Dysregulated proteome and N-glycoproteome in ALG1-deficient fibroblasts

Abstract

Asparagine-linked glycosylation 1 protein is a β-1,4-mannosyltransferase, is encoded by the ALG1 gene, which catalyzes the first step of mannosylation in N-glycosylation. Pathogenic variants in ALG1 cause a rare autosomal recessive disorder termed as ALG1-CDG. We performed a quantitative proteomics and N-glycoproteomics study in fibroblasts derived from patients with one homozygous and two compound heterozygous pathogenic variants in ALG1. Several proteins that exhibited significant upregulation included insulin-like growth factor II and pleckstrin, whereas hyaluronan and proteoglycan link protein 1 was downregulated. These proteins are crucial for cell growth, survival and differentiation. Additionally, we observed a decrease in the expression of mitochondrial proteins and an increase in autophagy-related proteins, suggesting mitochondrial and cellular stress. N-glycoproteomics revealed the reduction in high-mannose and complex/hybrid glycopeptides derived from numerous proteins in patients explaining that defect in ALG1 has broad effects on glycosylation. Further, we detected an increase in several short oligosaccharides, including chitobiose (HexNAc₂) trisaccharides (Hex-HexNAc₂) and novel tetrasaccharides (NeuAc-Hex-HexNAc₂) derived from essential proteins including LAMP1, CD44 and integrin. These changes in glycosylation were observed in all patients irrespective of their gene variants. Overall, our findings not only provide novel molecular insights into understanding ALG1-CDG but also offer short oligosaccharide-bearing peptides as potential biomarkers.

Keywords: ALG1‐CDG; LC‐MS/MS; biomarker; congenital disorders of glycosylation; glycoproteomics; proteomics.

Figure 1. An overview of N-glycosylation biosynthetic pathway and schematic workflow for proteomic and glycoproteomic analyses of patient derived ALG1 fibroblasts.

(A) Schematic representation showing the key steps involved in N-linked glycosylation pathway with the ALG1-dependent step highlighted with a red “X”. (B) The skin fibroblast cells from ALG1-CDG and control fibroblasts were lysed, proteins were extracted and digested with trypsin. Equal amounts of peptides were labeled with tandem mass tags (TMT) prior to pooling. Pooled TMT labeled samples were then split into two aliquots, where one aliquot was subjected to basic reversed-phase liquid chromatography (bRPLC) for proteomic analysis and the other part was used to enrich glycopeptides using size exclusion chromatography (SEC) for glycoproteomic analysis prior to LC-MS/MS.

Figure 2. Quantitative proteomic changes in patient derived ALG1 fibroblasts.

(A) Volcano plot depicting the differentially abundant proteins in ALG1-CDG patients. X-axis is log₂ fold-change (ALG1-CDG/controls) and Y-axis is the negative logarithm of the p-value from a t test for significance as indicated. The horizontal dashed red line represents the cutoff for significance (<0.05). Some of the highly changing proteins are marked in red circles with the corresponding proteins indicated. (B) Heatmap of all significantly changing proteins showing the differential expression in ALG1-CDG patients. The genotypes for each patient are indicated. The pattern is color coded and gene symbols are given. (C) Principal component analysis (PCA) based on reporter ion intensities for all identified proteins of ALG1-CDG patients and controls. The percentage of total variance associated with each component is shown in brackets with the axis label. (D) Dot plot showing reporter ion intensities of ALG1 protein in ALG1-CDG patient fibroblasts. Y-axis is the reporter ion intensity of TMT channels.

Figure 3. Gene ontology analysis of biological processes involving differentially expressed proteins.

(A) Top upregulated enriched biological processes in ALG1-CDG patients. Y-axis is the negative logarithm of p-values for significance. (B) Top downregulated enriched biological processes in ALG1-CDG patients. Y-axis is the negative logarithm of p-values for significance. (C) Dot plots showing reporter ion intensities for top changing proteins related to autophagy. Y-axis is the reporter ion intensity of TMT channels. Each dot in the plots represent the individual control or patient sample. p < 0.05(*) and p < 0.01(**). (D) Volcano plot depicting the differentially abundant mitochondrial proteins in patient derived ALG1-deficient fibroblasts. X-axis is log₂ fold-change (ALG1-CDG/controls) and Y-axis is the negative logarithm of p-value from a t test for significance as indicated. The horizontal dashed red line represents the cutoff for significance (<0.05). Some of the highly changing proteins are marked in red circles and protein names are provided.

Figure 4. Site-specific glycosylation changes in patient derived ALG1-CDG fibroblasts.

(A) A bar graph showing the global view of glycosylation in fibroblasts. (B) Glycopeptides that are significantly differentially expressed (p < 0.05) with different glycan classes are drawn in a stacked bar graph according to their relative expression in ALG1-CDG patients. (C) Volcano plot depicting the differentially expressed glycopeptides in ALG1-CDG patients. X-axis is log₂ fold-change (ALG1-CDG/controls) and Y-axis is the negative logarithm of p-value from a t test for significance as indicated. The horizontal dashed red line represents the cutoff for significance (<0.05). Some of the changing glycopeptides are marked in red circles and glycoproteins’ names, glycosylation sites and glycan structures are drawn. Heatmap of significantly changing glycopeptides (p-value <0.05) with different (D) complex or hybrid glycan composition and (E) high-mannose glycan moieties. The genotypes for each patient are indicated. The pattern is color coded. (F) Dot plots showing reporter ion intensities of four downregulated regulated glycopeptides derived from LAMP1, CD44, and ITB1 in ALG1-CDG. Y-axis is the reporter ion intensity of TMT channels. Each dot in the plots represent the individual control or patient sample. (G) Differential chord diagram depicting the site-specific glycosylation changes for the proteins encompassing the oligosaccharides in ALG1-CDG as compared to control fibroblasts. Proteins with different glycosylation sites are indexed on the right of the diagram and connected via chords to respective identified glycan structures on the left. Asn^x represents the asparagine at amino acid site “x” in the corresponding protein sequence. The fold-change pattern is color coded. Putative structures are shown using Symbol Nomenclature for Glycans (SNFG). p < 0.05 (*), p < 0.01 (**).

Figure 5

Site-specific alterations in abundance of various oligosaccharides bearing glycopeptides in ALG1-CDG. The dot plots depict the expression levels of glycopeptides associated with various oligosaccharides. Protein names, N-glycosylation sites and the oligosaccharide structures are indicated on the top of dot plots and X-axis depicts sample type while Y-axis is reporter ion intensity at the glycopeptide level. Asn^x represents the asparagine at amino acid site “x” in the corresponding protein sequence. Each dot in the plot corresponds to an individual sample from either the patient group or control. Putative structures are shown using Symbol Nomenclature for Glycans (SNFG). p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).