A blood-meal-induced lectin (agglutinin) with proteolytic activity was isolated from midgut extracts of Glossina longipennis by a two-step procedure involving anion-exchange chromatography. It is a glycoprotein [native molecular weight (M(r) 61,000 +/- 3000 da) composed of two noncovalently-linked subunits designated alpha (M(r), approximately 27,000 da) and beta (M(r), approximately 33,000 da). The trypsin activity and the glycosyl residues were present on the alpha- and beta-subunits, respectively. The native protein was capable of agglutinating both bloodstream-form and procyclic trypanosomes as well as rabbit red blood cells. This activity was strongly inhibited by D-glucosamine and weakly inhibited by N-acetyl-D-glucosamine. Similarly, soybean trypsin inhibitor abrogated agglutination of bloodstream-form parasites, whereas the procyclics were unaffected. The agglutination activity was sensitive to temperatures above 40 degrees C but was unaffected by chelators of metal ions. Antibodies raised against the protein were used in immunoblotting experiments to show the presence of a similar protein in several members of the Glossina species. However, no cross-reactivity was detected with midgut extracts prepared from sandflies, mosquitoes, or stable flies. It is proposed that this molecule might play an important role in differentiation of bloodstream-form trypanosomes into procyclic (midgut) forms.

No abstract

No abstract

The properties of a blood-meal-induced lectin (agglutinin) from the midgut of Glossina morsitans capable of agglutinating Trypanosoma brucei were studied in vitro. The midgut homogenate from flies that had been fed twice had the highest agglutination activity, followed by that from the once-fed flies and that from the unfed insects. As compared with the bloodstream-form trypanosomes, a much lower concentration of the midgut homogenate was required for agglutination of the procyclic parasites. Furthermore, the agglutination process was specifically inhibited by D-glucosamine. Soybean trypsin inhibitor abrogated agglutination of the bloodstream-form parasites, whereas the procyclics were unaffected. The agglutination process was temperature-sensitive, with little activity being evident between 4 degrees and 15 degrees C. Similarly, heating the midguts to 60 degrees-100 degrees C led to loss of activity. When the midgut homogenate was separated by anion-exchange chromatography, the agglutination activity co-eluted with trypsin activity at approximately 50% NaCl. These results suggest a very close relationship between midgut trypsin-like enzyme and the agglutinin. Since successful agglutination of bloodstream-form trypanosomes requires protease activity, it may be that the enzyme cleaves off some surface molecules on the parasite surface, thus exposing the lectin-binding sites.

We have previously described a bloodmeal-induced molecule (lectin-trypsin complex) from the midgut of the tsetse fly, Glossina longipennis, with both lectin and trypsin activities . In this paper, we report on the isolation of a similar molecule from the midguts of Glossina fuscipes fuscipes and provide direct evidence for its involvement in the development of African trypanosomes. The molecule (native M r -65,700) has two non-covalently linked subunits, M r -28,800 and M r -35,700. The native molecule was found to be capable of inducing differentiation of bloodstream-form trypanosomes into procyclic (midgut forms) in vitro. In the assays, specific antibodies against procyclin were used to monitor the transformation of the bloodstream-form trypanosomes into procyclic forms. This induction was specifically inhibited by D-glucosamine. Cisaconitate was also capable of inducing the transformation process with the same efficiency as that of the lectin-trypsin complex. While increasing the concentrations of the lectin-trypsin complex (>100 (.ig protein/ml) in the incubation assays resulted into higher transformation rates, it also led to high parasite mortality. These results provide evidence for the involvement of the midgut lectin-trypsin complex in the differentiation of bloodstream-form trypanosomes within tsetse midgut.Resume-Nous avons precedemment decrit une molecule induite par le repas de sang (un complexe lectine-trypsine) presente dans l'intestin moyen de la mouche tse-tse/G/oss/na longipennis, ayant une activite lectine et trypsine . Dans ce papier, nous decrivons l'isolement d'une molecule similaire presente dans l'intestin moyen de Glossina fuscipes fuscines et demontrons son implication dans le developpement des trypanosomes africains. La molecule (pure M r -65,700) presente deux sous unites liees non covalentes, M r -28,800 et M r -35,700. La molecule pure est capable d'induire la differenciation des formes sanguines du trypanosome en formes pro-cycliques (intestin moyen) in vitro. Lors des essais, des anticorps specifiques de la procycline ont ete utilises pour controler la transformation des formes sanguines du trypanosome en forme procycliques. Cette induction a ete inhibee specifiquement avec du D-glucosamine. Le ris-aconitate est egalement capable d'induire le processus de transformation avec une efficacite comparable a celle du complexe lectine-trypsine. Alors que l'augmentation des concentrations du complexe lectine-trypsine (>100 ug proteine/ml) dans les essais d'incubation permet d'augmenter les taux de transformation, il induit egalement une importante mortalite du parasite. Ces resultats demontrent la participation du complexe lectinetrypsine de l'intestin moyen dans la differenciation des formes sanguines des trypanosomes dans l'intestin moyen de la mouche tse-tse.