Characterization of the lectin from females of Phlebotomus duboscqi sand flies.
Lectin from females of the important sand fly vector, Phlebotomus duboscqi (Diptera: Psychodidae), was isolated by immunoaffinity chromatography using a minicolumn with immobilized anti-lectin immunoglobulins. Carbohydrate-binding specificity of active fractions corresponded to that of midgut and salivary gland lysates. Haemagglutination was inhibited by d-glucosamine, d-galactosamine and d-mannosamine. The homogeneity and molecular mass of the purified lectin was examined by SDS/PAGE in both reducing and nonreducing conditions. The active fractions showed one band strongly stained by Coomassie blue or silver nitrate; the molecular mass of the lectin was 42 kDa under nonreducing and 44 kDa under reducing conditions. SDS/PAGE of active fractions from the gel filtration revealed four to six protein bands, but the 42/44-kDa protein present in all active fractions was the only component reacting with specific antibodies in Western blots. Localization of the lectin in the gut of females was studied using indirect immunofluorescence on sections. The positive reaction of specific antibodies was localized in the lumen and along the microvillar surfaces of epithelial cells. The lectin was partially sequenced and characterized by MS. Peptide maps were obtained by MALDI-TOF MS, and several sequence tags were identified from tandem mass spectra on an ion trap. These sequences displayed high similarity to salivary protein precursors previously identified in a cDNA library of the sand flies Phlebotomus papatasi and Lutzomyia longipalpis. Two main hypotheses on the role of female lectin in Leishmania development are discussed.
Volf
Petr
P
Department of Parasitology, Charles University, Prague, Czech Republic. volf@cesnet.cz
Skarupová
Sona
S
Man
Petr
P
D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000378:metabolism; D004064:Digestive System / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism; D005260:Female; D015723:Gene Library; D006388:Hemagglutinins / Q000378:metabolism; D007136:Immunoglobulins / Q000378:metabolism; D037102:Lectins / Q000737:chemistry* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010691:Phlebotomus / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018076:DNA, Complementary; D006388:Hemagglutinins; D007136:Immunoglobulins; D037102:Lectins