Characterization of the Transmembrane Molecular Architecture of the Dystroglycan Complex in Schwann Cells*

Fumiaki SaitoDagger , Toshihiro Masaki§, Keiko Kamakura§, Louise V. B. Anderson, Sachiko FujitaDagger , Hiroko Fukuta-OhiDagger , Yoshihide SunadaDagger , Teruo ShimizuDagger , and Kiichiro MatsumuraDagger parallel

From the Dagger  Department of Neurology and Neuroscience, Teikyo University School of Medicine, Tokyo 173-8605, Japan, the § Third Department of Internal Medicine, National Defense Medical College, Saitama 359-8513, Japan, and the  University School of Neuroscience and Muscular Dystrophy Group Laboratories, Regional Neuroscience Centre, Newcastle General Hospital, Newcastle-upon-Tyne NE4 6BE, United Kingdom

    ABSTRACT
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ABSTRACT
INTRODUCTION
REFERENCES

We have demonstrated previously 1) that the dystroglycan complex, but not the sarcoglycan complex, is expressed in peripheral nerve, and 2) that alpha -dystroglycan is an extracellular laminin-2-binding protein anchored to beta -dystroglycan in the Schwann cell membrane. In the present study, we investigated the transmembrane molecular architecture of the dystroglycan complex in Schwann cells. The cytoplasmic domain of beta -dystroglycan was co-localized with Dp116, the Schwann cell-specific isoform of dystrophin, in the abaxonal Schwann cell cytoplasm adjacent to the outer membrane. beta -dystroglycan bound to Dp116 mainly via the 15 C-terminal amino acids of its cytoplasmic domain, but these amino acids were not solely responsible for the interaction of these two proteins. Interestingly, the beta -dystroglycan-precipitating antibody precipitated only a small fraction of alpha -dystroglycan and did not precipitate laminin and Dp116 from the peripheral nerve extracts. Our results indicate 1) that Dp116 is a component of the submembranous cytoskeletal system that anchors the dystroglycan complex in Schwann cells, and 2) that the dystroglycan complex in Schwann cells is fragile compared with that in striated muscle cells. We propose that this fragility may be attributable to the absence of the sarcoglycan complex in Schwann cells.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
REFERENCES

Dystroglycan is encoded by a single gene and cleaved into two proteins, an extracellular peripheral membrane glycoprotein alpha -dystroglycan and an integral membrane glycoprotein beta -dystroglycan, by posttranslational processing (1). In skeletal muscle, alpha -dystroglycan links laminin-2 and agrin in the basal lamina with beta -dystroglycan in the sarcolemma (1-4). On the cytoplasmic side of the sarcolemma, on the other hand, beta -dystroglycan is anchored to the cytoskeletal protein dystrophin (5, 6). These findings indicate that the dystroglycan complex, comprising alpha - and beta -dystroglycans, spans the sarcolemma and links the basal lamina with submembranous cytoskeleton, thus contributing to the mechanical stability of the sarcolemma. The recent findings that beta -dystroglycan interacts with Grb2, an adaptor protein, and rapsyn, a peripheral protein required for acetylcholine receptor clustering, also suggest that the dystroglycan complex may have additional functions in skeletal muscle (7, 8).

The dystroglycan complex is also expressed in nonmuscle tissues. For instance, the dystroglycan complex, but not the sarcoglycan complex, is expressed in peripheral nerve (9-11). In peripheral nerve, alpha - and beta -dystroglycans are expressed restricted to the Schwann cell outer membrane apposing the endoneurial basal lamina but not in the Schwann cell inner membrane or compact myelin, whereas laminin-2 and the nonneuronal isoform of agrin lacking acetylcholine receptor clustering activity are expressed in the endoneurial basal lamina (9-16). Recently, we have demonstrated that Schwann cell alpha -dystroglycan is a mucin-type glycoprotein, which links laminin-2 and agrin in the endoneurium with beta -dystroglycan in the Schwann cell outer membrane (11-13). Because peripheral myelination is greatly disturbed in congenital muscular dystrophy patients and dy mice deficient in laminin-2 (17-26), these findings implicate the interaction of the dystroglycan complex with laminin-2 in peripheral myelinogenesis. This is further supported by the recent revelation that Mycobacterium leprae invades Schwann cells by binding to alpha -dystroglycan via laminin-2, to cause leprosy, a disease characterized by peripheral nerve degeneration (27, 28).

Despite these recent discoveries, there remain a number of questions to be answered concerning the molecular organization of the dystroglycan complex in Schwann cells. For instance, the submembranous cytoskeletal system that anchors the dystroglycan complex in Schwann cells remains elusive, because the full-length 427-kDa dystrophin is not expressed in peripheral nerve (29). In the present study, we asked whether dystrophin isoform Dp116 is a component of this system, for two reasons. First, Dp116 is a Schwann cell-specific isoform of dystrophin, composed mainly of the hinge 4, cysteine-rich, and C-terminal domains containing the beta -dystroglycan binding site (5, 6, 29, 30). Second, it was reported recently that peripheral myelination was disturbed in a peculiar Duchenne muscular dystrophy patient lacking Dp116 in peripheral nerve because of a unique mutation at the 5' splice site of intron 69 of the dystrophin gene (31). In this study, we also investigated the stability of the interaction of the components of the dystroglycan complex in Schwann cells and discussed its implications for the pathogenesis of muscle cell degeneration in sarcoglycanopathy.

    EXPERIMENTAL PROCEDURES

Miscellaneous-- Monoclonal antibodies 43DAG/8D5 against the C terminus of beta -dystroglycan and DYS2 against the C terminus of dystrophin were characterized previously (9, 32). Alkaline and detergent extraction of crude bovine peripheral nerve membranes (5 mg/ml) were performed as described previously (10-12). 3-12% SDS-PAGE,1 immunoblotting, laminin blot overlay, and immunohistochemical analysis were performed as described previously (9-12).

Immunoelectron Microscopic Analysis-- All the following procedures were performed at 4 °C unless otherwise stated. The segments of bovine cauda equina were immersed for 3 h in a solution of periodate-lysine-paraformaldehyde (33) or for 16 h in 4% paraformaldehyde in 0.1 M phosphate buffer. The segments were then infiltrated with 30% sucrose in 0.1 M phosphate buffer overnight, mounted in OCT compound, quickly frozen in isopentane cooled in liquid nitrogen, and cut into 10-µm-thick sections on a cryostat. The frozen sections were placed on gelatin-coated slides and washed three times with phosphate-buffered saline (PBS; 0.01 M phosphate buffer, PH 7.4, containing 0.15 M NaCl). After blocking with PBS containing 5% bovine serum albumin and 0.05% Triton X-100 (buffer A) for 1 h, sections were incubated for 16 h with 43DAG/8D5 at a dilution of 1:30 or DYS2 at a dilution of 1:5 in buffer A. After washing five times in buffer A, the sections were incubated for 16 h with biotin-conjugated anti-mouse immunoglobulin (Pharmingen) at a dilution of 1:200 in buffer A. After washing five times in PBS, the sections were incubated for 4 h with Vectastain ABC reagent (Vector Laboratories). After washing five times in PBS, the sections were immersed in 1% glutaraldehyde in 0.1 M phosphate buffer. After washing three times in PBS, the sections were incubated for 30 min in diaminobenzidine solution (50 mM Tris-HCl buffer, pH 7.4, containing 0.05% 3,3'-diaminobenzidine tetrahydrochloride) at room temperature and then incubated for 3 min in diaminobenzidine solution supplemented with 0.01% H2O2. The sections were washed twice in PBS and were postfixed for 2 h in 2% OsO4 solution. After dehydration at room temperature in a series of increasing concentrations of ethanol, they were embedded in Epon 812. Ultrathin sections were made on an LKB Ultrotome and viewed on a JEOL JEM-1010 electron microscope without any contrast staining.

Preparation of Fusion Proteins-- The beta -dystroglycan binding motif of dystrophin has most recently been localized to amino acids 3054-3271, but additional amino acids located downstream have been shown to be important for maximum binding (5). Because these amino acids are common to both dystrophin and Dp116 (29, 30), we prepared, as a probe to study the interaction between Dp116 and beta -dystroglycan in peripheral nerve, the Dp116 fusion protein (Dp116C-Ter), which corresponds to amino acids 3054-3685 of dystrophin. With regard to beta -dystroglycan, fusion proteins corresponding to amino acids 654-750 (beta DGExt), 775-895 (beta DGCyt1), 775-880 (beta DGCyt2), 775-860 (beta DGCyt3), 775-840 (beta DGCyt4), 775-820 (beta DGCyt5), 775-800 (beta DGCyt6), 801-895 (beta DGCyt7), and 801-880 (beta DGCyt8) were prepared. The cleavage site of alpha - and beta -dystroglycans is localized to serine 654, and the transmembrane domain of beta -dystroglycan is localized to amino acids 751-774 (1, 34, 35) (Fig. 1). beta DGExt thus corresponds to the entire extracellular domain of beta -dystroglycan, whereas beta DGCyt1, beta DGCyt2, beta DGCyt3, beta DGCyt4, beta DGCyt5, beta DGCyt6, beta DGCyt7, and beta DGCyt8 correspond to various regions of the cytoplasmic domain of beta -dystroglycan (Fig. 1).


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Fig. 1.   Alignment of the beta -dystroglycan fusion proteins in the full length of bovine beta -dystroglycan. TM, transmembrane domain.

Human dystrophin cDNA corresponding to amino acids 3054-3685 (GenBank accession number M18533) and bovine beta -dystroglycan cDNAs corresponding to amino acids 654-750, 775-895, 775-880, 775-860, 775-840, 775-820, 775-800, 801-895, and 801-880 (GenBank accession number AB009079) were amplified by polymerase chain reaction and subcloned into pGEX-2TK expression vector (Amersham Pharmacia Biotech). Escherichia coli DH5alpha cells were transformed with the glutathione S-transferase (GST) fusion protein constructs. Overnight cultures were grown, and the fusion proteins were induced with 1 mM isopropyl-1-thio-beta -D-galactopyranoside. The cell cultures were spun down, resuspended in 50 mM Tris-HCl, pH 8.0, 1% Triton X-100, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 0.75 mM benzamidine, and 0.1 mM phenylmethylsulfonyl fluoride, and sonicated 10 times for 10 s. The sonicated material was centrifuged at 13,800 × g for 10 min. The supernatant containing the GST fusion protein was incubated with glutathione-agarose beads. After extensive wash, the glutathione-agarose beads were eluted with 10 mM glutathione in 50 mM Tris-HCl, pH 8.0.

Blot Overlay Assay-- Proteins were separated by 3-12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon). The PVDF transfers were blocked with 10 mM triethanolamine, pH 7.6, 140 mM NaCl, 1 mM CaCl2, and 1 mM MgCl2 containing 5% non-fat dry milk (MLBB) and then incubated with either the MLBB containing 1 mM dithiothreitol and 0.2 µg/ml (unless stated otherwise) fusion protein Dp116C-Ter overnight at room temperature or the alkaline extracts of the crude bovine peripheral nerve membranes for two nights at 4 °C. After extensive washing, the Dp116C-Ter or the endogenous bovine peripheral nerve Dp116, which bound to the PVDF transfers, was detected with monoclonal antibody DYS2.

Immunoprecipitation-- Immunoprecipitation was performed using monoclonal antibody 43DAG/8D5. Two parts of digitonin or Triton X-100 extracts of crude bovine peripheral nerve membranes (10-12) were incubated with one part of crude culture medium of 43DAG/8D5 for 1 h at 4 °C. The resultant immune complex was precipitated with protein G-Sepharose (Amersham Pharmacia Biotech). The precipitates and the voids were analyzed by 3-12% SDS-PAGE and immunoblotting.

    RESULTS

To determine the localization of Dp116 and beta -dystroglycan in bovine peripheral nerve, we performed immunohistochemical and immunoelectron microscopic analyses using monoclonal antibodies DYS2 and 43DAG/8D5. We have reported previously that DYS2 detects Dp116 in peripheral nerve (9-11). 43DAG/8D5 is directed against the C terminus of beta -dystroglycan and thus recognizes the cytoplasmic domain of beta -dystroglycan (1, 32). The results of immunohistochemical analysis are shown in Fig. 2. Dp116 and the cytoplasmic domain of beta -dystroglycan were both localized surrounding the outermost layer of myelin sheath of peripheral nerve fibers (Fig. 2). The results of immunoelectron microscopic analysis are shown in Fig. 3. Dp116 and the cytoplasmic domain of beta -dystroglycan were both localized in the abaxonal Schwann cell cytoplasm (Fig. 3). The distribution of these proteins was most prominent adjacent to the outer membrane (Fig. 3).


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Fig. 2.   Immunohistochemical analysis of beta -dystroglycan and Dp116 in bovine peripheral nerve. beta -Dystroglycan and Dp116 were detected with 43DAG/8D5 and DYS2, respectively. Scale bar, 15 µm.


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Fig. 3.   Immunoelectron microscopic analysis of beta -dystroglycan and Dp116 in bovine peripheral nerve. beta -Dystroglycan and Dp116 were detected with 43DAG/8D5 and DYS2, respectively. OM and CM, Schwann cell outer membrane and compact myelin, respectively. Scale bar, 0.5 µm.

To test whether Dp116 interacts with beta -dystroglycan, we prepared domain-specific fusion proteins of Dp116 and beta -dystroglycan (Figs. 1 and 4). The PVDF transfer of beta -dystroglycan fusion proteins was overlaid with Dp116C-Ter (Fig. 4, a and c). Dp116C-Ter bound to beta DGCyt1 corresponding to the cytoplasmic domain of beta -dystroglycan but not to beta DGExt corresponding to the extracellular domain of beta -dystroglycan or control GST (Fig. 4c). As we have reported previously (10, 11), Dp116 was enriched in the alkaline extracts of crude bovine peripheral nerve membranes (Fig. 4b). To see whether the endogenous bovine peripheral nerve Dp116 binds to beta -dystroglycan fusion proteins, the PVDF transfer of beta -dystroglycan fusion proteins was overlaid with the alkaline extracts of crude bovine peripheral nerve membranes, and the Dp116, which bound to beta -dystroglycan fusion proteins on the PVDF membranes, was detected with DYS2. The endogenous bovine peripheral nerve Dp116 bound to beta DGCyt1 but not to beta DGExt or control GST (Fig. 4c). These results demonstrate that Dp116 binds to the cytoplasmic domain of beta -dystroglycan.


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Fig. 4.   Blot overlay analysis of the binding of Dp116 fusion protein or the endogenous bovine peripheral nerve Dp116 with domain-specific beta -dystroglycan fusion proteins. a, preparations of Dp116 fusion protein Dp116C-Ter were separated by 3-12% SDS-PAGE and transferred to a PVDF membrane. CB and Anti-Dp116, SDS gel stained with Coomassie Blue and the PVDF transfer reacted with DYS2, respectively. The 110-kDa band that reacted with DYS2 strongly had the expected molecular size of the fusion protein, indicating that it is Dp116C-Ter. b, the crude bovine peripheral nerve membranes were extracted at pH 11 as described previously (10, 11). The extracts (Alk Ext) and pellets (Alk Plt) were separated by 3-12% SDS-PAGE, transferred to a PVDF membrane and reacted with DYS2 (Anti-Dp116). c, domain-specific beta -dystroglycan fusion proteins were separated by 3-12% SDS-PAGE and transferred to PVDF membranes. The PVDF transfers were overlaid with Dp116C-Ter (Dp116C-Ter O/L) or the alkaline extracts of crude bovine peripheral nerve membranes (Alk Ext O/L). The Dp116C-Ter or the endogenous bovine peripheral nerve Dp116 that bound to the beta -dystroglycan fusion proteins on the PVDF membranes was detected with DYS2. GST, control GST fusion protein having no inserts; CB, SDS gel stained with Coomassie Blue. Molecular mass standards (Da × 10-3) are shown on the left.

Dystrophin has been reported to bind to the 15 C-terminal amino acids of beta -dystroglycan (5). We asked whether these amino acids were also responsible for the binding of Dp116. Surprisingly, beta DGCyt2 corresponding to the cytoplasmic domain of beta -dystroglycan but lacking the 15 C-terminal amino acids bound both Dp116C-Ter and the endogenous bovine peripheral nerve Dp116, albeit with much lower binding affinity than beta DGCyt1 (Fig. 4c). The difference between beta DGCyt1 and beta DGCyt2 binding to the endogenous bovine peripheral nerve Dp116 appeared less marked than that to Dp116C-Ter (Fig. 4c). It is possible that the native protein might have slightly altered binding properties from the fusion protein or that the binding properties of Dp116 might be modified in the presence of the Dp116-binding proteins other than beta -dystroglycan in the peripheral nerve extracts (discussed below; also see Fig. 7).

We tested the effects of a synthetic peptide corresponding to the 15 C-terminal amino acids of beta -dystroglycan on the binding of Dp116C-Ter. The binding of Dp116C-Ter with beta DGCyt1 was greatly reduced, but not completely abolished, by the peptide, whereas the binding with beta DGCyt2 was not affected (Fig. 5). In the presence of the peptide in the overlay medium, furthermore, Dp116C-Ter bound to beta DGCyt1 and beta DGCyt2 with similar affinity (Fig. 5). The results indicate that the 15 C-terminal amino acids of beta -dystroglycan are involved in the high affinity binding of Dp116 but that they are not solely responsible for the interaction of beta -dystroglycan with Dp116. It was suggested recently that the sequence of PPPY in the 15 C-terminal amino acids of beta -dystroglycan might directly interact with the WW domain in hinge 4 of dystrophin (36, 37). However, a synthetic peptide, PPPY, did not significantly affect the binding of Dp116C-Ter with beta DGCyt1 (Fig. 5), suggesting that these amino acids may not be crucial for the binding.


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Fig. 5.   Inhibition of the binding of Dp116 fusion protein with domain-specific beta -dystroglycan fusion proteins by synthetic peptides. Domain-specific beta -dystroglycan fusion proteins were separated by 3-12% SDS-PAGE and transferred to PVDF membranes. The PVDF transfers of beta DGCyt1 and beta DGCyt2 were overlaid with Dp116C-Ter in the presence (15 C-Ter Peptide (+)) or absence (15 C-Ter Peptide (-)) of 500,000 times molar excess of synthetic amino acids corresponding to the 15 C-terminal amino acids of beta -dystroglycan. The PVDF transfers of beta DGCyt1 were overlaid with Dp116C-Ter in the presence (PPPY Peptide (+)) or absence (PPPY Peptide (-)) of 500,000 times molar excess of synthetic amino acids PPPY. The Dp116C-Ter that bound to the beta -dystroglycan fusion proteins on the PVDF membranes was detected with DYS2. CB, SDS gel stained with Coomassie Blue. Molecular mass standards (Da × 10-3) are shown on the left.

The above results suggest the presence of a Dp116 binding sequence other than the 15 C-terminal amino acids of beta -dystroglycan. To see whether this is true, we prepared fusion proteins corresponding to beta -dystroglycan cytoplasmic domains lacking various lengths of C-terminal amino acids and tested whether they bind Dp116C-Ter. Dp116C-Ter bound to all of beta DGCyt3, beta DGCyt4, beta DGCyt5, and beta DGCyt6 (Fig. 6). In addition, the binding affinity of Dp116C-Ter with beta DGCyt3, beta DGCyt4, beta DGCyt5, and beta DGCyt6 was similar to that with beta DGCyt2 and much weaker than that with beta DGCyt1 (Fig. 6), suggesting that the 26 N-terminal amino acids of the cytoplasmic domain of beta -dystroglycan may also be involved in the binding of Dp116. To confirm this, we prepared the beta -dystroglycan cytoplasmic domain fusion protein lacking the 26 N-terminal amino acids (beta DGCyt7) or lacking both the 26 N-terminal and 15 C-terminal amino acids (beta DGCyt8). The binding of Dp116C-Ter with beta DGCyt7 was slightly weaker than that with beta DGCyt1, and the binding with beta DGCyt8 was not detectable (Fig. 6), indicating that the 26 N-terminal amino acids of the cytoplasmic domain of beta -dystroglycan are involved in the binding of Dp116.


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Fig. 6.   Blot overlay analysis of the binding of Dp116 fusion protein with domain-specific beta -dystroglycan fusion proteins. Domain-specific beta -dystroglycan fusion proteins were separated by 3-12% SDS-PAGE and transferred to PVDF membranes. The PVDF transfers were overlaid with Dp116C-Ter. The Dp116C-Ter that bound to the beta -dystroglycan fusion proteins on the PVDF membranes was detected with DYS2. CB, SDS gel stained with Coomassie Blue. Molecular mass standards (Da × 10-3) are shown on the left.

The binding of Dp116C-Ter with beta DGCyt1 and beta DGCyt2 was maximal at the physiological salt concentration (results not shown). The binding of Dp116C-Ter with beta DGCyt1 and beta DGCyt2 was not inhibited by EDTA, indicating that it is independent of divalent cations (results not shown). We have also observed that beta DGCyt1 and beta DGCyt2 in solution do not bind to Dp116C-Ter separated by SDS-PAGE and transferred to PVDF membranes (results not shown). Together with the aforementioned results, this suggests that the beta -dystroglycan binding site of Dp116 is vulnerable to denaturation with SDS, whereas the Dp116 binding site of beta -dystroglycan is not.

To identify the endogenous bovine peripheral nerve proteins that interact with Dp116, we prepared the PVDF transfers of crude bovine peripheral nerve membranes. When the PVDF transfer was reacted with 43DAG/8D5, 43- and 30-kDa bands were detected (Fig. 7). As reported previously, the 43-kDa band corresponds to the full-length beta -dystroglycan, whereas the 30-kDa band is presumed to correspond to the unglycosylated form or the proteolytic fragment of beta -dystroglycan (11). When the identical PVDF transfer was overlaid with Dp116C-Ter, these 43- and 30-kDa bands bound Dp116C-Ter, in addition to the 60- and 35-kDa bands of undetermined origin (Fig. 7). Furthermore, the 43- and 30-kDa beta -dystroglycan bands immunoprecipitated by 43DAG/8D5 bound Dp116C-Ter (Fig. 8a). These results demonstrate that Dp116 binds to the endogenous bovine peripheral nerve beta -dystroglycan.


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Fig. 7.   Blot overlay analysis of the binding of Dp116 fusion protein with bovine peripheral nerve membrane proteins. The crude bovine peripheral nerve membranes were extracted with Triton X-100 as described previously (10-12). The extracts (Triton Extract) and pellets (Triton Pellet) were separated by 3-12% SDS-PAGE, transferred to PVDF membranes, and overlaid with 25 µg/ml Dp116C-Ter. The Dp116C-Ter that bound to bovine peripheral nerve membrane proteins on the PVDF membranes was detected with DYS2 (Dp116C-Ter O/L). Arrows, bands that bound Dp116C-Ter. CB, Anti-beta DG, and Anti-Dp116, SDS gel stained with Coomassie Blue and the identical PVDF transfers reacted with 43DAG/8D5 and DYS2, respectively. As reported previously, the 43-kDa band (43beta DG) corresponds to the full-length beta -dystroglycan, whereas the 30-kDa band (30beta DG) is presumed to correspond to the unglycosylated form or the proteolytic fragment of beta -dystroglycan (11). Molecular mass standards (Da × 10-3) are shown on the left.


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Fig. 8.   Immunoprecipitation of beta -dystroglycan from crude bovine peripheral nerve membranes. a, the extracts of crude bovine peripheral nerve membranes were incubated with 43DAG/8D5. The resultant immune complex was precipitated with protein G-Sepharose, separated by 3-12% SDS-PAGE, and transferred to PVDF membranes (Anti-beta DG Beads). Anti-beta DG, Lam O/L, Anti-Dp116, and Dp116C-Ter O/L, PVDF transfer reacted with 43DAG/8D5, overlaid with laminin-1, reacted with DYS2, and overlaid with Dp116C-Ter, respectively; alpha DG, 43beta DG, and 30beta DG, alpha -dystroglycan and the 43- and 30-kDa bands of beta -dystroglycan, respectively. Arrows, bands of mouse IgG detected by anti-mouse IgG secondary antibody. b, the extracts of crude bovine peripheral nerve membranes were incubated with or without 43DAG/8D5 (Anti-beta DG Beads and Control Beads, respectively). After precipitation with protein G-Sepharose, the voids were separated by 3-12% SDS-PAGE and transferred to PVDF membranes. Anti-beta DG, Lam O/L, and Anti-Dp116, PVDF transfer reacted with 43DAG/8D5, overlaid with laminin-1, and reacted with DYS2, respectively; CB, SDS gel stained with Coomassie blue; Lam, bands of laminin subunits detected by anti-laminin antibody used in laminin-1 overlay assay. Molecular mass standards (Da × 10-3) are shown on the left.

We performed immunoprecipitation of bovine peripheral nerve membrane extracts using 43DAG/8D5. Both the 43- and 30-kDa bands of beta -dystroglycan were precipitated completely by 43DAG/8D5 (Fig. 8, a and b). As expected, alpha -dystroglycan was also precipitated by 43DAG/8D5 (Fig. 8a). However, the amount of alpha -dystroglycan remaining in the voids after precipitation was only slightly reduced compared with control (Fig. 8b), indicating that only a small fraction, but not the vast majority, of alpha -dystroglycan was precipitated. To our surprise, laminin was not precipitated by 43DAG/8D5 (Fig. 8, a and b) (Note that the bands of laminin subunits are not detectable in the immunoprecipitates in Fig. 8a, Lam O/L.) Dp116 was not precipitated either (Fig. 8, a and b), but this could be attributable to the fact that 43DAG/8D5 is directed against the 15 C-terminal amino acids of beta -dystroglycan involved in the high affinity binding of Dp116 as described above. The results were identical using digitonin (Fig. 8) or Triton X-100 (results not shown) for extraction of peripheral nerve membranes.

    DISCUSSION

When combined together, the data presented here indicate that Dp116 is a component of the submembranous cytoskeletal system that anchors the dystroglycan complex in Schwann cells. We have found that the 15 C-terminal amino acids of the cytoplasmic domain of beta -dystroglycan are involved in the high affinity binding of Dp116 but that these amino acids are not solely responsible for the interaction of beta -dystroglycan with Dp116. We have found that the 26 N-terminal amino acids of the cytoplasmic domain of beta -dystroglycan are also involved in the low affinity binding of Dp116. In this respect, it is noteworthy that the 26 N-terminal, as well as the 15 C-terminal, amino acids of the cytoplasmic domain of beta -dystroglycan are completely conserved among all the species investigated so far, human, cow, rabbit and mouse, suggesting that this sequence may have an important function. Our finding is consistent with this expectation. In addition to beta -dystroglycan, furthermore, we have found that the 60- and 35-kDa bovine peripheral nerve proteins bind Dp116. Although the identification of these proteins awaits future studies, our results suggest that they may interact with Dp116 in the submembranous cytoskeletal system in Schwann cells. Recently, it was reported that M. leprae invades Schwann cells by binding to the dystroglycan complex via the G domain of laminin alpha 2 chain (28). Interestingly, the dystroglycan complex starts aggregating on the Schwann cell membrane in response to the challenge with the laminin alpha 2 chain-coated M. leprae, suggesting that the submembranous cytoskeleton anchoring the dystroglycan complex is reorganized to permit the entry of bacteria into the cytoplasm (28). Our results raise the possibility that Dp116 may play a role in this process.

In this study, we have found that the interaction of the components of the dystroglycan complex is unstable in Schwann cells. First, the finding that the beta -dystroglycan-precipitating antibody 43DAG/8D5 precipitated a small fraction, but not a major fraction, of alpha -dystroglycan from the peripheral nerve membrane extracts indicates that a substantial amount of alpha -dystroglycan was dissociated from beta -dystroglycan during biochemical procedures. This is in sharp contrast to the striated muscle cells, where alpha - and beta -dystroglycans are tightly complexed (2, 38, 39). Second, the finding that laminin was not precipitated by 43DAG/8D5 at all indicates that laminin was completely dissociated from alpha -dystroglycan during biochemical procedures. In this respect, it is noteworthy that the sarcoglycan complex, comprising alpha -, beta -, gamma -, and delta -sarcoglycans as a whole, is not expressed in peripheral nerve (9-11). Recent evidence indicates that, by binding to both alpha - and beta -dystroglycans, the sarcoglycan complex reinforces their interaction in striated muscle cells (40). We presume that, because of the absence of the sarcoglycan complex, the interaction between alpha - and beta -dystroglycans in Schwann cells is not as stable as in striated muscle cells. The absence of the sarcoglycan complex may somehow destabilize the interaction of laminin with alpha -dystroglycan as well. In any case, the link between the basal lamina and submembranous cytoskeleton via the dystroglycan complex is presumed to be fragile in Schwann cells.

    ACKNOWLEDGEMENTS

Bovine dystroglycan and human dystrophin cDNAs were kindly provided by Drs. Haruaki Ninomiya (Kyoto University) and Michihiro Imamura (National Center for Neurological and Psychiatric Disorders), respectively.

    FOOTNOTES

* This work was supported by the grants from Kato Memorial Bioscience Foundation, the Cell Science Research Foundation, and the Science Research Promotion Fund from Japan Private School Promotion Foundation, Research Grants 8A-1 and 8A-2 for Nervous and Mental Disorders from the Ministry of Health and Welfare, a Health Sciences research grant for research on brain science from the Ministry of Health and Welfare, and Research Grants 08457195, 09470156, 09770460, 09877121, and 10044319 from the Ministry of Education, Science, Sports and Culture.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed: Dept. of Neurology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan. Tel.: 81-3-3964-1211; Fax: 81-3-3964-6394; E-mail: k-matsu{at}med.teikyo-u.ac.jp.

    ABBREVIATIONS

The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; GST, glutathione S-transferase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
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