Identification of an 80 kDa glycoprotein located at sites of close apposition between skeletal muscle cells

An 80 kDa glycoprotein was isolated from adult frog skeletal muscle by concanavalin (Con A) affinity chromatography and electrophoretic separation by molecular mass. Characteristics of the 80 kDa glycoprotein are that it: 1) binds non-covalently to gelatin-agarose or some other protein(s) bound to gelatin-agarose, 2) does not bind wheat germ agglutinin, 3) appears only at 80 kDa in both reducing and non-reducing electrophoretic separations, 4) is present in skeletal muscle but absent in smooth muscle and cardiac muscle, 5) is not collagenase or hyaluronidase-sensitive, and 6) is antigenically similar to a protein in embryonic chicken skeletal muscle. It was used to generate a polyclonal antiserum which was affinity-purified and used for immunolocalization. Indirect immunofluorescence procedures showed the antigen to be present on the surface of the skeletal muscle cells and concentrated at sites where cells are closely apposed to one another. Preparations in which adult muscle cells were depleted of basement membrane and endomysial proteins did not reduce the amount of 80 kDa protein present in skeletal muscle. These data indicate that this is a cell surface glycoprotein that may mediate attachment of the cell to extracellular proteins at sites where adjacent skeletal muscle cells are apposed.     

Specific Association of Growth-associated Protein 43 with Calcium Release Units in Skeletal Muscles of Lower Vertebrates

Growth-associated protein 43 (GAP43), is a strictly conserved protein among vertebrates implicated in neuronal development and neurite branching. Since GAP43 structure contains a calmodulin-binding domain, this protein is able to bind calmodulin and gather it nearby membrane network, thus regulating cytosolic calcium and consequently calcium-dependent intracellular events. Even if for many years GAP43 has been considered a neuronal-specific protein, evidence from different laboratories described its presence in myoblasts, myotubes and adult skeletal muscle fibers. Data from our laboratory showed that GAP43 is localized between calcium release units (CRUs) and mitochondria in mammalian skeletal muscle suggesting that, also in skeletal muscle, this protein can be a key player in calcium/calmodulin homeostasis. However, the previous studies could not clearly distinguish between a mitochondrion- or a triad-related positioning of GAP43. To solve this question, the expression and localization of GAP43 was studied in skeletal muscle of Xenopus and Zebrafish known to have triads located at the level of the Z-lines and mitochondria not closely associated with them. Western blotting and immunostaining experiments revealed the expression of GAP43 also in skeletal muscle of lower vertebrates (like amphibians and fishes), and that the protein is localized closely to the triad junction. Once more, these results and GAP43 structural features, support an involvement of the protein in the dynamic intracellular Ca²⁺ homeostasis, a common conserved role among the different species.Key words: GAP43, RyR, skeletal muscle, Xenopus, Zebrafish     

Identification of a putative collagen-binding protein from chicken skeletal muscle as glycogen phosphorylase.

We have purified and generated antisera to a 95 kDa skeletal muscle protein that constitutes the largest mass fraction of gelatin-agarose binding proteins in skeletal muscle. Preliminary results indicated that this 95 kDa chicken skeletal muscle protein bound strongly to gelatin-agarose and type IV collagen-agarose, suggesting a possible function in muscle cell adhesion to collagen. However, N-terminal sequencing of proteolytic fragments of the 95 kDa protein indicates that it is the chicken skeletal muscle form of glycogen phosphorylase, the binding of which to gelatin-agarose is unlikely to be biologically relevant. Further characterization showed that the skeletal muscle form of glycogen phosphorylase is immunologically distinct from the liver and brain forms in the chicken, and suggests that, unlike mammalian skeletal muscle, chicken skeletal muscle may have two phosphorylase isoforms. Furthermore, immunolocalization data and solubility characteristics of glycogen phosphorylase in muscle extraction experiments suggest the enzyme may interact strongly with an unidentified component of the muscle cytoskeleton. Thus, this study yields a novel purification technique for skeletal muscle glycogen phosphorylase, provides new information on the distribution and isoforms of glycogen phosphorylase, and provides a caveat for using gelatin affinity chromatography as a primary step in purifying collagen-binding proteins from skeletal muscle.     

Structural and functional characterization of human NAD kinase

NADP is essential for biosynthetic pathways, energy, and signal transduction. Its synthesis is catalyzed by NAD kinase. Very little is known about the structure, function, and regulation of this enzyme from multicellular organisms. We identified a human NAD kinase cDNA and the corresponding gene using available database information. A cDNA was amplified from a human fibroblast cDNA library and functionally overexpressed in Escherichia coli. The obtained cDNA, slightly different from that deposited in the database, encodes a protein of 49 kDa. The gene is expressed in most human tissues, but not in skeletal muscle. Human NAD kinase differs considerably from that of prokaryotes by subunit molecular mass (49 kDa vs 30-35 kDa). The catalytically active homotetramer is highly selective for its substrates, NAD and ATP. It did not phosphorylate the nicotinic acid derivative of NAD (NAAD) suggesting that the potent calcium-mobilizing pyridine nucleotide NAADP is synthesized by an alternative route.     

Fusion and differentiation of murine C2C12 skeletal muscle cells that express Trichinella spiralis p43 protein

The ability of a 43 kDa stichocyte protein from Trichinella spiralis (Tsp43) to interfere with mammalian skeletal muscle gene expression was investigated. A MYC-tagged Tsp43 construct was expressed as a recombinant protein in C2C12 myoblasts. Transfection with low amounts of expression plasmid was required for successful expression of the protein. This construct had apparent toxic effects on transfected myoblasts and ectopic green fluorescent protein expression was suppressed in myoblasts co-transfected with the Tsp43 construct. These effects may result from similarities of Tsp43 to DNase II. Use of the general DNase inhibitor aurintricarboxylic acid (ATA) enhanced expression of MYC-Tsp43 in transfected muscle cells. Myoblasts transfected with Tsp43 did not fuse well when cultured under differentiation conditions without ATA. In contrast, transfected myoblasts transiently cultured with ATA underwent fusion and differentiation. Under short-term differentiation conditions without ATA, unfused myoblasts nevertheless expressed both MYC-Tsp43 and myosin heavy chain. Collectively, the results support that Tsp43 has a role in the T. spiralis life cycle that is distinct from repressing muscle gene expression during the muscle phase of infection. While the function of Tsp43 as a DNase is under debate, the effects of ATA on transfected muscle cells were consistent with this possibility.     

Functional properties of acetylcholine receptors coexpressed with the 43K protein in heterologous cell systems.

The nicotinic acetylcholine (ACh) receptor is an integral membrane protein which mediates synaptic transmission at the skeletal neuromuscular junction. A key event in the development of the neuromuscular junction is the formation of high density aggregates of ACh receptors in the postsynaptic membrane. Receptor clustering has been attributed, in part, to their association with a peripheral membrane protein of Mr 43,000 (43K protein). We have addressed whether the association of the 43K protein can alter the single channel properties of the ACh receptor, and thus influence neuromuscular transmission at developing synapses, by expressing ACh receptors with and without the 43K protein in heterologous expression systems. We found that coexpression of the 43K protein with the receptor did not significantly alter either its single channel conductance or its mean channel open time. This was true in oocytes and also in COS cells where it was possible to localize 43K-induced clusters by fluorescence microscopy and to record from those clustered receptors. These data are in agreement with previous single channel studies which have shown that the properties of diffusely distributed and clustered receptors in native muscle cells from both mice and Xenopus do not differ.     

Identification of the mouse muscle 43,000-dalton acetylcholine receptor-associated protein (RAPsyn) by cDNA cloning

The nicotinic acetylcholine receptor and a receptor-associated protein of 43 kDa are the major proteins present in postsynaptic membranes isolated from Torpedo electric organ. Immunochemical analyses indicated that a protein sharing antigenic determinants with the receptor-associated protein is also present at receptor clusters of muscle cell lines and postsynaptic membranes of vertebrate neuromuscular junctions. We now provide definitive proof that a homolog of the 43-kDa protein exists in mammals. Complimentary DNA clones encoding the complete protein sequence have been isolated from the mouse muscle cell line, BC3H1. We heretofore refer to these proteins as nicotinic receptor-associated proteins at synapses or N-RAP-syns. The deduced sequence of mouse RAPsyn has 412 amino acids and a molecular mass of 46,392 daltons. The overall identity with Torpedo RAPsyn is 70%; some regions are extremely well conserved and are therefore postulated to be functionally important. Important domains, including the amino terminus and a cAMP-dependent protein kinase phosphorylation site, are conserved between species. Several structural features are consistent with the proposal that RAPsyn is a peripheral membrane protein that associates with membranes by virtue of covalently bound myristate. Although multiple mRNAs were previously identified in Torpedo electric organ, RNA blot analysis reveals a single polyadenylated RAPsyn mRNA of approximately equal to 2.0 kilobases in newborn and 4-week-old mouse muscle. Finally, genomic DNA blot analysis indicates that a single N-RAPsyn gene is present in the mouse genome.     

Treatments that extract the 43K protein from acetylcholine receptor clusters modify the conformation of cytoplasmic domains of all subunits of the receptor.

The nicotinic acetylcholine receptor (AChR) of Torpedo electric organ and vertebrate skeletal muscle is closely associated with a Mr 43,000 protein (43K). In this study, we have examined the effects on the AChR of treatments which remove the 43K protein. We used semiquantitative fluorescence techniques to measure the binding of antibodies to clustered AChR in cultured rat myotubes. We found that labeling by antibodies to the cytoplasmic portions of each of the four receptor polypeptides increased significantly upon extraction of the 43K protein. Labeling by an antibody to an extracellular epitope of the alpha subunits was not affected by removal of the 43K protein, suggesting that changes were restricted to the cytoplasmic domains of the AChR. Increases in labeling by antibodies were more limited following protease treatment, which removes most cytoskeletal structures but leaves the 43K protein bound to the membrane. Competition between an antibody to the beta subunit and an antibody to the gamma and delta subunits suggests that the cytoplasmic portion of the AChR still retains a degree of native structure in the absence of the 43K protein. Our results suggest that, although some of these changes may be due to simply exposing additional epitopes on the AChR, the cytoplasmic portions of all the subunits of the AChR undergo significant conformational changes upon extraction of the 43K protein.     

Phosphorylase kinase from bovine stomach smooth muscle: a Ca2(+)-dependent protein kinase associated with an actin-like molecule

Phosphorylase kinase was purified (110-fold) from bovine stomach smooth muscle by a procedure involving DEAE-cellulose chromatography, ammonium sulfate fractionation and glycerol density ultracentrifugation. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) the final enzyme preparation shows a single protein band of 43 kDa. The purified protein exhibits a close similarity with bovine aortic actin, as revealed by amino acid analysis and sequencing of a tryptic decapeptide fragment, although it differs widely from actin in several respects. In our effort to separate phosphorylase kinase activity from the 43 kDa protein we used a variety of chromatographic procedures, but in all cases the catalytic activity (when eluted) was accompanied by the 43 kDa protein band. Bovine stomach phosphorylase kinase exhibits an apparent molecular mass of 950 kDa, it shows a low Vmax value for phosphorylase b (85 nmol.min-1.mg-1), a pH 6.8/8.2 activity ratio of 0.23, it has an absolute requirement for Ca2+ and it is activated 1.8-fold by Ca2+/calmodulin. Furthermore, the protein kinase activity is neither inhibited by antibodies against rabbit skeletal muscle phosphorylase kinase nor activated by protein phosphorylation. These results suggest that bovine stomach phosphorylase kinase is tightly bound to an aggregate of actin-like molecules.     

Molecular characterization of 1,4-dihydropyridine-sensitive calcium channels of chick heart and skeletal muscle

A monoclonal antibody designated as MAC-L1 immunoprecipitated [3H]PN200-110-labeled calcium channels of chick cardiac and skeletal muscle. On specific immunoprecipitation of 125I-labeled proteins, two large polypeptides (Mr 197,000 and 139,000 for heart, and 172,000 and 135,000 for skeletal muscle, under reducing conditions) were identified as the major components of these channels. Both polypeptides were found to exist together as a complex in 1% digitonin, but to become separated from each other in 1% Triton X-100. The 197 and 172 kDa peptides of cardiac and skeletal muscles, respectively, were photolabeled with [3H]azidopine. Under nonreducing conditions, the 139 kDa polypeptide of heart and the 135 kDa polypeptide of skeletal muscle took on larger molecular weights of 192,000 and 190,000, respectively. The 139 kDa but not the 197 kDa component of the heart was capable of binding to wheat germ agglutinin-Sepharose. Among the polypeptides specifically precipitated by MAC-L1, a 165 kDa peptide of skeletal muscle was phosphorylated by cAMP-dependent protein kinase. In contrast, a minor 99 kDa polypeptide, but not the major 197 kDa polypeptide, of the heart was phosphorylated by this kinase. These results suggest that the dihydropyridine-sensitive cardiac calcium channel has alpha 1 and alpha 2 subunits that are homologous but not identical to those of the skeletal muscle calcium channel.     

Serum and growth factors regulate expression of a 43 kDa protein in smooth muscle cell cultures

Smooth muscle cells respond to injury and the presence of serum factors by modulating from a quiescent contractile cell to a motile synthetic phenotype. To evaluate the biochemical response to serum exposure, we examined the proteins synthesized and secreted in response to serum. The most prominent effect of serum was the rapid production of a protein with an apparent molecular weight of 43 kDa. Removal of serum from the culture environment led to a cessation of 43 kDa protein production. The effect of exogenous heparin on 43 kDa protein production was also evaluated. Neither the 43 kDa protein nor a previously described 38 kDa protein was induced by heparin. Further, heparin treatment did not counteract the effects of serum. These studies demonstrate that an early response of vascular smooth muscle cells to serum is the production of this previously undescribed protein and that other modifications of the culture conditions did not affect its synthesis.     

A second catalytic subunit of type-2A protein phosphatase from rabbit skeletal muscle

A cDNA clone encoding a second type-2A protein phosphatase catalytic subunit (2A beta) was isolated from a rabbit skeletal muscle cDNA library constructed in lambda gt10. The deduced protein sequence (309 residues, 35.59 kDa) was 97% identical to that of phosphatase 2A alpha (309 residues, 35.58 kDa). At the nucleotide level, the two clones showed only 82% identity in the coding region. The results indicate the presence of at least two isoforms of protein phosphatase 2A in skeletal muscle.     

Kinase activity and protein phosphorylation in control and malignant hyperthermic skeletal muscle

1. Native 6% Laemmli gels were used to resolve 7 protein kinase activity bands in control and malignant hyperthermia (MH)-susceptible porcine and human skeletal muscle extracts. 2. MH-susceptible samples were consistently more active than the controls. 3. Following halothane treatment, a 43 kDa component displayed increased phosphorylation by a calcium-calmodulin dependent kinase in MH-susceptible vs control human samples. 4. Increased phosphorylation of additional endogenous protein components of molecular mass 116 and 60 kDa was observed.     

Cytochrome P-450 omega-hydroxylase: a potential O(2) sensor in rat arterioles and skeletal muscle cells

The purposes of this study were to 1) further evaluate the possible role that vasoconstrictor metabolites of cytochrome P-450 (CYP) omega-hydroxylase plays in O(2)-induced constriction of arterioles in the rat skeletal muscle microcirculation, 2) determine whether omega-hydroxylases are expressed in rat cremaster muscle, and 3) determine whether the enzyme is located in the parenchyma or the arterioles. O(2)-induced constriction of third-order arterioles in the in situ cremaster muscle of Sprague-Dawley rats was significantly inhibited by the CYP inhibitors N-methyl-sulfonyl-12,12-dibromododec-11-enamide (DDMS; 50 microM) and 17-octadecynoic acid (ODYA; 10 microM). Immunoblot analysis with antibody raised against CYP4A protein indicated the presence of immunoreactive proteins in the cremaster muscle and in isolated arterioles and muscle fibers from this tissue. However, the molecular mass of the immunoreactive proteins was 85 kDa instead of the expected 50--52 kDa for CYP4A omega-hydroxylase isolated from rat liver or kidney. Treatment of the cremaster muscle with deglycosidases shifted the bands to the expected range which indicates that these proteins are likely glycosylated in skeletal muscle. Immunohistochemistry revealed intense staining of both muscle fibers and microvessels in the cremaster muscle. The results of this study indicate that O(2) sensing in the skeletal muscle microcirculation may be mediated by CYP4A omega-hydroxylases in both arterioles and parenchymal cells.     

Detection and isolation of a 30 kDa abnormal protein in avian dystrophic muscle

We have studied the protein composition of the pectoralis superficialis muscle of genetically dystrophic (New Hampshire line 413) and normal control (line 412) chickens by one- and two-dimensional gel electrophoresis. A protein, referred to hereafter as the 30 kDa abnormal protein, was specifically detected in the affected muscle. It was purified to homogeneity, and its molecular properties were studied. It is a monomer with a molecular mass of approximately 30 kDa and an isoelectric point of about pI 8.4. We have screened by Western blotting a variety of muscles from line 412 and line 413 chickens for the presence of the 30 kDa protein. While the pattern of total protein is very similar in all cases, the 30 kDa protein was not detected in the pectoralis superficialis muscle of line 412 chickens. However, the immunoreactive bands were detected in the sartorius muscle and the tensor fasciae latae muscle from dystrophic and normal chickens. Interestingly, the immunoreactive bands of normal skeletal muscles are smaller in molecular weight than those of dystrophic skeletal muscles. To determine the early time sequence of the appearance of the abnormal protein, we studied muscles from embryos and post-hatched chickens at various ages. The abnormal protein was detected in dystrophic muscles as early as 15 days ex ovo and occurred throughout development up to six months ex ovo. Although the implication of the dystrophy-associated appearance of the 30 kDa protein in the affected muscle is not clear at present, it would be of particular interest to elucidate the biochemical functions of the 30 kDa protein in the affected muscle (pectoralis superficialis muscle) of genetically dystrophic chicken.     

A segment corresponding to amino acids Gln199-Lys208 of murine IL-1α cross-reacts with an antigenic determinant localized in the Z-line of Drosophila melanogaster myofibrils

Immunofluorescence microscopic observations indicated that a monoclonal antibody, Vmp 18, raised against the peptide 199–208 of murine interleukin 1α, cross-reacted with an antigenic determinant of Drosophila thorax muscles. Immunoelectron microscopic analysis showed that the gold particles were mainly localized in the Z-line which is the attachment site of thin filaments from adjacent sarcomeres. On the contrary, the antibody failed to mark the Z-line in vertebrate skeletal muscle. A Western blot of total protein extract from Drosophila thorax muscles bound a protein of 43 kDa. Our observations suggest that the Vmp 18 antibody could contribute to clarify the composition of the Z-line in insect's flight muscles.     

Sequence analysis of the ryanodine receptor: possible association with a 12K, FK506-binding immunophilin/protein kinase C inhibitor

A computer-assisted sequence analysis of the ryanodine receptor pointed to a 15-residue peptide, "KC7", reported to have been purified from a proteolytic digest of the 565 kDa rabbit skeletal muscle protein. Sequence comparisons, however, showed that this peptide probably originated from a much smaller protein which copurified with the ryanodine receptor. Peptide KC7 (excluding its unknown N-terminal residue) was identical to the N-terminus of a 12 kDa immunophilin (immunosupressant-binding protein), human T-cell FK506- binding protein (FKBP), which has recently been identified as an inhibitor of protein kinase C. There was no other sequence similarity between FKBP and the ryanodine receptor. It is suggested that in vivo interaction of the ryanodine receptor and FKBP may play a role in the modulation of calcium release in muscle.     

Immunoaffinity isolation of Na+ channels from rat skeletal muscle. Analysis of subunits

Polyclonal antiserum and monoclonal antibodies raised against the sodium channel from rat skeletal muscle sarcolemma have been immobilized on Sepharose and used to immunoaffinity purify this channel directly from skeletal muscle without the intervening purification of surface membranes. These antibodies isolate a approximately 260-kDa protein from whole muscle, although each purifies predominantly a 150-kDa component when isolated sarcolemmal membranes are used as starting material. A 45-kDa band is also found in the material purified from sarcolemma but not that obtained from whole muscle. In addition, these immunoaffinity columns isolate a 38-kDa band from both whole muscle and sarcolemma that copurifies with the 260-kDa protein. In some preparations this component appears as two closely spaced bands of 37 and 39 kDa. These small subunits coelute with the 260-kDa subunit when thiocyanate gradients are used to displace protein bound to the immunoaffinity columns and behave as integral components of the sodium channel. Estimates of stoichiometry were made for the large and small subunits of the muscle channel protein. After correction for labeling efficiency, values consistent with a ratio of one 260-kDa subunit to one 38-kDa subunit were obtained. We conclude that the rat skeletal muscle sodium channel contains a large alpha subunit of approximately 260 kDa that is sensitive to proteolytic nicking during the isolation of sarcolemmal membranes. In addition, at least one 38-kDa beta subunit is associated with each alpha subunit in the native channel.     

A cofilin-like protein is involved in the regulation of actin assembly in developing skeletal muscle

An actin-binding protein of 20 kDa (called 20K protein) was purified from the sarcoplasmic fraction of embryonic chicken skeletal muscle. The properties of this protein were very similar to cofilin, which was discovered in porcine brain (Nishida et al. (1984) Biochemistry, 23, 5307-5313): it bound to both G- and F-actin, inhibited actin polymerization in a pH-dependent manner, inhibited binding of tropomyosin to F-actin, and had almost the same molecular size and pI as cofilin. A specific monoclonal antibody to 20K protein (MAB-22) was prepared to examine the expression and location of 20K protein during skeletal muscle development. When the whole protein lysates of embryonic and post-hatched chicken skeletal muscles were examined by means of immunoblotting combined with SDS-PAGE, 20K protein was detected in skeletal muscle through the developmental stages. Location of 20K protein in the cells differed between the embryonic and adult tissues; immunofluorescence staining of the cryosections of embryonic muscle with MAB-22 visualized irregular dot-like structures, but adult muscle sections were stained faintly and uniformly. 20K protein was present as a complex with actin in embryonic muscle, as judged by the ability to bind to a DNase I affinity column, while the same protein was free from actin in the cytoplasm of adult muscle. From these results, it is suggested that 20K protein regulates actin assembly transiently in developing skeletal muscle.