Brickman M J, Balber A E
Department of Immunology, Duke University Medical Center, Durham, North Carolina 27710.
Exp Parasitol. 1993 Jun;76(4):329-44. doi: 10.1006/expr.1993.1041.
Bloodstream forms of Trypanosoma brucei rhodesiense take up macromolecules in endocytic vesicles that form in a large coated pit called the flagellar pocket. Glycoproteins that bind to ricin are concentrated in the flagellar pocket and in intracellular vesicles. We purified Triton X-100-soluble ricin-binding glycoproteins by lectin affinity chromatography and immunized mice to generate hybridomas. Monoclonal antibody produced by the CB1 hybridoma recognized heterodisperse trypanosome components migrating with M(r) 84-140 kDa in immunoblots. CB1 binding was specifically inhibited by lactose. The CB1-reactive material was purified by sequential affinity chromatography on ricin- and CB1-Sepharose. N-Glycosidase F, but not endoglycosidase H, digestion destroyed CB1-reactivity of purified material. This suggests that N-linked oligosaccharides contribute to the CB1 epitope. Glycosidase digestion of biosynthetically radiomethionine-labeled, affinity purified, CB1-reactive material yielded two radiolabeled polypeptides, p57 and p42. Thirteen methionyl peptides were resolved in one-dimensional peptide maps of V8 protease digests of p57; p42 had 10 methionyl peptides with mobilities indistinguishable from those of peptides of p57. This suggests that p57 and p42 are closely related. In cryoimmunoelectron microscopy studies CB1 specifically labeled the interior surface of tubular and vesicular membranes located between the nucleus and the flagellar pocket. These membranes were morphologically identical to structures that have been previously identified as trans Golgi, lysosomal, and endosomal elements. In double-labeling studies endocytosed serum albumen-gold complexes were found in the lumen of vesicles that had CB1-reactive material in their membranes. This provides direct evidence that vesicles containing high levels of CB1-reactive material are part of the lysosome/endosomal system. Some CB1-reactive material was also detected in the flagellar pocket by cryoimmunoelectron microscopy. Corrolated flow cytofluorimetry and immunofluorescence analysis showed that 85-96% of the total CB1-reactive material was intracellular and inaccessible to antibody in living cells. The 4-15% of the total CB1-reactive material accessible to antibody in living cells was localized in the flagellar pocket. Bloodstream forms of Trypanosoma brucei brucei, Trypanosoma brucei gambiense, and T.b. rhodesiense all expressed the CB1 epitope. However, expression of this epitope is developmentally regulated during the parasite life cycle, for no CB1-reactive material was detected in procyclic forms. The trypanosome proteins detected by CB1 show some similarities to vertebrate lysosomal and endosomal membrane proteins.
罗德西亚布氏锥虫的血流形式在称为鞭毛袋的大型被膜小窝中形成的内吞小泡中摄取大分子。与蓖麻毒素结合的糖蛋白集中在鞭毛袋和细胞内小泡中。我们通过凝集素亲和层析纯化了Triton X-100可溶性蓖麻毒素结合糖蛋白，并免疫小鼠以产生杂交瘤。CB1杂交瘤产生的单克隆抗体在免疫印迹中识别迁移分子量为84 - 140 kDa的异质锥虫成分。乳糖可特异性抑制CB1结合。通过在蓖麻毒素和CB1 - 琼脂糖上的顺序亲和层析纯化CB1反应性物质。N - 糖苷酶F而非内切糖苷酶H的消化破坏了纯化物质的CB1反应性。这表明N - 连接寡糖对CB1表位有贡献。对生物合成放射性甲硫氨酸标记、亲和纯化的CB1反应性物质进行糖苷酶消化产生了两种放射性标记多肽，p57和p42。在p57的V8蛋白酶消化产物的一维肽图中解析出13个甲硫氨酰肽段；p42有10个甲硫氨酰肽段，其迁移率与p57的肽段无法区分。这表明p57和p42密切相关。在冷冻免疫电子显微镜研究中，CB1特异性标记位于细胞核和鞭毛袋之间的管状和囊泡膜的内表面。这些膜在形态上与先前鉴定为反式高尔基体、溶酶体和内体成分的结构相同。在双重标记研究中，发现内吞的血清白蛋白 - 金复合物存在于其膜中有CB1反应性物质的小泡腔中。这提供了直接证据，表明含有高水平CB1反应性物质的小泡是溶酶体/内体系统的一部分。通过冷冻免疫电子显微镜在鞭毛袋中也检测到一些CB1反应性物质。相关的流式细胞荧光测定和免疫荧光分析表明，活细胞中总CB1反应性物质的85 - 96%位于细胞内且抗体无法接近。活细胞中可被抗体接近的总CB1反应性物质的4 - 15%定位于鞭毛袋。布氏布氏锥虫、布氏冈比亚锥虫和罗德西亚布氏锥虫的血流形式均表达CB1表位。然而，该表位的表达在寄生虫生命周期中受到发育调控，因为在原循环形式中未检测到CB1反应性物质。CB1检测到的锥虫蛋白与脊椎动物溶酶体和内体膜蛋白有一些相似之处。
