Die Trans-Sialidase von :i:Trypanosoma congolense:/i: - Isolierung, Charakterisierung und Molekularbiologie

Abstract

:i:Trypanosoma congolense:/i: is the agent of Nagana, the trypanosomiasis in African ruminants. During its insect stage the parasite expresses an enzyme called trans-sialidase (TS), which enables the parasites to transfer sialic acids from the environment onto trypanosomal surface molecules and is believed to play an important role in maintaining the pathogenicity of the parasites. Thus far, only two TS proteins and their sequences have been characterised in detail, one from the American trypanosome :i:T. cruzi:/i: and one from the African trypanosome :i:T. b. brucei:/i:. :p:This study describes the purification and characterisation of two TS forms from the African trypanosome :i:T. congolense:/i:. The purification of the two TS forms using a combination of anion exchange chromatography, isoelectric focusing, gel filtration and, subsequently, antibody affinity chromatography resulted, in both cases, in the isolation of a 90 kDa monomer on SDS-PAGE which was identified as TS using micro-sequencing. Monoclonal antibody 7/23, which bound and partially inhibited TS activity, was found in both cases to bind to a 90 kDa protein. Both TS forms possessed sialidase and transfer activity, but markedly differed in their activity ratios. Interestingly, GARP, the surface glycoprotein, was co-purified with TS-form 1 suggesting an association between both proteins. :p: This study also describes the identification of two partial TS gene sequences from :i:T. congolense:/i: utilising a PCR-based approach. Both :i:T. congolense:/i: TS genes, TS1 and TS2, which share only 50 % identity with each other, show significant similarity with known viral, bacterial and trypanosomal sialidases and TS. In both partial sequences most of the critical active site residues common to other trypanosomal sialidases and TS are conserved. Moreover, a peptide sequence derived from the native, active TS from :i:T. congolense:/i: was found within the sequence of TS1