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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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Purification of DAG-125 and its identification as biglycan. a, Blot overlay analysis of DAG-125 in selected purification steps. DAG-125 was purified using biochemical and affinity methods before performing peptide microsequencing. Represented are the initial (synaptic membrane protein, lane 1), middle (input to chondroitin sulfate column, lane 2), and final (concentrated eluate from heparin sulfate column, lane 3) steps of purification. See Materials and Methods for details of the purification scheme. The final purified product was subjected to SDS-PAGE and blotted to PVDF. An upper (U) and a lower (L) region of the Ponceau-stained membrane was excised and digested with trypsin. The released peptides were analyzed by HPLC using a C8 column and UV detection. The column profiles were virtually identical, indicating that the polydisperse band is due to the presence of a single, heterogeneously glycosylated protein. b, Peptide microsequencing of DAG-125. Three peptides from the trypsin digest were collected as fractions from the HPLC analysis and subjected to automated Edman degradation. The sequences obtained were compared with public databases. The alignment of the Torpedo DAG-125 peptides to the deduced sequence of human biglycan is shown (aa 241–249, 258–266, and 330–348). c, Domain structure of human biglycan (Fisher et al. 1989; Hocking et al. 1998). Biglycan is one of a family of small leucine-rich repeat proteins. It consists of a prepropeptide that is not present in the mature polypeptide. This domain is followed by a short unique sequence with two chondroitin sulfate attachment sites (shown as stacked beads). There are two pairs and one pair of disulfide-linked cysteines at the NH2- and COOH-terminal domains, respectively. Finally, the bulk of the protein is comprised of ten (or 11, depending upon the classification of the region within the COOH-terminal cysteine pair) leucine-rich repeats. The position of the three Torpedo peptides relative to the human sequence is indicated by horizontal lines.
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Figure 3: Purification of DAG-125 and its identification as biglycan. a, Blot overlay analysis of DAG-125 in selected purification steps. DAG-125 was purified using biochemical and affinity methods before performing peptide microsequencing. Represented are the initial (synaptic membrane protein, lane 1), middle (input to chondroitin sulfate column, lane 2), and final (concentrated eluate from heparin sulfate column, lane 3) steps of purification. See Materials and Methods for details of the purification scheme. The final purified product was subjected to SDS-PAGE and blotted to PVDF. An upper (U) and a lower (L) region of the Ponceau-stained membrane was excised and digested with trypsin. The released peptides were analyzed by HPLC using a C8 column and UV detection. The column profiles were virtually identical, indicating that the polydisperse band is due to the presence of a single, heterogeneously glycosylated protein. b, Peptide microsequencing of DAG-125. Three peptides from the trypsin digest were collected as fractions from the HPLC analysis and subjected to automated Edman degradation. The sequences obtained were compared with public databases. The alignment of the Torpedo DAG-125 peptides to the deduced sequence of human biglycan is shown (aa 241–249, 258–266, and 330–348). c, Domain structure of human biglycan (Fisher et al. 1989; Hocking et al. 1998). Biglycan is one of a family of small leucine-rich repeat proteins. It consists of a prepropeptide that is not present in the mature polypeptide. This domain is followed by a short unique sequence with two chondroitin sulfate attachment sites (shown as stacked beads). There are two pairs and one pair of disulfide-linked cysteines at the NH2- and COOH-terminal domains, respectively. Finally, the bulk of the protein is comprised of ten (or 11, depending upon the classification of the region within the COOH-terminal cysteine pair) leucine-rich repeats. The position of the three Torpedo peptides relative to the human sequence is indicated by horizontal lines.

Mentions: We next identified DAG-125. Although DAG-125 copurified with postsynaptic membranes, it was insoluble in all nonionic detergents tested, including Triton X-100 and N-octyl-β-d-glucopyranoside, both of which efficiently extract α-/β-dystroglycan from these membranes (Bowe et al. 1994; Deyst et al. 1995). We determined that even without detergent, ∼50% of DAG-125 could be extracted at pH 11, and near-complete solubilization was achieved by a short pH 12 treatment (see Fig. 1 a). Importantly, DAG-125 remained soluble when returned to neutral pH. We developed a purification protocol (see Materials and Methods) based upon these properties and the findings that DAG-125 binds to both heparin and chondroitin sulfate columns (data not shown). We estimated the final purity of DAG-125 to be ∼30%. This material was separated by SDS-PAGE, blotted to PVDF, and two regions of the DAG-125 band were excised and digested with trypsin. HPLC analysis showed that the two regions (Fig. 3 a, see U and L) had identical peptide maps (data not shown). This finding established the purity of the DAG-125 in these regions and also indicated that the polydisperse DAG-125 band arises from the heterogeneous glycosylation of a common polypeptide core. We then sequenced three tryptic peptides and found that all were highly homologous to mammalian biglycan, with an overall 76% identity (Fig. 3 b). We thus conclude that DAG-125 is a Torpedo orthologue of mammalian biglycan.


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)

Purification of DAG-125 and its identification as biglycan. a, Blot overlay analysis of DAG-125 in selected purification steps. DAG-125 was purified using biochemical and affinity methods before performing peptide microsequencing. Represented are the initial (synaptic membrane protein, lane 1), middle (input to chondroitin sulfate column, lane 2), and final (concentrated eluate from heparin sulfate column, lane 3) steps of purification. See Materials and Methods for details of the purification scheme. The final purified product was subjected to SDS-PAGE and blotted to PVDF. An upper (U) and a lower (L) region of the Ponceau-stained membrane was excised and digested with trypsin. The released peptides were analyzed by HPLC using a C8 column and UV detection. The column profiles were virtually identical, indicating that the polydisperse band is due to the presence of a single, heterogeneously glycosylated protein. b, Peptide microsequencing of DAG-125. Three peptides from the trypsin digest were collected as fractions from the HPLC analysis and subjected to automated Edman degradation. The sequences obtained were compared with public databases. The alignment of the Torpedo DAG-125 peptides to the deduced sequence of human biglycan is shown (aa 241–249, 258–266, and 330–348). c, Domain structure of human biglycan (Fisher et al. 1989; Hocking et al. 1998). Biglycan is one of a family of small leucine-rich repeat proteins. It consists of a prepropeptide that is not present in the mature polypeptide. This domain is followed by a short unique sequence with two chondroitin sulfate attachment sites (shown as stacked beads). There are two pairs and one pair of disulfide-linked cysteines at the NH2- and COOH-terminal domains, respectively. Finally, the bulk of the protein is comprised of ten (or 11, depending upon the classification of the region within the COOH-terminal cysteine pair) leucine-rich repeats. The position of the three Torpedo peptides relative to the human sequence is indicated by horizontal lines.
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Related In: Results  -  Collection

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Figure 3: Purification of DAG-125 and its identification as biglycan. a, Blot overlay analysis of DAG-125 in selected purification steps. DAG-125 was purified using biochemical and affinity methods before performing peptide microsequencing. Represented are the initial (synaptic membrane protein, lane 1), middle (input to chondroitin sulfate column, lane 2), and final (concentrated eluate from heparin sulfate column, lane 3) steps of purification. See Materials and Methods for details of the purification scheme. The final purified product was subjected to SDS-PAGE and blotted to PVDF. An upper (U) and a lower (L) region of the Ponceau-stained membrane was excised and digested with trypsin. The released peptides were analyzed by HPLC using a C8 column and UV detection. The column profiles were virtually identical, indicating that the polydisperse band is due to the presence of a single, heterogeneously glycosylated protein. b, Peptide microsequencing of DAG-125. Three peptides from the trypsin digest were collected as fractions from the HPLC analysis and subjected to automated Edman degradation. The sequences obtained were compared with public databases. The alignment of the Torpedo DAG-125 peptides to the deduced sequence of human biglycan is shown (aa 241–249, 258–266, and 330–348). c, Domain structure of human biglycan (Fisher et al. 1989; Hocking et al. 1998). Biglycan is one of a family of small leucine-rich repeat proteins. It consists of a prepropeptide that is not present in the mature polypeptide. This domain is followed by a short unique sequence with two chondroitin sulfate attachment sites (shown as stacked beads). There are two pairs and one pair of disulfide-linked cysteines at the NH2- and COOH-terminal domains, respectively. Finally, the bulk of the protein is comprised of ten (or 11, depending upon the classification of the region within the COOH-terminal cysteine pair) leucine-rich repeats. The position of the three Torpedo peptides relative to the human sequence is indicated by horizontal lines.
Mentions: We next identified DAG-125. Although DAG-125 copurified with postsynaptic membranes, it was insoluble in all nonionic detergents tested, including Triton X-100 and N-octyl-β-d-glucopyranoside, both of which efficiently extract α-/β-dystroglycan from these membranes (Bowe et al. 1994; Deyst et al. 1995). We determined that even without detergent, ∼50% of DAG-125 could be extracted at pH 11, and near-complete solubilization was achieved by a short pH 12 treatment (see Fig. 1 a). Importantly, DAG-125 remained soluble when returned to neutral pH. We developed a purification protocol (see Materials and Methods) based upon these properties and the findings that DAG-125 binds to both heparin and chondroitin sulfate columns (data not shown). We estimated the final purity of DAG-125 to be ∼30%. This material was separated by SDS-PAGE, blotted to PVDF, and two regions of the DAG-125 band were excised and digested with trypsin. HPLC analysis showed that the two regions (Fig. 3 a, see U and L) had identical peptide maps (data not shown). This finding established the purity of the DAG-125 in these regions and also indicated that the polydisperse DAG-125 band arises from the heterogeneous glycosylation of a common polypeptide core. We then sequenced three tryptic peptides and found that all were highly homologous to mammalian biglycan, with an overall 76% identity (Fig. 3 b). We thus conclude that DAG-125 is a Torpedo orthologue of mammalian biglycan.

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