Abstract
Chagas' disease, caused by Trypanosoma cruzi, affects about 18 million people in Latin America, and no effective treatment is available to date. To acquire sialic acid from the host glycoconjugates, T. cruzi expresses an unusual surface sialidase with trans-sialidase activity (TcTS) that transfers the sugar to parasite mucins. Surface sialic acid was shown to have relevant functions in protection of the parasite against the lysis by complement and in mammalian host cell invasion. The recently determined 3D structure of TcTS allowed a detailed analysis of its catalytic site and showed the presence of a lactose-binding site where the beta-linked galactose accepting the sialic acid is placed. In this article, the acceptor substrate specificity of lactose derivatives was studied by high pH anion-exchange chromatography with pulse amperometric detection. The lactose open chain derivatives lactitol and lactobionic acid, as well as other derivatives, were found to be good acceptors of sialic acid. Lactitol, which was the best of the ones tested, effectively inhibited the transfer of sialic acid to N-acetyllactosamine. Furthermore, lactitol inhibited parasite mucins re-sialylation when incubated with live trypanosomes and TcTS. Lactitol also diminished the T. cruzi infection in cultured Vero cells by 20-27%. These results indicate that compounds directed to the lactose binding site might be good inhibitors of TcTS.
Highlights
To acquire sialic acid from the host glycoconjugates, Trypanosoma cruzi expresses a glycosylphosphatidilinositolanchored trans-sialidase (TcTS) on its surface
Lactitol was able to inhibit the trans-sialidase reaction toward conventional substrates in vitro and to interfere with parasite infection in cultured cells. These results suggest that the lactose-binding site of Trypanosoma cruzi trans-sialidase (TcTS) might be used for the design of inhibitors to be tested against trypanosomal infections
Under the conditions used for high pH anion-exchange chromatography (HPAEC), the lactose formed in the reaction and the lactitol that remained not sialylated were eluted before 2 min
Summary
To acquire sialic acid from the host glycoconjugates, Trypanosoma cruzi expresses a glycosylphosphatidilinositolanchored trans-sialidase (TcTS) on its surface The sialic acid (sub)site is present in all sialidases but in TcTS is somewhat different. The sugar enters in a tilted position compared with other sialidases, partially explaining the lack of inhibition observed with Neu2en5Ac. On the other hand, the lactose (sub)site is absent in all known sialidases and is formed on binding of substrate. Binding of the sialic acid moiety of the donor substrate in the TcTS reactive center triggers a conformational switch that creates the sugar acceptor-binding site for a terminal b-galactose. On binding of the donor substrate, the Tyr119 side chain suffers a displacement leaving the sialic acid binding cleft and forming a stacking interaction with lactose, which is placed against the Trp312. In addition to creating the acceptor-binding site, a second consequence of the binding of sialic acid is a conformational switch of a Tyr residue at Glycobiology vol 14 no. In addition to creating the acceptor-binding site, a second consequence of the binding of sialic acid is a conformational switch of a Tyr residue at Glycobiology vol 14 no. 7 # Oxford University Press 2004; all rights reserved
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