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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 (A & C) and Western Blot (B) analysis of muscular dystrophy patients. Panel A shows 2D DIGE gel images of C1 esterase inhibitor in control, Patient S with an MAN1B1 N- & O-linked disorder and patient L with a LARGE mutation. A low molecular weight glycoform is observed in both patients. This particular lower molecular weight glycoform is not observed in N-linked CDG disorders. Panel B shows 1D western blot images of C1 esterase inhibitor for PMM2-CDG where a lower molecular weight form is observed. This glycoform indicates the degree of mass change for C1 esterase inhibitor in an N-linked only disorder. However in the muscular dystrophy patient with a LARGE mutation, an even greater lower molecular weight band is observed (red arrow) as well as in an MD patient with an unknown mutation. No molecular weight glycoforms are observed for other MD mutations FKRP and POMGNT1. Panel C shows an overlaid 2D DIGE image of α1-antichymotrypsin (top chain) and α-2-HS-glycoprotein (bottom chain) of a patient Y an MD patient with a B3GALNT2 mutation. No changes were observed for any N-linked proteins in patient Y however a clear lower molecular weight glycoform of α2-HS-glycoprotein is seen. α2-HS-glycoprotein is N- & O-glycosylated indicating that O-glycosylation maybe affected in this patient.
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f0020: 2D DIGE (A & C) and Western Blot (B) analysis of muscular dystrophy patients. Panel A shows 2D DIGE gel images of C1 esterase inhibitor in control, Patient S with an MAN1B1 N- & O-linked disorder and patient L with a LARGE mutation. A low molecular weight glycoform is observed in both patients. This particular lower molecular weight glycoform is not observed in N-linked CDG disorders. Panel B shows 1D western blot images of C1 esterase inhibitor for PMM2-CDG where a lower molecular weight form is observed. This glycoform indicates the degree of mass change for C1 esterase inhibitor in an N-linked only disorder. However in the muscular dystrophy patient with a LARGE mutation, an even greater lower molecular weight band is observed (red arrow) as well as in an MD patient with an unknown mutation. No molecular weight glycoforms are observed for other MD mutations FKRP and POMGNT1. Panel C shows an overlaid 2D DIGE image of α1-antichymotrypsin (top chain) and α-2-HS-glycoprotein (bottom chain) of a patient Y an MD patient with a B3GALNT2 mutation. No changes were observed for any N-linked proteins in patient Y however a clear lower molecular weight glycoform of α2-HS-glycoprotein is seen. α2-HS-glycoprotein is N- & O-glycosylated indicating that O-glycosylation maybe affected in this patient.

Mentions: C1 esterase inhibitor shows only a very small molecular weight change for what would be predicted in PMM2-CDG (Supplementary Fig. S1 panel Cb) However as described previously, it is very difficult to confirm at this very low pI range due to breakdown of IEF resolution therefore C1 esterase inhibitor is not an ideal glycoprotein to assess N-glycosylation. However patients S, L, J and K did show a much lower molecular weight glycoform (Fig. 4). Fig. 4 shows representative images of C1 esterase inhibitor from a 2D DIGE image of patients with MAN1B1 deficiency and a LARGE mutation. This glycoform was low in abundance and analysis by in-gel digestion and LC-MS/MS confirmed this protein was C1 esterase inhibitor. Further confirmation was obtained using western blotting for C1 esterase inhibitor performed on PMM2-CDG and several confirmed muscular dystrophy patient serum samples (Fig. 4B). The Western blot data confirmed the presence of a smaller MW glycoform (appx 5–10 kDa less) in PMM2-CDG serum as observed by 2D DIGE. An even smaller and fainter MW glycoform of appx 10–20 kDa less was observed for patient L and in patient M with an unknown mutation. Profiles of C1 esterase inhibitor for other MD patient samples with known mutations were however observed to be normal (Fig. 4B). The smaller C1 esterase inhibitor glycoform observed in patient L by Western blotting confirmed the identity of the lower molecular weight glycoform observed by 2D DIGE. This glycoform is not observed in typical PMM2-CDG and CDG-IIx samples and has only been observed in patients with known affected N- & O-linked glycosylation and apo-CIII positive results (patients S, & K). This indicates strongly that this C1 esterase inhibitor glycoform is present due to altered O-glycosylation.


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 (A & C) and Western Blot (B) analysis of muscular dystrophy patients. Panel A shows 2D DIGE gel images of C1 esterase inhibitor in control, Patient S with an MAN1B1 N- & O-linked disorder and patient L with a LARGE mutation. A low molecular weight glycoform is observed in both patients. This particular lower molecular weight glycoform is not observed in N-linked CDG disorders. Panel B shows 1D western blot images of C1 esterase inhibitor for PMM2-CDG where a lower molecular weight form is observed. This glycoform indicates the degree of mass change for C1 esterase inhibitor in an N-linked only disorder. However in the muscular dystrophy patient with a LARGE mutation, an even greater lower molecular weight band is observed (red arrow) as well as in an MD patient with an unknown mutation. No molecular weight glycoforms are observed for other MD mutations FKRP and POMGNT1. Panel C shows an overlaid 2D DIGE image of α1-antichymotrypsin (top chain) and α-2-HS-glycoprotein (bottom chain) of a patient Y an MD patient with a B3GALNT2 mutation. No changes were observed for any N-linked proteins in patient Y however a clear lower molecular weight glycoform of α2-HS-glycoprotein is seen. α2-HS-glycoprotein is N- & O-glycosylated indicating that O-glycosylation maybe affected in this patient.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

f0020: 2D DIGE (A & C) and Western Blot (B) analysis of muscular dystrophy patients. Panel A shows 2D DIGE gel images of C1 esterase inhibitor in control, Patient S with an MAN1B1 N- & O-linked disorder and patient L with a LARGE mutation. A low molecular weight glycoform is observed in both patients. This particular lower molecular weight glycoform is not observed in N-linked CDG disorders. Panel B shows 1D western blot images of C1 esterase inhibitor for PMM2-CDG where a lower molecular weight form is observed. This glycoform indicates the degree of mass change for C1 esterase inhibitor in an N-linked only disorder. However in the muscular dystrophy patient with a LARGE mutation, an even greater lower molecular weight band is observed (red arrow) as well as in an MD patient with an unknown mutation. No molecular weight glycoforms are observed for other MD mutations FKRP and POMGNT1. Panel C shows an overlaid 2D DIGE image of α1-antichymotrypsin (top chain) and α-2-HS-glycoprotein (bottom chain) of a patient Y an MD patient with a B3GALNT2 mutation. No changes were observed for any N-linked proteins in patient Y however a clear lower molecular weight glycoform of α2-HS-glycoprotein is seen. α2-HS-glycoprotein is N- & O-glycosylated indicating that O-glycosylation maybe affected in this patient.
Mentions: C1 esterase inhibitor shows only a very small molecular weight change for what would be predicted in PMM2-CDG (Supplementary Fig. S1 panel Cb) However as described previously, it is very difficult to confirm at this very low pI range due to breakdown of IEF resolution therefore C1 esterase inhibitor is not an ideal glycoprotein to assess N-glycosylation. However patients S, L, J and K did show a much lower molecular weight glycoform (Fig. 4). Fig. 4 shows representative images of C1 esterase inhibitor from a 2D DIGE image of patients with MAN1B1 deficiency and a LARGE mutation. This glycoform was low in abundance and analysis by in-gel digestion and LC-MS/MS confirmed this protein was C1 esterase inhibitor. Further confirmation was obtained using western blotting for C1 esterase inhibitor performed on PMM2-CDG and several confirmed muscular dystrophy patient serum samples (Fig. 4B). The Western blot data confirmed the presence of a smaller MW glycoform (appx 5–10 kDa less) in PMM2-CDG serum as observed by 2D DIGE. An even smaller and fainter MW glycoform of appx 10–20 kDa less was observed for patient L and in patient M with an unknown mutation. Profiles of C1 esterase inhibitor for other MD patient samples with known mutations were however observed to be normal (Fig. 4B). The smaller C1 esterase inhibitor glycoform observed in patient L by Western blotting confirmed the identity of the lower molecular weight glycoform observed by 2D DIGE. This glycoform is not observed in typical PMM2-CDG and CDG-IIx samples and has only been observed in patients with known affected N- & O-linked glycosylation and apo-CIII positive results (patients S, & K). This indicates strongly that this C1 esterase inhibitor glycoform is present due to altered O-glycosylation.

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