Glossina Proteolytic Lectin Does Not Require a Carbohydrate Moiety for Enzymatic or Trypanosome-Transforming Activities

• Published in: Journal of medical entomology 2006-03, Vol.43 (2), p.301-308
• Main Authors: Amin, Daniel N., Kamita, Shizuo G., Muluvi, Geoffrey M., Machuka, Jesse, Hammock, Bruce D., Osir, Ellie O.
• Format: Article
• Language: English
• Subjects: agglutination; Amino Acid Sequence; amino acid sequences; Animals; Baculoviridae - genetics; Base Sequence; Biological and medical sciences; biological development; Cell Line; Chromatography, Affinity; enzyme activity; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Glossina; Glossina austeni; glycosylation; Hemagglutination; insect vectors; Insect Vectors - chemistry; Insect Vectors - physiology; lectins; Lectins - biosynthesis; Lectins - chemistry; Lectins - physiology; Male; Medically important nuisances and vectors, pests of stored products and materials: population survey and control; midgut; Molecular Sequence Data; nucleotide sequences; proteolysis; proteolytic lectin; Rabbits; Rats; Rats, Wistar; recombinant proteins; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Sequence Alignment; Spodoptera; structure-activity relationships; Trypanosoma; Trypanosoma brucei brucei - growth & development; trypanosome; Trypsin - metabolism; Tsetse Flies - parasitology; Tsetse Flies - physiology; tsetse fly; VECTOR/PATHOGEN/HOST INTERACTION, TRANSMISSION; Vectors. Intermediate hosts
• ISSN: 0022-2585; 1938-2928
• DOI: 10.1603/0022-2585%282006%29043%5B0301%3AGPLDNR%5D2.0.CO%3B2

The developmental cycle of the cyclically transmitted African trypanosome involves an obligatory passage through the tsetse fly, Glossina spp. This intricate relationship requires the presence of molecules within the insect vector, including a midgut lectin, that interact with the trypanosome. Recently, a gene encoding for a proteolytic lectin, with trypanosome-transforming activity, was isolated from a midgut cDNA library of Glossina fuscipes fuscipes Austen in our laboratory. Using the same approach, we have identified a similar gene from a midgut cDNA library of Glossina austeni (Newstead). The protein encoded by this gene was expressed in bacteria and a baculovirus-based expression system. The baculovirus-expressed 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 baculovirus-expressed protein also was glycosylated despite the absence of classical O-linked and N-linked sugar attachment motifs. Both the baculovirus- and bacterium-expressed lectin proteins were shown to agglutinate trypanosomes and rabbit red blood cells in vitro. This agglutination was strongly inhibited by d-glucosamine. d-Glucosamine also inhibited the action of the authentic and recombinant lectins upon the chromogenic substrate Chromozym TRY. Interestingly, both baculovirus- and bacterium-expressed lectins showed no significant differences in terms of these activities, indicating that a sugar moiety is not essential for biological activity. Our results provide an important molecular tool for further characterization of Glossina proteolytic lectin.