Abstract:
Trypanosomiasis continues to be a serious problem in sub-Saharan Africa. The inadequacies of existing control strategies necessitate the development of alternative control measures. In most cases, the causative agents of African trypanosomiasis, Trypanosoma spp., require an obligatory passage through the tsetse, Glossina spp (Diptera; Glossinidae ), vector. This developmental step of the parasite leads to vector-parasite interactions, especially within the midgut. Among the vector molecules highly implicated in the interplay are carbohydrate-binding proteins known as lectins. This thesis describes studies on the midgut proteolytic lectin of Glossina austeni. The purification of a proteolytic lectin from the midgut homogenate of G. austeni was achieved through a 2-step chromatographic procedure, involving anion exchange and affinity columns. The purified protein was shown to cause the agglutination of Trypanosoma brucei brucei (Kinetoplastida, Trypanosomatidae) and washed rabbit red cells in vitro. It also showed some trypsin activity when the chromogenic substrate, chromozym-TRY, was used. Polyclonal antibodies to this molecule were used to isolate a putative gene encoding the protein from a G. austeni midgut cDNA library. The sequence of the isolated gene (GenBank Accession Number DQ060150) showed very high similarity to another gene previously obtained from G. fuscipes fuscipes midgut cDNA library. Analysis of the gene sequence, using basic bioinformatics tools, showed that the translated protein with 274 amino acids contains a signal peptide region and signature motifs for the serine protease trypsin family. The recombinant protein was expressed in E. coli BL 21 (DE2) cells and the purified product used to raise polyclonal antibodies. The recombinant Glossina proteolytic lectin was further expressed in Spodoptera frugiperda (St) 21 cell lines by the baculovirus expression system. This is the first G/ossina protein to be expressed with this system. The baculovirus-expressed recombinant lectin was found in the medium of baculovirus-infected Sf-21 cell cultures indicating that the tsetse fly-derived signal peptide was recognized and cleaved by the Sf-21 cells. The recombinant protein was purified by immuno-affinity chromatography and shown by Periodic Acid Schiff stain to be post-translationally modified by glycosylation. Moreover, the glycosylation pattern was not via the classical 0-linked and N-linked sugar attachment motifs as these were absent from the nucleotide sequence. Both the baculovirus- and bacteria- expressed lectin proteins showed agglutination and enzymatic properties. The two recombinant forms of the expressed lectins showed no significant differences in terms of biological activity indicating that the sugar moiety may not be crucial for these functions. It is plausible that the physiological relevance of the sugar moiety is to act in synergy with the signal peptide for proper targeting of the secreted protein and also enhancing its solubility within the midgut region of the fly. The findings provide an important contribution in the characterisation of Glossina proteolytic lectin. The constructed recombinant baculovirus, designated AcMNP.gpl, constitutes a very useful molecular tool for further investigation of both the structure of the protein as well as the mechanisms of action of Glossina proteolytic lectin (Gpl) upon African trypanosomes. Preliminary data from the present study has led to the suggestion that the initial part of interaction between Glossina proteolytic lectin and D-glucosamine, which is binding, may rely more on structural complementarities between the two molecules, in a pattern similar to a lock and key mechanism.