The transferrin receptor (TfR) of the bloodstream form (BSF) of Trypanosoma brucei is a heterodimer comprising glycosylphosphatidylinositol (GPI)-anchored expression site-associated gene 6 (ESAG6 or E6) and soluble ESAG7. Mature E6 has five N-glycans, consisting of three oligomannose and two unprocessed paucimannose structures. Its GPI anchor is modified by the addition of 4-6 α-galactose residues. TfR binds tomato lectin (TL), specific for N-acetyllactosamine (LacNAc) repeats, and previous studies have shown transport-dependent increases in E6 size consistent with post-glycan processing in the endoplasmic reticulum. Using pulse-chase radiolabeling, peptide-N-glycosidase F treatment, lectin pulldowns, and exoglycosidase treatment, we have now investigated TfR N-glycan and GPI processing. E6 increased ∼5 kDa during maturation, becoming reactive with both TL and Erythrina cristagalli lectin (ECL, terminal LacNAc), indicating synthesis of poly-LacNAc on paucimannose N-glycans. This processing was lost after exoglycosidase treatment and after RNAi-based silencing of TbSTT3A, the oligosaccharyltransferase that transfers paucimannose structures to nascent secretory polypeptides. These results contradict previous structural studies. Minor GPI processing was also observed, consistent with α-galactose addition. However, increasing the spacing between E6 protein and the GPI ω-site (aa 4-7) resulted in extensive post-translational processing of the GPI anchor to a form that was TL/ECL-reactive, suggesting the addition of LacNAc structures, confirmed by identical assays with BiPNHP, a non-N-glycosylated GPI-anchored reporter. We conclude that BSF trypanosomes can modify GPIs by generating structures reminiscent of those present in insect-stage trypanosomes and that steric constraints, not stage-specific expression of glycosyltransferases, regulate GPI processing.

中文翻译：

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布鲁氏锥虫中糖基磷脂酰肌醇锚的空间约束控制加工。

布氏锥虫血流形式 (BSF) 的转铁蛋白受体 (TfR) 是一种异二聚体，包含糖基磷脂酰肌醇 (GPI)-锚定表达位点相关基因 6（ESAG6 或 E6）和可溶性 ESAG7。成熟的 E6 有五个 N-聚糖，由三个低聚甘露糖和两个未加工的寡甘露糖结构组成。它的 GPI 锚通过添加 4-6 个 α-半乳糖残基进行修饰。TfR 结合番茄凝集素 (TL)，对 N-乙酰乳糖胺 (LacNAc) 重复具有特异性，之前的研究表明 E6 大小的转运依赖性增加与内质网中的聚糖后加工一致。使用脉冲追踪放射性标记、肽-N-糖苷酶 F 处理、凝集素下拉和外切糖苷酶处理，我们现在研究了 TfR N-聚糖和 GPI 加工。E6 在成熟过程中增加了 ~5 kDa，变得与 TL 和 Erythrina cristagalli 凝集素（ECL，末端 LacNAc）发生反应，表明在寡甘露糖 N-聚糖上合成了 poly-LacNAc。这种加工在外切糖苷酶处理后和基于 RNAi 的 TbSTT3A 沉默后丢失，TbSTT3A 是将寡糖结构转移到新生分泌多肽的寡糖转移酶。这些结果与以前的结构研究相矛盾。还观察到轻微的 GPI 加工，与 α-半乳糖添加一致。然而，增加 E6 蛋白和 GPI ω 位点 (aa 4-7) 之间的间距导致 GPI 锚的广泛翻译后加工成为 TL/ECL 反应性的形式，表明添加了 LacNAc 结构，确认通过与 BiPNHP 相同的测定，BiPNHP 是一种非 N-糖基化 GPI 锚定报告基因。