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Fukutin is prerequisite to ameliorate muscular dystrophic phenotype by myofiber-selective LARGE expression.

Ohtsuka Y, Kanagawa M, Yu CC, Ito C, Chiyo T, Kobayashi K, Okada T, Takeda S, Toda T - Sci Rep (2015)

Bottom Line: However, the in vivo therapeutic benefit of using LARGE activity is controversial.Furthermore, forced expression of Large in fukutin-deficient embryonic stem cells also failed to recover α-DG glycosylation, however coexpression with fukutin strongly enhanced α-DG glycosylation.Together, our data demonstrated that fukutin is required for LARGE-dependent rescue of α-DG glycosylation, and thus suggesting new directions for LARGE-utilizing therapy targeted to myofibres.

View Article: PubMed Central - PubMed

Affiliation: Division of Neurology/Molecular Brain Science, Kobe University Graduate School of Medicine, Kobe, 650-0017, Japan.

ABSTRACT
α-Dystroglycanopathy (α-DGP) is a group of muscular dystrophy characterized by abnormal glycosylation of α-dystroglycan (α-DG), including Fukuyama congenital muscular dystrophy (FCMD), muscle-eye-brain disease, Walker-Warburg syndrome, and congenital muscular dystrophy type 1D (MDC1D), etc. LARGE, the causative gene for MDC1D, encodes a glycosyltransferase to form [-3Xyl-α1,3GlcAβ1-] polymer in the terminal end of the post-phosphoryl moiety, which is essential for α-DG function. It has been proposed that LARGE possesses the great potential to rescue glycosylation defects in α-DGPs regardless of causative genes. However, the in vivo therapeutic benefit of using LARGE activity is controversial. To explore the conditions needed for successful LARGE gene therapy, here we used Large-deficient and fukutin-deficient mouse models for MDC1D and FCMD, respectively. Myofibre-selective LARGE expression via systemic adeno-associated viral gene transfer ameliorated dystrophic pathology of Large-deficient mice even when intervention occurred after disease manifestation. However, the same strategy failed to ameliorate the dystrophic phenotype of fukutin-conditional knockout mice. Furthermore, forced expression of Large in fukutin-deficient embryonic stem cells also failed to recover α-DG glycosylation, however coexpression with fukutin strongly enhanced α-DG glycosylation. Together, our data demonstrated that fukutin is required for LARGE-dependent rescue of α-DG glycosylation, and thus suggesting new directions for LARGE-utilizing therapy targeted to myofibres.

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α-DG glycosylation in fukutin- ES cells after fukutin or LARGE expression.Fukutin (FLAG-tagged) and/or LARGE (myc-tagged) were expressed in wild-type or fukutin- mouse ES cells and α-DG glycosylation status was analysed by western blotting. Exogenous LARGE expression produced highly glycosylated α-DG in wild-type but not fukutin- ES cells, indicated by IIH6 staining. Co-transfection of fukutin and Large yielded LARGE-dependent glycosylation of α-DG in fukutin- ES cells. Arrow and asterisk indicate LARGE protein and non-specific signals, respectively. The full-length blots with α-DG (IIH6), LARGE (myc), fukutin (FLAG), and β-DG are presented in Supplementary Figure S2i-l, respectively.
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f4: α-DG glycosylation in fukutin- ES cells after fukutin or LARGE expression.Fukutin (FLAG-tagged) and/or LARGE (myc-tagged) were expressed in wild-type or fukutin- mouse ES cells and α-DG glycosylation status was analysed by western blotting. Exogenous LARGE expression produced highly glycosylated α-DG in wild-type but not fukutin- ES cells, indicated by IIH6 staining. Co-transfection of fukutin and Large yielded LARGE-dependent glycosylation of α-DG in fukutin- ES cells. Arrow and asterisk indicate LARGE protein and non-specific signals, respectively. The full-length blots with α-DG (IIH6), LARGE (myc), fukutin (FLAG), and β-DG are presented in Supplementary Figure S2i-l, respectively.

Mentions: The amount of LARGE protein expressed in the AAV-treated Myf5-fukutin-cKO mice was comparable to that in AAV-treated Largemyd (Fig. 3a). α-DG glycosylation was recovered in Largemyd skeletal muscle after AAV9-MCK-Large treatment, suggesting something other than protein expression levels is responsible for the failure of glycosylation recovery in Myf5-fukutin-cKO mice. We hypothesized that complete loss of fukutin caused failure to build the part of post-phosphoryl moiety, which may be required for LARGE-dependent glycosylation; therefore, even excess LARGE protein could not form the [-3Xyl-α1,3GlcAβ1-] polymer on α-DG. To test this hypothesis, we expressed LARGE in fukutin- embryonic stem (ES) cells. Transfection of the fukutin cDNA restored IIH6 reactivity in fukutin- ES cells, but transfection of the Large cDNA failed to restore α-DG glycosylation, although LARGE expression in wild-type ES cells produced strong IIH6-reactivity (Fig. 4). When the fukutin and Large cDNAs were co-transfected into fukutin- ES cells, we observed increases in IIH6-reactivity in comparison to fukutin singly transfected cells although expression levels of both fukutin and LARGE were much lower than they were in each single transfection (Fig. 4, lanes 6–8). These data show that fukutin-dependent modification is a prerequisite for LARGE-dependent formation of [-3Xyl-α1,3GlcAβ1-] repeating units.


Fukutin is prerequisite to ameliorate muscular dystrophic phenotype by myofiber-selective LARGE expression.

Ohtsuka Y, Kanagawa M, Yu CC, Ito C, Chiyo T, Kobayashi K, Okada T, Takeda S, Toda T - Sci Rep (2015)

α-DG glycosylation in fukutin- ES cells after fukutin or LARGE expression.Fukutin (FLAG-tagged) and/or LARGE (myc-tagged) were expressed in wild-type or fukutin- mouse ES cells and α-DG glycosylation status was analysed by western blotting. Exogenous LARGE expression produced highly glycosylated α-DG in wild-type but not fukutin- ES cells, indicated by IIH6 staining. Co-transfection of fukutin and Large yielded LARGE-dependent glycosylation of α-DG in fukutin- ES cells. Arrow and asterisk indicate LARGE protein and non-specific signals, respectively. The full-length blots with α-DG (IIH6), LARGE (myc), fukutin (FLAG), and β-DG are presented in Supplementary Figure S2i-l, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4321163&req=5

f4: α-DG glycosylation in fukutin- ES cells after fukutin or LARGE expression.Fukutin (FLAG-tagged) and/or LARGE (myc-tagged) were expressed in wild-type or fukutin- mouse ES cells and α-DG glycosylation status was analysed by western blotting. Exogenous LARGE expression produced highly glycosylated α-DG in wild-type but not fukutin- ES cells, indicated by IIH6 staining. Co-transfection of fukutin and Large yielded LARGE-dependent glycosylation of α-DG in fukutin- ES cells. Arrow and asterisk indicate LARGE protein and non-specific signals, respectively. The full-length blots with α-DG (IIH6), LARGE (myc), fukutin (FLAG), and β-DG are presented in Supplementary Figure S2i-l, respectively.
Mentions: The amount of LARGE protein expressed in the AAV-treated Myf5-fukutin-cKO mice was comparable to that in AAV-treated Largemyd (Fig. 3a). α-DG glycosylation was recovered in Largemyd skeletal muscle after AAV9-MCK-Large treatment, suggesting something other than protein expression levels is responsible for the failure of glycosylation recovery in Myf5-fukutin-cKO mice. We hypothesized that complete loss of fukutin caused failure to build the part of post-phosphoryl moiety, which may be required for LARGE-dependent glycosylation; therefore, even excess LARGE protein could not form the [-3Xyl-α1,3GlcAβ1-] polymer on α-DG. To test this hypothesis, we expressed LARGE in fukutin- embryonic stem (ES) cells. Transfection of the fukutin cDNA restored IIH6 reactivity in fukutin- ES cells, but transfection of the Large cDNA failed to restore α-DG glycosylation, although LARGE expression in wild-type ES cells produced strong IIH6-reactivity (Fig. 4). When the fukutin and Large cDNAs were co-transfected into fukutin- ES cells, we observed increases in IIH6-reactivity in comparison to fukutin singly transfected cells although expression levels of both fukutin and LARGE were much lower than they were in each single transfection (Fig. 4, lanes 6–8). These data show that fukutin-dependent modification is a prerequisite for LARGE-dependent formation of [-3Xyl-α1,3GlcAβ1-] repeating units.

Bottom Line: However, the in vivo therapeutic benefit of using LARGE activity is controversial.Furthermore, forced expression of Large in fukutin-deficient embryonic stem cells also failed to recover α-DG glycosylation, however coexpression with fukutin strongly enhanced α-DG glycosylation.Together, our data demonstrated that fukutin is required for LARGE-dependent rescue of α-DG glycosylation, and thus suggesting new directions for LARGE-utilizing therapy targeted to myofibres.

View Article: PubMed Central - PubMed

Affiliation: Division of Neurology/Molecular Brain Science, Kobe University Graduate School of Medicine, Kobe, 650-0017, Japan.

ABSTRACT
α-Dystroglycanopathy (α-DGP) is a group of muscular dystrophy characterized by abnormal glycosylation of α-dystroglycan (α-DG), including Fukuyama congenital muscular dystrophy (FCMD), muscle-eye-brain disease, Walker-Warburg syndrome, and congenital muscular dystrophy type 1D (MDC1D), etc. LARGE, the causative gene for MDC1D, encodes a glycosyltransferase to form [-3Xyl-α1,3GlcAβ1-] polymer in the terminal end of the post-phosphoryl moiety, which is essential for α-DG function. It has been proposed that LARGE possesses the great potential to rescue glycosylation defects in α-DGPs regardless of causative genes. However, the in vivo therapeutic benefit of using LARGE activity is controversial. To explore the conditions needed for successful LARGE gene therapy, here we used Large-deficient and fukutin-deficient mouse models for MDC1D and FCMD, respectively. Myofibre-selective LARGE expression via systemic adeno-associated viral gene transfer ameliorated dystrophic pathology of Large-deficient mice even when intervention occurred after disease manifestation. However, the same strategy failed to ameliorate the dystrophic phenotype of fukutin-conditional knockout mice. Furthermore, forced expression of Large in fukutin-deficient embryonic stem cells also failed to recover α-DG glycosylation, however coexpression with fukutin strongly enhanced α-DG glycosylation. Together, our data demonstrated that fukutin is required for LARGE-dependent rescue of α-DG glycosylation, and thus suggesting new directions for LARGE-utilizing therapy targeted to myofibres.

Show MeSH
Related in: MedlinePlus