Tiengwe Calvin, Bush Peter J, Bangs James D
Department of Microbiology & Immunology, School of Medicine and Biomedical Sciences, University at Buffalo (SUNY), Buffalo, New York, United States of America.
South Campus Instrument Center, School of Dental Medicine, University at Buffalo (SUNY), Buffalo, New York, United States of America.
PLoS Pathog. 2017 May 1;13(5):e1006366. doi: 10.1371/journal.ppat.1006366. eCollection 2017 May.
Bloodstream-form African trypanosomes encode two structurally related glycosylphosphatidylinositol (GPI)-anchored proteins that are critical virulence factors, variant surface glycoprotein (VSG) for antigenic variation and transferrin receptor (TfR) for iron acquisition. Both are transcribed from the active telomeric expression site. VSG is a GPI2 homodimer; TfR is a GPI1 heterodimer of GPI-anchored ESAG6 and ESAG7. GPI-valence correlates with secretory progression and fate in bloodstream trypanosomes: VSG (GPI2) is a surface protein; truncated VSG (GPI0) is degraded in the lysosome; and native TfR (GPI1) localizes in the flagellar pocket. Tf:Fe starvation results in up-regulation and redistribution of TfR to the plasma membrane suggesting a saturable mechanism for flagellar pocket retention. However, because such surface TfR is non-functional for ligand binding we proposed that it represents GPI2 ESAG6 homodimers that are unable to bind transferrin-thereby mimicking native VSG. We now exploit a novel RNAi system for simultaneous lethal silencing of all native TfR subunits and exclusive in-situ expression of RNAi-resistant TfR variants with valences of GPI0-2. Our results conform to the valence model: GPI0 ESAG7 homodimers traffick to the lysosome and GPI2 ESAG6 homodimers to the cell surface. However, when expressed alone ESAG6 is up-regulated ~7-fold, leaving the issue of saturable retention in the flagellar pocket in question. Therefore, we created an RNAi-resistant GPI2 TfR heterodimer by fusing the C-terminal domain of ESAG6 to ESAG7. Co-expression with ESAG6 generates a functional heterodimeric GPI2 TfR that restores Tf uptake and cell viability, and localizes to the cell surface, without overexpression. These results resolve the longstanding issue of TfR trafficking under over-expression and confirm GPI valence as a critical determinant of intracellular sorting in trypanosomes.
血流形式的非洲锥虫编码两种结构相关的糖基磷脂酰肌醇（GPI）锚定蛋白，它们是关键的毒力因子，即用于抗原变异的可变表面糖蛋白（VSG）和用于获取铁的转铁蛋白受体（TfR）。两者均从活跃的端粒表达位点转录。VSG是GPI2同型二聚体；TfR是GPI锚定的ESAG6和ESAG7的GPI1异型二聚体。GPI价态与血流锥虫中的分泌进程和命运相关：VSG（GPI2）是表面蛋白；截短的VSG（GPI0）在溶酶体中降解；天然TfR（GPI1）定位于鞭毛袋。转铁蛋白：铁饥饿导致TfR上调并重新分布到质膜，这表明存在一种用于鞭毛袋保留的饱和机制。然而，由于这种表面TfR对配体结合无功能，我们提出它代表无法结合转铁蛋白的GPI2 ESAG6同型二聚体，从而模拟天然VSG。我们现在利用一种新型RNAi系统同时致死性沉默所有天然TfR亚基，并特异性原位表达具有GPI0 - 2价态的RNAi抗性TfR变体。我们的结果符合价态模型：GPI0 ESAG7同型二聚体转运至溶酶体，GPI2 ESAG6同型二聚体转运至细胞表面。然而，当单独表达时，ESAG6上调约7倍，这使得鞭毛袋中饱和保留的问题存在疑问。因此，我们通过将ESAG6的C末端结构域与ESAG7融合，创建了一种RNAi抗性GPI2 TfR异型二聚体。与ESAG6共表达产生功能性异型二聚体GPI2 TfR，其恢复了转铁蛋白摄取和细胞活力，并定位于细胞表面，而不会过度表达。这些结果解决了长期存在的TfR在过表达情况下的转运问题，并证实GPI价态是锥虫细胞内分选的关键决定因素。
