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ABSTRACT: Trypanosoma cruzi expresses a trans-sialidase on its surface, which catalyzes the transfer of sialic acid from mammalian host glycans to its own surface glycoproteins. It has been proposed that the enzyme consists of three domains prior to a long C-terminal repeating sequence that is not required for enzyme activity. The first of these domains shares significant sequence identity with bacterial sialidases which catalyse the hydrolysis of sialic acid. Here we report the sequence of the N-terminal domains of the TS19y trans-sialidase gene, which was expressed in bacteria with the same specific activity as natural enzyme of T. cruzi. Various deletion mutants of TS19y, without the C-terminal tandem repeat, have been cloned and expressed and their trans-sialidase and sialidase activities measured. These experiments show that all three N-terminal domains are required for full trans-sialidase activity, though only the first is necessary for sialidase activity. Some transferase activity is observed, however, even with the shortest construct comprising the first N-terminal domain. Deletion mutants to probe the role of the N-terminal residues of the first domain suggest that the first 33 residues are also required for trans-sialidase activity, but not for sialidase activity. Molecular modelling of the first N-terminal domain of TS19y based on our structures of bacterial sialidases and site-directed mutations suggests the location of a galactose-binding site within this domain.
Biochemical and Biophysical Research Communications 09/1999; 262(2):549-56. · 2.48 Impact Factor
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ABSTRACT: Trypanosoma cruzi, the etiological agent of Chagas' disease, expresses a trans-sialidase at highest levels in infective trypomastigotes, where it attaches to the plasma membrane by a glycophosphoinositol linkage. Bound enzyme sheds into the extracellular milieu in a soluble form. Experiments performed in vitro suggest that the trans-sialidase participates in several parameters of T. cruzi-host interactions, like cell adhesion and complement resistance. However, the role that membrane-bound and soluble trans-sialidase plays in the infection of mammals is not understood. To begin to study the role the enzyme may play in vivo, T. cruzi trypomastigotes were inoculated subcutaneously into mice that had been sensitized for various times with the purified protein. A single dose of either endogenous or recombinant trans-sialidase injected into the connective tissues of BALB/c mice greatly enhanced parasitemia and mortality. Maximum enhancement was achieved with 1-2-h priming. Injection of the enzyme after the parasites had been established in the inoculation site had little, if any, consequence in modifying virulence. The enhancement did not seem to be through a direct effect of the enzyme on trypomastigote-host cell interactions because it occurred when the sites of trans-sialidase sensitization and parasite inoculation were physically separate. Rather, virulence enhancement seemed to depend on inflammatory cells, since priming with trans-sialidase had no significant effect in severe combined immunodeficiency mice, which lack functional T and B lymphocytes. However, antibody response to T. cruzi in the trans-sialidase-primed BALB/c mice was the same as in the control animals. Virulence enhancement was specific for the trans-sialidase because it did not occur in mice primed with Newcastle virus sialidase, which has the same substrate specificity as the T. cruzi enzyme, or with the sialidase from the bacterium Vibrio cholerae, whose substrate specificity is broader than the trypanosome sialidase. Furthermore, no enhancement of virulence occurred after sensitization with another adhesion protein (penetrin) purified from T. cruzi trypomastigotes and engineered bacteria, nor with bacterial lipopolysaccharide. The virulence-promoting activity of soluble trans-sialidase in the mouse model may be physiologically relevant because it was achieved with tiny doses, approximately 1-2 microgram/kg, raising the possibility that neutralization of the enzyme with specific probes could impair the development of Chagas' disease. In fact, a monoclonal antibody specific for the tandem repeat in the trans-sialidase COOH terminus enhanced infection of BALB/c mice, in agreement with earlier experiments in vitro, whereas antibodies against an amino acid sequence in the Cys region had the opposite effect.
Journal of Experimental Medicine 06/1995; 181(5):1693-703. · 13.85 Impact Factor
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ABSTRACT: Trypanosoma cruzi attaches and invades a large variety of mammalian cells. The nature of the cell receptors and of the corresponding parasite counter-receptors that mediate T. cruzi-host cell interaction are not known. Three sialic acid-deficient mutants of Chinese hamster ovary (CHO) cells were used to probe the role of host sialyl residues in T. cruzi infection. All three mutants supported adhesion and infection to a much lower extent than the parental CHO cells. One of the mutants, Lec2, contains sugar chains terminating in non-reducing beta Gal residues, which are acceptors for sialylation by the T. cruzi trans-sialidase. Re-sialylation of Lec2 cells restored T. cruzi adhesion and invasion to about the same extent as wild-type cells. Digestion of wild-type cells with bacterial sialidase reduced T. cruzi interaction but after re-sialylation, the cells were almost as good as control, naturally sialylated parental cells. These results suggest that T. cruzi recognizes sialyl residues on the surface of host cells during invasion. On the other hand, affinity-purified trans-sialidase blocked T. cruzi adherence and invasion of sialylated cells, and had no effect on parasite interaction with sialic acid-deficient Lec2 mutant. Furthermore, 2,3-sialyllactose, a substrate for the trans-sialidase, competitively inhibited T. cruzi invasion of sialylated parental K1 cells, but 2,6-sialyllactose, which does not react with the trans-sialidase, was without effect, as were other sugars that do not contain alpha 2,3 sialyl residues. These results suggest that the trans-sialidase functions as a counter-receptor for trypomastigote binding to alpha 2,3-sialyl receptors on host cells as a prelude to T. cruzi invasion.
Molecular and Biochemical Parasitology 07/1993; 59(2):243-52. · 2.55 Impact Factor
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ABSTRACT: The substrate specificity, physico-chemical, and kinetic properties of the trans-sialidase from Trypanosoma cruzi have been investigated. The enzyme demonstrates activity towards a wide range of saccharide, glycolipid, and glycoprotein acceptors which terminate with a beta-linked galactose residue, and synthesizes exclusively an alpha 2-3 sialosidic linkage. Oligosaccharides which terminate in Gal beta 1-4(Fuc alpha 1-3)GlcNAc, Gal beta 1-3(Fuc alpha 1-4)GlcNAc, or Gal alpha 1- are not acceptor-substrates. The enzyme utilizes alpha 2,3-linked sialic acid when the donor species is an oligosaccharide and can also transfer, at a low rate, sialic acid from synthetic alpha-sialosides such as p-nitrophenyl-alpha-N-acetylneuraminic acid, but NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)Glc is not a donor-substrate. The trans-sialidase has an apparent pH optimum of 7.9 and a temperature optimum of 13 degrees C. The kinetic properties of the enzyme suggest that the trans-sialylation reaction may occur via a rapid equilibrium random or steady-state ordered mechanism. A method for immobilizing the enzyme is described together with examples of its use for the synthesis of oligosaccharide and glycoprotein precursors of sialyl-Lewis and sialyl-Lewis.
Journal of Biological Chemistry 06/1993; 268(13):9886-91. · 4.77 Impact Factor
