Biochemical Diversity in the Trypanosoma congolense Trans-sialidase Family

Abstract

Trans-sialidases are key enzymes in the life cycle of African trypanosomes in both, mammalian host and insect vector and have been associated with the disease trypanosomiasis, namely sleeping sickness and nagana. Besides the previously reported TconTS1, we have identified three additional active trans-sialidases, TconTS2, TconTS3 and TconTS4, and three trans-sialidase like genes in Trypanosoma congolense. At least TconTS1, TconTS2 and TconTS4 are found in the bloodstream of infected animals. We have characterised the enzymatic properties of recombinant proteins expressed in eukaryotic fibroblasts using fetuin as model blood glycoprotein donor substrate. One of the recombinant trans-sialidases, TconTS2, had the highest specific activity reported thus far with very low sialidase activity. The active trans-sialidases share all the amino acids critical for the catalytic reaction with few variations in the predicted binding site for the leaving or acceptor glycan. However, these differences cannot explain the orders of magnitudes between their transfer activities, which must be due to other unidentified structural features of the proteins or substrates selectivity. Interestingly, the phylogenetic relationships between the lectin domains correlate with their specific trans-sialylation activities. This raises the question whether and how the lectin domains regulate the trans-sialidase reaction. The identification and enzymatic characterisation of the trans-sialidase family in T. congolense will contribute significantly towards the understanding of the roles of these enzymes in the pathogenesis of Animal African Trypanosomiasis.

Carbohydrate Recognition Specificity of Trans-sialidase Lectin Domain from Trypanosoma congolense

“…The enzymatic activities of TconTSs were previously characterised [ 30 , 31 ] using recombinant proteins expressed in CHO-Lec1 cells that therefore contain N -glycans of the high-mannose-type, similar to the recombinant huS2-Fc used here in binding/inhibition assays. N -glycosylation site prediction analysis revealed 8–9 potential sites in TconTSs, and none for the attached SNAP-tag ( S3 Fig ).…”

“…In contrast to TcTS, only a few studies have investigated the enzymatic activities of T . brucei TS (TbTS) [ 26 – 29 ] and TconTS [ 6 , 30 , 31 ]. However, given the overall high primary sequence similarity of all TS at the catalytic site it can be assumed that the molecular mechanisms of the African TS are similar to those described for TcTS.…”

“…Three additional T . congolense TS family members, TconTS2, TconTS3 and TconTS4, have recently been described, sharing more then 40% amino acid identity [ 31 ]. Furthermore, kinetic data show that all TconTS investigated so far have different affinities for glycoprotein and oligosaccharide substrates [ 30 , 31 ].…”

“…Interestingly, a detailed phylogenetic analysis comparing TS domains revealed that the TconTS-CDs grouped the highly active TconTS together with the less active enzymes. In contrast, when aligning TconTS-LDs, the highly active TconTS grouped together [ 31 ], indicating a potential role of TconTS-LD in modulating enzyme activities.…”

Production, purification and crystallization of atrans-sialidase fromTrypanosoma vivax

“…The members of this enzyme class use host glycoconjugates as sialyl donors, but transfer the SA to parasite acceptor molecules rather than water. Coinciding with their highly conserved sialidase-like active site, it has been shown that trans-sialidases exhibit residual sialidase activity in the absence of the appropriate acceptor (Cross & Takle, 1993;Harrison et al, 2001;Gbem et al, 2013). In contrast to sialidases, it is interesting to note that the structural and mechanistic features of trans-sialidases remain understudied, as only a handful of these enzymes have been characterized (Cheng et al, 2008(Cheng et al, , 2009Gut et al, 2008;Newstead et al, 2004).…”