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

Glycosylation-deficient Chinese hamster ovary 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 Chinese hamster ovary Lec5 and Lec9 cells.

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Publication Details

Journal
Journal of Biological Chemistry
Published
2024-10-10
DOI
https://doi.org/10.1016/j.jbc.2024.107875
Citations
4
Primary Topic
Viral Infectious Diseases and Gene Expression in Insects
Type
article
Field-Weighted Citation Impact
0.94

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article

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

Guido T. Bommer, Gert Matthijs, Matthew P. Wilson, Erika L. Souche et al.
4 citations
Journal of Biological Chemistry
Viral Infectious Diseases and Gene Expression in Insects
0.94
article

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

Guido T. Bommer, Gert Matthijs, Matthew P. Wilson, Erika L. Souche, Francesco Caligiore, Julie N. Graff, Joseph N. Contessa, Céline Schulz, Takfarinas Kentache, Charlotte R. Althoff, Pamela Stanley, Emile Van Schaftingen, François Foulquier, E. Pieters
article en
4 citations

Abstract

Glycosylation-deficient Chinese hamster ovary 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 Chinese hamster ovary Lec5 and Lec9 cells.

Journal of Biological ChemistryVol. 300(12)
Centre National de la Recherche Scientifique (FR), Albert Einstein College of Medicine (US), Université de Lille (FR), Yale University (US), VIB-KU Leuven Center for Cancer Biology (BE), Center for Human Genetics (US), de Duve Institute (BE), Unité de Glycobiologie Structurale et Fonctionnelle (FR), UCLouvain (BE), KU Leuven (BE)
Université Catholique de Louvain, Mizutani Foundation for Glycoscience, Agence Nationale de la Recherche, Fonds De La Recherche Scientifique - FNRS, Fonds Wetenschappelijk Onderzoek, KU Leuven, Onderzoeksraad, KU Leuven, Centre National de la Recherche Scientifique, Fonds Baillet Latour, Walloon excellence in life sciences and biotechnology, Fondation Médicale Reine Elisabeth, National Institutes of Health, Université Catholique de Louvain, Universitaire Ziekenhuizen Leuven, KU Leuven, HORIZON EUROPE European Research Council
Zero hunger
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Viral Infectious Diseases and Gene Expression in Insects
0.94
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