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A new patient-derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of ?-dystroglycan.

ABSTRACT: Dystroglycan, an extracellular matrix receptor, has essential functions in various tissues. Loss of ?-dystroglycan-laminin interaction due to defective glycosylation of ?-dystroglycan underlies a group of congenital muscular dystrophies often associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic human dystroglycanopathy cell models has limited our ability to test potential drugs in a human- and neural-specific context. Here, we generated induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous FKRP (fukutin-related protein gene) mutation. We showed that CRISPR/Cas9-mediated gene correction of FKRP restored glycosylation of ?-dystroglycan in iPSC-derived cortical neurons, whereas targeted gene mutation of FKRP in wild-type cells disrupted this glycosylation. In parallel, we screened 31,954 small molecule compounds using a mouse myoblast line for increased glycosylation of ?-dystroglycan. Using human FKRP-iPSC-derived neural cells for hit validation, we demonstrated that compound 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile (4BPPNit) significantly augmented glycosylation of ?-dystroglycan, in part through upregulation of LARGE1 glycosyltransferase gene expression. Together, isogenic human iPSC-derived cells represent a valuable platform for facilitating dystroglycanopathy drug discovery and therapeutic development.

SUBMITTER: Kim J

PROVIDER: S-EPMC6832011 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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A new patient-derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α-dystroglycan.

Kim Jihee J Lana Beatrice B Torelli Silvia S Ryan David D Catapano Francesco F Ala Pierpaolo P Luft Christin C Stevens Elizabeth E Konstantinidis Evangelos E Louzada Sandra S Fu Beiyuan B Paredes-Redondo Amaia A Chan Aw Edith AE Yang Fengtang F Stemple Derek L DL Liu Pentao P Ketteler Robin R Selwood David L DL Muntoni Francesco F Lin Yung-Yao YY

EMBO reports 20190930 11

Dystroglycan, an extracellular matrix receptor, has essential functions in various tissues. Loss of α-dystroglycan-laminin interaction due to defective glycosylation of α-dystroglycan underlies a group of congenital muscular dystrophies often associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic human dystroglycanopathy cell models has limited our ability to test potential drugs in a human- and neural-specific context. Here, we generated induced pluripote ...[more]

PMID: 31566294

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Project description:?-dystroglycan (?-DG) is a peripheral membrane protein that is an integral component of the dystrophin-glycoprotein complex. In an inherited subset of muscular dystrophies known as dystroglycanopathies, ?-DG has reduced glycosylation which results in lower affinity binding to several extracellular matrix proteins including laminins. The glycosylation status of ?-DG is normally assessed by the binding of the ?-DG antibody IIH6 to a specific glycan epitope on ?-DG involved in laminin binding. Immunocytochemistry and immunoblotting are two of the most widely used methods to detect the amount of ?-DG glycosylation in muscle. While the interpretation of the presence or absence of the epitope on muscle using these techniques is straightforward, the assessment of a mild defect can be challenging. In this study, flow cytometry was used to compare the amount of IIH6-reactive glycans in fibroblasts from dystroglycanopathy patients with defects in genes known to cause ?-DG hypoglycosylation to the amount in fibroblasts from healthy and pathological control subjects. A total of twenty one dystroglycanopathy patient fibroblasts were assessed, as well as fibroblasts from three healthy controls and seven pathological controls. Control fibroblasts have clearly detectable amounts of IIH6-reactive glycans, and there is a significant difference in the amount of this glycosylation, as measured by the mean fluorescence intensity of an antibody recognising the epitope and the percentage of cells positive for the epitope, between these controls and dystroglycanopathy patient fibroblasts (p<0.0001 for both). Our results indicate that the amount of ?-DG glycosylation in patient fibroblasts is comparable to that in patient skeletal muscle. This method could complement existing immunohistochemical assays in skeletal muscle as it is quantitative and simple to perform, and could be used when a muscle biopsy is not available. This test could also be used to assess the pathogenicity of variants of unknown significance in genes involved in dystroglycanopathies.

Project description: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 ?1-antitrypsin, ?1-antichymotrypsin, ?2-HS-glycoprotein, ceruloplasmin, and ?1-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.

Dataset Information

A new patient-derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of ?-dystroglycan.

Publications

A new patient-derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α-dystroglycan.

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