Absence of the dolichol synthesis gene DHRSX leads to N-glycosylation defects in Lec5 and Lec9 Chinese hamster ovary cells.

IF 4 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Takfarinas Kentache,Charlotte R Althoff,Francesco Caligiore,Erika Souche,Céline Schulz,Julie Graff,Eline Pieters,Pamela Stanley,Joseph N Contessa,Emile Van Schaftingen,Gert Matthijs,François Foulquier,Guido T Bommer,Matthew P Wilson
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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.
中国仓鼠卵巢细胞 Lec5 和 Lec9 中缺少 Dolichol 合成基因 DHRSX 会导致 N-糖基化缺陷。
糖基化缺陷的中国仓鼠卵巢(CHO)细胞系在发现 N-糖基化机制方面发挥了重要作用。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,尽管这两个细胞系由多酚形成的多立醇的缺陷已被证实。我们最近发现,多聚甲酚合成多分三步:多聚甲酚通过 DHRSX 转化为多聚甲醛;多聚甲醛通过 SRD5A3 还原为多立卡醛;多立卡醛通过 DHRSX 还原为多立酚。这促使我们研究 Lec5 和 Lec9 细胞中存在缺陷的多立醇合成。这两种细胞系都显示出多酚及其衍生物水平的升高,与此同时,多立醇和衍生物的水平下降,但多酚醛的水平没有变化,这表明 DHRSX 缺乏。因此,N-聚糖的合成和多异戊二烯水平的变化通过与人 DHRSX 的互补而不是 SRD5A3 的互补得到了纠正。此外,在来自 Lec5 和 Lec9 细胞的膜制备物中,DHRSX 的典型多聚异戊烯醇脱氢酶和多聚异戊烯醛还原酶活性缺失,而 SRD5A3 催化的多聚异戊烯醛还原为多聚异戊烯醛的活性未受影响。对 Lec5 和 Lec9 细胞进行的长读全基因组测序没有发现 SRD5A3 的 ORF 发生突变,但含有 DHRSX 的基因组区域缺失。最后,我们确定了中国仓鼠 DHRSX 的序列,并验证了该蛋白具有与人酶相似的动力学特性。因此,我们的研究工作确定了 CHO Lec5 和 Lec9 细胞中多立醇合成缺陷的基础。
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来源期刊
Journal of Biological Chemistry
Journal of Biological Chemistry Biochemistry, Genetics and Molecular Biology-Biochemistry
自引率
4.20%
发文量
1233
期刊介绍: The Journal of Biological Chemistry welcomes high-quality science that seeks to elucidate the molecular and cellular basis of biological processes. Papers published in JBC can therefore fall under the umbrellas of not only biological chemistry, chemical biology, or biochemistry, but also allied disciplines such as biophysics, systems biology, RNA biology, immunology, microbiology, neurobiology, epigenetics, computational biology, ’omics, and many more. The outcome of our focus on papers that contribute novel and important mechanistic insights, rather than on a particular topic area, is that JBC is truly a melting pot for scientists across disciplines. In addition, JBC welcomes papers that describe methods that will help scientists push their biochemical inquiries forward and resources that will be of use to the research community.
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