Identification of Intercellular Cell Adhesion Molecule 1 (ICAM-1) as a Hypoglycosylation Marker in Congenital Disorders of Glycosylation Cells

He, Ping; Ng, Bobby G.; Losfeld, Marie-Estelle; Zhu, Wenhong; Freeze, Hudson H.

doi:10.1074/jbc.m112.355677

Cited by 34 publications

(31 citation statements)

References 28 publications

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“…Importantly, complementation of primary fibroblasts from CDG-0359 with a plasmid containing wild-type SSR3 cDNA completely restored the expression of SSR3 protein and the integrity of the TRAP complex as determined by the reappearance of SSR1 and SSR4 protein levels seen in control fibroblasts (Figure 1). Finally, we show that primary fibroblast from CDG-0359 have abnormal glycosylation of two previously reported marker proteins, GP130 and ICAM1 18,19 (Figure 2). These results along with the abnormal CDT indicate that the mutation in SSR3 compromises N-linked glycosylation.…”

Section: Western Blot Analysissupporting

confidence: 59%

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Mutations in the translocon‐associated protein complex subunit SSR3 cause a novel congenital disorder of glycosylation

Lourenço²,

Losfeld

et al. 2019

J of Inher Metab Disea

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The translocon‐associated protein (TRAP) complex facilitates the translocation of proteins across the endoplasmic reticulum membrane and associates with the oligosaccharyl transferase (OST) complex to maintain proper glycosylation of nascent polypeptides. Pathogenic variants in either complex cause a group of rare genetic disorders termed, congenital disorders of glycosylation (CDG). We report an individual who presented with severe intellectual and developmental disabilities and sensorineural deafness with an unsolved type I CDG, and sought to identify the underlying genetic basis. Exome sequencing identified a novel homozygous variant c.278_281delAGGA [p.Glu93Valfs*7] in the signal sequence receptor 3 (SSR3) subunit of the TRAP complex. Biochemical studies in patient fibroblasts showed the variant destabilized the TRAP complex with a complete loss of SSR3 protein and partial loss of SSR1 and SSR4. Importantly, all subunit levels were corrected by expression of wild‐type SSR3. Abnormal glycosylation status in fibroblasts was confirmed using two markers proteins, GP130 and ICAM1. Our findings confirm mutations in SSR3 cause a novel CDG. A novel frameshift variant in the translocon associated protein, SSR3, disrupts the stability of the TRAP complex and causes a novel Congenital Disorder of Glycosylation.

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Section: Western Blot Analysissupporting

confidence: 59%

“…Western blot analysis was carried out as previously described using two commercially available control fibroblast lines GM‐00038, GM‐03348 (Coriell cell repository) and CDG‐0359 to determine the protein levels for SSR1‐4 (Novus Biologicals) . GP130 and ICAM1 western blots were performed as previously described …”

Section: Methodsmentioning

confidence: 99%

Mutations in the translocon‐associated protein complex subunit SSR3 cause a novel congenital disorder of glycosylation

Lourenço²,

Losfeld

et al. 2019

J of Inher Metab Disea

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“…We additionally performed flow cytometry with the fluorescently labeled lectins GNA and Con A. Con A also preferentially binds to high-mannose-type N-glycans ( 44 ) and is not known to bind to O-glycans ( 68 ). This analysis was performed on intact cells and showed reduced levels of cell surface exposed high-mannose-type N-glycans, which could be a consequence of membrane protein hypoglycosylation or reduced incorporation of glycoproteins into the plasma membrane, as recently shown for hypoglycosylated ICAM-1 ( 69 ). By spiking-in a defined reference glycoprotein (asialofetuin) to our samples, we here present the application of xCGE-LIF for quantitative comparison between different samples.…”

Section: Discussionmentioning

confidence: 60%

Glycomic Characterization of Induced Pluripotent Stem Cells Derived from a Patient Suffering from Phosphomannomutase 2 Congenital Disorder of Glycosylation (PMM2-CDG)

Thiesler

Cajic

Hoffmann

et al. 2016

Molecular & Cellular Proteomics

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PMM2-CDG, formerly known as congenital disorder of glycosylation-Ia (CDG-Ia), is caused by mutations in the gene encoding phosphomannomutase 2 (PMM2). This disease is the most frequent form of inherited CDG-diseases affecting protein N-glycosylation in human. PMM2-CDG is a multisystemic disease with severe psychomotor and mental retardation. In order to study the pathophysiology of PMM2-CDG in a human cell culture model, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of a PMM2-CDG-patient (PMM2-iPSCs). Expression of pluripotency factors and in vitro differentiation into cell types of the three germ layers was unaffected in the analyzed clone PMM2-iPSC-C3 compared with nondiseased human pluripotent stem cells (hPSCs), revealing no broader influence of the PMM2 mutation on pluripotency in cell culture. Analysis of gene expression by deep-sequencing did not show obvious differences in the transcriptome between PMM2-iPSC-C3 and nondiseased hPSCs. By multiplexed capillary gel electrophoresis coupled to laser induced fluorescence detection (xCGE-LIF) we could show that PMM2-iPSC-C3 exhibit the common hPSC N-glycosylation pattern with high-mannose-type N-glycans as the predominant species. However, phosphomannomutase activity of PMM2-iPSC-C3 was 27% compared with control hPSCs and lectin staining revealed an overall reduced protein glycosylation. In addition, quantitative assessment of N-glycosylation by xCGE-LIF showed an up to 40% reduction of high-mannose-type N-glycans in PMM2-iPSC-C3, which was in concordance to the observed reduction of the Glc3Man9GlcNAc2 lipid-linked oligosaccharide compared with control hPSCs. Thus we could model the PMM2-CDG disease phenotype of hypoglycosylation with patient derived iPSCs in vitro. Knock-down of PMM2 by shRNA in PMM2-iPSC-C3 led to a residual activity of 5% and to a further reduction of the level of N-glycosylation. Taken together we have developed human stem cell-based cell culture models with stepwise reduced levels of N-glycosylation now enabling to study the role of N-glycosylation during early human development.

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“…Additionally, cis-PTase activity (Figure 4B) and mannose incorporation into proteins (Figure 4C) was markedly lower in NgBR R290H fibroblasts compared to control. We also examined defective glycosylation of proteins in patient fibroblasts by Western blotting for two known glycoproteins, LAMP-1 and ICAM-1 (He et al, 2012; Xiang et al, 2013). Both LAMP-1 and ICAM-1 were hypo-glycosylated in the patient fibroblasts (Figure 4D).…”

Section: Resultsmentioning

confidence: 99%

Mutation of Nogo-B Receptor, a Subunit of cis-Prenyltransferase, Causes a Congenital Disorder of Glycosylation

et al. 2014

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Summary Dolichol is an obligate carrier of glycans for N-linked protein glycosylation, O-mannosylation, and GPI anchor biosynthesis. Cis-prenyltransferase (cis-PTase) is the first enzyme committed to the synthesis of dolichol. However, the proteins responsible for mammalian cis-PTase activity have not been delineated. Here we show that Nogo-B receptor (NgBR) is a subunit required for dolichol synthesis in yeast, mice and man. Moreover, we describe a family with a congenital disorder of glycosylation caused by a loss of function mutation in the conserved C terminus of NgBR-R290H and show that fibroblasts isolated from patients exhibit reduced dolichol profiles and enhanced accumulation of free cholesterol identically to fibroblasts from mice lacking NgBR. Mutation of NgBR-R290H in man and orthologs in yeast proves the importance of this evolutionarily conserved residue for mammalian cis-PTase activity and function. Thus, these data provides a genetic basis for the essential role of NgBR in dolichol synthesis and protein glycosylation.

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Identification of Intercellular Cell Adhesion Molecule 1 (ICAM-1) as a Hypoglycosylation Marker in Congenital Disorders of Glycosylation Cells

Cited by 34 publications

References 28 publications

Mutations in the translocon‐associated protein complex subunit SSR3 cause a novel congenital disorder of glycosylation

Mutations in the translocon‐associated protein complex subunit SSR3 cause a novel congenital disorder of glycosylation

Glycomic Characterization of Induced Pluripotent Stem Cells Derived from a Patient Suffering from Phosphomannomutase 2 Congenital Disorder of Glycosylation (PMM2-CDG)

Mutation of Nogo-B Receptor, a Subunit of cis-Prenyltransferase, Causes a Congenital Disorder of Glycosylation

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