Dataset Information

CSF glycoproteomics reveals brain-specific changes in glyco-sylation in congenital disorders of glycosylation

ABSTRACT: In this project, CSF samples from healthy volunteers and patients diagnosed with congenital disorders of glycosylation (CDG) were analyzed with MS-based glycoproteomics to characterize the glycoproteome in the two cohorts. In order to identify highly truncated glycopeptides and glycopeptides that show the complete loss of glycosylation, we chose to not perform a glycopeptide enrichment. Using the high identification rate of the timsTOF Pro including PASEF fragmentation, we were able to detect over 800 unique glycopeptides. We show that changes in protein N-glycosylation of CDG patients can be different on brain-derived proteins compared to blood derived proteins.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cerebrospinal Fluid

DISEASE(S): Congenital Disorder Of Glycosylation

SUBMITTER: Melissa Baerenfaenger

LAB HEAD: Melissa Baerenfaenger

PROVIDER: PXD038787 | Pride | 2023-01-18

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	CSF_CDG_16_16_1_2_20201112_Slot1-12_1_5026.mzML	Mzml
	CSF_CDG_16_16_1_2_20201112_Slot1-12_1_5027.mzML	Mzml
	CSF_CDG_3_30_1_2_20201112_Slot1-08_1_5012.mzML	Mzml
	CSF_CDG_3_30_1_2_20201112_Slot1-08_1_5013.mzML	Mzml
	CSF_CDG_3_64_1_2_20201112_Slot1-10_1_5022.mzML	Mzml

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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.

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CSF glycoproteomics reveals brain-specific changes in glyco-sylation in congenital disorders of glycosylation

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