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The small leucine-rich repeat proteoglycan biglycan binds to alpha-dystroglycan and is upregulated in dystrophic muscle.

Bowe MA, Mendis DB, Fallon JR - J. Cell Biol. (2000)

Bottom Line: The dystrophin-associated protein complex (DAPC) is necessary for maintaining the integrity of the muscle cell plasma membrane and may also play a role in coordinating signaling events at the cell surface.Biglycan binding to alpha-dystroglycan was confirmed by coimmunoprecipitation with both native and recombinant alpha-dystroglycan.These findings reveal a novel binding partner for alpha-dystroglycan and demonstrate a novel avenue for interaction of the DAPC and the extracellular matrix.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Brown University, Providence, Rhode Island 02912, USA.

ABSTRACT
The dystrophin-associated protein complex (DAPC) is necessary for maintaining the integrity of the muscle cell plasma membrane and may also play a role in coordinating signaling events at the cell surface. The alpha-/beta-dystroglycan subcomplex of the DAPC forms a critical link between the cytoskeleton and the extracellular matrix. A ligand blot overlay assay was used to search for novel dystroglycan binding partners in postsynaptic membranes from Torpedo electric organ. An approximately 125-kD dystroglycan-binding polypeptide was purified and shown by peptide microsequencing to be the Torpedo ortholog of the small leucine-rich repeat chondroitin sulfate proteoglycan biglycan. Biglycan binding to alpha-dystroglycan was confirmed by coimmunoprecipitation with both native and recombinant alpha-dystroglycan. The biglycan binding site was mapped to the COOH-terminal third of alpha-dystroglycan. Glycosylation of alpha-dystroglycan is not necessary for this interaction, but binding is dependent upon the chondroitin sulfate side chains of biglycan. In muscle, biglycan is detected at both synaptic and nonsynaptic regions. Finally, biglycan expression is elevated in muscle from the dystrophic mdx mouse. These findings reveal a novel binding partner for alpha-dystroglycan and demonstrate a novel avenue for interaction of the DAPC and the extracellular matrix. These results also raise the possibility of a role for biglycan in the pathogenesis, and perhaps the treatment, of muscular dystrophy.

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Analysis of Torpedo biglycan glycosylation. Alkaline-solubilized synaptic membrane proteins were digested as indicated, in the presence of protease inhibitors, and then analyzed for dystroglycan binding by blot overlay assay with 35S-DG345-653. Both chondroitinase ABC and chondroitinase AC greatly reduced the binding of dystroglycan to DAG-125/Torpedo biglycan. Chondroitinase B, which degrades dermatan sulfate, had a much smaller effect. See also Table .
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Figure 4: Analysis of Torpedo biglycan glycosylation. Alkaline-solubilized synaptic membrane proteins were digested as indicated, in the presence of protease inhibitors, and then analyzed for dystroglycan binding by blot overlay assay with 35S-DG345-653. Both chondroitinase ABC and chondroitinase AC greatly reduced the binding of dystroglycan to DAG-125/Torpedo biglycan. Chondroitinase B, which degrades dermatan sulfate, had a much smaller effect. See also Table .

Mentions: Mammalian biglycan is often substituted with chondroitin sulfate. We therefore asked if Torpedo biglycan is also a chondroitin sulfate proteoglycan, and whether glycosylation is important for its binding to α-dystroglycan. We digested DAG-125 with various glycosidases and glycosaminoglycanases and analyzed the products by α-dystroglycan ligand blot overlay (Fig. 4; Table ). Removal of chondroitin sulfate side chains abolished the binding to α-dystroglycan. Chondroitinase B (specific for dermatan sulfate) had a much smaller effect compared with chondroitinases whose activity included chondroitin sulfate A and C. No other glycosidase or glycosaminoglycanase treatment had a detectable effect on α-dystroglycan binding (Table ). Several lines of evidence indicate that the effects of chondroitinase digestion are due to chondroitinase activity and not to contaminating proteases: the digestions were performed in a cocktail of protease inhibitors (see Materials and Methods); the same result was seen with four different preparations of chondroitinase, including two which had been affinity-purified to remove proteases; and the effect was prevented by addition of 5 mM Zn2+, an inhibitor of chondroitinase, but not of proteases. We conclude that biglycan from Torpedo synaptic membranes is substituted with chondroitin sulfate chains, which are predominantly chondroitin sulfate A and/or C. Finally, chondroitin sulfate substitution of biglycan is necessary for binding to dystroglycan.


The small leucine-rich repeat proteoglycan biglycan binds to alpha-dystroglycan and is upregulated in dystrophic muscle.

Bowe MA, Mendis DB, Fallon JR - J. Cell Biol. (2000)

Analysis of Torpedo biglycan glycosylation. Alkaline-solubilized synaptic membrane proteins were digested as indicated, in the presence of protease inhibitors, and then analyzed for dystroglycan binding by blot overlay assay with 35S-DG345-653. Both chondroitinase ABC and chondroitinase AC greatly reduced the binding of dystroglycan to DAG-125/Torpedo biglycan. Chondroitinase B, which degrades dermatan sulfate, had a much smaller effect. See also Table .
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169361&req=5

Figure 4: Analysis of Torpedo biglycan glycosylation. Alkaline-solubilized synaptic membrane proteins were digested as indicated, in the presence of protease inhibitors, and then analyzed for dystroglycan binding by blot overlay assay with 35S-DG345-653. Both chondroitinase ABC and chondroitinase AC greatly reduced the binding of dystroglycan to DAG-125/Torpedo biglycan. Chondroitinase B, which degrades dermatan sulfate, had a much smaller effect. See also Table .
Mentions: Mammalian biglycan is often substituted with chondroitin sulfate. We therefore asked if Torpedo biglycan is also a chondroitin sulfate proteoglycan, and whether glycosylation is important for its binding to α-dystroglycan. We digested DAG-125 with various glycosidases and glycosaminoglycanases and analyzed the products by α-dystroglycan ligand blot overlay (Fig. 4; Table ). Removal of chondroitin sulfate side chains abolished the binding to α-dystroglycan. Chondroitinase B (specific for dermatan sulfate) had a much smaller effect compared with chondroitinases whose activity included chondroitin sulfate A and C. No other glycosidase or glycosaminoglycanase treatment had a detectable effect on α-dystroglycan binding (Table ). Several lines of evidence indicate that the effects of chondroitinase digestion are due to chondroitinase activity and not to contaminating proteases: the digestions were performed in a cocktail of protease inhibitors (see Materials and Methods); the same result was seen with four different preparations of chondroitinase, including two which had been affinity-purified to remove proteases; and the effect was prevented by addition of 5 mM Zn2+, an inhibitor of chondroitinase, but not of proteases. We conclude that biglycan from Torpedo synaptic membranes is substituted with chondroitin sulfate chains, which are predominantly chondroitin sulfate A and/or C. Finally, chondroitin sulfate substitution of biglycan is necessary for binding to dystroglycan.

Bottom Line: The dystrophin-associated protein complex (DAPC) is necessary for maintaining the integrity of the muscle cell plasma membrane and may also play a role in coordinating signaling events at the cell surface.Biglycan binding to alpha-dystroglycan was confirmed by coimmunoprecipitation with both native and recombinant alpha-dystroglycan.These findings reveal a novel binding partner for alpha-dystroglycan and demonstrate a novel avenue for interaction of the DAPC and the extracellular matrix.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Brown University, Providence, Rhode Island 02912, USA.

ABSTRACT
The dystrophin-associated protein complex (DAPC) is necessary for maintaining the integrity of the muscle cell plasma membrane and may also play a role in coordinating signaling events at the cell surface. The alpha-/beta-dystroglycan subcomplex of the DAPC forms a critical link between the cytoskeleton and the extracellular matrix. A ligand blot overlay assay was used to search for novel dystroglycan binding partners in postsynaptic membranes from Torpedo electric organ. An approximately 125-kD dystroglycan-binding polypeptide was purified and shown by peptide microsequencing to be the Torpedo ortholog of the small leucine-rich repeat chondroitin sulfate proteoglycan biglycan. Biglycan binding to alpha-dystroglycan was confirmed by coimmunoprecipitation with both native and recombinant alpha-dystroglycan. The biglycan binding site was mapped to the COOH-terminal third of alpha-dystroglycan. Glycosylation of alpha-dystroglycan is not necessary for this interaction, but binding is dependent upon the chondroitin sulfate side chains of biglycan. In muscle, biglycan is detected at both synaptic and nonsynaptic regions. Finally, biglycan expression is elevated in muscle from the dystrophic mdx mouse. These findings reveal a novel binding partner for alpha-dystroglycan and demonstrate a novel avenue for interaction of the DAPC and the extracellular matrix. These results also raise the possibility of a role for biglycan in the pathogenesis, and perhaps the treatment, of muscular dystrophy.

Show MeSH
Related in: MedlinePlus