PDF(Pubmed)
Abstract：
The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake, i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis.
摘要：
布鲁氏锥虫的血流形式在糖蛋白子集的复杂N-聚糖上表达大的聚N-乙酰乳糖胺（pNAL）链。已经假设pNAL可能是受体介导的内吞作用所必需的。非洲锥虫含有一个独特的糖基转移酶家族，GT67家族其中两个，TbGT10和TbGT8已被证明参与血液中的pNAL生物合成，同时删除两种酶可能会取消pNAL生物合成，并为pNAL功能和/或必要性提供线索。在本文中,我们描述了包含单个TbGT8等位基因的TbGT10空突变体的创建，该等位基因可以在添加雷帕霉素后切除，由此,TbGT10和TbGT8双无效突变体。通过凝集素印迹分析这些突变体，糖肽甲基化连锁分析和流式细胞术。数据显示突变体是有缺陷的,但没有废除，在pNAL合成中，表明其他GT67家族成员可以在一定程度上补偿TbGT10和TbGT8的损失。尽管在这些突变体中有残余的pNAL合成，某些糖蛋白似乎特别受影响。这些包括溶酶体CBP1B丝氨酸羧肽酶，细胞表面ESAG2和必需寄生虫转铁蛋白受体（TfR）的ESAG6亚基。突变体中pNAL缺陷的TfR在蛋白质稳定性方面继续正常发挥功能。转铁蛋白结合，受体介导的转铁蛋白胞吞作用和亚细胞定位。此外,pNAL缺陷突变体在体外和体内小鼠感染实验中与野生型寄生虫一样存活。尽管我们能够用高浓度的pNAL结构类似物（N-乙酰壳寡糖）重现对转铁蛋白摄取的抑制作用，这种效应在低浓度下消失，仍然抑制番茄凝集素的吸收，即，在能够胜过凝集素-pNAL结合的浓度下。基于这些发现,我们建议修订pNAL依赖性受体介导的细胞内吞假说.
