Balber A E, Frommel T O
Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710.
J Protozool. 1988 May;35(2):214-9. doi: 10.1111/j.1550-7408.1988.tb04326.x.
We have measured binding of fluorescein-conjugated succinyl-concanavalin A (Fl-s-Con A) to bloodstream and procyclic forms of Trypanosoma brucei gambiense and to bloodstream forms of T. b. rhodesiense by flow cytofluorimetry. Bloodstream forms bound an order of magnitude less lectin than procyclic forms. Trypsin-treating cells enhanced binding of Fl-s-Con A to bloodstream forms 3-16-fold depending on the strain and the length of trypsinization but had little effect on Fl-s-Con A binding by procyclics. The trypsinization protocol used did not remove major common glycoproteins detected on lectin blots of either life cycle form but removed greater than 95% of the variant specific glycoprotein and fragments derived from this protein of bloodstream forms. Microscopically detectable Fl-s-Con A binding to bloodstream forms was confined to the flagellar pocket. Trypsinized bloodstream forms and procyclics bound Fl-s-Con A in the flagellar pocket, on the flagellum, and on the cell surface. Lectin remained cell associated but appeared to redistribute towards the flagellum and pocket when cells that had bound lectin on ice were subsequently incubated at physiological temperatures. The Fl-s-Con A binding had specificity characteristic of the interaction between the lectin and oligosaccharides. These results are consistent with the hypothesis that the variant specific surface glycoprotein blocks binding of the lectin to surface glycoproteins of bloodstream forms and suggest that concanavalin A-binding glycoproteins are abundant in the flagellar pocket of both life cycle forms.
我们通过流式细胞荧光术测定了荧光素偶联的琥珀酰伴刀豆球蛋白A（Fl-s-Con A）与布氏冈比亚锥虫的血流型和前循环型以及罗德西亚锥虫的血流型的结合情况。血流型结合的凝集素比前循环型少一个数量级。用胰蛋白酶处理细胞可使Fl-s-Con A与血流型的结合增强3至16倍，具体倍数取决于菌株和胰蛋白酶处理的时长，但对前循环型的Fl-s-Con A结合影响不大。所采用的胰蛋白酶处理方案并未去除在两种生命周期形式的凝集素印迹上检测到的主要常见糖蛋白，但去除了超过95%的血流型变体特异性糖蛋白及其衍生片段。显微镜下可检测到的Fl-s-Con A与血流型的结合局限于鞭毛袋。经胰蛋白酶处理的血流型和前循环型在鞭毛袋、鞭毛和细胞表面结合Fl-s-Con A。凝集素仍与细胞相关，但当在冰上结合了凝集素的细胞随后在生理温度下孵育时，凝集素似乎会重新分布到鞭毛和鞭毛袋。Fl-s-Con A的结合具有凝集素与寡糖相互作用的特异性特征。这些结果与以下假设一致，即变体特异性表面糖蛋白会阻断凝集素与血流型表面糖蛋白的结合，并表明伴刀豆球蛋白A结合糖蛋白在两种生命周期形式的鞭毛袋中都很丰富。
