans-sialidase activity was preferred.[89] To monitor the hydroly- Brener, Dm28c and Tulahuen strains.[98–103] The O-glycans on sis of sialylated donors and the transfer of sialic acid from the cell surface were excellent sialic acid acceptors for trans- donor to acceptor molecules when using recombinant T. cruzi sialidase.[77,78,104,105] This was shown with, among others, azido- trans-sialidase, an extensive NMR study with a variety of modified Neu5Ac, which was readily transferred to galactose donors and acceptors was carried out.[62,90] As confirmed by residues of surface mucins of living parasites.[106] This sialic acid NMR spectroscopy, T. cruzi trans-sialidase catalyses the hydroly- derivative was found to be also transferred to a complex pat- sis of the sialyl glycosidic linkage with retention of configura- tern of glycoproteins on mouse thymocytes, lymphocytes and tion.[62,90, 91] spleen cells by parasite trans-sialidase shed into the blood The acceptor substrate specificity of T. cruzi trans-sialidase stream; CD45 was the main acceptor, followed by integrins. for lactose variants was evaluated for, among others, lactitol This sialylation of both parasite and host membrane glycopro- and lactobionic acid.[92] With Neu5Ac(a2–3)Gal(b1–4)Glc as teins by trans-sialidase has great biological implications, espe- donor, both turned out to be good acceptors for sialic acid. cially on the immune system, as will be discussed below. In Lactitol effectively inhibited the transfer of Neu5Ac to N-acetyl- this context, it is of interest to mention that sialidase-treated lactosamine; when incubated with live trypanosomes and sheep and human erythrocytes could be resialylated up to T. cruzi trans-sialidase, it also inhibited the resialylation of the 50% using T. cruzi trans-sialidase and 3’-sialyllactose, and that parasite mucins. Also Gal(b1–3)Ara and Gal(b1–3)Ara-ol were resialylation of sheep erythrocytes restored their resistance to shown to be good acceptors. Remarkably, when Neu5Ac(a2– lysis by human complement.[107] 3)lactitol, Neu5Ac(a2–3)lactobionic acid and Neu5Ac(a2–3)-N- To get a detailed insight into the terminal Gal residues that acetyllactosamine were tested as donor substrates with lactose can be sialylated, the established major O-glycan structure of as acceptor, the lactitol variant was not active at all. This con- the G strain, Galp(b1–2)[Galp(b1–3)]Galp(b1–6)[Galf(b1– trasted with a study that demonstrates that Neu5Ac(a2–3)lacti- 4)]GlcpNAc, and the corresponding alditol and benzyl a-glyco- tol can act as donor in the sialylation of lactose.[27,93] A repeti- side were synthesised and subjected to sialic acid transfer by tion of the experiments with Neu5Ac(a2-3)lactitol by the au- using recombinant T. cruzi trans-sialidase with Neu5Ac(a2– thors that found no activity showed some transfer of Neu5Ac 3)Gal(b1–4)Glc as the donor.[108] It was found that selective to lactose.[94] (a2–3)-sialylation occurred only at the less hindered terminal Series of (a2–3)-sialylated glycans were prepared with re- Galp(b1–3) unit. However, when using the terminal Galp(b1– combinant T. cruzi trans-sialidase by using Gal(b1–3)GalNAc- 2)[Galp(b1–3)]Gal fragment in its free, alditol, or benzyl a-gly- (a1–O)Ser/Thr, lactosides and lactosamide derivatives as ac- coside form, only the flexible alditol form was sialylated, at ceptors, and pNP-aNeu5Ac or MU-aNeu5Ac as donors.[95] At- either one of the two terminal Gal units (disialylation, if pres-

2254 www.chembiochem.org 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2011, 12, 2246 – 2264 Trypanosomal trans-Sialidases ent, was negligible).[109] All three compounds were effective in- (a2–3)Gal(b1–4)GlcNAc and fetuin), sialylated milk oligosac- hibitors of the T. cruzi trans-sialidase-mediated (a2–3)-sialyla- charide mixtures and k-caseine-derived glycomacropeptide tion of N-acetyllactosamine. Additionally, in these studies the also proved to be good donors. A series of neutral milk oligo- rates of sialylation of the major synthetic product, and a series saccharides were good acceptors, especially when the tetra- of related fragments (as free oligosaccharide, alditol, and meric T. congolense trans-sialidase subform was used, but in benzyl a-glycoside) were compared, with the aim of relating agreement with the findings for T. cruzi trans-sialidase, T. con- the presence of Galf with the ability of these compounds to golense trans-sialidase preferred the terminal Gal(b1–4)GlcNAc act as substrates in the T. cruzi trans-sialidase-catalysed transfer sequence over the terminal Gal(b1–3)GlcNAc sequence. reaction. Although there were differences, all fragments were [105,108,110–113] shown to be good acceptor molecules. In the re- trans-Sialidase Inhibitors combinant T. cruzi trans-sialidase-mediated (a2–3) sialylation of a synthetic T. cruzi-related glycopeptide fragment, Thr-Thr- Inhibitors of trans-sialidases are gaining great significance as (LacNAc-Thr)-Thr-Thr-Gly with Gal(b1–4)GlcNAc (=LacNAc) cou- possible drugs, for example, against Chagas disease. trans-Siali- pled a-glycosidically (natural linkage) and b-glycosidically (non- dases are only weakly inhibited by substances that are known natural substrate), with calf serum fetuin as a sialic acid donor, as potential inhibitors of classical sialidases,[2,5,115,116] such as 2- it turned out that the linkage between GlcNAc and Thr does deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu2en5Ac), 4- not present a problem for good yield.[114] amino-Neu2en5Ac, 4-guanidino-Neu2en5Ac and N-(4-nitrophe- Trypanosoma brucei trans-sialidase: The native T. brucei nyl)oxamic acid.[2,31, 33] These molecules can inhibit trans-siali- trans-sialidase from procyclic trypomastigotes has been shown dases from both the African and American pathogenic trypa- to transfer sialic acids from a variety of sialoglycoconjugates nosomes by 50 %, but only in the higher millimolar range. In (i.e., serum glycoconjugates, human and bovine erythrocytes) particular, Neu2en5Ac, which is more widely used as a sialidase to terminal bGal residues of oligosaccharides and glycoconju- inhibitor than the oxamic acid derivative, can inhibit verte- gates (i.e., sialidase-treated erythrocytes) by (a2–3) linkages.[31] brate, bacterial and viral sialidases at micromolar concentra- With [Glc-1-14C]lactose as acceptor, of the range of tested tions to various extents. Free Neu5Ac is a weak inhibitor of donors, Neu5Ac(a2–3)Gal(b1–4)Glc and Neu5Ac(a2–3)Gal(b1– classical sialidases,[2] but does not act at all on the trans-siali- 4)Glc-ol, followed by fetuin (all of which (a2–3) linkages) and dases. Correspondingly, the 2,3-difluoro derivative of Neu5Ac, MU-aNeu5Ac, were the best donors. Compounds with (a2–6) by forming covalent intermediates with sialidases and trans- linkages, such as Neu5Ac(a2–6)Gal(b1–4)Glc, a1-acid glycopro- sialidases, was required at high concentrations (20 mm)toin- tein, and bovine submandibular gland mucin, or with (a2–8) activate the enzyme completely.[71] It turned out to be possible linkages, such as in colominic acid, and bovine brain ganglio- to make 3-fluorosialyl fluoride more specific as an inhibitor for sides with (a2–3) and (a2–8) linkages, showed only minimal T. cruzi trans-sialidase over the human sialidase Neu2 by incor- transfer of sialic acid. Free lactose, lactitol, bGal1Me, a dianten- poration of an aromatic group at C9.[117] These substances in- nary asialo N-glycan, and asialo-fetuin were found to be good teract hydrophobically with the phenyl side chain of Tyr119, acceptors, whereas the Lewisx determinant was a poor sialic and thus impair the binding of the lactoside acceptors. acid acceptor. Flavonoid and anthraquinone derivatives, found in a natural Trypanosoma congolense trans-sialidase: T. congolense product library, represent another new class of relatively trans-sialidase from procyclic trypomastigotes also uses prefer- strong trans-sialidase inhibitors. 6-Chloro-9,10-dihydro-4,5,7-tri- entially (a2–3)-linked sialic acid (both Neu5Ac and Neu5Gc) for hydroxy-9,10-dioxo-2-anthracenecarboxylic acid was revealed transfer to terminal bGal residues, thus generating Sia(a2– to be an excellent and quite specific trans-sialidase inhibitor at 3)Gal(b1- elements.[32,73] Of all tested donors with lactose as ac- 0.58 mm.[118] Sulfonamide-containing hydroxylated chalcone and ceptor, Neu5Ac(a2–3)Gal(b1–4)Glc and Neu5Gc(a2–3)Gal(b1– quinolinone derivatives are also strong T. cruzi trans-sialidase- m [119] 4)Glc turned out to be the best, with fetuin and MU-aNeu5Ac specific inhibitors with Ki values between 2.0 and 0.2 m . second. Neu5Ac(a2–6)Gal(b1–4)Glc and Neu5Ac(a2–8)Neu5Ac The tested compounds did not show any significant inhibition showed only minimal activity, whereas bovine brain ganglio- of human sialidase Neu2. It was suggested that the anionic sides, collocalia mucin and bovine submandibular gland mucin form of these substances binds to the enzyme’s site that is did not lead to Neu5Ac transfer. At the acceptor side, lactose, normally occupied by the carboxylate group of sialic acids. The 4MU-bGal, pNP-bGal, a diantennary asialo N-glycan and asialo- 2-difluoromethyl-4-nitrophenyl glycoside of aNeu5Ac is a fetuin were good acceptors, whereas bGal1Me and the Lewisx mechanism-based irreversible inhibitor of trans-sialidase with [120] structure were moderate acceptors. Surprisingly, a lactose–BSA an IC50 of 0.6 mm. This substance inhibits infection of mam- conjugate was a very moderate acceptor. Similar experiments malian cells by T. cruzi and thus might be suitable for chemo- were repeated with two subforms of the T. congolense trans- therapy against Chagas disease. When testing a library of 1,2,3- sialidase, which markedly differed in catalytic efficiency.[33] In triazole-substituted galactose derivatives synthesised by “click this study, it was found for both T. congolense trans-sialidase chemistry”, substances were found that inhibited T. cruzi trans- subforms that sialic acids in (a2–6) linkage also served as rea- sialidase modestly, but had trypanocidal activity in cell cul- sonable donors (e.g., Neu5Ac(a2–6)Gal(b1–4)Glc), although tures.[121] Chiral heterocyclic systems (2,3-dihydro-1,4-benzodi- only (a2–3) linkages were created in the transfer reaction. Be- thiine and methyl-2,3-dihydro-1,4-benzodithiine) on carbohy- sides the usual substrates (Neu5Ac(a2–3)Gal(b1–4)Glc, Neu5Ac- drate templates were found to be strongly toxic for the blood-

ChemBioChem 2011, 12, 2246 – 2264 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chembiochem.org 2255 R. Schauer and J. P. Kamerling

[122] [130] stream form of T. brucei with an IC50 value of 11 mm. C-Sialo- year. It is an old human disease, as T. cruzi DNA was found sides, which have aromatic residues in an a-configuration, in ~9000-year-old mummies in Chile and Peru.[131] Recently, were also revealed to be promising T. cruzi trans-sialidase in- Chagas disease has been spread by travellers to other coun- hibitors, by targeting Tyr119 and Trp312 of the acceptor bind- tries, such as North America, Europe, Australia and Japan.[131] In ing region.[123] Europe, for example, about 80 000 people are reported to carry As trans-sialidases possess a lactose-binding site in their the parasite. T. cruzi was discovered to be the causative agent active centre, lactose and its derivatives, especially lactitol, in- by Carlos Chagas in Brasil 102 years ago.[132] These parasites hibit sialic acid transfer by the T. cruzi enzyme.[124–128] In order enter the body through skin lesions and mucous membranes, to construct inhibitors of T. cruzi trans-sialidase with a longer and cause sometimes acute but mostly mild fever, swelling of life-time and better bioavailability, poly(ethylene glycol) (PEG) lymph nodes and other tissues and skin lesions.[130] In many pa- was conjugated with lactose (reductive amination by PEGami- tients, the parasites persist mainly in cells of the host, and no), lactobionolactone (amide formation from a carboxyl func- cause a chronic phase, which often remains undetected for tion and PEGamino), and Gal(b1–6)GlcN(a1–O)Bn (amide for- about 30 years, until severe complications arise that can lead mation from an amino function and PEG-NHS ester).[125] Inhibi- to sudden heart failure and death. Hypertrophy of the heart tion by these compounds was tested against the recombinant and colon (“megacolon”) are the predominant final symptoms T. cruzi trans-sialidase-mediated transfer reaction of Neu5Ac of this disease. from 3’-sialyllactose to [Glc-1-14C]lactose. Although the Gal(b1– The parasites are transmitted through skin lesions from 6)GlcN(a1–O)Bn-PEG conjugate gave good results, comparable faeces-contaminated blood-sucking insects, mainly Triatoma in- with the free saccharide, there was rapid clearance from the festans, T. dimidiada, and Rhodnius prolixus (the “kissing bug”). blood. These species can transfer the protozoa between humans and Oligosaccharides from the mucins of T. cruzi can also inhibit from animals to man. Thus, Chagas disease can be considered the transfer of sialic acid to substrates such as N-acetyllactos- as a zoonosis. The animal hosts are various mammals, includ- amine.[105] It should be noted that heavy metal ions, especially ing rats and dogs. However, as trypanosomes can also pene- mercury, are potent inhibitors of trans-sialidases, which do not trate mucous epithelia, for example, eyes and mouth, infection require calcium ions, unlike some sialidases.[2] Furthermore, is also possible by food or, in the case of babies, at birth. potent inhibitors of T. cruzi propagation in vitro and in vivo are In the mammalian stage, T. cruzi mainly lives and multiplies antibodies directed against trans-sialidase or the a-galactosyl inside the cells of many tissues. The life cycle is shown in residues of trypanosomal mucins. Inhibition of sialylation by Figure 6.[130,133,134] The parasites are acquired as trypomasti- anti-trans-sialidase antibodies increases the killing of the para- gotes from the insect and penetrate into the host’s cells, sites by anti-a-galactosyl antibodies, because normally the where they multiply as amastygotes. After transformation into mucin is heavily sialylated (ca. 107 sialic acid residues per cell), trypomastigotes and cell lysis, they enter the blood stream and which has a strong protective effect for the trypanosome.[126] are taken up by blood-sucking insects. In the insect’s gut they Incubation of human myoblastoma cells (86-HG-39) with N- become epimastigotes and multiply in the midgut. trans-Siali- propionylmannosamine or other N-acyl-mannosamines was dase is expressed in all stages, but with different enzymatic found to appreciably attenuate sialylation of T. cruzi parasites and biological activities. and correspondingly the infection of the myoblastoma cells, Six separate groups of T. cruzi are known, and these occur in because the N-acyl-modified sialic acids were transferred by distinct areas of South and North America. The phylogenetic trans-sialidase at reduced rates.[129] With a newly developed a- mapping of proteomic diversity has been mentioned above.[69] galactose-based vaccine, complete protection of mice from The diagnosis of Chagas disease, including in the silent T. cruzi infection was achieved.[127] It was also possible to pro- phase, is possible by using serum antibodies and PCR reac- tect mice from a lethal challenge of T. cruzi by genetic vaccina- tions. Although drugs are available for the treatment of tion with a pool of trans-sialidase genes.[128] In contrast, vacci- Chagas disease, new drugs with less toxicity are under investi- nation with trypanosomal mucin genes was not successful. gation. One target of such medicaments is believed to be the trans-sialidase because it has been revealed to be a crucial Biology and Pathology of trans-Sialidases pathogenic factor involved in spreading and survival of trypanosomes in the host. This research is being pursued in several The trans-sialidases of African and American trypanosomes are laboratories (see “trans-Sialidase Inhibitors” above). strong virulence factors involved in both Chagas disease and In order to understand the biochemical role of trans-sialidase sleeping sickness and in a variety of animal diseases. Here, the in the life-cycle of T. cruzi, the expression of this enzyme was epidemiology, vectors and medical aspects of these diseases studied in the insect vector and during the blood stage and in- will be summarised, followed by some cell biological mecha- tracellular life in the mammalian host. It was found that trans- nisms of the pathogenesis. sialidase seems to play a minor role in the insect’s gut but is very important for replication and persistence in the host. This is quite in contrast to the behaviour of T. brucei, which does Chagas disease not invade cells (see below). After the blood meal, T. cruzi epi- Chagas disease, caused by T. cruzi, is endemic to Latin America: mastigotes adhere to the endothelial cells of the insect’s pos- 10–12 million people are affected, and about 15000 die each terior midgut, as investigated with Rhodnius prolixus.[135] Perimi-

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Figure 6. Life cycle of the South American trypanosome T. cruzi. Trypomastigotes enter the human body through skin lesions or mucous membranes. After cell invasion, amastigotes multiply by binary fission, and trypomastigotes are released into the blood stream, and thereby infect other tissues; they can be taken 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.

crovillar membrane glycoproteins of the insect’s intestine and and development in the vector is suggested by the observa- hydrophobic proteins of the trypanosome surface seem to tion that the parasites grew well when feeding various insect play roles in this interaction. The binding can be inhibited by species with a blood-free diet.[138] various N-acetylhexosamines, galactose, mannose and sialic It should be noted that during the insect stage (epimasti- acid. Neu5Ac exhibited the strongest inhibitory potency. Ac- gotes) the trans-sialidase of TS + parasites lacks the carboxy ter- cordingly, sialylated glycans seem to be involved in the bind- minus tandem repeats of the enzyme found in the vertebrate ing of trypanosomes before leaving with the insect faeces. stage.[136] This protein motif is expressed in the trans-sialidase Studies have been reported that have questioned the role of of T. cruzi trypomastigotes and influences their immunological trans-sialidase and sialic acids for the survival of T. cruzi in the properties. insect.[136] The T. cruzi Silvio strain has two subpopulations, a In the last 20 years, many studies have examined the role of smaller one that expresses trans-sialidase (TS+) and a larger trans-sialidase as a strong virulence factor during the life and one without enzyme activity (TS). Surprisingly, when infecting multiplication of T. cruzi in the mammalian host.[49,136] These R. prolixus with these subpopulations, the absence of trans-sial- showed that sialylation events catalysed by trans-sialidase on idase activity led to a higher parasite load in the insect. The either parasite or host cells have many effects and finally addition of monoclonal anti-trans-sialidase antibody to TS + enable T. cruzi to survive for a long time—often several de- cells stimulated their multiplication, while the addition of cades—in their hosts. Chagas disease can have a rather chronic trans-sialidase to TS trypanosomes reduced parasite numbers course. in the insect. These studies suggest a different biological role The trans-sialidase activity helps the parasites to better resist for TS+ and TS subpopulations in the insect vector and verte- the host’s innate and acquired immune systems, to enter cells, brate host. TS parasites might more easily reproduce in the and even to manipulate the host’s immune system and the insect, whereas TS + trypanosomes are better suited to invasion biology of various other cell types; this enables the parasites’ of vertebrate cells.[136,137] Thus, the polymorphism of T. cruzi long residency in their hosts, and thus supplies a constant try- might increase their fitness in different host environments. Fur- panosome reservoir for further distribution.[139] Some character- ther evidence that sialic acid is not essential for T. cruzi survival

ChemBioChem 2011, 12, 2246 – 2264 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chembiochem.org 2257 R. Schauer and J. P. Kamerling istic examples from the wealth of publications will be present- thrombocytes,[153] and this is known to lead to their rapid elimi- ed in the following. nation by phagocytosis.[154] Active or inactive trans-sialidase The importance of trans-sialidases during the time of infec- can bind to mammalian cells, for example, heart cells or T lym- tion of the host by T. cruzi is mirrored by a ten- to 20-fold phocytes, in a lectin-like manner.[155, 156] Of special importance higher expression of this enzyme in trypomastigotes relative to in the biology of Chagas disease is the binding of trypano- that in the epimastigotes of the vector stage.[25,140] Intracellular somes by their mucin-bound sialic acid coat to siglecs (sialic amastigotes were found not to express significant trans-siali- acid-binding immunoglobulin-like lectins). In mice, the binding dase activity. At the site of the insect’s bite, metacyclic trypa- of sialoadhesin (siglec-1)-positive macrophages to sialylated li- nosomes (Figure 6) begin to invade many kinds of cells by, for gands on T. cruzi cells might be important in the initial trypo- example, activating signal transduction pathways, hindering mastigote infection.[104,157] Siglec-E of mouse phagocytic and apoptosis, the mobilisation of Ca2+, and the engagement of ly- dendritic cells was found to be the target of sialic acid binding sosomes.[141–144] It was found that this process involves active for the T. cruzi Tulahuen strain that expresses high trans-siali- and inactive members of the trans-sialidase superfamily (GP85/ dase activity (Figure 7).[104,158] This event suppresses secretion TS) and sialylated members of the mucin superfamily on the of the pro-inflammatory cytokine IL-12 and consequent T-cell trypanosome surface, as well as different components of the activation. The molecular mechanism behind this phenomenon extracellular matrix of the host tissue, such as fibronectin, lami- might be disruption of cis interactions between siglecs and nin, collagen, galectin-3, and other substances.[142,145,146] All sialic acids on immune-competent host cells. T. cruzi trans-siali- members of the GP85/TS glycoprotein superfamily contain the dase activity mediates the transfer of the host cell’s sialic acid conserved sequence VTVXNVFLYNR, of which the FLY motif to the parasite cell by its trans-sialidase activity. The resulting was found to strongly enhance parasite entry into mammalian trans interaction with the parasite can impair the immune re- cells by stimulating a signalling cascade.[147] For example, trans- sponse, similarly to the inhibition of the antibacterial function sialidase triggers NF-kB activation, thereby leading to the ex- of neutrophils after the ligation to siglec-9 of sialylated glycans pression of adhesion molecules and thus trypanosome entry by group B Streptococcus.[159] Such sialic acid-mediated molecu- into host cells.[148] After endocytosis, the parasitophorous va- lar mimicry seems to be the basic mechanism by which para- cuole is degraded with the aid of trypanosomal proteases. It sites sabotage host cytokine-secretion.[160] The outstanding role has been found that expression of trans-sialidase facilitates of trans-sialidase in these events, aimed at weakening the parasite escape from its vacuole.[140] In the cytosol the trypano- host’s defence system and improving the parasite’s chances of somes differentiate into amastigotes and start to divide. In this survival, is mirrored by an increase in virulence of Leishmania compartment they can again differentiate into trypomastigotes major into which trans-sialidase had been transfected.[160,161] before they are liberated into the intercellular space or the Correspondingly, trans-sialidase is a kind of cytokine mimetic, a blood stream, where they infect other cells or are ingested by “parasitokine”.[160] Sensitising mice with small doses of trans- an insect with a blood meal,[142] thus starting a new trypano- sialidase turned them into highly susceptible hosts for T. cruzi some lifecycle. infection.[161] This virulence-enhancing activity can in part be The crucial role of trans-sialidase in the infection mechanism explained by polyclonal lymphocyte activation and hypergam- of T. cruzi has been well established since the finding that sialic maglobulinemia, as well as the loss of self-tolerance of lym- acid deficient CHO(Lcc2) cells are relatively resistant to trypa- phocytes observed in the acute phase of Chagas disease. Thus, nosome invasion.[149] However, there are still many open ques- trans-sialidase is a T-cell-independent B-cell mitogen and indu- tions concerning how this enzyme and sialylated structures on ces nonspecific Ig secretion. the parasite and host cells manage to exploit important cellular processes in target tissues, and to compromise the immune system to favour long survival. As well as the trans-sialidase on the parasite’s surface, the soluble enzyme shed into the blood serum has to be considered.[141] Its peptide repeats, which are not involved in catalysis, are players in this complex infection mechanism too, because they generate a strong immune response.[144,150] Stimulation of interleukin 6 (IL-6) by this protein motif was observed in human intestinal microvascular endothelial cells and in peripheral blood mononuclear cells.[151] Fur- thermore, the peptide repeats raise the half-life of trans-sialidase activity in the blood;[152] such stabilising effects can also be achieved by genetic fusion of simple amino acid sequences [152] from other proteins. Figure 7. Representation of the interaction of siglecs with T. cruzi cells. Most The following will summarise typical pathologies (mostly of siglecs are involved in cis interactions by binding to sialylated glycans on the shed, soluble trans-sialidase), which might be involved in cell surfaces. Removal of sialic acid from the cell surface glycans by trypano- the complex pathogenesis of Chagas disease. Pronounced somal trans-sialidase leads to release of siglecs from their cis interactions. The trans-sialidase-mediated sialylation by T. cruzi parasites generates li- thrombocytopenia is observed during the acute phase of the gands for trans interactions. Reproduced from ref. [104] with permission. disease, because trans-sialidase activity reduces sialylation of Copyright: Elsevier, 2010.

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Cross-reactive epitopes located in the catalytic region of tion in various in vitro neuronal insult models. At the molecular trans-sialidase were reported to diminish the elicitation of level, trans-sialidase binds to the NGF tyrosine kinase receptor- trans-sialidase-neutralising antibodies.[162] This cross-reactivity is A (TrkA), hydrolyses (a2–3)-linked sialyl residues of the recep- believed to delay the humoral response that would otherwise tor, and thereby leads to receptor internalisation and activa- result from the highly immunogenic SAPA repeats located at tion, and to neuronal differentiation.[167] The interaction of the trans-sialidase C terminus. trans-sialidase with the b1-adrenergic receptor (involved in The biology of T-cells is also strongly disturbed in T. cruzi in- neurotransmission) is assumed to also influence the pathology fection. trans-Sialidase has been shown to lead to depletion by of Chagas disease.[169] apoptosis of mouse thymocytes in a nurse cell complex.[163] It should finally be mentioned that trans-sialidase might Supposedly, the enzyme influences sialylation of the maturing gain clinical significance. The addition of trans-sialidase to cul- thymocytes, thus mimicking the expression of endogenous tured human monocytes from the peripheral blood of healthy sialyltransferase. These are most important during develop- donors or from mice inhibited proliferation of these cells, simi- ment of the cells, although at the wrong place and wrong lar to that observed with lymphoid organs.[170] The exploitation time this might lead to a decrease in the supply of mature of trans-sialidases for immunosuppression in organ transplan- T lymphocytes. trans-Sialidase can also subvert T-cell sialylation tation medicine was discussed. trans-Sialidase is also being and might compromise antigen-specific CD8+ T-cell respons- considered as a therapeutic tool for the treatment of chronic es.[164] Normally, these cytotoxic lymphocytes are desialylated inflammatory diseases with fibrosis, for example atherosclero- upon activation, thereby increasing their effector activity. trans- sis.[171] The enzyme is possibly active against mycoplasma and Sialidase can resialylate CD8+ T-cells and thus reduce the anti- chlamydia, often found as co-infecting agents in affected gen-specific response of these cells, and consequently might tissue. This was deduced from the observation that chagasic favour persistence of the parasites in the host. CD43, which is patients usually do not show severe atherosclerosis and pres- involved in lymphocyte signalling, is a candidate target of this ent less mycoplasma in the heart than persons not infected by distorted sialylation. In patients with mild Chagas symptoms a T. cruzi. higher frequency of interferon-gamma (IFN-g)-producing T-cells After the injection of T. cruzi trans-sialidase into rats infected specific for T. cruzi was identified than in individuals with with Mycoplasma pulmonis and Chlamydia pneumoniae, clinical severe symptoms.[165] A multitude of HLA supertype-binder epi- symptoms such as pneumonia improved.[171] Also, in tumour topes encoded within multiple trans-sialidase genes were cell culture trans-sialidase reduced the infection rate of M. pul- tested for their ability to stimulate a recall CD8+ T cell re- monis; in atherosclerotic rabbits trans-sialidase together with sponse in peripheral blood, irrespective of the HLA haplotype anti-oxidants diminished intimal atherosclerotic thickness and of the patients. It could be shown with some of these HLA-re- increased the medial layer thickness. The reason for this reduc- stricted trans-sialidase peptides that the CD8+ T-cell compart- tion of the infectivity of M. pulmonis may be explained by a re- ment specific for T. cruzi exhibits a very low level of polyfunc- duction of the sialylation by trans-sialidase of cell membrane tional cytokine response characteristic for chronic T. cruzi infec- sites required for Chlamydia binding and infection. tion. These peptides might become useful for monitoring immune competence and other parameters of disease status Sleeping sickness in this illness. T. cruzi follows another strategy to optimize the host–para- The main aspects of this disease, caused by various subspecies site equilibrium by reducing the damage of nerve cells. It had of T. brucei in Africa, affects both humans and domesticated attracted attention that in the long intermittent, often symp- animals. The animal form is called Nagana. The following infor- tom-less phase of Chagas disease, the average numbers of mation is mostly taken from very informative reviews.[133, 172,173] neurons in both cardiac and gastrointestinal ganglia increase Trypanosomiasis, caused by the trypanosomes mentioned, is with the age of the patients, compared to those in non- endemic in sub-Saharan Africa. It mainly affects remote and Chagas individuals.[166] Also, signs of neurite development, undeveloped rural regions, and influences the socioeconomy axon regeneration and sprouting of sympathetic and parasym- in villages. Nagana causes the loss of livestock. Sick people are pathetic nerve fibres of the heart and colon were observed in not often found in modern towns. Travellers can also carry the rodents. Furthermore, it was found that T. cruzi trans-sialidase disease to other continents, although there it cannot spread induces neurite outgrowth of pheochromocytoma PC12 cells further. and reduces apoptosis caused by growth factor deprivation.[166] The prevalence of African trypanosomiasis declined during The enzyme interacts especially with ciliary neurotrophic factor the last 100 years due to control, intervention programmes (CNTF) and the leukaemia inhibitory factor (LIF), neurocyto- and medical treatment. The disease was almost eliminated in kines of the IL-6 family produced by Schwann cells, to rescue the mid-1960s, but surged in the late 1990s. It is on the decline neurons from death. Extension of these studies into the neuro- again and presently about 70000 sick people are known to be protective effects of trans-sialidase[167,168] confirmed that trans- infected, although it is difficult to identify all patients in sialidase can mimic neurotrophic factors such as nerve growth remote areas. Sleeping sickness is a chronic, severe disease, factor (NGF) and brain-derived neurotrophic factor (BDNF); this which leads to death within three years if untreated. Various was evident in cell survival responses against oxidative stress, types exist in different African regions. The main form occurs hypoxia-reduced neurite retraction and serum/glucose depriva- in the central and western parts of Africa and is caused by

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T. brucei gambiense (West African trypanosomiasis). In the more south-eastern regions T. brucei rhodesiense prevails. It causes a more acute illness, which is fatal within weeks or months (East African trypanosomiasis). Two stages are observed in sleeping sickness. After the bite by a tsetse fly, an inflamed area of the skin (a “chancre”) is formed, from where the trypanosomes enter the blood and lymph system and initiate the haemolymphatic stage, which has intermittent fever and a variety of symptoms. Later the parasites cross the blood–brain barrier and in this second, meningoencephalitic, stage can be found in the cerebrospinal fluid. In contrast to T. cruzi, these parasites usually do not enter cells. Sleep disturbances and neuropsychiatric disorders pre- dominate in this phase, and the disease concludes with a terminal coma. Trypanosomiasis can be diagnosed, even if asymptomatic, by immunological means, PCR, proteomic fingerprinting and, best, by microscopy search for parasites in body fluids. Various medicaments are available for effective treatment, although vaccines 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 the sterile insect technique.[133,172] T. brucei gambiense, the main causative agent of human Figure 8. Life cycle of the African trypanosome, T. brucei. In man the blood- sleeping sickness, is transmitted mainly from human to human, stream forms show a polymorphism with A) dividing (bent arrows) slender forms, B) intermediate forms, and C) “stumpy” forms. In the tsetse fly vector, while for T. brucei rhodesiense, cattle, which themselves do not bloodstream forms transform to D) dividing midgut forms, then to E) the mi- fall sick, are an important reservoir. T. brucei brucei, T. congo- grating epimastigote forms, which develop in the salivary glands to F) the lense,andT. evansi are animal-only pathogenic. infective metacyclic forms, which are injected during the next blood meal In contrast to T. cruzi, the trans-sialidases of some African try- into the mammalian host. Reproduced from refs. [172], and [181] with permission. Copyright: Karger Verlag. panosome species are expressed only in the insect stage.[31,32,133,172] Trypanosomes from the blood meal develop in the midgut of the tsetse fly to the procyclic form (Figure 8), glycoproteins or PARP and, from cells like erythrocytes and which multiplies and expresses trans-sialidase. It is interesting even the trypanosomes themselves was demonstrated not to note (and this points to its function) that this enzyme is only with solubilised enzyme but also with trans-sialidase still only expressed in those trypanosomes that exhibit this type of bound to procyclic trypanosomes in culture.[31,32] developmental cycle in the vector, such as T. brucei group and It is not known whether trans-sialidase is additionally re- T. congolense.[32] The other pathogenic (for animals only) Afri- quired to sialylate molecules of the insect’s gut, or whether can trypanosomes T. equiperdum and T. evansi do not express sialic acids help the trypanosomes to attach to the fly’s epithe- trans-sialidase activity. They are transferred between mammali- lia by lectins. It is more feasible, based on knowledge about an hosts and lack the intestinal stage in the tsetse fly. Also, the manyfold functions of sialic acids,[2,175–178] that coating of T. vivax lacks transformation in the fly’s gut and corresponding- the trypanosomes with sialic acids might protect them during ly trans-sialidase activity. It expresses classical sialidase activity their intestinal life from destruction by proteases and other di- in the host’s blood stream.[32,38] This distribution of trans-siali- gestive enzymes, by complement factors, immunoglobulins, dase activity leads to the assumption of a crucial role of this trypanocidal lectins, and antimicrobial peptides.[31,32, 179] Some enzyme and of sialic acids for its life in the insect. Culturing of these agents might be derived from the blood meal. Fur- trypanosomes in sialic acid containing medium revealed sialy- thermore, it is conceivable that sialic acids are required for lation of their cell surface, but only for (sub-)species with an transformation of the procyclic trypanosomes into the epimas- insect stage.[31,32] The site of sialylation was found to be the tigotic and metacyclic forms in the fly’s salivary glycolipid anchor of the developmentally regulated PARP (a gland.[29,31, 133,172] The latter form is infective and is transferred procyclin, “procyclic acidic repetitive protein”) in T. brucei.[31] It into the host’s skin during the insect’s bite. From the develop- was estimated that there are five sialic acid residues in this ing shancre the trypanosomes enter the blood stream and membrane anchor, with a polylactosamine structure.[174] Al- loose trans-sialidase activity. It is, however, not known whether though trypanosomal trans-sialidases have a narrow specificity trans-sialidase activity persists in the salivary gland or is re- regarding the sialic acid accepting sugar and the linkage quired during the short stay of the trypanosome in inflamed formed, they exhibit broad specificity concerning the sialic skin after the bite. acid donor and acceptor glycans. Transfer of sialic acids from Strong evidence exists to support the theory that sialic acids various oligosaccharides, glycoconjugates, for example, serum are required for trypanosome survival in fly vectors,[179] and it

2260 www.chembiochem.org 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2011, 12, 2246 – 2264 Trypanosomal trans-Sialidases has been shown that disruption of the linkage of procyclin or of many glycan structures—a promising biotechnological trans-sialidase to the glycolipid anchor, or disturbance of GPI application. synthesis by gene knockout (KO) with cultured procyclic trypanosomes, resulted in less procyclin or trans-sialidase activity. These strongly reduced their survival in the fly’s midgut. After Acknowledgements transfection of GPI8KO cells, which lack GPI transamidase activity, with a truncated gene that expressed a soluble form of We thank Ralf Schwanbeck, Biochemisches Institut, Christian-Al- T. brucei trans-sialidase, partial restoration of the viability of the brechts-Universitt Kiel, for help in preparing the figures. parasites was obtained, possibly a consequence of acquisition of sialic acids by the secreted trans-sialidase from the incuba- Keywords: donor–acceptor systems · Chagas disease · tion medium. This confirms the hypothesis that an intact sialo- inhibitors · reaction mechanisms · sialic acids · sleeping glycocalyx coat is essential for the life cycle of African trypano- sickness · trans-sialidases · trypanosomes somes that require a procyclic insect stage. Therefore, inhibitors of GPI synthesis or trans-sialidase activity could prevent parasite transmission, although this is rather [1] R. Schauer, Cell Biology Monographs, Vol. 10: Sialic Acids: Chemistry, Me- tabolism and Function, Springer, Wien, 1982. hypothetical as the chemotherapeutic targets are expressed [2] R. Schauer, J. P. Kamerling in New Comprehensive Biochemistry, Vol. 29b: only in the tsetse fly and therefore not easily accessible. Glycoproteins II (Eds.: J. Montreuil, J. F. G. Vliegenthart, H. Schachter), In the host, the trypanosome starts to express variable sur- Elsevier, Amsterdam, 1997, pp. 243– 402. face glycoproteins (VSG), for which they possess a large gene [3] J. P. Kamerling, G. J. Gerwig in Comprehensive Glycoscience: From Chemistry to Systems Biology, Vol. 2 (Eds.: J. P. Kamerling, G.-J. Boons, [32,133,172,180] number. These variable antigens, which are not sialy- Y. C. Lee, A. Suzuki, N. Taniguchi, A. G. J. Voragen), Elsevier, Amsterdam, lated represent a potent defence mechanism that enables 2007, pp. 1– 68. these African trypanosomes to survive in the mammalian host [4] A. P. Corfield, R. Schauer in Cell Biology Monographs, Vol. 10: Sialic Acid: and to cause the symptoms of chronic and very severe illness. Chemistry, Metabolism and Function (Ed.: R. Schauer), Springer, Wien, 1982, pp. 195–261. [5] E. Monti, E. Bonten, A. D’Azzo, R. Bresciani, B. Venerando, G. Borsani, R. Schauer, G. Tettamanti, Adv. Carbohydr. Chem. Biochem. 2010, 64, 403– 479. Conclusion [6] T. Miyagi, Trends Glycosci. Glycotechnol. 2010, 22, 162– 172. [7] T. Corfield, Glycobiology 1992, 2, 509 –521. trans-Sialidases are rare and unique enzymes. They were dis- [8] E. R. Vimr, K. A. Kalivoda, E. L. Deszo, S. M. Steenbergen, Microbiol. Mol. covered first in trypanosomes about 20 years ago, and were Biol. Rev. 2004, 68, 132– 153. found to be expressed by only a few microorganisms. Trypano- [9] S. Kim, D.-B. Oh, H. A. Kang, O. Kwon, Appl. Microbiol. Biotechnol. 2011, somal trans-sialidases have attracted great attention, and have 91, 1– 15. [10] I. Maru, Y. Ohta, K. Okamoto, S. Suzuki, K. Kakehi, Y. 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Bovin, Bioorg. site growth in the insect stage, the corresponding enzyme of Med. Chem. Lett. 2004, 14, 5161 – 5164. [21] H. Yu, H. Chokhawala, R. Karpel, H. Yu, B. Wu, J. Zhang, Y. Zhang, Q. T. cruzi exerts its function mainly in the vertebrate host: for Jia, X. Chen, J. Am. Chem. Soc. 2005, 127, 17618 –17619. uptake into cells, exploitation of cellular processes, controlled [22] J. Cheng, H. Yu, K. Lau, S. Huang, H. A. Chokhawala, Y. Li, V. K. Tiwari, X. weakening of the host’s immune system, and prevention of Chen, Glycobiology 2008, 18, 686– 697. significant damage of the nerve system. This in vivo property [23] J. Cheng, S. Huang, H. Yu, Y. Li, K. Lau, X. Chen, Glycobiology 2010, 20, 260 –268. of T. cruzi trans-sialidases might also be considered for use in [24] R. Schauer, G. Reuter, H. Mhlpfordt, A. F. B. Andrade, M. E. A. Pereira, the treatment of immunological, inflammatory or neurological Hoppe-Seyler’s Z. Physiol. Chem. 1983, 364, 1053 –1057. diseases. [25] M. E. A. 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