Purification and characterization of trypanoagglutinin from the midgut of tsetse fly, glossina longipennis

Abstract

The tsetse fly is an insect of great economic importance to man as a vector of both human and animal trypanosomiasis. Trypanosomes are ingested by the tsetse fly vector with a bloodmeal taken from an infected host. In the midgut, these parasites are exposed to a hostile environment which comprises of lectins/ trypanoagglutinins, proteolytic enzymes, trypanolysins and other unknown factors. Lectins or agglutinins are a group of proteins of non-immune in origin, that bind carbohydrates specifically. Recent investigations have shown that trypsin, the most predominant protease in the midgut, appears to be very closely related to the midgut lectins. It was therefore considered pertinent to resolve the relationship between the lectins/trypanoagglutinins and trypsin or trypsin-like enzymes in the tsetse midgut. In this study, the trypanoagglutinin was purified from the midgut of Glossina longipennis and its properties studied in vitro. Purification was achieved by anion exchange chromatography using Fast Protein Liquid Chromatography (FPLC). It co-eluted with trypsin -like activity at approximately 225 mM NaCI gradient. The molecule was capable of agglutinating Trypanosoma brucei brucei and rabbit erythrocytes. The release of the molecule was induced by a bloodmeal since unfed midguts showed negligible agglutination activity. Compared to the bloodstream parasites, a much lower concentration of trypanoagglutinin was required to agglutinate procyclics and erythrocytes. Furthermore, agglutination of the parasites as well as of the erythrocytes was specifically inhibited by glucosamine. Similarly, the soybean trypsin inhibitor abrogated the agglutination of bloodstream parasites while agglutination of erythrocytes was partially inhibited. In contrast, agglutination of procyclic parasites was not affected by this inhibitor. The molecule was found to be thermo-labile since 90% of the agglutination activity was lost by heating at 60-100° C for 10 minutes. Analysis on native polyacrylamide gel electrophoresis (PAGE) revealed one band of M, - 61,000 which also showed trypsin activity. However, electrophoresis under denaturing conditions gave two subunits (M, - 33,000 and 27,000). Only the 27,000 subunit showed trypsin activity. It is thus postulated that both the agglutination and trypsin-like activity exist in the same molecule. Antibodies raised against the isolated trypanoagglutinin inhibited agglutination as well as the trypsin activities of the molecule. Out of the bloodsucking insect species tested for immunological cross-reactivity, only members of the Glossina family gave a positive reaction. The two bands in G. m. morsitans comigrated with those from G. longipennis, suggesting that this protein could be unique to tsetse fly.