Apical expression of three RhopH1/Clag proteins as components of the Plasmodium falciparum RhopH complex

The Plasmodium falciparum high molecular mass rhoptry protein (`PfRhopH') complex is important for parasite growth and comprises three distinct gene products: RhopH1, RhopH2 and RhopH3. We have previously shown that P. falciparum RhopH1 is encoded by either PFC0110w (clag3.2) or PFC0120w (clag3.1), members of the previously-named clag (cytoadherence-linked asexual gene) multigene family. In this report, we have further characterized rhoph1/clag members in terms of gene structure, transcription and protein expression. The cDNA sequences for all five rhoph1/clag members were determined, confirming previous in silico predictions of intron-exon boundaries. All member genes were transcribed in HB3 and 3D7 parasite lines, but clag3.2 was not transcribed in Dd2 parasites. The peak abundance of transcripts for all genes was observed during the late schizont stage. Antisera specific to Clag2 and Clag3.1 localized these proteins to the apical end of merozoites in segmented schizonts, and both proteins are found to be components of the PfRhopH complex. PfRhopH complex that was immunoprecipitated with anti-Clag9 antibody contained neither Clag2 nor Clag3.1, thereby suggesting that PfRhopH complexes contain only individual rhoph1/clag gene products. Since the PfRhopH complex binds the erythrocyte surface, and RhopH2 and RhopH3 are encoded by single copy genes, the RhopH1/Clag proteins may serve to confer some degree of specificity to the roles of the individual complexes. (c) 2005 Elsevier B.V. All rights reserved.

恶性疟原虫高分子量棒状体蛋白（“PfRhopH”）复合物对寄生虫的生长很重要，它由三种不同的基因产物组成：RhopH1、RhopH2和RhopH3。我们先前已经表明，恶性疟原虫的RhopH1由PFC0110w（clag3.2）或PFC0120w（clag3.1）编码，它们是先前命名的clag（细胞黏附相关无性基因）多基因家族的成员。在本报告中，我们从基因结构、转录和蛋白质表达方面进一步对rhoph1/clag成员进行了表征。确定了所有五个rhoph1/clag成员的cDNA序列，证实了先前对内含子 - 外显子边界的计算机预测。所有成员基因在HB3和3D7寄生虫株系中都有转录，但clag3.2在Dd2寄生虫中没有转录。在裂殖体晚期观察到所有基因转录本的丰度峰值。针对Clag2和Clag3.1的特异性抗血清将这些蛋白质定位在分节裂殖体中裂殖子的顶端，并且发现这两种蛋白质都是PfRhopH复合物的组成成分。用抗 - Clag9抗体免疫沉淀的PfRhopH复合物既不包含Clag2也不包含Clag3.1，因此表明PfRhopH复合物仅包含单个的rhoph1/clag基因产物。由于PfRhopH复合物结合红细胞表面，并且RhopH2和RhopH3由单拷贝基因编码，RhopH1/Clag蛋白质可能在一定程度上赋予单个复合物作用的特异性。（c）2005 Elsevier B.V. 保留所有权利。