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Glycobiology Advance Access published online on June 9, 2004
Glycobiology, doi:10.1093/glycob/cwh108
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Received April 2, 2004
Revised May 21, 2004
Accepted June 2, 2004

ORIGINAL ARTICLES

NMR spectroscopic and molecular modelling investigations of the trans-sialidase from Trypanosoma cruzi

Thomas Haselhorst 1, Jennifer C. Wilson 1, Angela Liakatos 1, Milton J. Kiefel 1, Jeffrey C. Dyason 1, Mark von Itzstein 1*

1 Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Qld., 9726, Australia

* To whom correspondence should be addressed. E-mail: m.vonitzstein{at}griffith.edu.au.


  Abstract

NMR spectroscopy was used to investigate the transfer of sialic acid from a range of sialic acid donor compounds to acceptor molecules, catalysed by Trypanosoma cruzi trans-sialidase (TcTS). We demonstrate here that NMR spectroscopy is a powerful tool to monitor the trans-sialidase enzyme reaction for a variety of donor and acceptor molecules. The hydrolysis or transfer reactions that are catalysed by TcTS1 were also investigated using a range of N-acetylneuraminosyl- based donor substrates and asialo acceptor molecules. These studies showed that the synthetic N-acetylneuraminosyl donor 4-methylumbelliferyl {alpha}-D-N-acetylneuraminide (MUN) is hydrolysed by the enzyme approximately 3 to 5 times faster than either the disaccharide Neu5Ac{alpha}(2,3)Galb1Me or the trisaccharide Neu5Ac{alpha}(2,3)Lac{beta}1Me. In the transfer reaction, we show that Neu5Ac{alpha}(2,3)Lac{beta}1Me is the most favourable substrate for TcTS, and is a better substrate than the naturally-occurring N-acetylneuraminosyl donor {alpha}1-acid glycoprotein. In the case of MUN as the donor molecule, the transfer of Neu5Ac to different acceptors is significantly slower than when other N-acetylneuraminosyl donors are used. We hypothesise that when MUN is bound by the enzyme, the orientation and steric bulk of the umbelliferyl aglycon moiety may restrict the access for the correct positioning of an acceptor molecule. AutoDock studies support our hypothesis and show that the umbelliferyl aglycon moiety undergoes a strong pi-stacking interaction with Trp-312.

The binding properties of TcTS towards acceptor (lactose) and donor substrate (Neu5Ac) molecules have also been investigated using saturation transfer difference (STD) NMR experiments. These experiments have clearly demonstrated that lactose in the absence of other co-ligands does not bind to the TcTS active site or other binding domains. However, in the presence of the sialic acid donor, lactose (an asialo acceptor) was observed by NMR spectroscopy to interact with the enzyme's active site. The association of the asialo acceptor with the active site is an absolute requirement for the transfer reaction to proceed.

Key words: Trypanosoma cruzi, Chagas disease, NMR spectroscopy, sialic acid

1 TcTS - Trypanosoma cruzi trans-sialidase; MUN - 4-methylumbelliferyl {alpha}-D-N-acetylneuraminide; MU - 4-methylumbelliferyl; Neu5Ac - N-acetylneuraminic acid; STD - saturation transfer difference; NMR - nuclear magnetic resonance spectroscopy; CMP - cytidine monophosphate; PNP - para-nitrophenol; Gal - D-galactose; Lac - D-lactose; Neu5Ac2en - 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enoic acid; HPLC - high pressure liquid chromatography; Tris-tris[hydroxymethyl]aminomethane; SDS-PAGE - sodium dodecylsulfate polyacrylamide gel electrophoresis


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