PXD052706-1

PXD052706 is an original dataset announced via ProteomeXchange.

Title	Absence of the dolichol synthesis gene DHRSX leads to N-glycosylation defects in Lec5 and Lec9 Chinese hamster ovary cells
Description	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.
HostingRepository	PRIDE
AnnounceDate	2024-10-15
AnnouncementXML	Submission_2024-10-15_01:50:06.539.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD052706
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Francesco Caligiore
SpeciesList	scientific name: Cricetulus griseus (Chinese hamster) (Cricetulus barabensis griseus); NCBI TaxID: 10029;
ModificationList	Hex7HexNAc2 N4-glycosylated asparagine; Hex3HexNAc2 N4-glycosylated asparagine; Hex2HexNAc2 N4-glycosylated asparagine; Hex9HexNAc2 N4-glycosylated asparagine; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2024-05-30 03:32:03	ID requested
⏵ 1	2024-10-15 01:50:07	announced
2	2024-10-22 07:01:46	announced	2024-10-22: Updated project metadata.

10.1016/J.JBC.2024.107875;

10.6019/PXD052706;

submitter keyword: Congenital Disorders of Glycosylation, Lec9, DHRSX, N-glycosylation, Dolichal, polyprenal, Lec5, polyprenol,Dolichol

Guido Bommer
contact affiliation	de Duve Institute UCLouvain and WELBIO
contact email	guido.bommer@uclouvain.be
lab head
Francesco Caligiore
contact affiliation	UCLouvain
contact email	francesco.caligiore@uclouvain.be
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD052706
     1. Label: PRIDE project
     2. Name: Absence of the dolichol synthesis gene DHRSX leads to N-glycosylation defects in Lec5 and Lec9 Chinese hamster ovary cells