PDF(Pubmed)
Abstract：
Glycosylation-deficient Chinese hamster ovary (CHO) cell lines have been instrumental in the discovery of N-glycosylation machinery. Yet, the molecular causes of the glycosylation defects in the Lec5 and Lec9 mutants have been elusive, even though for both cell lines a defect in dolichol formation from polyprenol was previously established. We recently found that dolichol synthesis from polyprenol occurs in three steps consisting of the conversion of polyprenol to polyprenal by DHRSX, the reduction of polyprenal to dolichal by SRD5A3 and the reduction of dolichal to dolichol, again by DHRSX. This led us to investigate defective dolichol synthesis in Lec5 and Lec9 cells. Both cell lines showed increased levels of polyprenol and its derivatives, concomitant with decreased levels of dolichol and derivatives, but no change in polyprenal levels, suggesting DHRSX deficiency. Accordingly, N-glycan synthesis and changes in polyisoprenoid levels were corrected by complementation with human DHRSX but not with SRD5A3. Furthermore, the typical polyprenol dehydrogenase and dolichal reductase activities of DHRSX were absent in membrane preparations derived from Lec5 and Lec9 cells, while the reduction of polyprenal to dolichal, catalyzed by SRD5A3, was unaffected. Long-read whole genome sequencing of Lec5 and Lec9 cells did not reveal mutations in the ORF of SRD5A3, but the genomic region containing DHRSX was absent. Lastly, we established the sequence of Chinese hamster DHRSX and validated that this protein has similar kinetic properties to the human enzyme. Our work therefore identifies the basis of the dolichol synthesis defect in CHO Lec5 and Lec9 cells.
摘要：
糖基化缺陷的中国仓鼠卵巢（CHO）细胞系在N-糖基化机制的发现中发挥了重要作用。然而，Lec5和Lec9突变体中糖基化缺陷的分子原因一直难以捉摸，即使对于这两种细胞系，以前都已建立了聚丙炔醇形成多力酚的缺陷。我们最近发现，由聚丙炔醇合成的多利康醇发生在三个步骤中，包括通过DHRSX将聚丙炔醇转化为聚丙炔醇，SRD5A3将多基因还原为dolichal，将dolichal还原为dolichol，再次由DHRSX。这导致我们研究了Lec5和Lec9细胞中缺陷的dolichol合成。两种细胞系都显示出增加的多烯醇及其衍生物水平，伴随着dolichol和衍生物水平的降低，但是多核水平没有变化，提示DHRSX缺乏。因此，通过与人DHRSX而不是SRD5A3互补来校正N-聚糖合成和聚异戊二烯水平的变化。此外，来自Lec5和Lec9细胞的膜制剂中不存在DHRSX的典型的多普瑞诺脱氢酶和dolichal还原酶活性，虽然将多基因减少为dolichal，SRD5A3催化，不受影响。Lec5和Lec9细胞的长读全基因组测序没有发现SRD5A3的ORF突变，但含有DHRSX的基因组区域不存在。最后,我们建立了中国仓鼠DHRSX的序列，并验证了该蛋白具有与人类酶相似的动力学特性。因此，我们的工作确定了CHOLec5和Lec9细胞中dolichol合成缺陷的基础。
