1 Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, 1428, Argentina, and 2 Instituto de Investigaciones Biotecnologicas, Universidad Nacional de Gral. San Martin, INTI, Av. General Paz entre Albarellos y Constituyentes, Edificio 24, 1650 San Martin, Buenos Aires, Argentina
Received on June 29, 2004; accepted on June 30, 2004
We read the letter of Dr. Previato with regard to one experiment in the publication by Agusti et al. (2004). As indicated by the title of the article, the aim of our work was to study the properties of lactose derivatives as substrates and inhibitors of the Trypanosoma cruzi trans-sialidase (TcTS), and is based in the 3D structure of TcTS (Amaya et al., 2003
; Buschiazzo et al., 2000
, 2002
). In these articles, it was described that glucose in the lactose also interacts with the enzyme. Thus our study was oriented in this direction for clarifying the requirements of the enzyme. As an additional experiment we decided to compare the ability of the sialylated derivatives of lactitol, lactobionic acid, and N-acetyllactosamine to act as donors of the trans-sialidase reaction. In Figure 4B of our article (Agusti et al., 2004
) it was shown that sialyllactobionic acid was completely consumed and sialyllactose and lactobionic acid were formed. Also in the case of sialyllactosamine as donor, a considerable amount of sialyllactose was formed.
It is worthwhile to point out that this experiment was not quantitative and that the amount of sialyl donor, which was obtained by collecting the fraction from the high-performance anion exchange chromatography (see Figure 2 in Agusti et al., 2004), was probably not even in the same order that the amount of lactose used. This is the likely explanation for the lack of detection of sialyllactose when sialyllactitol was used as the donor. However, the amounts of sialylated donors were similar in all the three experiments performed, allowing us to state that sialyllactitol was a poor donor when compared with sialyllactobionic acid or with the sialylated derivative of the conventional substrate N-acetyllactosamine. We repeated the reaction using quantified sialyllactitol obtained by reduction of sialyllactose, and we also observed some transference of sialic acid to lactose when both substrates of the reaction were in the same concentration range.
Our competition experiment, using equal molar concentrations of the donor sialyllactose and the acceptors N-acetyllactosamine and lactitol (see Figure 5A in Agusti et al., 2004), showed that most of the sialic acid is taken by lactitol. Also in the experiment performed by Previato, it can still be seen that a considerable amount of sialyllactitol is in the presence of a large excess of the substrate lactose. It is likely that when a low amount of sialyllactitol is present, sialic acid is rapidly transferred backward to lactitol because TcTS has a higher affinity for lactitol than for lactose as we state in the discussion of our article (Agusti et al., 2004
).
References
Agusti, R., Paris, G., Ratier, L., Frasch, A.C., and de Lederkremer, R.M. (2004) Lactose derivatives are inhibitors of Trypanosoma cruzi trans-sialidase activity toward conventional substrates in vitro and in vivo. Glycobiology, 14, 659670.
Amaya, M.F., Buschiazzo, A., Nguyen, T., and Alzari, P.M. (2003) The high resolution structures of free and inhibitor-bound Trypanosoma rangeli sialidase and its comparison with T. cruzi trans-sialidase. J. Mol. Biol., 325, 773784.[CrossRef][ISI][Medline]
Buschiazzo, A., Tavares, G.A., Campetella, O., Spinelli, S., Cremona, M.L., Paris, G., Amaya, M.F., Frasch, A.C., and Alzari, P.M. (2000) Structural basis of sialyltransferase activity in trypanosomal sialidases. EMBO J., 19, 1624.
Buschiazzo, A., Amaya, M.F., Cremona, M.L., Frasch, A.C., and Alzari, P.M. (2002) The crystal structure and mode of action of trans-sialidase, a key enzyme in Trypanosoma cruzi pathogenesis. Mol. Cell, 10, 757768.[ISI][Medline]
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