Relationship Between Lectins And Trypsin In The Midgut Of The Tsetse Fly, Glossina Morsitans Morsitans

Abstract

Tsetse flies are haematophagous arthropods which feed on a diverse range of vertebrates. The Glossina morsii ans group of tsetse flies are among the important vectors of the African trypanosomes. The African trypanosomes are the causative agent of the diseases, sleeping sickness in man and nagana in livestock. The trypanosomes are protozoal parasites with a complicated life cycle both within the vertebrate host and the tsetse fly vector. These diseases, are generally fatal unless treated. Once a tsetse fly ingests trypanosomes during feeding, the parasites pass through the oesophagus to the midgut. Here, the parasites are exposed to a hostile environment which consists of the digestive enzymes like trypsin, a trypsin-like enzyme, chymotrypsin, carboxypeptidase A and B, and an aminopeptidase. Out of the six digestive enzymes, trypsin is the most abundant involved in digestion. The tsetse fly midgut trypsin has been implicated in the lysis of the trypanosomes as well as their transformation into procyclic forms. Similarly, "lectins", a group of proteins that bind carbohydrates specifically, ~aye been reported to be secreted by tsetse midgut in response to a bloodmeal. These "lectins" which are specific for glucosamine have been proposed to have two functions. Firstly, they mediate lysis of the parasites that enter the fly midgut .

Secondly, they provide the signal for the surviving parasites to differentiate to procyclic forms. Furthermore, in vivo studies have shown that flies infected with parasites and maintained on a diet of blood containing D-glucosamine develop elevated levels of midgut infections. It might be argued that the reason for the increased infections is due to inhibition of the "lectins" by D-glucosamine. Since the "lectins" and trypsin appear to be induced by the same factors, and mediate similar events, it was considered pertinent to explore the relationship between "lectins" and trypsin in midgut of the tsetse fly, Glossina morsitans morsitans. In these studies, it was shown that the sugar D-glucosamine specifically and reversibly inhibited trypsin and trypsin-like enzyme activity. In contrast, galactosamine, mannose, glucose, fructose, galactose, Inositol, glucosamine pentacetate. N-acetyl-glucosamine, methyl-a- Dglucopyranoside, methyl-E-Dvglucopyranoside, a-D-mannosamine had no inhibitory effect on tsetse fly trypsin activity even at concentrations as high as 700 mM. The inhibition by glucosamine was determined to be partial, as shown by non linear Dixon plots. The Lineweaver-Burk plots intersected on the y axis, a pattern characteristic or competitive inhibition. The Kjwas estimated to be 68 + 3 mM. Clucosarnine also had similar inhibition effect on bovine pancreas trypsin. However, glucosamine did

not inhibit tsetse fly midgut aminopeptidase activity. Interestingly, glucosamine at a concentration of 100 mM both in vivo and in vitro, inhibited transformation of Trypanosoma brucei brucei bloodstream forms to procyclic forms. The inhibition of transformation was fairly constant, and 90% of the parasites were still in the bloodstream forms 8 hours after feeding. On the other hand, transformation proceeded normally in the absence of glucosamine with 80-90% of the parasites in the procyclic form 8 hours after feeding. The results suggest a close relationship exist between tsetse fly midgut lectins which are specifically inhibited by glucosamine and the enzymes, trypsin and trypsin-like. In addition Glossina morsitans morsitans midgut proteases, trypsin and trypsin like were purified and partially characterized. The purification process involved anion exchange, gel filtration, affinity chromatography and electroelution. The two enzymes, trypsin and trypsin-like enzyme had the molecular weight of - 24 ;r<D. They differed in charge, trypsin being positively charged at pH 8.0 and trypsin-like enzyme had a net negative charge at the same pH. Soybean trypsin inhibitor (STI) and N-p- Tosyl-lysine chloromethyl ketone .(TLCK) had similar inhibitory effect on

the trypsin and trypsin-like enzyme. The Kjs' for trypsin and trypsin-like enzyme were determined to be 0.295, 0.24 ~g/ml for STI and 0.12, 0.15 mg/ml for TLCK respectively. The tsetse fly midgut trypsin was 2-3 times more active than the trypsin-like enzyme.