Bishnu D. Sanwal

Cloning of a human collagen-binding protein, and its homology with rat gp46, chick hsp47 and mouse J6 proteins

Several cDNA clones encoding a collagen-binding protein were isolated from human fibroblasts. The cDNA encoded a 417 amino acid protein, containing two potential N-linked oligosaccharide binding sites and a C-terminal RDEL sequence, which has been shown to act as an endoplasmic retention signal in other systems. The derived amino acid sequence of the protein shows close homology with gp46 from rat skeletal myoblasts, J6 protein from mouse F9 embryonal carcinoma cells and hsp47 from chick embryo fibroblasts. It also shows sequence similarity with members of the serpin family.

Developmental changes in gangliosides during myogenesis of a rat myoblast cell line and its drug resistant variants.

Abstract Cloned cells of a myoblast line show the presence of GM3, GM2, GM1 and GD1a gangliosides. The amount of GM3, GM2 and GM1 gangliosides does not vary significantly during the differentiation of myoblasts to myotubes. However, the concentration of GD1a transiently increases almost 3-fold just prior to the fusion of myoblasts and returns to the basal levels in the myotubes. Mutant myoblasts selected for 5-azacytidine resistance and unable to fuse produce only GM3 and traces of GM2. We conclude that GD1a probably participates in the fusion process through yet unknown mechanism.

Inhibition of myoblast fusion by tunicamycin and pantomycin.

Abstract The formation of myotubes by a continuous rat myoblast line, L6, can be inhibited by non-toxic concentrations of tunicamycin and pantomycin. The effect of tunicamycin, an inhibitor of UDP-N-acetyl-glucosamine: dolichol phosphate N-acetylglucosaminyltransferase, could be reversed by N-acetylglucosamine but not by mannose, glucose or UDP-N-acetylglucosamine.

A collagen-binding protein involved in the differentiation of myoblasts recognizes the Arg―Gly―Asp sequence

We had earlier demonstrated that a 46-kDa glycoprotein is involved in the differentiation of rat skeletal myoblasts. We now show that the binding of this glycoprotein to collagen and gelatin is disrupted by Arg-Gly-Asp (RGD) containing peptide but not by Arg-Gly-Glu (RGE). The former peptide also selectively elutes the 46-kDa glycoprotein bound to gelatin-Sepharose. Since all other proteins which bind RGD sequences have been found at the cell surface, we attempted to localize the 46-kDa glycoprotein by means of immuno fluorescent staining and radioiodine labeling. Surprisingly, the majority of the protein was found to be localized in the endoplasmic reticulum. Protease treatment of a microsomal fraction revealed that the protein is in the interior of the reticulum. Immunoprecipitation experiments, using a polyclonal antibody against the 46-kDa protein, demonstrated that no closely related proteins exist in myoblasts and also confirmed that the protein was not a fragment of a cell-surface localized protein. These findings suggest that the RGD sequence is also used in protein recognition within the cell.

Partial characterization of a collagen-binding, differentiation-related glycoprotein from skeletal myoblasts

A 46-kDa glycoprotein, gp46, which binds collagen has been purified to homogeneity from L6 rat skeletal myoblasts. The procedure involves extraction of crude myoblast membranes with 1% sodium dodecyl sulfate followed by concanavalin A affinity chromatography and preparative gel electrophoresis. The sequence of 15 N-terminal amino acids had some resemblance to a sequence in myosin light chains. The oligosaccharide chains of the glycoprotein can be released by treatment with endoglycosidase H, suggesting that gp46 has high-mannose type of glycans. Galactose and sialic acid are not detected in the purified protein. gp46 is widely distributed and conserved in different cell lines as determined by immunoblotting using a monoclonal anti-gp46 antibody. High levels of gp46 were found in several fibroblastic and myogenic cell lines, but not in a hematopoietic cell line. Undifferentiated F9 embryonal carcinoma cells lacked gp46 but the glycoprotein was induced when the cells were made to differentiate in the presence of retinoic acid. Broad survey of gp46 in different cell lines also suggests that it is present mainly in those cell lines which attach to the substratum and produce collagens. Although the function of gp46 is not yet known, the evidence suggests that it is developmentally regulated and is probably involved in the synthesis or assembly of collagen in the endoplasmic reticulum.

Apparent dominance of serine auxotrophy and the absence of expression of muscle-specific proteins in rat myoblast × mouse L-cell hybrids

Hybrids obtained from crosses of rat myoblasts and mouse L-cells are unable to differentiate into myotubes. The synthesis of muscle-specific proteins, creatine phosphokinase (CPK), myosin, and adenyl cyclase is also suppressed in the hybrids. Adenyl cyclase produced in the hybrids is not activated by isoproterenol. The glycine auxotrophy of myoblasts is phenotypically expressed as a dominant trait in hybrids. Genotypically, however, all the enzymes of the phosphorylated pathway of serine biosynthesis and serine hydroxymethyltransferase, the enzyme inter converting serine and glycine, are present in normal concentrations in the hybrids, just as they are in the mouse L-cells. Unlike either of the parental lines, the hybrid cells are unable to grow on serine-supplemented medium, but do so when glycine is also added. Glycine by itself supports growth of the hybrids in the absence of added serine. All the curious effects of glycine/serine on growth of hybrids are rationalized and accounted for by the hypothesis that in these cells the enzyme serine hydroxy-methyltransferase, for unknown reasons, does not function in vivo.

Membrane bound substrate recognition components of the dicarboxylate transport system in Escherichia coli

Summary Membranes isolated from Escherichia coli and solubilized with Lubrol 17A-10 contain two proteins, designated SBP 1 and SBP 2 which can be separated and purified on aspartate-coupled Sepharose 4B. Both of the proteins bind succinate, malate and fumarate but not aspartate, malonate, tartarate and acetate. The dissociation constants of SBP 1 and SBP 2 for succinate are 23 μM and 2.3 μM, respectively. The binding of succinate to these components is inhibited by sulfhydryl reagents. A mutant of Escherichia coli ( dct A ) which is unable to transport succinate and other dicarboxylic acids has very small amounts of the membrane bound SBP 2 protein. Genetic and biochemical evidence strongly suggests that SBP 1 and SBP 2 cooperate in some unknown way in the transport of succinate in vivo .

The noncoordinate expression of muscle-specific proteins in mutant rat skeletal myoblasts and reinitiation of differentiation in hybrids

In rat skeletal myoblasts which are resistant to 5-azacytidine and fusion-incompetent, the muscle-specific protein, creatine phosphokinase, is produced but muscle-specific myosin is not. In human diploid fibroblast X rat myoblast hybrids, myoblast-specific properties are extinguished. Clones can be selected from the hybrids after a number of doublings which reexpress fusion but do not produce creatine phosphokinase. The conclusion is drawn that the expression of muscle-specific phenotype is not coordinate and fusion of myoblasts is not an essential requirement for the appearance of myoblastspecific proteins.

Colligin, A Collagen Binding Serpin

Colligin is a major glycoprotein in many cells and has been discovered several times by different approaches and hence given different names. Kurkinen et al. (1984) first found it as a collagen-binding protein in parietal endoderm cells and proposed the name‘colligin’ but it was later detected as a differentiation related protein in myoblasts (‘gp46’) (Cates et al., 1984) and as a heat shock responsive protein in chick fibroblasts (HSP47) (Nagata & Yamada, 1986). Sequencing later confirmed the identity of the various proteins. Each of the routes of discovery delivered clues about the function of the protein, leading eventually to the current idea of a role as a molecular chaperone in collagen synthesis.

Glycoprotein glycans may not be necessary for the differentiation of skeletal myoblasts

Previous work using glycosylation inhibitors has suggested that high-mannose type but not complex type oligosaccharides on the surface of cells may play a role in the differentiation of skeletal myoblasts. Earlier, we had shown that a concanavalin A-resistant mutant derived from an L6 myoblast line fails to differentiate in a medium containing 10% horse serum. Here we show that one such concanavalin A-resistant mutant (D-1) which was reported to have oligosaccharides of the type Man(3-5)G1cNAc2, shows significant fusion ability when grown in media containing 1% horse serum. Lowering the serum concentration did not alter the dolichol-phosphate mannosyltransferase activity in D-1 which remained at low levels compared to L6. The incorporation of [3H]mannose in D-1 was found to be 60% of L6 in 10% serum whereas in 1% serum the incorporation into D-1 was further reduced to 30% of L6. [3H]mannose-labeled ConA-binding proteins isolated from L6 were quantitatively and qualitatively similar in cells grown in either 10 or 1% serum. However, in D-1 cells a further decrease in the ConA-binding ability of these glycoproteins was observed. Biochemical differentiation also occurs in D-1 upon fusion in 1% serum as seen by the increase in mRNA levels of the muscle-specific markers myosin light chain and troponin T. These results suggest the high-mannose type of oligosaccharides may not be involved in myoblast differentiation.