Jean L, Grosclaude J, Labbé M, Tomley F, Péry P
Unité de Virologie et d'Immunologie Moléculaires, INRA, Domaine de Vilvert, 78352, Cedex, Jouy-en-Josas, France.
Int J Parasitol. 2000 Sep;30(10):1099-107. doi: 10.1016/s0020-7519(00)00099-0.
Aspartyl proteinases are essential for the survival of many pathogens. A single copy gene in species of Eimeria encodes an aspartyl proteinase, which we propose should be called eimepsin to conform to the commonly used names of this family of proteinases. An epitope map, constructed using BIAcore technology, confirmed the specificity of 14 mAbs for eimepsin and defined four antigenic domains, which were conserved between native and recombinant forms of eimepsin. In resting sporozoites, mAb defining antigenic domains I and II stained the refractile body organelles, whereas those defining antigenic domains III and IV stained cytoplasmic granules. During host cell invasion, the staining patterns of mAb defining antigenic domains I, III and IV changed dramatically with the apical tips of invading sporozoites becoming strongly stained. In contrast, mAb defining antigenic domain II continued to stain only the refractile bodies. During early schizogony, mAb to all four domains stained the single fused refractile body, but when schizonts matured, mAb to antigenic domains I, III and IV stained the apical tip of merozoites whereas those to antigenic domain II continued to follow the developmental redistribution of the refractile body. Irrespective of localisation, mAb to three antigenic domains recognised a polypeptide of 49 kDa, which from N-terminal sequencing corresponds to a mature form of eimepsin. Staining with fluorescent pepstatin localised a mature, active form of eimepsin to the refractile bodies of the sporozoite, schizont and first generation merozoite. It remains to be determined whether eimepsin has a catalytic function within the refractile body or whether the activated enzyme is stored in the refractile body so that it can be rapidly redistributed to the apical tip during parasite invasion.
天冬氨酰蛋白酶对许多病原体的生存至关重要。艾美耳球虫属物种中的一个单拷贝基因编码一种天冬氨酰蛋白酶，我们提议将其命名为艾美psin，以符合该蛋白酶家族常用的命名方式。使用BIAcore技术构建的表位图谱证实了14种单克隆抗体对艾美psin的特异性，并确定了四个抗原结构域，这些结构域在天然和重组形式的艾美psin之间是保守的。在静止的子孢子中，定义抗原结构域I和II的单克隆抗体对折光体细胞器进行染色，而定义抗原结构域III和IV的单克隆抗体对细胞质颗粒进行染色。在宿主细胞入侵期间，定义抗原结构域I、III和IV的单克隆抗体的染色模式发生了显著变化，入侵子孢子的顶端强烈染色。相比之下，定义抗原结构域II的单克隆抗体继续仅对折光体进行染色。在早期裂殖生殖期间，针对所有四个结构域的单克隆抗体对单个融合的折光体进行染色，但当裂殖体成熟时，针对抗原结构域I、III和IV的单克隆抗体对裂殖子的顶端进行染色，而针对抗原结构域II的单克隆抗体继续跟踪折光体的发育重新分布。无论定位如何，针对三个抗原结构域的单克隆抗体识别出一种49 kDa的多肽，从N端测序来看，它对应于艾美psin的成熟形式。用荧光胃蛋白酶抑制剂染色将艾美psin的成熟、活性形式定位到子孢子、裂殖体和第一代裂殖子的折光体中。艾美psin在折光体内是否具有催化功能，或者活化的酶是否储存在折光体内以便在寄生虫入侵期间能够迅速重新分布到顶端，还有待确定。
