Biochemical characterisation of trans-sialidases from Trypanosoma congolense

Abstract

The work compiled here aimed at biochemically characterising trans-sialidase (TS) genes from Trypanosoma congolense, the most prevalent causative agent of animal African trypanosomiasis, also called nagana. Using published partial TS sequences as starting queries, database at the Welcome Trust Sanger Institute (WTSI) was queried and TS1 genes identified. This is a multi-copy gene group comprising 11 members. Members were cloned and recombinant protein expressed in fibroblasts and purified proteins assayed for enzyme activities (Publication 1/Chapter 1). All TS1 variants were found to be active TS enzymes, transferring Sia from donor fetuin to the lactose acceptor substrate and could resialylate asialofetuin up to 50%. Use of synthetic substrates revealed poor specific activities. Further searches on WTSI using TconTS1 gene variants as queries revealed 3 other TS genes, TconTS2, TconTS3 and TconTS4, all conserving the critical amino acids required in activity. A further 3 distantly related genes, this latter group is presumed inactive due to lack of conservation of critical amino acids (Publication 2/Chapter 3). TS/sialidase ratios confirmed the former to be active trans-sialidases but with differing specific activities. A mouse monoclonal antibody raised from native proteins probably containing other TconTS and not only TconTS1 reacted with both TconTS1 and TconTS2, two of the proteins with the highest specific activities. Incidentally, the epitope for the antibody is localised on the lectin domain (LD) in both enzymes. Phylogenetic analysis using the LD grouped TconTS1 and TconTS2 together while the use of catalytic domain (CD) grouped them separately. This suggests a possible role for the LD in enzyme activities and hence pathology of nagana. To gain knowledge on the role of TS in pathogenesis of nagana, blood glycoconjugates were employed as substrates and activities of TS gene characterised (Manuscript/Chapter 4). All TconTS proteins transferred Sia from serum-bound glycoconjugates to lactose. In absence of lactose, TconTS1 and TconTS2 released free Sia from the serum-glycoconjugates, possibly explaining the high amounts of free Sia observed in blood and serum of animals suffering nagana. TconTS3 showed an unidentified product peak in presence of serum-bound Sia and lactose, while 3 unidentified signals probably representing serum-glycoconjugates inherent in serum were altered by TconTS2. Collectively, the above indicate the possibility of acceptor and donor preferences and show that expressing more than one TS gene at a time could be beneficial to the parasite. Since TS genes are expressed in two different hosts systems; the Glossina insect vector and the mammalian hosts with different pH systems, pH optima for the enzymes was studied. TconTS2 exhibited a wide pH optima that would make it active in both host systems.