Pimenta P F, Pinto da Silva P, Rangarajan D, Smith D F, Sacks D L
Laboratory of Parasitic Diseases, NIAID, NIH, Bethesda, MD 20892.
Exp Parasitol. 1994 Nov;79(3):468-79. doi: 10.1006/expr.1994.1108.
The lipophosphoglycan (LPG) of Leishmania promastigotes forms a dense glycocalyx which effectively covers the entire surface of the cell, and which undergoes structural modifications during the differentiation of promastigotes to the infective or metacyclic stage. Recently, the first protein marker for metacyclic promastigotes of Leishmania major has been characterized. This protein, termed gene B protein, is located on the cell surface, yet it lacks any hydrophobic sequence for membrane attachment. It does contain an unusual amino acid repeat that is related to the peptidoglycan binding domain of protein A from Staphylococcus aureus, suggesting that the protein might interact with metacyclic LPG via this domain for attachment to the cell. We have studied the distribution of LPG, gene B protein, and the major surface protease, gp63, by labeling them with immunogold or immunofluorescence prior to and during capping events. Thin sections of double-labeled parasites revealed that the gene B protein-gold particles were colocalized with the LPG-gold particles in the LPG capping structures at the extremities of the cell. Cocapping of LPG and gene B protein was also observed with two-color fluorescence. No similar redistribution was seen in gp63 or with integral membrane proteins. In contrast to the gene B protein, gp63 could only be immunogold labeled on the metacyclic surface after capping and shedding of the LPG, providing further that it and other membrane-associated proteins are normally buried under the LPG coat. The unusual surface exposure of the gene B protein is consistent with its hydrophilic and LPG binding properties, which allow it to become incorporated into the cell coat and to localize to the most external aspects of the cell.
利什曼原虫前鞭毛体的脂磷壁酸聚糖（LPG）形成一层致密的糖萼，有效地覆盖细胞的整个表面，并且在前鞭毛体向感染性或循环后期阶段分化过程中会发生结构修饰。最近，已鉴定出大利什曼原虫循环后期前鞭毛体的首个蛋白质标志物。这种蛋白质称为基因B蛋白，位于细胞表面，但它缺乏任何用于膜附着的疏水序列。它确实含有一个不寻常的氨基酸重复序列，该序列与金黄色葡萄球菌蛋白A的肽聚糖结合结构域相关，这表明该蛋白质可能通过此结构域与循环后期LPG相互作用以附着于细胞。我们通过在帽化事件之前和期间用免疫金或免疫荧光标记LPG、基因B蛋白和主要表面蛋白酶gp63，研究了它们的分布。双标记寄生虫的薄片显示，基因B蛋白 - 金颗粒与细胞末端LPG帽化结构中的LPG - 金颗粒共定位。用双色荧光也观察到LPG和基因B蛋白的共帽化。在gp63或整合膜蛋白中未观察到类似的重新分布。与基因B蛋白相反，gp63只有在LPG帽化和脱落之后才能在循环后期表面进行免疫金标记，这进一步证明它和其他膜相关蛋白通常被埋在LPG包膜之下。基因B蛋白不寻常的表面暴露与其亲水和LPG结合特性一致，这使其能够掺入细胞包膜并定位到细胞的最外部。
