Large Induces Functional Glycans in an O-Mannosylation Dependent Manner and Targets GlcNAc Terminals on Alpha-Dystroglycan

Hu, Yihong; Li, Zhifang; Wu, Xiaohua; Lu, Qilong

doi:10.1371/journal.pone.0016866

Cited by 20 publications

(18 citation statements)

References 36 publications

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“…However some forms of muscular dystrophy have been reported to have defects in the glycosylation pathway which affect not just O-mannosyl but also GlcNAc O-glycosylation. One such defect is related to LARGE mutations, which have recently been described to affect both of these forms of O-glycosylation [14]. This has been confirmed in our own laboratories, as LARGE patient serum demonstrated a reduced molecular weight form of the heavily N and O-glycosylated protein C1-inhibitor by western blotting [15].…”

Section: Introductionsupporting

confidence: 54%

The Emerging Field of Diagnostics in Glycosylation Disorders

Heywood¹,

Clayton²,

Mills³

et al. 2013

Pediat Therapeut

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Protein glycosylation is an important post-translational modification. It enhances the functional diversity of proteins, half-life and influences their biological activity. Defective glycosylation often leads to multisystem disease and adds itself to the expanding group of 'Congenital disorders or glycosylation' which are predominantly disorders of N-linked glycosylation. Another rapidly growing group of disorders are defects in O-linked glycosylation, including a subset of dystroglycanopathies. Current diagnostic strategies for glycosylation disorders are compounded by the multivariate clinical phenotype of many of the diseases. Biochemical tests such as the isoelectric focusing of transferrin and apolipoprotein CIII are used to assess a patient's glycoform profile before in depth enzyme and genetic analysis is initiated. Whilst the glycoform profiling has been instrumental in screening for many glycosylation disorders, there is a need for a more sensitive and informative test. This short review gives an overview of the recent methods used in glycobiology research that could be used to devise such a test, which alongside currently used diagnostic tests should further facilitate the delineation of CDG subtypes. It provides a view to a potential strategy using marker glycopeptides to develop a mass spectrometry based assay that could be implemented into clinical diagnostic laboratories.

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Section: Introductionsupporting

confidence: 54%

The Emerging Field of Diagnostics in Glycosylation Disorders

Heywood¹,

Clayton²,

Mills³

et al. 2013

Pediat Therapeut

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“…Early studies reported that LARGE could modify complex N-and mucine O-glycans on a-DG to induce laminin binding (Patnaik and Stanley, 2005). LARGE overexpression competes to modify GlcNAc terminals with galactosylation to generate the functional glycans on both O-linked and Nlinked glycans (Hu et al, 2011). LARGE also facilitates functional phosphoryl glycosylation of O-linked mannose and N-linked glycans on proteins other than a-DG (Zhang and Hu, 2012).…”

mentioning

confidence: 99%

Adeno-Associated Virus-Mediated Overexpression of LARGE Rescues α-Dystroglycan Function in Dystrophic Mice with Mutations in the Fukutin-Related Protein

Vannoy

Keramaris

et al. 2014

Human Gene Therapy Methods

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Multiple genes (e.g., POMT1, POMT2, POMGnT1, ISPD, GTDC2, B3GALNT2, FKTN, FKRP, and LARGE) are known to be involved in the glycosylation pathway of a-dystroglycan (a-DG). Mutations of these genes result in muscular dystrophies with wide phenotypic variability. Abnormal glycosylation of a-DG with decreased extracellular ligand binding activity is a common biochemical feature of these genetic diseases. While it is known that LARGE overexpression can compensate for defects in a few aforementioned genes, it is unclear whether it can also rescue defects in FKRP function. We examined adeno-associated virus (AAV)-mediated LARGE or FKRP overexpression in two dystrophic mouse models with loss-of-function mutations: (1) Large myd (LARGE gene) and (2) FKRP P448L (FKRP gene). The results agree with previous findings that overexpression of LARGE can ameliorate the dystrophic phenotypes of Large myd mice. In addition, LARGE overexpression in the FKRP P448L mice effectively generated functional glycosylation (hyperglycosylation) of a-DG and improved dystrophic pathologies in treated muscles. Conversely, FKRP transgene overexpression failed to rescue the defect in glycosylation and improve the phenotypes of the Large myd mice. Our findings suggest that AAVmediated LARGE gene therapy may still be a viable therapeutic strategy for dystroglycanopathies with FKRP deficiency.

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“…However, another study showed that stable expression of LARGE1 in the O -mannosylation-deficient mutant CHO cells failed to alter the processing of α-DG, with only transient overexpression that causes extremely high levels of LARGE1 expression able to produce the functional modification (Hu et al 2011). We speculate that when the expression level of LARGE1 is artificially high, this enzyme can modify unnatural acceptors such as N - or O -glycans, and possibly non-α-DG proteins.…”

Section: Discussionmentioning

confidence: 99%

LARGE2-dependent glycosylation confers laminin-binding ability on proteoglycans

et al. 2016

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Both LARGE1 (formerly LARGE) and its paralog LARGE2 are bifunctional glycosyltransferases with xylosy- and glucuronyltransferase activities, and are capable of synthesizing polymers composed of a repeating disaccharide [-3Xylα1,3GlcAβ1-]. Post-translational modification of the O-mannosyl glycan of α-dystroglycan (α-DG) with the polysaccharide is essential for it to act as a receptor for ligands in the extracellular matrix (ECM), and both LARGE paralogs contribute to the modification in vivo. LARGE1 and LARGE2 have different tissue distribution profiles and enzymatic properties; however, the functional difference of the homologs remains to be determined, and α-DG is the only known substrate for the modification by LARGE1 or LARGE2. Here we show that LARGE2 can modify proteoglycans (PGs) with the laminin-binding glycan. We found that overexpression of LARGE2, but not LARGE1, mediates the functional modification on the surface of DG−/−, Pomt1−/− and Fktn−/− embryonic stem cells. We identified a heparan sulfate-PG glypican-4 as a substrate for the LARGE2-dependent modification by affinity purification and subsequent mass spectrometric analysis. Furthermore, we showed that LARGE2 could modify several additional PGs with the laminin-binding glycan, most likely within the glycosaminoglycan (GAG)-protein linkage region. Our results indicate that LARGE2 can modify PGs with the GAG-like polysaccharide composed of xylose and glucuronic acid to confer laminin binding. Thus, LARGE2 may play a differential role in stabilizing the basement membrane and modifying its functions by augmenting the interactions between laminin globular domain-containing ECM proteins and PGs.

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Large Induces Functional Glycans in an O-Mannosylation Dependent Manner and Targets GlcNAc Terminals on Alpha-Dystroglycan

Cited by 20 publications

References 36 publications

The Emerging Field of Diagnostics in Glycosylation Disorders

The Emerging Field of Diagnostics in Glycosylation Disorders

Adeno-Associated Virus-Mediated Overexpression of LARGE Rescues α-Dystroglycan Function in Dystrophic Mice with Mutations in the Fukutin-Related Protein

LARGE2-dependent glycosylation confers laminin-binding ability on proteoglycans

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