Dystrophin-associated proteins are greatly reduced in skeletal muscle from mdx mice

Dystrophin, the protein product of the human Duchenne muscular dystrophy gene, exists in skeletal muscle as a large oligomeric complex that contains four glycoproteins of 156, 50, 43, and 35 kD and a protein of 59 kD. Here, we investigated the relative abundance of each of the components of the dystrophin-glycoprotein complex in skeletal muscle from normal and mdx mice, which are missing dystrophin. Immunoblot analysis using total muscle membranes from control and mdx mice of ages 1 d to 30 wk found that all of the dystrophin-associated proteins were greatly reduced (80-90%) in mdx mouse skeletal muscle. The specificity of the loss of the dystrophin-associated glycoproteins was demonstrated by the finding that the major glycoprotein composition of skeletal muscle membranes from normal and mdx mice was identical. Furthermore, skeletal muscle membranes from the dystrophic dy/dy mouse exhibited a normal density of dystrophin and dystrophin-associated proteins. Immunofluorescence microscopy confirmed the results from the immunoblot analysis and showed a drastically reduced density of dystrophin-associated proteins in mdx muscle cryosections compared with normal and dy/dy mouse muscle. Therefore, our results demonstrate that all of the dystrophin-associated proteins are significantly reduced in mdx skeletal muscle and suggest that the loss of dystrophin-associated proteins is due to the absence of dystrophin and not due to secondary effects of muscle fiber degradation.     

Restoration of dystrophin-associated proteins in skeletal muscle of mdx mice transgenic for dystrophin gene

Duchenne muscular dystrophy (DMD) patients and mdx mice are characterized by the absence of dystrophin, a membrane cytoskeletal protein. Dystrophin is associated with a large oligomeric complex of sarcolemmal glycoproteins, including dystroglycan which provides a linkage to the extarcellular matrix component, laminin. The finding that all of the dystrophin-associated proteins (DAPs) are drastically reduced in DMD and mdx skeletal muscle supports the primary function of dystrophin as an anchor of the sarcolemmal glycoprotein complex to the subsarcolemmal cytoskeleton. These findings indicate that the efficacy of dystrophin gene therapy will depend not only on replacing dystrophin but also on restoring all of the DAPs in the sarcolemma. Here we have investigated the status of the DAPs in the skeletal muscle of mdx mice transgenic for the dystrophin gene. Our results demonstrate that transfer of dystrophin gene restores all of the DAPs together with dystrophin, suggesting that dystrophin gene therapy should be effective in restoring the entire dystrophin-glycoprotein complex.     

Mild deficiency of dystrophin-associated proteins in Becker muscular dystrophy patients having in-frame deletions in the rod domain of dystrophin.

The dystrophin-glycoprotein complex spans the sarcolemma to provide a linkage between the subsarcolemmal cytoskeleton and the extracellular matrix in skeletal muscle. In Duchenne muscular dystrophy (DMD), the absence of dystrophin leads to a drastic reduction in all of the dystrophin-associated proteins in the sarcolemma, thus causing the disruption of the dystrophin-glycoprotein complex and the loss of the linkage to the extracellular matrix. The resulting sarcolemmal instability is presumed to render muscle fibers susceptible to necrosis. In the present study, we investigated the status of the dystrophin-associated proteins in the skeletal muscle from patients with Becker muscular dystrophy (BMD), a milder allelic form of DMD. BMD patients having in-frame deletions in the rod domain of dystrophin showed a mild to moderate reduction in all of the dystrophin-associated proteins in the sarcolemma, but this reduction was not as severe as that in DMD patients. The reduction of the immunostaining for the dystrophin-associated proteins showed a good correlation with that for dystrophin in both intensity and distribution. Our results indicate that (1) the abnormality of the sarcolemmal glycoprotein complex, which is similar to but milder than that in DMD patients, also exists in these BMD patients and (2) the rod domain of dystrophin is not crucial for the interaction with the dystrophin-associated proteins.     

Dystrophin and Utrophin Complexed with Different Associated Proteins in Cardiac Purkinje Fibres

Abnormal dystrophin expression is directly responsible for Duchenne and Becker muscular dystrophies. In skeletal muscle, dystrophin provides a link between the actin network and the extracellular matrix via the dystrophin-associated protein complex. In mature skeletal muscle, utrophin is a dystrophin-related protein localized mainly at the neuromuscular junction, with the same properties as dystrophin in terms of linking the protein complex. Utrophin could potentially overcome the absence of dystrophin in dystrophic skeletal muscles. In cardiac muscle, dystrophin and utrophin were both found to be present with a distinct subcellular distribution in Purkinje fibres, i.e. utrophin was limited to the cytoplasm, while dystrophin was located in the cytoplasmic membrane.In this study, we used this particular characteristic of cardiac Purkinje fibres and demonstrated that associated proteins of dystrophin and utrophin are different in this structure. We conclude, contrary to skeletal muscle, dystrophin-associated proteins do not form a complex in Purkinje fibres. In addition, we have indirect evidence of the presence of two different 400kDa dystrophins in Purkinje fibres.     

A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin

The dystrophin-glycoprotein complex was tested for interaction with several components of the extracellular matrix as well as actin. The 156-kD dystrophin-associated glycoprotein (156-kD dystroglycan) specifically bound laminin in a calcium-dependent manner and was inhibited by NaCl (IC50 = 250 mM) but was not affected by 1,000-fold (wt/wt) excesses of lactose, IKVAV, or YIGSR peptides. Laminin binding was inhibited by heparin (IC50 = 100 micrograms/ml), suggesting that one of the heparin-binding domains of laminin is involved in binding dystroglycan while negatively charged oligosaccharide moieties on dystroglycan were found to be necessary for its laminin-binding activity. No interaction between any component of the dystrophin- glycoprotein complex and fibronectin, collagen I, collagen IV, entactin, or heparan sulfate proteoglycan was detected by 125I-protein overlay and/or extracellular matrix protein-Sepharose precipitation. In addition, laminin-Sepharose quantitatively precipitated purified dystrophin-glycoprotein complex, demonstrating that the laminin-binding site is accessible when dystroglycan is associated with the complex. Dystroglycan of nonmuscle tissues also bound laminin. However, the other proteins of the striated muscle dystrophin-glycoprotein complex appear to be absent, antigenically dissimilar or less tightly associated with dystroglycan in nonmuscle tissues. Finally, we show that the dystrophin-glycoprotein complex cosediments with F-actin but does not bind calcium or calmodulin. Our results support a role for the striated muscle dystrophin-glycoprotein complex in linking the actin- based cytoskeleton with the extracellular matrix. Furthermore, our results suggest that dystrophin and dystroglycan may play substantially different functional roles in nonmuscle tissues.     

Syntrophins and dystrobrevins: defining the dystrophin scaffold at synapses

Dystrophin and its associated proteins were originally identified in skeletal muscle, where the complex provides mechanical stabilization to the sarcolemma during contraction. However, the dystrophin complex is also present at membrane specializations in many non-muscle cells, including synaptic sites in neurons. The function of the dystrophin complex at these sites is still unknown, but emerging results suggest that the dystrophin complex can function as a scaffold for signaling proteins. In this review, we examine the growing body of evidence that suggests the dystrophin complex may have a dual function: membrane stabilization and transmembrane signaling. We focus on the role of two dystrophin-associated proteins, syntrophin and dystrobrevin, in the formation of a signaling scaffold and review evidence suggesting a role in synapse formation and maintenance.     

A PDZ-containing scaffold related to the dystrophin complex at the basolateral membrane of epithelial cells

Membrane scaffolding complexes are key features of many cell types, serving as specialized links between the extracellular matrix and the actin cytoskeleton. An important scaffold in skeletal muscle is the dystrophin-associated protein complex. One of the proteins bound directly to dystrophin is syntrophin, a modular protein comprised entirely of interaction motifs, including PDZ (protein domain named for PSD-95, discs large, ZO-1) and pleckstrin homology (PH) domains. In skeletal muscle, the syntrophin PDZ domain recruits sodium channels and signaling molecules, such as neuronal nitric oxide synthase, to the dystrophin complex. In epithelia, we identified a variation of the dystrophin complex, in which syntrophin, and the dystrophin homologues, utrophin and dystrobrevin, are restricted to the basolateral membrane. We used exogenously expressed green fluorescent protein (GFP)-tagged fusion proteins to determine which domains of syntrophin are responsible for its polarized localization. GFP-tagged full-length syntrophin targeted to the basolateral membrane, but individual domains remained in the cytoplasm. In contrast, the second PH domain tandemly linked to a highly conserved, COOH-terminal region was sufficient for basolateral membrane targeting and association with utrophin. The results suggest an interaction between syntrophin and utrophin that leaves the PDZ domain of syntrophin available to recruit additional proteins to the epithelial basolateral membrane. The assembly of multiprotein signaling complexes at sites of membrane specialization may be a widespread function of dystrophin-related protein complexes.     

Dystroglycan is not required for localization of dystrophin, syntrophin, and neuronal nitric-oxide synthase at the sarcolemma but regulates integrin alpha 7B expression and caveolin-3 distribution.

Dystroglycan is part of the dystrophin-associated protein complex, which joins laminin in the extracellular matrix to dystrophin within the subsarcolemmal cytoskeleton. We have investigated how mutations in the components of the laminin-dystroglycan-dystrophin axis affect the organization and expression of dystrophin-associated proteins by comparing mice mutant for merosin (alpha(2)-laminin, dy), dystrophin (mdx), and dystroglycan (Dag1) using immunohistochemistry and immunoblots. We report that syntrophin and neuronal nitric-oxide synthase are depleted in muscle fibers lacking both dystrophin and dystroglycan. Some fibers deficient in dystroglycan, however, localize dystrophin at the cell surface at levels similar to that in wild-type muscle. Nevertheless, these fibers have signs of degeneration/regeneration including increased cell surface permeability and central nuclei. In these fibers, syntrophin and nitric-oxide synthase are also localized to the plasma membrane, whereas the sarcoglycan complex is disrupted. These results suggest a mechanism of membrane attachment for dystrophin independent of dystroglycan and that the interaction of sarcoglycans with dystrophin requires dystroglycan. The distribution of caveolin-3, a muscle-specific component of caveolae recently found to bind dystroglycan, was affected in dystroglycan- and dystrophin-deficient mice. We also examined alternative mechanisms of cell-extracellular matrix attachment to elucidate how the muscle basement membrane may subsist in the absence of dystroglycan, and we found the alpha(7B) splice variant of the alpha(7) integrin receptor subunit to be up-regulated. These results support the possibility that alpha(7B) integrin compensates in mediating cell-extracellular matrix attachment but cannot rescue the dystrophic phenotype.     

Creatine kinase, cell membrane and Duchenne muscular dystrophy

In 1958 Professor Setsuro Ebashi found that serum creatine kinase activity is increased in patients suffering from various muscular dystrophies, especially Duchenne muscular dystrophy (DMD). He and others proposed that creatine kinase passes through the cell membrane as it is released from DMD muscle fibers.Since then, it has been found that dystrophin and dystrophin-associated proteins are connected to several other components, including the basal lamina and subsarcolemmal cytoskeletal networks on the cell membrane, while dystrophin anchors these dystrophin-associated proteins to the actin filaments inside the muscle cell. In DMD muscle, dystrophin has been found to be absent and dystroglycans and sarcoglycans decreased. However, how creatine kinase molecules can pass through the DMD muscle cell membrane still remains unanswered.On the basis of recent findings on the structure of the protein layers which sandwich the lipid bilayer of muscle cell membranes, this essay stresses the importance of these lipid bilayers in protecting creatine kinase release from protoplasma in normal muscle. It further indicates the possibility that the absence of dystrophin in DMD muscle during muscle contraction may result in temporal damage to the lipid bilayer.     

Dystrophin complex functions as a scaffold for signalling proteins

Dystrophin is a 427 kDa sub-membrane cytoskeletal protein, associated with the inner surface membrane and incorporated in a large macromolecular complex of proteins, the dystrophin-associated protein complex (DAPC). In addition to dystrophin the DAPC is composed of dystroglycans, sarcoglycans, sarcospan, dystrobrevins and syntrophin. This complex is thought to play a structural role in ensuring membrane stability and force transduction during muscle contraction. The multiple binding sites and domains present in the DAPC confer the scaffold of various signalling and channel proteins, which may implicate the DAPC in regulation of signalling processes. The DAPC is thought for instance to anchor a variety of signalling molecules near their sites of action. The dystroglycan complex may participate in the transduction of extracellular-mediated signals to the muscle cytoskeleton, and β-dystroglycan was shown to be involved in MAPK and Rac1 small GTPase signalling. More generally, dystroglycan is view as a cell surface receptor for extracellular matrix proteins. The adaptor proteins syntrophin contribute to recruit and regulate various signalling proteins such as ion channels, into a macromolecular complex. Although dystrophin and dystroglycan can be directly involved in signalling pathways, syntrophins play a central role in organizing signalplex anchored to the dystrophin scaffold. The dystrophin associated complex, can bind up to four syntrophin through binding domains of dystrophin and dystrobrevin, allowing the scaffold of multiple signalling proteins in close proximity. Multiple interactions mediated by PH and PDZ domains of syntrophin also contribute to build a complete signalplex which may include ion channels, such as voltage-gated sodium channels or TRPC cation channels, together with, trimeric G protein, G protein-coupled receptor, plasma membrane calcium pump, and NOS, to enable efficient and regulated signal transduction and ion transport. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

The structure of the N-terminal actin-binding domain of human dystrophin and how mutations in this domain may cause Duchenne or Becker muscular dystrophy

BACKGROUND: Dystrophin is an essential component of skeletal muscle cells. Its N-terminal domain binds to F-actin and its C terminus binds to the dystrophin-associated glycoprotein (DAG) complex in the membrane. Dystrophin is therefore thought to serve as a link from the actin-based cytoskeleton of the muscle cell through the plasma membrane to the extracellular matrix. Pathogenic mutations in dystrophin result in Duchenne or Becker muscular dystrophy. RESULTS: The crystal structure of the dystrophin actin-binding domain (ABD) has been determined at 2.6 A resolution. The structure is an antiparallel dimer of two ABDs each comprising two calponin homology domains (CH1 and CH2) that are linked by a central alpha helix. The CH domains are both alpha-helical globular folds. Comparisons with the structures of utrophin and fimbrin ABDs reveal that the conformations of the individual CH domains are very similar to those of dystrophin but that the arrangement of the two CH domains within the ABD is altered. The dystrophin dimer reveals a change of 72 degrees in the orientation of one pair of CH1 and CH2 domains (from different monomers) relative to the other pair when compared with the utrophin dimer. The dystrophin monomer is more elongated than the fimbrin ABD. CONCLUSIONS: The dystrophin ABD structure reveals a previously uncharacterised arrangement of the CH domains within the ABD. This observation has implications for the mechanism of actin binding by dystrophin and related proteins. Examining the position of three pathogenic missense mutations within the structure suggests that they exert their effects through misfolding of the ABD, rather than through disruption of the binding to F-actin.     

Glycoprotein complex anchoring dystrophin to sarcolemma

We found six groups of proteins, A0-A5, besides dystrophin itself in a dystrophin preparation obtained by the reported method [Campbell, K.P. & Kahl, S.D.(1989) Nature 338, 259-262] with some modifications. Their molecular weights were 94, 62, 52, 43, 36, and 24 kDa, respectively. Their molar ratios to dystrophin were 0.14, 2.2, 0.88, 0.90, 1.7, and 0.34, respectively. Each of A1, A3, and A4 was split into several bands. But each group of bands except A3 seemed to behave like the same kind of protein. The doublet of A3 was subdivided into A3a and A3b in the decreasing order of molecular weight. All the A-proteins except A2 were cross-linked with dystrophin molecule by a cross-linker, bis(sulfosuccinimidyl)suberate, suggesting them to be dystrophin-associated proteins. When dystrophin preparation was treated with KI, which is known to break membrane cytoskeletal interactions, as described by Campbell and Kahl, A2, A3, and A4 were absorbed by wheat germ lectin (WGL) Sepharose, but the dystrophin molecule and A1 were not absorbed. On the other hand, A2 and A3b reacted with biotinyl WGL but A3a and A4 did not in blotting analysis. This apparent discrepancy can be explained if we postulate that A3a and/or A4 would associate with A2 and/or A3b. On the basis of these results including stoichiometric considerations, we are of the opinion that the complex of A2.A4 among various possible ones is the most important to anchor dystrophin to sarcolemma. In this A2.A4 complex, A4 but not A2 is directly associated with dystrophin.     

Forced expression of dystrophin deletion constructs reveals structure- function correlations

Dystrophin plays an important role in skeletal muscle by linking the cytoskeleton and the extracellular matrix. The amino terminus of dystrophin binds to actin and possibly other components of the subsarcolemmal cytoskeleton, while the carboxy terminus associates with a group of integral and peripheral membrane proteins and glycoproteins that are collectively known as the dystrophin-associated protein (DAP) complex. We have generated transgenic/mdx mice expressing "full-length" dystrophin constructs, but with consecutive deletions within the COOH- terminal domains. These mice have enabled analysis of the interaction between dystrophin and members of the DAP complex and the effects that perturbing these associations have on the dystrophic process. Deletions within the cysteine-rich region disrupt the interaction between dystrophin and the DAP complex, leading to a severe dystrophic pathology. These deletions remove the beta-dystroglycan-binding site, which leads to a parallel loss of both beta-dystroglycan and the sarcoglycan complex from the sarcolemma. In contrast, deletion of the alternatively spliced domain and the extreme COOH terminus has no apparent effect on the function of dystrophin when expressed at normal levels. The proteins resulting from these latter two deletions supported formation of a completely normal DAP complex, and their expression was associated with normal muscle morphology in mdx mice. These data indicate that the cysteine-rich domain is critical for functional activity, presumably by mediating a direct interaction with beta-dystroglycan. However, the remainder of the COOH terminus is not required for assembly of the DAP complex.     

Correlation of glycosylation forms with position in amino acid sequence

We surveyed published reports on about 50 glycoproteins whose amino acid sequence, glycosylation sites, and type of glycosylation at a particular site have been established. We note that high-mannose substances were rarely found at the N-terminal side of a previously glycosylated complex site. There was a very definite distribution of complex sites about the N-terminal region. Furthermore, secreted glycoproteins usually contained only complex oligosaccharides whereas membrane proteins contained both types. We suggest that the position of the glycosylation site with respect to the N-terminus affects the extent of oligosaccharide processing and subsequent presentation of complex or high-mannose structures in the mature glycoprotein. This review relates glycosylation type to its position in the known sequence of given proteins and discusses these observations in light of known glycosylation processing reactions.     

Protein trafficking and anchoring complexes revealed by proteomic analysis of inward rectifier potassium channel (Kir2.x)-associated proteins

Inward rectifier potassium (Kir) channels play important roles in the maintenance and control of cell excitability. Both intracellular trafficking and modulation of Kir channel activity are regulated by protein-protein interactions. We adopted a proteomics approach to identify proteins associated with Kir2 channels via the channel C-terminal PDZ binding motif. Detergent-solubilized rat brain and heart extracts were subjected to affinity chromatography using a Kir2.2 C-terminal matrix to purify channel-interacting proteins. Proteins were identified with multidimensional high pressure liquid chromatography coupled with electrospray ionization tandem mass spectrometry, N-terminal microsequencing, and immunoblotting with specific antibodies. We identified eight members of the MAGUK family of proteins (SAP97, PSD-95, Chapsyn-110, SAP102, CASK, Dlg2, Dlg3, and Pals2), two isoforms of Veli (Veli-1 and Veli-3), Mint1, and actin-binding LIM protein (abLIM) as Kir2.2-associated brain proteins. From heart extract purifications, SAP97, CASK, Veli-3, and Mint1 also were found to associate with Kir2 channels. Furthermore, we demonstrate for the first time that components of the dystrophin-associated protein complex, including alpha1-, beta1-, and beta2-syntrophin, dystrophin, and dystrobrevin, interact with Kir2 channels, as demonstrated by immunoaffinity purification and affinity chromatography from skeletal and cardiac muscle and brain. Affinity pull-down experiments revealed that Kir2.1, Kir2.2, Kir2.3, and Kir4.1 all bind to scaffolding proteins but with different affinities for the dystrophin-associated protein complex and SAP97, CASK, and Veli. Immunofluorescent localization studies demonstrated that Kir2.2 co-localizes with syntrophin, dystrophin, and dystrobrevin at skeletal muscle neuromuscular junctions. These results suggest that Kir2 channels associate with protein complexes that may be important to target and traffic channels to specific subcellular locations, as well as anchor and stabilize channels in the plasma membrane.     

In vivo sulfation of the contact site A glycoprotein of Dictyostelium discoideum

During the development of Dictyostelium discoideum from the growth phase to the aggregation stage, a glycoprotein with an apparent mol. wt. of 80 kd is known to be expressed on the cell surface. This glycoprotein, referred to as contact site A, has been implicated in the formation of species-specific, EDTA-stable contacts of aggregating cells. When developing cells were labeled in vivo with [³⁵S]sulfate, the 80-kd glycoprotein was found to be the most prominently sulfated protein. Another strongly sulfated protein had an apparent mol. wt. of 130 kd and was, like the 80-kd glycoprotein, developmentally regulated and associated with the particulate fraction of the cells. The [³⁵S]sulfate incorporated into the 80-kd and 130-kd proteins was not present as tyrosine-O-sulfate, a modified amino acid found in many proteins of mammalian cells. D. discoideum cells incubated with [³⁵S]sulfate in the presence of tunicamycin, an inhibitor of N-glycosylation, produced a 66-kd protein that reacted with monoclonal antibodies raised against the 80-kd glycoprotein, but no longer contained [³⁵S]sulfate. These results suggest that sulfation of the 80-kd glycoprotein occurred on carbohydrate residues. The possible importance of sulfation for a role of the 80-kd glycoprotein in cell adhesion is discussed.     

Dysferlin in membrane trafficking and patch repair

The muscular dystrophies are a heterogeneous group of inherited disorders, defined by progressive muscle weakness and atrophy. Following the discovery of dystrophin, remarkable progress has been made in defining the molecular properties of proteins involved in the various dystrophies. This has underlined the importance of the dystrophin-associated protein complex as a cell membrane scaffold, providing structural stability to muscle cells (McNeil PL, Khakee R. Disruptions of muscle fiber plasma membranes. Role in exercise-induced damage. Am J Pathol 1992;140:1097-1109). While the dystrophies linked to loss of function of dystrophin and its associated proteins are caused by diminished membrane integrity, it is now believed that a new class of dystrophies arises because of a diminished capacity for rapid muscle membrane repair after injury. Dysferlin is the first identified member of a putative muscle-specific repair complex that permits rapid resealing of membranes disrupted by mechanical stress. Membrane resealing is a function conserved by most cells and is mediated by a mechanism closely resembling regulated, Ca2+-dependent exocytosis. A primary role for dysferlin in this pathway, as a Ca2+-regulated fusogen, has been suggested, and a number of candidate partner proteins have been identified. This review outlines the current understanding of the role of dysferlin in membrane repair and the evolving picture of dysferlin-related signaling pathways in muscle cell physiology and pathology.     

Mass spectrometric identification of dystrophin isoform Dp427 by on-membrane digestion of sarcolemma from skeletal muscle

Although the membrane cytoskeletal protein dystrophin of 427 kDa and its tightly associated glycoprotein complex are drastically affected in muscular dystrophy, recent large-scale proteomic investigations did not identify full-length dystrophin in muscle preparations and were unable to determine its molecular fate in dystrophinopathy. Because conventional two-dimensional gel electrophoresis underrepresents many low-abundance and membrane-associated protein species and in-gel trypsination is often hampered by an inefficient digestion of certain target proteins, here we have applied direct on-membrane digestion of one-dimensional blots of the sarcolemma-enriched fraction and the isolated dystrophin-glycoprotein complex. This method succeeded in the mass spectrometric identification of dystrophin isoform Dp427 and associated glycoproteins as well as sarcolemmal dysferlin. In addition, protein bands representing established signature molecules of cross-contaminating membrane systems, such as the voltage-sensing dihydropyridine receptor of transverse tubules, the ryanodine receptor Ca²⁺-release channel of triad junctions, and the Ca²⁺-ATPase of the sarcoplasmic reticulum, were identified by mass spectrometry. Thus, proteomic approaches using on-membrane digestion might be suitable for future studies of low-abundance proteins, integral proteins, peripheral membrane proteins, and high-molecular-mass proteins. On-membrane digestion has the potential to develop into the method of choice for studying these classes of proteins, whose presence is otherwise missed by conventional gel electrophoresis-based proteomics.     

Embryonic expression patterns of the Drosophila dystrophin-associated glycoprotein complex orthologs

Mutations in genes encoding proteins of the human dystrophin-associated glycoprotein complex (DGC) cause the Duchenne, Becker and limb-girdle muscular dystrophies. Subsets of the DGC proteins form tissue-specific complexes which are thought to play structural and signaling roles in the muscle and at the neuromuscular junction. Furthermore, mutations in the dystrophin gene that lead to Duchenne muscular dystrophy are frequently associated with cognitive and behavioral deficits, suggesting a role for dystrophin in the nervous system. Despite significant progress over the past decade, many fundamental questions about the roles played by dystrophin and the other DGC proteins in the muscle and peripheral and central nervous systems remain to be answered. Mammalian models of DGC gene function are complicated by the existence of fully or partially redundant genes whose functions can mask effects of the inactivation of a given DGC gene. The genome of the fruitfly Drosophila melanogaster encodes a single ortholog of the majority of the mammalian DGC protein subclasses, thus potentially simplifying their functional analysis. We report here the embryonic mRNA expression patterns of the individual DGC orthologs. We find that they are predominantly expressed in the nervous system and in muscle. Dystrophin, dystrobrevin-like, dystroglycan-like, syntrophin-like 1, and all three sarcoglycan orthologs are found in the brain and the ventral nerve cord, while dystrophin, dystrobrevin-like, dystroglycan-like, syntrophin-like 2, sarcoglycan alpha and sarcoglycan delta are expressed in distinct and sometimes overlapping domains of mesoderm-derived tissues, i.e. muscles of the body wall and around the gut.     

Changes in macrophage membrane proteins in relation to protein deficiency in rats

Resident and thioglycollate (TG) macrophages were isolated from rats fed 20 and 4% protein diets. These cells were cultured for 2 and 18 hr; further the membrane proteins were separated by SDSPAGE. Though basically all the samples had a similar banding pattern, the protein profile was very complex in nature. TG elicited macrophages from the protein fed group had increased contents of high molecular weight proteins compared to the resident cells from both the groups as well as TG cells from the protein deficient group. The difference increased with prolonged incubation. Polypeptides at 56.5 and 46 kd which were prominently present in the control samples, was very low in the protein deficient ones. Bands at 34, 32, 27 to 19.5 kd were reduced in the TG cells from the protein fed group. The protein deficient samples exhibited a strong band at 43 kd (presumably actin) while it was present in very small amounts in the cells from the protein fed groups. These observations indicate that the protein restriction had down regulated TG induced modulation of macrophage membrane proteins to the level of resident cells which are physiologically in a lower state of activation. These changes could reflect on their reduced functional properties.