Howard R F, Reese R T
Agouron Institute, La Jolla, California 92037.
Exp Parasitol. 1990 Oct;71(3):330-42. doi: 10.1016/0014-4894(90)90038-e.
We have previously described several monoclonal antibodies (McAbs) which specifically recognize antigens in the rhoptries of Plasmodium falciparum and which immunoprecipitate polypeptides of 82, 70, 67, 39, and 37 kDa. We now show that only p82, p70, p67, and a 86-kDa precursor (Pr86) of p82 possessed epitopes for these McAbs. These four proteins were not synthesized until schizogony. These results and proteolysis experiments indicated that Pr86, p82, p70, and p67 were the products of the same gene, whereas the dissimilar digestion patterns of p39 and p37 suggested that p39 was encoded by a second gene and p37 by yet another. Complexes of these proteins (termed RI complexes) are maintained by noncovalent interactions since the ionic detergent SDS was sufficient to dissociate them into individual polypeptides. Sucrose gradient centrifugation demonstrated that RI complex formation was not dependent on the presence of antibody and that these complexes had higher sedimentation rates than the 185-kDa P. falciparum merozoite glycoprotein. Covalent crosslinking with the reversible, homobifunctional, primary amine-specific reagent 3,3'-dithiobis(sulfosuccinimidylpropionate) followed by RI McAb immunoprecipitation resulted in purification of intact complexes which were not dissociable by SDS alone. Immunodepletion experiments with a subtype of RI McAb which does not immunoprecipitate p37 suggested that the binding of p39 and p37 to the other RI proteins was mutually exclusive. Therefore, the minimal composition of the RI complexes is one molecule of Pr86, p82, p70, or p67 and one of p39 or p37. The epitopes of Pr86, p82, p70, and p67 for the RI McAbs were sensitive to disulfide bond reduction. Surprisingly, reduction increased their electrophoretic mobilities. This enhanced mobility could not be accounted for by post-translational glycosylation, phosphorylation, or acylation, or by covalent attachment via the sulfhydryl moiety of cysteine residues to additional parasite proteins. We suggest that, due to an asymmetric distribution of amino acids in the Pr86-class molecules, SDS binding results in a lower charge to mass ratio in the native folded polypeptides and a higher charge to mass ratio upon disulfide bond reduction and unfolding of the polypeptides.
我们之前描述过几种单克隆抗体（McAbs），它们能特异性识别恶性疟原虫滋养体中的抗原，并免疫沉淀82、70、67、39和37 kDa的多肽。我们现在发现，只有p82、p70、p67以及p82的86 kDa前体（Pr86）具有这些McAbs的表位。这四种蛋白质直到裂体生殖阶段才开始合成。这些结果以及蛋白水解实验表明，Pr86、p82、p70和p67是同一基因的产物，而p39和p37不同的消化模式表明p39由第二个基因编码，p37由另一个基因编码。这些蛋白质的复合物（称为RI复合物）通过非共价相互作用维持，因为离子去污剂SDS足以将它们解离成单个多肽。蔗糖梯度离心表明，RI复合物的形成不依赖于抗体的存在，并且这些复合物的沉降速率比185 kDa的恶性疟原虫裂殖子糖蛋白更高。用可逆的、同双功能的、伯胺特异性试剂3,3'-二硫代双（磺基琥珀酰亚胺丙酸酯）进行共价交联，随后进行RI McAb免疫沉淀，得到了完整的复合物，这些复合物不能仅通过SDS解离。用一种不免疫沉淀p37的RI McAb亚型进行免疫去除实验表明，p39和p37与其他RI蛋白的结合是相互排斥的。因此，RI复合物的最小组成是一个Pr86、p82、p70或p67分子以及一个p39或p37分子。RI McAbs识别的Pr86、p82、p70和p67的表位对二硫键还原敏感。令人惊讶的是，还原增加了它们的电泳迁移率。这种增强的迁移率不能用翻译后糖基化、磷酸化或酰化来解释，也不能用通过半胱氨酸残基的巯基部分与其他寄生虫蛋白的共价连接来解释。我们认为，由于Pr86类分子中氨基酸的不对称分布，SDS结合导致天然折叠多肽中的电荷与质量比降低，而在二硫键还原和多肽展开后电荷与质量比升高。
