Dataset Information

Proteome and glycoproteome analysis of fibroblasts from patients with congenital disorders of glycosylation

ABSTRACT: In the biological systems, several genes are involved in protein glycosylation pathway. Congenital Disorders of Glycosylation (CDG) is known to be a multisystem disorder. Pathogenic variants in the glycosylation genes lead to abnormal N- or O-linked glycosylation that causes cellular stress. We used TMT-based N-glycoproteomics and proteomics on different patient-derived CDG and control fibroblasts to characterize the alteration in glycoproteome and proteome. 12 fractions of enriched glycopeptides after size exclusion chromatography (SEC) and 12 fractions after basic reverse phase liquid chromatography (bRPLC) were analyzed by LC-MS/MS for glycoproteomics and proteomics, respectively. A site-specific aberrant glycosylation was observed for several proteins such as mitochondrial, autophagy, extracellular matrix and cell adhesion proteins. By using quantitative proteomics, we observed alteration in abundances of several proteins separated the affected individuals and controls. The glycoproteomics and proteomics analysis in CDG patients provides the potential biomarkers and will increase our general understanding of its pathogenesis.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Fibroblast

SUBMITTER: Akhilesh Pandey

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD044327 | Pride | 2023-10-24

REPOSITORIES: Pride

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Publications

Interplay of Impaired Cellular Bioenergetics and Autophagy in PMM2-CDG.

Ligezka Anna N AN Budhraja Rohit R Nishiyama Yurika Y Fiesel Fabienne C FC Preston Graeme G Edmondson Andrew A Ranatunga Wasantha W Van Hove Johan L K JLK Watzlawik Jens O JO Springer Wolfdieter W Pandey Akhilesh A Morava Eva E Kozicz Tamas T

Genes 20230804 8

Congenital disorders of glycosylation (CDG) and mitochondrial disorders are multisystem disorders with overlapping symptomatology. Pathogenic variants in the PMM2 gene lead to abnormal N-linked glycosylation. This disruption in glycosylation can induce endoplasmic reticulum stress, contributing to the disease pathology. Although impaired mitochondrial dysfunction has been reported in some CDG, cellular bioenergetics has never been evaluated in detail in PMM2-CDG. This prompted us to evaluate mit ...[more]

PMID: 37628636

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Project description:Phosphomannomutase 2 (PMM2) converts mannose-6-phospahate to mannose-1-phosphate; the substrate for GDP-mannose, a building block of the glycosylation biosynthetic pathway. Pathogenic variants in the PMM2 gene have been shown to be associated with protein hypoglycosylation causing PMM2-congenital disorder of glycosylation (PMM2-CDG). While mannose supplementation improves glycosylation in vitro, but not in vivo, we hypothesized that liposomal delivery of mannose-1-phosphate could increase the stability and delivery of the activated sugar to enter the targeted compartments of cells. Thus, we studied the effect of liposome-encapsulated mannose-1-P (GLM101) on global protein glycosylation and on the cellular proteome in skin fibroblasts from individuals with PMM2-CDG, as well as in individuals with two N-glycosylation defects early in the pathway, namely ALG2-CDG and ALG11-CDG. We leveraged multiplexed proteomics and N-glycoproteomics in fibroblasts derived from different individuals with various pathogenic variants in PMM2, ALG2 and ALG11 genes. Proteomics data revealed a moderate but significant change in the abundance of some of the proteins in all CDG fibroblasts upon GLM101 treatment. On the other hand, N-glycoproteomics revealed the GLM101 treatment enhanced the expression levels of several high-mannose and complex/hybrid glycopeptides from numerous cellular proteins in individuals with defects in PMM2 and ALG2 genes. Both PMM2-CDG and ALG2-CDG exhibited several-fold increase in glycopeptides bearing Man6 and higher glycans and a decrease in Man5 and smaller glycan moieties, suggesting that GLM101 helps in the formation of mature glycoforms. These changes in protein glycosylation were observed in all individuals irrespective of their genetic variants. ALG11-CDG fibroblasts also showed increase in high mannose glycopeptides upon treatment; however, the improvement was not as dramatic as the other two CDG. Overall, our findings suggest that treatment with GLM101 overcomes the genetic block in the glycosylation pathway and can be used as a potential therapy for CDG with enzymatic defects in early steps in protein N-glycosylation.

Project description:The most common congenital disorder of glycosylation (CDG) is caused by pathogenic variants in PMM2 (PMM2-CDG) that reduce phosphomannomutase activity and impair N-glycan synthesis. Patients present early in life with multi-system disabilities. Traditional diagnosis relies on measuring carbohydrate-deficient transferrin (CDT) and confirmation by genetic testing. However, tests for CDT can be normal in some patients, or normalize with age. Because patients also show subtle alterations in serum glycan structures, we considered that site-specific glycosylation changes might be diagnostic. We analyzed serum from 35 individuals with PMM2-CDG for discovery and validation of glycoproteomic changes. We carried out mass spectrometry-based discovery studies to profile the glycoproteome in an initial set of well-characterized affected individuals and matched controls. Next, we developed a streamlined method for analyzing additional affected individuals. Finally, targeted mass spectrometry was used to validate selected N-glycopeptides in an independent set of samples in a blinded fashion. Of the 3,342 N-glycopeptides derived from 284 glycoproteins identified in discovery experiments, individuals with PMM2-CDG exhibited a general decrease in complex-type N-glycans and an increase in truncated, mannose-rich and hybrid species. We identified a glycopeptide from complement C4 carrying the glycan Man5GlcNAc2, which was not detected in controls. Notably, this glycopeptide was also detected in five affected individuals with normal CDT, including one individual after liver transplant, two individuals with a known genetic variant associated with mild disease and two individuals with well characterized variants, indicating that this glycopeptide is more sensitive than CDT. Finally, this marker was also detected by targeted analysis in two individuals with variants of uncertain significance (VUS) in PMM2 gene. Overall, we believe that C4-derived Man5GlcNAc2 glycopeptide could serve as a novel next-generation biomarker for more accurate diagnosis and tracking progress of patients in therapeutic trials.

			Action	DRS
	CDG_Fibro_NGP.xlsx	Xlsx
	CDG_Fibro_NGP_B1-B2.raw	Raw
	CDG_Fibro_NGP_B11-B12.raw	Raw
	CDG_Fibro_NGP_B3-B4.raw	Raw
	CDG_Fibro_NGP_B5-B6.raw	Raw