Glossina proteolytic lectin does not require a carbohydrate moiety for enzymatic or trypanosome-transforming activities.
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.
Amin
Daniel N
DN
International Center of Insect Physiology and Ecology, P.O. Box 30772, Nairobi, Kenya.
Kamita
Shizuo G
SG
Muluvi
Geoffrey M
GM
Machuka
Jesse
J
Hammock
Bruce D
BD
Osir
Ellie O
EO
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D006384:Hemagglutination; D007303:Insect Vectors / Q000737:chemistry / Q000502:physiology; D037102:Lectins / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D008297:Male; D008969:Molecular Sequence Data; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D016415:Sequence Alignment; D018411:Spodoptera; D014346:Trypanosoma brucei brucei / Q000254:growth & development*; D014357:Trypsin / Q000378:metabolism; D014370:Tsetse Flies / Q000469:parasitology* / Q000502:physiology*
D037102:Lectins; D011994:Recombinant Proteins; D014357:Trypsin