Enzymatically Inactive trans-Sialidase from Trypanosoma cruzi Binds Sialyl and β-Galactopyranosyl Residues in a Sequential Ordered Mechanism

Abstract

Host/parasite interaction mediated by carbohydrate/ lectin recognition results in the attachment to and invasion of host cells and immunoregulation, enabling parasite replication and establishment of infection. Trypanosoma cruzi, the protozoan responsible for Chagas disease, expresses on its surface a family of enzymatically active and inactive trans-sialidases. The parasite uses the active trans-sialidase for glycoprotein sialylation in an unusual trans-glycosylation reaction. Inactive trans-sialidase is a sialic acid-binding lectin that costimulates host T cells through leucosialin (CD43) engagement. The co-mitogenic effect of trans-sialidase can be selectively abrogated by N-acetyllactosamine, suggesting the presence of an additional carbohydrate binding domain for galactosides, in addition to that for sialic acid. Here we investigated the interaction of inactive trans-sialidase in the presence of ␤-galactosides. By using NMR spectroscopy, we demonstrate that inactive trans-sialidase has a ␤-galactoside recognition site formed following a conformational switch induced by sialoside binding. Thus prior positioning of a sialyl residue is required for the ␤-galactoside interaction. When an appropriate sialic acid-containing molecule is available, both sialoside and ␤-galactoside are simultaneously accommodated in the inactive trans-sialidase binding pocket. This is the first report of a lectin recognizing two distinct ligands by a sequential ordered mechanism. This uncommon binding behavior may play an important role in several biological aspects of T. cruzi/host cell interaction and could shed more light into the catalytic mechanism of the sialic acid transfer reaction of enzymatically active trans-sialidase.Trypanosoma cruzi is the etiologic agent of Chagas disease or American trypanosomiasis that affects ϳ18 million people in Central and South America. Another 100 million people are at risk of infection (1). Mammalian cell invasion is crucial for T. cruzi survival (2). Elucidation of molecular components regulating the initiation of the parasitic infection is critical for understanding the pathogenesis of Chagas disease and will enable the development of novel, effective, and selective treatments.Initial communication between T. cruzi trypomastigotes and mammalian cells requires contact of soluble or membranebound parasite molecules with host ligands. T. cruzi expresses on its surface a family of glycosylphosphatidylinositol-anchored active and inactive trans-sialidase (TS) 1 proteins (3-5), which contain a Tyr or a His residue, respectively, at position 342 (4). The parasite uses active TS to sialylate its surface glycoproteins by a trans-sialidase reaction (6). Thus, TS activity is capable of extensively remodeling the T. cruzi cell surface by using host glycoconjugates as sialyl donor. Alternatively, the enzyme may sialylate host cell glycomolecules to generate receptors used by the trypanosome for adherence to and penetration of target cells. Results with sialic acid-deficient mutants of Chinese hamster ov...

Evidence of Ternary Complex Formation in Trypanosoma cruzi trans-Sialidase Catalysis

“…Using DOSY-NMR spectroscopy, we demonstrated that TS Y342H and TS, but not TS D247A , interacted with Me-Lac only after sialoside binding, which supports our MD data and previous work (6). Furthermore, the conformational switch triggered by sialic acid donor binding in TS was indeed confirmed by the differences in chemical shifts observed in 1 H-15 N heteronuclear single quantum coherence spectra.…”

“…Active site rearrangement following the binding of sialoside was further proposed for the active TcTS (23). Results of TS Y342H incubated with ␣2,6-sialyllactose in the presence of lacto-N-tetraose show that incorrect fitting of sialoside into the binding site of TS Y342H does not trigger ␤-Galp binding, which corroborates this hypothesis (6). Additionally, surface plasmon resonance results show that lactose (Lac) binds to the inactive mutant TS D59N in the presence of ␣2,3-sialyllactose (3-SL) (24).…”

“…Y342H Mutation Affects the Structure of Protein-Carbohydrate Complex-Given that histidine is a residue frequently found in reaction mechanisms, functioning either as a nucleophile (40) or as a basic catalyst (41,42), the inactivation of enzy- matic activity due to the Y342H mutation was unexpected although extensively reported (6,(43)(44)(45).…”

“…Classical (19) and hybrid quantum mechanics/MD (20) simulations combined with mutagenesis studies (19,21) have identified other key residues that probably contribute to the plasticity of the binding site. Moreover, experimental evidence for such plasticity arose from the observation that the non-functional variant of the trans-sialidase (TS Y342H ) (22) undergoes conformational changes upon sialoside binding, suggesting the opening of a second binding site that accommodates a ␤-Galp moiety (6). Active site rearrangement following the binding of sialoside was further proposed for the active TcTS (23).…”

Endothelial cell signalling induced by trans-sialidase from Trypanosoma cruzi

“…Both active and inactive forms of TS are able to translocate NF-kB into the nucleus, indicating that sialic acid transfer or hydrolysis is not required for this effect in agreement with prior work (Todeschini et al, 2004). Using STD NMR spectroscopy we previously demonstrated that riTS has two carbohydrate-binding domains: one for a2,3-linked sialosides and one carbohydrate recognition domain for b-Galp residue, which is formed only after a conformational switch, triggered by prior sialoside binding.…”