The trans‐sialidase from the african trypanosome Trypanosoma brucei

Abstract

Trypanosoma brucei is the cause of the diseases known as sleeping sickness in humans (T. brucei ssp. gambiense and ssp. rhodesiense) and ngana in domestic animals (T. brucei brucei) in Africa. Procyclic trypomastigotes, the tsetse vector stage, express a surface‐bound trans‐sialidase that transfers sialic acid to the glycosylphosphatidylinositol anchor of procyclin, a surface glycoprotein covering the parasite surface. Trans‐sialidase is a unique enzyme expressed by a few trypanosomatids that allows them to scavenge sialic acid from sialylated compounds present in the infected host. The only enzyme extensively characterized is that of the American trypanosome T. cruzi (TcTS). In this work we identified and characterized the gene encoding the trans‐sialidase from T. brucei brucei (TbTS). TbTS genes are present at a small copy number, at variance with American trypanosomes where a large gene family is present. The recombinant TbTS protein has both sialidase and trans‐sialidase activity, but it is about 10 times more efficient in transferring than in hydrolysing sialic acid. Its N‐terminus contains a region of 372 amino acids that is 45% identical to the catalytic domain of TcTS and contains the relevant residues required for catalysis. The enzymatic activity of mutants at key positions involved in the transfer reaction revealed that the catalytic sites of TcTS and TbTS are likely to be similar, but are not identical. As in the case of TcTS and TrSA, the substitution of a conserved tryptophanyl residue changed the substrate specificity rendering a mutant protein capable of hydrolysing both α‐(2,3) and α‐(2,6)‐linked sialoconjugates.

Procyclic Trypanosoma brucei Expresses Separate Sialidase and trans-Sialidase Enzymes on Its Surface Membrane

“…A related American parasite, Trypanosoma rangeli, expresses a homologous protein (TrSA) with 70% amino acid identity to TcTS, but this enzyme is devoid of trans-glycosidase activity and is strictly a hydrolase (12). In the case of T. brucei, the trans-sialidase has been purified and characterized (13)(14)(15), and information on the parasite gene encoding the protein is available (16). It should also be mentioned that the T. brucei and T. cruzi trans-sialidases have been found to be essentially trans-glycosidases.…”

“…However, in the absence of acceptors of sialic acid in the milieu, they are able to release free sialic acid and thus are, to a much lower extent, hydrolytic enzymes. A comparison of the crystal structures of TcTS and TrSA (17) and information derived from several mutagenesis approaches (16,18,19) show that trypanosomal sialidases and trans-sialidases share a similar active site architecture in which several amino acid residues critical for enzyme function are conserved. In the T. cruzi and T. rangeli enzymes, a conserved tryptophan residue (Trp-313) was shown to be implicated in the binding of substrate and to be necessary for the specificity of the enzyme for ␣-(2,3)-linkage sialic acid (18).…”

“…T. cruzi has ϳ140 transsialidase genes, half of which encode proteins lacking activity due to a substitution of Tyr-342 by a histidine residue (20). In contrast, it has been postulated that T. brucei has a much lower number of trans-sialidase genes (16). Recently, two different partial sequences of putative trans-sialidase genes were identified in another African trypanosome species, Trypanosoma congolense (21), supporting the hypothesis that the number of gene copies may be lower in African trypanosomes.…”

The Suppression of Galactose Metabolism in Procylic Form Trypanosoma brucei Causes Cessation of Cell Growth and Alters Procyclin Glycoprotein Structure and Copy Number

“…These are the largest and most complex known and are characterized by the presence of large poly-disperse branched poly-N-acetyl-lactosamine (Gal␤1-4GlcNAc)-containing side chains (with an average of about 8 -12 repeats, depending on the preparation) that can terminate with ␣2-3-linked sialic acid residues (6,20). Sialic acid is transferred from serum sialoglycoconjugates to terminal ␤-galactose residues by the action of a cell surface trans-sialidase enzyme (21)(22)(23) and trans-sialylation of surface components plays a role in the successful colonization of the tsetse fly (9). In vivo, the N termini of the procyclins are removed by tsetse fly gut proteases, and it is thought that the underlying (protease-resistant) anionic repeat units and associated GPI anchor side chains might protect the parasite from the approach tsetse fly gut hydrolases (19).…”

The surface charge of trypanosomatids

“…There are some evidences that sialic acid residues present on the surface of trypomastigote forms of T. cruzi play important roles on the invasion of host cell, on the circulation of parasites in the extracellular matrix or bloodstream and on the resistance of parasite lysis by the alternative pathway of complement (Kipnis et al 1981, Araújo-Jorge and De Souza 1984, Schenkman and Eichinger 1993. Besides the epimastigote and trypomastigote forms of T. cruzi trans-sialidase is also found in insect forms of T. brucei (Pontes de Carvalho et al 1993, Montagna et al 2002 and Endotrypanum (Medina-Acosta et al 1994).…”