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Global serum glycoform profiling for the investigation of dystroglycanopathies & Congenital Disorders of Glycosylation.

Heywood WE, Bliss E, Mills P, Yuzugulen J, Carreno G, Clayton PT, Muntoni F, Worthington VC, Torelli S, Sebire NJ, Mills K, Grunewald S - Mol Genet Metab Rep (2016)

Bottom Line: These biomarkers do not always detect complex or subtle defects present in older patients, therefore there is a need to investigate additional glycoproteins in some cases.In addition, we could identify abnormal serum glycoproteins in LARGE and B3GALNT2-deficient muscular dystrophies.The technique has further potential in monitoring patients for future treatment strategies.

View Article: PubMed Central - PubMed

Affiliation: Centre for Inborn Errors of Metabolism, Great Ormond Street Hospital, Great Ormond Street, London WC1N 3JH, UK; Centre for Translational Omics, UCL Institute of Child Health & Great Ormond Street Hospital NHS Foundation Trust, London WC1N 1EH, UK.

ABSTRACT
The Congenital Disorders of Glycosylation (CDG) are an expanding group of genetic disorders which encompass a spectrum of glycosylation defects of protein and lipids, including N- & O-linked defects and among the latter are the muscular dystroglycanopathies (MD). Initial screening of CDG is usually based on the investigation of the glycoproteins transferrin, and/or apolipoprotein CIII. These biomarkers do not always detect complex or subtle defects present in older patients, therefore there is a need to investigate additional glycoproteins in some cases. We describe a sensitive 2D-Differential Gel Electrophoresis (DIGE) method that provides a global analysis of the serum glycoproteome. Patient samples from PMM2-CDG (n = 5), CDG-II (n = 7), MD and known complex N- & O-linked glycosylation defects (n = 3) were analysed by 2D DIGE. Using this technique we demonstrated characteristic changes in mass and charge in PMM2-CDG and in charge in CDG-II for α1-antitrypsin, α1-antichymotrypsin, α2-HS-glycoprotein, ceruloplasmin, and α1-acid glycoproteins 1&2. Analysis of the samples with known N- & O-linked defects identified a lower molecular weight glycoform of C1-esterase inhibitor that was not observed in the N-linked glycosylation disorders indicating the change is likely due to affected O-glycosylation. In addition, we could identify abnormal serum glycoproteins in LARGE and B3GALNT2-deficient muscular dystrophies. The results demonstrate that the glycoform pattern is varied for some CDG patients not all glycoproteins are consistently affected and analysis of more than one protein in complex cases is warranted. 2D DIGE is an ideal method to investigate the global glycoproteome and is a potentially powerful tool and secondary test for aiding the complex diagnosis and sub classification of CDG. The technique has further potential in monitoring patients for future treatment strategies. In an era of shifting emphasis from gel- to mass-spectral based proteomics techniques, we demonstrate that 2D-DIGE remains a powerful method for studying global changes in post-translational modifications of proteins.

No MeSH data available.


Related in: MedlinePlus

2D DIGE analysis of ceruloplasmin in control, PMM2-CDG and CDG-II. Representative overlaid (top panels) and 3D images of ceruloplasmin illustrating typical changes observed in CDG-II and PMM2-CDG. The overlaid image shows normal pink and overlaid with either CDG as green. CDG-II patients show a subtle charge change as typically seen with many serum glycoproteins which is highlighted by the red ring. The overall concentration, charge and mass change is significantly affected in PMM2-CDG and indicated by the red rings either side of the ceruloplasmin chain.
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f0015: 2D DIGE analysis of ceruloplasmin in control, PMM2-CDG and CDG-II. Representative overlaid (top panels) and 3D images of ceruloplasmin illustrating typical changes observed in CDG-II and PMM2-CDG. The overlaid image shows normal pink and overlaid with either CDG as green. CDG-II patients show a subtle charge change as typically seen with many serum glycoproteins which is highlighted by the red ring. The overall concentration, charge and mass change is significantly affected in PMM2-CDG and indicated by the red rings either side of the ceruloplasmin chain.

Mentions: Ceruloplasmin is a glycoprotein with 6 N-linked glycans and is involved in the transport of copper in serum. 2D DIGE analysis revealed ceruloplasmin to be characteristically altered in PMM2-CDG and CDG-II (Fig. 3).


Global serum glycoform profiling for the investigation of dystroglycanopathies & Congenital Disorders of Glycosylation.

Heywood WE, Bliss E, Mills P, Yuzugulen J, Carreno G, Clayton PT, Muntoni F, Worthington VC, Torelli S, Sebire NJ, Mills K, Grunewald S - Mol Genet Metab Rep (2016)

2D DIGE analysis of ceruloplasmin in control, PMM2-CDG and CDG-II. Representative overlaid (top panels) and 3D images of ceruloplasmin illustrating typical changes observed in CDG-II and PMM2-CDG. The overlaid image shows normal pink and overlaid with either CDG as green. CDG-II patients show a subtle charge change as typically seen with many serum glycoproteins which is highlighted by the red ring. The overall concentration, charge and mass change is significantly affected in PMM2-CDG and indicated by the red rings either side of the ceruloplasmin chain.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834675&req=5

f0015: 2D DIGE analysis of ceruloplasmin in control, PMM2-CDG and CDG-II. Representative overlaid (top panels) and 3D images of ceruloplasmin illustrating typical changes observed in CDG-II and PMM2-CDG. The overlaid image shows normal pink and overlaid with either CDG as green. CDG-II patients show a subtle charge change as typically seen with many serum glycoproteins which is highlighted by the red ring. The overall concentration, charge and mass change is significantly affected in PMM2-CDG and indicated by the red rings either side of the ceruloplasmin chain.
Mentions: Ceruloplasmin is a glycoprotein with 6 N-linked glycans and is involved in the transport of copper in serum. 2D DIGE analysis revealed ceruloplasmin to be characteristically altered in PMM2-CDG and CDG-II (Fig. 3).

Bottom Line: These biomarkers do not always detect complex or subtle defects present in older patients, therefore there is a need to investigate additional glycoproteins in some cases.In addition, we could identify abnormal serum glycoproteins in LARGE and B3GALNT2-deficient muscular dystrophies.The technique has further potential in monitoring patients for future treatment strategies.

View Article: PubMed Central - PubMed

Affiliation: Centre for Inborn Errors of Metabolism, Great Ormond Street Hospital, Great Ormond Street, London WC1N 3JH, UK; Centre for Translational Omics, UCL Institute of Child Health & Great Ormond Street Hospital NHS Foundation Trust, London WC1N 1EH, UK.

ABSTRACT
The Congenital Disorders of Glycosylation (CDG) are an expanding group of genetic disorders which encompass a spectrum of glycosylation defects of protein and lipids, including N- & O-linked defects and among the latter are the muscular dystroglycanopathies (MD). Initial screening of CDG is usually based on the investigation of the glycoproteins transferrin, and/or apolipoprotein CIII. These biomarkers do not always detect complex or subtle defects present in older patients, therefore there is a need to investigate additional glycoproteins in some cases. We describe a sensitive 2D-Differential Gel Electrophoresis (DIGE) method that provides a global analysis of the serum glycoproteome. Patient samples from PMM2-CDG (n = 5), CDG-II (n = 7), MD and known complex N- & O-linked glycosylation defects (n = 3) were analysed by 2D DIGE. Using this technique we demonstrated characteristic changes in mass and charge in PMM2-CDG and in charge in CDG-II for α1-antitrypsin, α1-antichymotrypsin, α2-HS-glycoprotein, ceruloplasmin, and α1-acid glycoproteins 1&2. Analysis of the samples with known N- & O-linked defects identified a lower molecular weight glycoform of C1-esterase inhibitor that was not observed in the N-linked glycosylation disorders indicating the change is likely due to affected O-glycosylation. In addition, we could identify abnormal serum glycoproteins in LARGE and B3GALNT2-deficient muscular dystrophies. The results demonstrate that the glycoform pattern is varied for some CDG patients not all glycoproteins are consistently affected and analysis of more than one protein in complex cases is warranted. 2D DIGE is an ideal method to investigate the global glycoproteome and is a potentially powerful tool and secondary test for aiding the complex diagnosis and sub classification of CDG. The technique has further potential in monitoring patients for future treatment strategies. In an era of shifting emphasis from gel- to mass-spectral based proteomics techniques, we demonstrate that 2D-DIGE remains a powerful method for studying global changes in post-translational modifications of proteins.

No MeSH data available.


Related in: MedlinePlus