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

Autor:	Kentache T; Metabolic Research Group, de Duve Institute & WELRI, Université Catholique de Louvain, Brussels, Belgium., Althoff CR; Laboratory for Molecular Diagnosis, Center for Human Genetics, KU Leuven, Leuven, Belgium; Univ. Lille, CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, Lille, France., Caligiore F; Metabolic Research Group, de Duve Institute & WELRI, Université Catholique de Louvain, Brussels, Belgium., Souche E; Laboratory for Cytogenetics and Genome Research, Department of Human Genetics, KU Leuven, Leuven, Belgium., Schulz C; Univ. Lille, CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, Lille, France., Graff J; Metabolic Research Group, de Duve Institute & WELRI, Université Catholique de Louvain, Brussels, Belgium., Pieters E; Laboratory for Molecular Diagnosis, Center for Human Genetics, KU Leuven, Leuven, Belgium., Stanley P; Department of Cell Biology, Albert Einstein College of Medicine, New York, New York., Contessa JN; Department of Therapeutic Radiology, Yale School of Medicine, New Haven, Connecticut; Department of Pharmacology, Yale School of Medicine, New Haven, Connecticut., Van Schaftingen E; Metabolic Research Group, de Duve Institute & WELRI, Université Catholique de Louvain, Brussels, Belgium., Matthijs G; Laboratory for Molecular Diagnosis, Center for Human Genetics, KU Leuven, Leuven, Belgium., Foulquier F; Univ. Lille, CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, Lille, France., Bommer GT; Metabolic Research Group, de Duve Institute & WELRI, Université Catholique de Louvain, Brussels, Belgium. Electronic address: guido.bommer@uclouvain.be., Wilson MP; Laboratory for Molecular Diagnosis, Center for Human Genetics, KU Leuven, Leuven, Belgium. Electronic address: matthew.wilson@kuleuven.be.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2024 Oct 10; Vol. 300 (12), pp. 107875. Date of Electronic Publication: 2024 Oct 10.
DOI:	10.1016/j.jbc.2024.107875
Abstrakt:	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. Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article. (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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