Human Molecular Genetics, 2000, Vol. 9, No. 20 3091-3100
© 2000 Oxford University Press
A sarcoglycan–dystroglycan complex anchors Dp116 and utrophin in the peripheral nervous system
1Department of Cell Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi-cho, Kodaira, Tokyo 187-8502, Japan and 2Inheritance and Variation Group, PRESTO, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan
Received 4 September 2000; Revised and Accepted 20 October 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The dystrophin-associated membrane-integrated protein complex anchors dystrophin in the sarcolemma of striated muscles and is composed of two glycoprotein subcomplexes, the dystroglycan and the sarcoglycan (SG) complexes, and a small membrane protein termed sarcospan (SPN). The SG complex consists of four transmembrane glycoproteins,
-SG, ß-SG, 
-SG and 
-SG. We found that ß-SG and -SG were co-expressed with 
-SG, a -SG homolog, in the peripheral nerve, but not with -SG or -SG. SPN, which tightly links to the SG complex in the muscle cell membrane, was absent in the peripheral nerve. These peripheral nerve SGs were colocalized at the outermost layer of the myelin sheath of nerve fibers together with the dystroglycan complex, utrophin, and a short dystrophin isoform (Dp116). Immunocytochemical analysis using SG-deficient animals showed that a defect in ß- or -SG led to a great reduction of all residual SGs, but not of the other proteins, i.e., dystroglycans, Dp116 and utrophin, in the peripheral nerve. This observation suggests that the -, ß- and -SG molecules form a complex behaving as a single unit similar to the SG complex in muscle cells. An immunoprecipitation study indicated that the SG complex is associated with the dystroglycan complex and Dp116 or utrophin. These results demonstrated that Dp116 and utrophin are anchored to a novel membrane protein architecture, which consists of the SG and dystroglycan complexes, but not SPN, in the Schwann cell membrane. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The dystrophin–dystrophin-associated protein (DAP) complex, a large membrane protein complex, is critical for the stability of the striated muscle cell membrane. This multi-protein complex consists of dystrophin,
- and ß-dystroglycan, -, ß-, - and -sarcoglycan (SG), sarcospan (SPN), syntrophins and dystrobrevins (1–3). Biochemical analysis with n-octyl ß-D-glucoside clearly showed that the complicated complex was divided into two glycoprotein subcomplexes, namely the dystroglycan and SG complexes, and a cytoplasmic protein subcomplex consisting of dystrophin and dystrophin-associated cytoplasmic proteins (syntrophins and dystrobrevins) (4).
Dystrophin, a gene product responsible for Duchenne muscular dystrophy, is a 427 kDa protein composed of four domains: an N-terminal actin-binding domain, a large rod domain, a cysteine-rich domain and a C-terminal domain. The latter two domains bind to transmembranous ß-dystroglycan, intercellular syntrophins and dystrobrevins (5–8). Dystrophin associates with the muscle sarcolemma via ß-dystroglycan, which in turn binds to -dystroglycan (4,7,8). -Dystroglycan tightly binds to laminin (9), a major component of the basal lamina, indicating that dystrophin and the dystroglycan complex form a cross bridge between the extracellular matrix (ECM) and the actin-based cytoskeletal network. Four SGs, , ß-, - and -SG, comprise a heterotetramer complex that is tightly associated with the dystroglycan complex and SPN (4,10–12). Analyses of striated muscles of SG-deficient animals revealed that the SG complex plays a role in reinforcing the molecular linkage from -dystroglycan to dystrophin through the membrane-spanning ß-dystroglycan (13–15). It is considered that the reinforcement of molecular interactions by the SG complex is essential for protecting the muscle cell membrane against contraction-induced mechanical stress (3,16). On the other hand, besides the structural role, the SG complex was suggested to play a role in scaffolding for signal-transduction cascades (12). It remains important to determine whether or not the SG complex is specific for muscle cells in order to understand the physiological roles of the entire dystrophin–DAP complex as well as the SG complex. Until now, no SG complex-like structure has been found in non-contracting cells in spite of attempts to find one (17,18).
Dystroglycan complexes are widely expressed in a variety of nonmuscle cells as well as in muscle cells. Utrophin and short isoforms of dystrophin are also expressed in these nonmuscle cells. Utrophin (395 kDa) is a homolog of dystrophin and conserves actin- and dystroglycan-binding domains at its N-and C-terminal regions, respectively (19–22). Short dystrophin isoforms having molecular masses of 260 kDa (Dp260), 140 kDa (Dp140), 116 kDa (Dp116) and 70–80 kDa (Dp71 or apo-dystrophin-1) are translated from transcripts derived from the 3' region of the dystrophin gene (23–28). These isoforms lack the domain for actin binding but commonly contain the domains for ß-dystroglycan binding. Due to the fact that binding analyses using recombinant fusion proteins demonstrated the association between the ß-dystroglycan and utrophin/dystrophin isoforms (8,21,22,29), it is thought that the utrophin and dystrophin isoforms are simply anchored to the ß-dystroglycan in nonmuscle cells. However, the molecular organization of these proteins in vivo has not been clarified.
-SG, a homolog of -SG, was recently discovered and reported to form a new type of SG complexes by replacing -SG (17,30–33). In contrast to the -SG expression, which is specific for striated muscles, -SG is widely expressed in nonmuscle cells as well as in muscle cells. This observation raised the possibility that -SG forms a novel SG complex in nonmuscle cells. Therefore, we focused on -SG and searched for the expression of other SG subunits in nonmuscle tissues that express -SG. In this study, we found expression of ß- and -SG in peripheral nerve highly expressing -SG. Morphological and biochemical analyses showed that these three SGs form a complex together with the dystroglycan complex in the Schwann cell membrane, which anchors Dp116 or utrophin. Our data suggested that the Schwann cell-type SG complex stabilizes a molecular linkage from -dystroglycan to Dp116/utrophin through ß-dystroglycan, although this reinforcement of the linkage may be weaker than that by the skeletal muscle-type SG complex. Our findings will provide molecular bases for the functional study of the Dp116/utrophin-DAP complex in the peripheral nervous system.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Tissue expression of
-SG-SG forms a complex with ß- and -SG in smooth muscles and with ß-, -SG and -SG in skeletal muscles (17,30,33). The -SG protein has been shown to be widely expressed in a variety of nonmuscle tissues (31,32). In order to determine whether -SG forms the SG complex in nonmuscle cells, we examined the expression levels of other SGs (ß-, - and -SG) in nonmuscle tissues that highly express -SG.
Figure 1 shos the distribution pattern of -SG present in 10 µg of total proteins of various mouse tissue lysates. The 46 kDa -SG was expressed in all the tissues examined so far, i.e. cerebrum, cerebellum, sciatic nerve, lung, liver, kidney, spleen, testis, heart, skeletal muscle and intestine. Its prominent expression was found in peripheral nerve tissue. In the following study, we focused on the peripheral nerve and examined the expression of SGs and dystrophin-associated proteins.
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Expression and localization of dystrophin and dystrophin-associated membrane proteins in peripheral nerve
Immunofluorescence analysis of rabbit peripheral nerve cryosections revealed that ß-SG,
-SG, -SG, -dystroglycan and ß-dystroglycan were expressed along the outer region of nerve fibers, whereas -SG, -SG and SPN were not (Fig. 2). The cytoskeletal proteins, Dp116 (a short dystrophin isoform) and utrophin (a homolog of full-length dystrophin), were also present in the peripheral nerve outer region. Two monoclonal antibodies against dystrophin, DYS1 and DYS2, differentially stained peripheral nerve fibers; DYS2 stained them clearly, but DYS1 did not. This is due to the difference in their epitopes on the dystrophin molecule (34). DYS2 reacts with all types of dystrophin isoforms, whereas DYS1 only reacts with full-length dystrophin. This observation indicated that full-length dystrophin was not expressed in the peripheral nerve but short dystrophin isoforms were present, which was consistent with the result that the antibody specific to Dp116 stained the peripheral nerve. This was further confirmed by determining the molecular mass of the protein corresponding to the band that indicates a reaction with these antibodies by the immunoblot assay (Fig. 3B). In control staining using rabbit skeletal muscle sections, dystroglycans, four SGs (, ß, and ) and SPN were localized on the muscle cell membrane, but only a very weak expression of -SG was detected. DYS1 and DYS2 stained the sarcolemma, but the anti-Dp116 antibody did not. -SG expression was markedly detected in the capillary vasculature present in the muscle.
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All proteins detected in the peripheral nerve were located along the outer region of nerve fibers. To determine whether the locus stained with these antibodies is the axon surface or Schwann cells, we performed double-staining analysis of nerve fibers using antibodies against
-SG and neurofilament or laminin-B1 chain. The double staining clearly showed that -SG was present at a location distinct from the axon that was stained with an anti-neurofilament antibody (Fig. 4a–c). On the other hand, it was present very close to the basal lamina containing laminin (Fig. 4d–f). -SG was colocalized with ß- and -SGs at the outer region of the myelin sheath (Fig. 4g–l). -SG was also colocalized with -dystroglycan, ß-dystroglycan, utrophin and Dp116 (data not shown). Taken together with the fact that SGs are membrane-spanning glycoproteins, these results indicate that all the proteins examined were colocalized in the outermost layer of Schwann cells.
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Peripheral nerve SGs and dystroglycans form a complex that anchors Dp116 and utrophin
Immunocytochemical observations led us to consider a possibility that SGs (ß-,
- and -SGs), dystroglycans, Dp116 and utrophin assemble into a complex at the outermost layer of the myelin sheath constructed by Schwann cells. To investigate this possibility, we first examined whether these proteins fall into the wheat germ agglutinin (WGA)-bound fraction on WGA affinity chromatography because WGA fractionation is a standard method to isolate the dystrophin–DAP complex (1,2).
Figure 3A shows profiles obtained by WGA affinity chromatography using digitonin-solubilized rabbit skeletal muscle and peripheral nerve lysates. Immunoblots of the WGA-fractionated skeletal muscle lysate showed that almost all - and ß-dystroglycans were present in the WGA-bound fraction, whereas a small amount of these were detected in the void fraction. Likewise, almost all dystrophin and -SG were bound to the WGA–Sepharose resin. In the case of peripheral nerve lysate, all the -dystroglycan was detected in the WGA-bound fraction, whereas a large amount of the ß-dystroglycan was found in the WGA-bound fraction but a certain amount still remained in the void fraction. -SG showed a result similar to that of ß-dystroglycan. Immunoblotting revealed that Dp116 and utrophin were present in the WGA-bound fraction. However, the amount of Dp116 present in the WGA-bound fraction was almost the same as that in the void fraction, whereas the amount of utrophin present in the WGA-bound fraction was less than that in the void fraction. These differences in the amounts present in the WGA-bound and void fractions may be due to the differences in their binding affinities to ß-dystroglycan.
In the case of the skeletal muscle lysate, in addition to dystroglycans, 50 kDa -SG, 43 kDa ß-SG, 35 kDa -SG, 35 kDa -SG, 25 kDa SPN and dystrophin were clearly detected in WGA-bound fractions but 46 kDa -SG and Dp116 were not detected (Fig. 3B, standard). However, a weak -SG expression was observed in the blots when the detection sensitivity was raised (Fig. 3B, higher). In the peripheral nerve WGA-bound fraction, 43 kDa ß-SG, 35 kDa -SG, 46 kDa -SG, dystroglycans, Dp116 and utrophin were detected, but -SG, -SG and SPN were not detected even under a more sensitive condition for detection. DYS2 detected a signal corresponding to a protein of 116 kDa showing the same mobility as the band detected using an antibody specific to Dp116 in the peripheral nerve, although it also stained the full-length dystrophin (427 kDa) in the WGA-bound fraction of the skeletal muscle lysate. This result was in agreement with that of a previous report showing that Dp116 was specifically detected in a peripheral nerve homogenate but full-length dystrophin was not (25). In the detection of dystroglycans, ß-dystroglycan showed the same molecular size of 43 kDa both in peripheral nerve and skeletal muscle fractions, whereas the molecular mass of -dystroglycan in the peripheral nerve fraction was 120 kDa and that in the skeletal muscle fraction was 165 kDa (Fig. 3B). The staining pattern was in agreement with that in a previous study using rabbit peripheral nerve lysates (18). The molecular mass of the peripheral nerve -dystroglycan is similar to that of the brain -dystroglycan (35,36). The lower molecular mass of the brain -dystroglycan was reported to be due to its differential glycosylation (35,36). Presumably, this is also the case in the peripheral nerve -dystroglycan.
To further examine whether the proteins in the WGA-bound fraction associate with each other to form a protein complex, we analyzed the fraction by immunoprecipitation. Using an antibody against -SG and also antibodies against other SGs (ß- and -SGs) and ß-dystroglycan, ß-, - and -SGs, - and ß-dystroglycans, Dp116 and utrophin were immunoprecipitated from the peripheral nerve WGA-bound fraction (Fig. 5A). However, using an antibody against -SG, these proteins were not isolated from the peripheral nerve fraction. In contrast to that, the anti--SG antibody immunoprecipitated four SGs (-, ß-, - and -SGs), dystroglycans, SPN and dystrophin from the skeletal muscle WGA-bound fraction (Fig. 5B). This indicates that, in the peripheral nerve, ß-, - and -SGs associate with dystroglycans and also with Dp116 and utrophin. In this analysis, the amount of precipitated -dystroglycan relative to that of ß-dystroglycan was lower than that in the control immunoprecipitate from the skeletal muscle fraction, suggesting that the peripheral nerve -dystroglycan is easily separated from the complex as a result of the preparation process.
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Both Dp116 and utrophin have dystroglycan-binding structures, which were shown to bind to the same region of the ß-dystroglycan cytoplasmic domain (8,21,22,25,29). This suggests that these proteins bind to ß-dystroglycan in a competitive manner and that there are two types of SG–dystroglycan complexes that anchor Dp116 or utrophin in the peripheral nerve. Immunoprecipitation using an anti-Dp116 antibody co-precipitated three SGs (ß-,
- and -SGs), dystroglycans and Dp116 but not utrophin (Fig. 5A); this result supports the above assumption. On the other hand, in the immunoprecipitation using an anti-utrophin antibody, we only detected utrophin but not SGs, dystroglycans or Dp116. This observation suggested that the interaction of utrophin with ß-dystroglycan is weaker than that of Dp116. This idea was in agreement with the observation that fractionation by WGA affinity chromatography was not efficient in the recovery of utrophin from the peripheral nerve lysate (Fig. 3A). Another possible explanation of the result is that the binding of the antibody to utrophin may be sterically hindered or may disrupt interaction between utrophin and dystroglycan. Such a case was shown in an immunoprecipitation study of the SG complex using anti-SG antibodies (37).
Reduction in expression levels of SGs in the peripheral nerve of ß-SG- and -SG-deficient animals
A defect in any one of the four SG subunits (, ß, and ) leads to a great reduction or absence of the entire SG complex in the striated muscle cell membrane (38–43). We found a similar phenomenon in the peripheral nerve of SG-deficient animals (Fig. 6). Immunofluorescence analysis clearly showed that the expression levels of -SG and -SG were greatly reduced in the peripheral nerve of the ß-SG-deficient mouse, whereas those of - and ß-SGs were reduced in that of the -SG-deficient hamster. However, the other proteins, i.e. dystroglycans, Dp116 and utrophin, did not show significant changes in their expression levels between the wild-type and the SG-deficient mouse or hamster. The laminin-2 chain, the extracellular matrix component of the peripheral nerve, was also expressed at the same level between them.
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In vitro expression analysis for SG–dystroglycan complex formation
It was previously reported that the expression of all of the four SG proteins (
-, ß-, - and -SGs) was necessary for the formation of a stable SG complex at the cell membrane surface in Chinese hamster ovary (CHO) cells (44). However, in the peripheral nerve, three SG proteins (ß-, - and -SGs) were assembled into a complex with dystroglycans in the absence of -SG. Using in vitro expression analysis, we examined whether the three SGs could form a stable protein complex at the CHO cell membrane surface. We engineered mammalian expression vectors encoding full-length mouse ß-, - and -SG cDNAs and the vectors were introduced into CHO cells by electroporation. Cell membrane surface molecules were biotinylated and purified as avidin-bound fractions. As shown in Figure 7A, control CHO cells expressed small amounts of -SG and ß-dystroglycan, but the other four types of SGs (-, ß-, -, and -SG) were not detected. Thirty hours following cotransfection with a combination of two different SG expression vectors, i.e. ß- and -SG vectors, ß- and -SG vectors and - and -SG vectors, all the exogenous SG proteins were found in the digitonin-solubilized whole cell lysates. However, only in the cells cotransfected with ß- and -SG expression vectors, large amounts of ß-, - and -SGs were found in the avidin-bound cell surface fraction (Fig. 7A). Immunoprecipitation of the avidin-bound fraction using an anti--SG antibody showed that these SG proteins assemble into a complex together with endogenous ß-dystroglycan (Fig. 7B). In the absence of ß-SG or -SG, -SG did not associate with either exogenous SG proteins or endogenous ß-dystroglycan. It was noteworthy that endogenous -SG expression was markedly upregulated in the cells cotransfected with ß- and -SG expression vectors.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In this study, we found that ß-,
- and -SG proteins were co-expressed with dystroglycans, Dp116 and utrophin on the protoplasmic membrane of the outermost layer of Schwann cells (Figs 2 and 4). Immunoprecipitation analysis showed that the SG and dystroglycan proteins assembled into a complex which anchored Dp116 or utrophin (Fig. 5). This is the first report to demonstrate that SGs formed a dystrophin-associated glycoprotein complex (DAGC) together with dystroglycans in nonmuscle cells. We clearly showed that the expression levels of all SGs were greatly reduced in the peripheral nerve of ß-SG-deficient mouse and -SG-deficient hamster but that there was no obvious change in the expression levels of dystroglycans, Dp116 and utrophin (Fig. 6). This phenomenon was very similar to that observed in SG-deficient muscle cells (15), indicating that the three SG proteins (ß-, - and -SGs) in the peripheral nerve also form a single unit within the Dp116/utrophin–DAGC complex.
Using an extract of crude bovine peripheral nerve membranes, Saito et al. (45) showed that the linkage between the components of the dystroglycan was unstable. In an immunoprecipitation study using an antibody against ß-dystroglycan, they showed that ß-dystroglycan was detected only in the immunoprecipitate but that of -dystroglycan was detected in both the precipitate and the supernatant (45). Their findings are in agreement with our results obtained by WGA affinity chromatography and immunoprecipitation (Figs 3A and 5) although they did not observe the presence of a SG complex composed of ß-, - and -SGs. Since in their study a control experiment using skeletal muscle dystroglycan was not performed, it was unclear whether the observed molecular feature was specific for the peripheral nerve-type dystroglycan complex. In this study, we showed that the intermolecular interaction of peripheral nerve dystroglycans was weaker than that of skeletal muscle dystroglycans in comparative analyses using skeletal muscle samples by WGA affinity fractionation (Fig. 3A) and immunoprecipitation (Fig. 5).
The present data further suggest that the interaction of Dp116 and utrophin with ß-dystroglycan is also unstable. WGA affinity chromatography using peripheral nerve lysate revealed that ß-dystroglycan, -SG, Dp116 and utrophin remained in the void fraction, but the amounts of ß-dystroglycan and -SG were clearly smaller than those in the WGA-bound fraction (Fig. 3A). On the other hand, the amounts of Dp116 and utrophin in the void fraction were slightly larger than those in the bound fraction. Compared with these proteins, the amount of ß-dystroglycan present in the WGA-bound fraction was much larger than that in the void fraction. All -dystroglycan was found in the WGA-bound fraction. Since -dystroglycan is known to bind to WGA through its sugar chain, some amounts of ß-dystroglycan as well as Dp116 and utrophin in larger amounts may dissociate from -dystroglycan. These findings suggest that Dp116 and utrophin easily separate from ß-dystroglycan. An antibody specific to Dp116 coprecipitated dystroglycans, SGs and Dp116, but their amounts were smaller than those in the immunoprecipitates using antibodies against SGs and ß-dystroglycan. Furthermore, these membrane proteins were undetectable in immunoprecipitate using anti-utrophin antibody (Fig. 5A). These results seemed to correlate with those of WGA affinity chromatography on the percent amounts of these proteins remaining in the void fraction, i.e. the amount of utrophin remaining in the void fraction was relatively larger than that of Dp116. The interaction between utrophin and ß-dystroglycan may be weaker than that between Dp116 and ß-dystroglycan.
A significant structural difference between the dystrophin–DAP complexes of the Schwann cell and the skeletal muscle was found in terms of the composition of their SG subcomplexes. The peripheral nerve SG complex was composed of ß-, - and -SGs, whereas the skeletal muscle SG complex was composed of -, ß-, - and -SGs (10). The fragility of the Dp116/utrophin–DAP complex of the peripheral nerve may possibly be due to the protein composition of the SG subcomplex that contains -SG and/or no -SG. Durbeej and Campbell analyzed the interaction between dystroglycan and utrophin in the rabbit kidney, which has no SG complex (17). They barely detected utrophin in the dystroglycan fraction by sucrose gradient fractionation and did not show direct evidence of their association. This result suggests that the interaction between utrophin and dystroglycan was quite weak in the absence of the SG complex, even if they are able to associate. Furthermore, concerning Dp140, which is expressed in the kidney, they did not demonstrate any Dp140 interaction with dystroglycan. Here, we clearly showed that Dp116/utrophin and dystroglycans were co-isolated with the SG complex. The ß---SG complex probably plays a role in stabilizing the interaction between Dp116/utrophin and dystroglycan in Schwann cells although it is functionally inferior to the striated muscle-type SG complex.
Recently, two types of SG complexes containing -SG were found in skeletal muscle and smooth muscle cells (17,30,33). In skeletal muscle cells, -SG was shown to form a complex with ß-, - and -SGs, whereas three SGs, ß-, - and -SGs, form a complex in smooth muscle cells. The SG composition of the smooth muscle-type complex was identical to that of the peripheral nerve-type SG complex. RNA and protein analyses carried out by Campbell and colleagues using normal muscle and muscle cells from ß-SG-deficient mice (17,30,46) led them to propose that -SG is part of the smooth muscle complex. However, in the case of the peripheral nerve, we could not detect -SG either by immunohistochemistry or transcriptional analysis (data not shown). Therefore, the Schwann cell-type SG complex seems to be different from the reported smooth muscle-type SG complex, although our data do not exclude the possibility that a fourth unknown protein is contained in the Schwann cell-type SG complex.
Holt and Campbell reported that cotransfection with all four types of expression constructs encoding -, ß-, - and -SG cDNAs was necessary for stably retaining SG molecules in the CHO cell membrane (44). However, we showed the stable expression of ß- and -SGs in the cell membrane even when only two expression vectors encoding ß- and -SG cDNAs were co-transfected. Synthesis of the ß- and -SGs increased the expression level of the endogenous -SG protein in CHO cells but did not affect the expression level of endogenous dystroglycan (Fig. 7A). Exogenous ß- and -SGs possibly stabilize the -SG protein by forming a SG complex in the endoplasmic reticulum, and then the subcomplex associates with endogenous dystroglycan in the Golgi apparatus, as shown in a previous report using the myogenic cell line, C2/4 (47). The major difference between Holt and Campbell’s study and ours is in the design of cDNA expression constructs. They added all the previously reported SG cDNAs to a Myc epitope tag sequence for expressing the tag at their intracellular tails, whereas we did not add any extra sequences to our SG cDNAs. The artificial tail sequence may change some of the properties of the original SG molecules by causing some conformational changes.
Disruption of the entire SG complex eventually causes degeneration of skeletal and cardiac muscle cells (38–43). The loss of the SG complex in a ß- and -SG-deficient mouse was shown to induce vascular smooth muscle irregularities in the heart, diaphragm and kidney (46,48). On the other hand, we could not find apparent abnormalities in the peripheral nerves of ß-SG-deficient mouse and -SG-deficient hamster. A weak linkage between ECM and actin-cytoskeleton through the dystroglycan–utrophin complex in the reduction in the expression level of the SG complex does not seem to cause significant damage of Schwann cells in terms of function. Striated and smooth muscle cells are always subjected to mechanical stress induced by contraction and blood pressure, whereas Schwann cells are not. Possibly, the different circumstances between muscles and peripheral nerve may be related to their phenotypic appearance. The Schwann cell-type DAGC complex anchors Dp116 as well as utrophin. The Dp116–DAGC complex must play a role different from that of the utrophin–DAGC complex because Dp116 lacks an actin-binding domain and cannot link to ECM and cytoskeletal actin. It is of interest to consider the role of the SG complex in the Dp116–DAGC complex. Since it has been suggested that the SG complex plays a role in signal transduction in addition to its structural role (49), studies are necessary to elucidate the functional roles of the Schwann cell-type SG complex.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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WGA affinity chromatography
Sciatic, femoral and tibial nerves were dissected from a rabbit. The peripheral nerve was homogenized in 10 vol (w/v) of buffer A (1% digitonin, 20 mM HEPES–NaOH pH 7.5, 150 mM NaCl, 0.5 mM sodium vanadate, Calpain inhibitor II, 0.5 mM phenylmethyl sulfonylfluoride, protease inhibitor cocktail, 15 µM N-Acetyl-Leu-Leu-methioninal). After centrifugation at 50 000 g for 30 min, the supernatant was mixed with WGA–Sepharose beads and incubated for 2 h at 4°C. The Sepharose beads were then washed extensively with buffer A, and WGA-bound proteins were eluted with buffer A containing 0.5 M N-acetyl-D-glucosamine.
Antibodies
Mouse monoclonal antibodies against -SG (NCL-a-SARC), ß-SG (NCL-b-SARC), -SG (NCL-g-SARC), ß-dystroglycan (NCL-b-DG), dystrophin (NCL-DYS1, NCL-DYS2) and utrophin (NCL-DRP2) were purchased from Novo Castra Laboratories (Newcastle-upon-Tyne, UK). The other mouse monoclonal antibodies were anti--dystroglycan (VIA 4–1; Upstate Biotechnology, Lake Placid, NY), anti-laminin-B1 chain (MAB1921; Chemicon International, Temecula, CA) and neurofilament (RT97; YLEM, Rome, Italy). A mouse monoclonal antibody against -SG (DSG-1) was produced by Asahi Techno Glass (Tokyo, Japan) using its N-terminal synthetic peptide (50). A rat monoclonal antibody against the laminin-2 chain (4H8-2) was purchased from Alexis (Laufelfingen, Switzerland).
Affinity-purified rabbit antibodies against -SG, ß-SG and mouse SPN were previously described (15,47,50). A previously reported antiserum against utrophin, UT-2 (51), was affinity-purified with the antigen and used.
Rabbit antibodies against the cytoplasmic regions of ß-dystroglycan and -SG were raised against the recombinant human dystroglycan cytoplasmic region (amino acid positions 795–895) and the -SG cytoplasmic region (amino acid positions 320–413), respectively. The dystroglycan (0.5 kbp) and -SG cDNA fragments (0.4 kbp) were amplified from a human skeletal muscle cDNA library (Clontech, Palo Alto, CA) by PCR using the following oligonucleotide primer sets: 5'-GGGGGTGCCTATCATCTTTGC-3' (2776–2796) and 5'-GACCAGAGAGGGCGGGCTTGT-3' (3243–3223) for human dystroglycan (52) and 5'-GGAAGGCGTGGAAAAGAGAAACA-3' (1068–1690) and 5'-TCATGCATTATTGGAAGAGAAAA-3' (1440–1418) for human -SG (32). The amplification was carried out using LA-Taq (Takara, Kyoto, Japan) for 35 cycles, each cycle consisting of 94°C for 1 min, 55°C for 1 min and 72°C for 3 min. The amplified DNA fragments were subcloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA). Then, the EcoRI fragments of dystroglycan and -SG cDNA were purified from cloned pCR2.1 plasmid and ligated into the pGEX expression vector (Amersham Pharmacia Biotech, Little Chalfont, UK). Recombinant proteins were expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli and purified from the soluble fraction of cell lysates on a glutathione–Sepharose column as described by Suzuki et al. (8). These recombinant proteins were used as antigens. The obtained antisera against dystroglycan and -SG were purified using affinity columns coupled with the recombinant antigens. The purified anti-GST–dystroglycan and GST–-SG antibodies were absorbed with affinity columns coupled with GST and GST–-SG (15), respectively.
Rabbit antibodies to Dp116 and human SPN were prepared against the N-terminal unique sequence of rat Dp116, CSPRFKLKMLHRKTYHVKDLQ (amino acids 7–27) and the C-terminal sequence of human SPN, WKHRYQVFYVVRICSLTASEGPQQKI (amino acids 191–217), respectively. Each synthetic peptide coupled with keyhole limpet hemocyanine (KLH) was used to immunize rabbits. The antisera against the KLH-Dp116 peptide and KLH-human SPN were purified using affinity columns coupled with the synthetic peptide antigens.
Rat antibodies against -SG, -SG, mouse SPN and Dp116 were raised against the above described antigens for -SG and Dp116 and previously described antigens for -SG and mouse SPN (50). The rat antibody specific to -SG was purified from antiserum by the same procedure as the rabbit antibody purification described above. The other rat antibodies (to -SG, SPN and Dp116) were purified using affinity columns coupled with synthetic peptide antigens.
Immunocytochemistry
Cryosections (6 and 10 µm) were used for immunofluorescence staining of skeletal muscles and peripheral nerves, respectively. These cryosections were placed on slide glasses and fixed in cold acetone or phosphate-buffered saline (PBS) containing 3.5% formalin. After equilibration of the fixed sections with PBS, the sections were blocked in PBS containing 2% casein. Indirect immunofluorescence microscopy was performed as previously described (9), using the following primary antibodies at appropriate dilutions: mouse monoclonal antibodies against -SG at 1:100, ß-SG at 1:100, -SG at 1:100, -SG at 1:80, -dystroglycan at 1:100, ß-dystroglycan at 1:100, dystrophin (NCL-DYS1, NCL-DYS2) at 1:100, utrophin at 1:5, anti-laminin-B1 chain at 1:100, neurofilament at 1:100, rat monoclonal antibody against the laminin-2 chain at 1:100, affinity-purified rat polyclonal antibodies against -SG at 1:1000, -SG at 1:1000, Dp116 at 1:200, affinity-purified rabbit polyclonal antibodies against ß-SG at 1:500, ß-dystroglycan at 1:1000, -SG at 1:600 and utrophin at 1:600 dilution. For staining with anti--SG, anti--dystroglycan, anti-Dp116 and anti-laminin-B1 chain, formalin-fixed sections were used. Other antibodies were basically reacted to acetone-fixed tissue sections. As secondary antibodies, Alexa488-labeled anti-mouse (1:600) and anti-rabbit antibodies (1:600), Alexa568-labeled anti-mouse (1:1000) and anti-rabbit (1:1000) antibodies (Molecular Probes, Oregon, OR) and fluorescein isothiocyanate (FITC)-labeled anti-rat antibody (1:1000; Organon Teknika, Durham, NC) were used. Affinity-purified anti-human SPN and anti--SG rabbit antibodies were labeled with Alexa568 (Molecular Probes) and used at 1:500 dilution for direct immunofluorescence staining. Fluorescence signals on cryosections were observed under a confocal laser scanning microscope (Leica TCS SP; Leica, Heidelberg, Germany).
Cell culture and cDNA plasmid transfection by electroporation
Mouse full-length ß, and -SG cDNAs (50) were ligated to an EcoRI site downstream of the CAG promoter of the pCAGGS expression vector (53).
CHO cells were maintained in a Ham F12 medium supplemented with 10% fetal bovine serum. The cells were electroporated with SG-expression vectors at 340 V and 960 microfarads.
Cell surface biotinylation and purification of the biotinylated molecules
Thirty hours after transfection, the cell monolayers were washed three times with Dulbecco’s PBS. The cells were incubated with NHS–biotin (Pierce, Rockford, IL) in PBS (0.5 mg/ml) for 30 min at room temperature. To remove unreacted NHS–biotin, the cell monolayers were washed four times with PBS. The cells were removed from the culture dish by pipetting and collected by centrifugation. The cell pellet was lysed in buffer B (20 mM HEPES pH 7.5, 150 mM NaCl, 0.2 mM sodium vanadate, 0.5 mM phenylmethylsulfonyl fluoride, 15 µM N-acetyl-Leu-Leu-methioninal) containing 1.5% digitonin and an appropriate amount of protease inhibitor cocktail (Complete EDTA-free; Roche Diagnostics, Mannheim, Germany). Clarified solubilized fractions were incubated with 250 µl of monomeric avidin resin (Promega, Madison, WI) in an Eppendorf tube with rotation for 3 h at 4°C. The reaction tubes were centrifuged and supernatants were collected and used as avidin–resin unbound fractions. The avidin–resins were washed five times with 1 ml of buffer B containing 0.1% digitonin and an appropriate amount of protease inhibitor cocktail. The washed resin was incubated with 0.4 ml of the washing buffer containing 0.05 M biotin in an Eppendorf tube with rotation for 8 h at 4°C. To remove the avidin resin, the tube was centrifuged and the supernatant was collected as the avidin-bound fraction.
Other procedures
SDS lysates of mouse tissues were prepared as described previously (51). SDS–polyacrylamide electrophoresis (SDS–PAGE) and protein transfer to the PVDF membrane were performed as described by Laemmli (54) and Kyhse-Anderson (55), respectively. Immunoprecipitation and immunoblotting were performed as described previously (15). Immunoreactive protein bands in the immunoblotting were visualized using the chemiluminescence detection system for standard type (ECL; Amersham Pharmacia) or hypersensitive type (ECL+Plus; Amersham Pharmacia). Protein concentration was determined using a protein assay (BioRad, Hercules, CA) with bovine serum albumin as a standard.
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ACKNOWLEDGEMENTS |
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The authors thank Dr J. Miyazaki (Osaka University) for generously providing the mammalian expression vector pCAGGS. This work was supported by a grant from the COE program and Health Sciences Research Grants for Research on the Brain from the Ministry of Health and Welfare.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +81 423 46 1720; Fax: +81 423 46 1750; Email: imamura@ncnp.go.jp
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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