Effects of a homozygous missense mutation in the GNE gene p.V543M on cell phenotype and its mechanisms.
10.11817/j.issn.1672-7347.2025.240287
- Author:
Ruolan WU
1
;
Huilong LI
2
;
Pingyun WU
3
;
Qi YANG
3
;
Xueting WAN
3
;
Yuan WU
4
,
5
Author Information
1. Department of Clinical Laboratory, Third Xiangya Hospital, Central South University, Changsha 410013, China. 228311055@csu.edu.cn.
2. Department of Clinical Laboratory, Third Xiangya Hospital, Central South University, Changsha 410013, China. 218311063@csu.edu.cn.
3. Department of Clinical Laboratory, Third Xiangya Hospital, Central South University, Changsha 410013, China.
4. Department of Clinical Laboratory, Third Xiangya Hospital, Central South University, Changsha 410013, China. korchim@
5. com.
- Publication Type:Journal Article
- Keywords:
GNE myopathy;
cellular phenotype;
mitochondria;
p.V543M mutation;
sialic acid;
uridine diphospho-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase
- MeSH:
Humans;
Mutation, Missense;
HEK293 Cells;
Apoptosis/genetics*;
Phenotype;
Multienzyme Complexes/metabolism*;
Cell Proliferation;
Homozygote;
Cell Adhesion/genetics*;
Distal Myopathies/genetics*
- From:
Journal of Central South University(Medical Sciences)
2025;50(1):105-118
- CountryChina
- Language:English
-
Abstract:
OBJECTIVES:Uridine diphospho-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) myopathy is a progressive neurodegenerative disease associated with homozygous or compound heterozygous missense mutations in the GNE gene. This study aims to explore the impact of the homozygous p.V543M mutation in on cell phenotype and to gain preliminary insights into the underlying mechanisms.
METHODS:Human embryonic kidney 293T (HEK 293T) cells were used to construct wild-type (WT-GNE) and mutant (MUT-GNE) GNE overexpression models. Western blotting and immunofluorescence were used to assess GNE protein expression levels and subcellular localization. Cell adhesion, proliferation, apoptosis, and mitochondrial membrane potential were evaluated using the cell counting kit-8 (CCK-8) assay, crystal violet staining, flow cytometry, Hoechst 33342/propidium iodide (PI) staining, and tetramethylrhodamine ethyl ester (TMRE) staining. Sialic acid synthesis levels and GNE enzymatic activity were measured, and the mRNA expression of sialic acid biosynthesis-related enzymes was quantified by real-time PCR.
RESULTS:Western blotting confirmed successful establishment of GNE overexpression models. Immunofluorescence showed significantly reduced co-localization of GNE protein with Golgin-97 in the MUT-GNE group compared to WT-GNE (Pearson's correlation coefficient: 0.65±0.08 vs 0.83±0.06, P<0.05). Compared with WT-GNE, cells in the MUT-GNE group exhibited increased adhesion, decreased proliferation, and reduced mitochondrial membrane potential (P<0.05). No significant differences in apoptosis were observed between groups. The MUT-GNE group showed reduced sialic acid production, significantly decreased kinase activity, and downregulated transcription of sialic acid biosynthesis-related enzymes compared to WT-GNE (P<0.001).
CONCLUSIONS:The p.V543M mutation in the GNE gene alters cellular phenotype by reducing GNE enzymatic activity and the transcription of sialic acid biosynthesis enzymes, ultimately impairing sialic acid production.
