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Biomedical subjects

K Olden

Publications and source records attributed to K Olden.

At least 91 records · Page 5Linked to original sources
PubMed1982

Carbohydrates selectively protect a specific domain of fibronectin against proteases.

To determine how the carbohydrate moiety of fibronectin influences the susceptibility of protein to proteolytic degradation, we compared the effects of various proteases on glycosylated and nonglycosylated fibronectins. Nonglycosylated fibronectin, from tunicamycin-treated chicken embryo fibroblasts, was degraded more rapidly to acid-soluble products than glycosylated fibronectin by pronase, thermolysin, trypsin, and chymotrypsin. The absence of carbohydrate did not markedly affect overall patterns of proteolytic fragments identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Except for the expected increases in electrophoretic mobilities of the nonglycosylated peptides, the only important difference was that of the nonglycosylated fragment corresponding to the carbohydrate-rich, collagen-binding domain, was completely digested by the proteases in 60 min at 30 degrees C. In contrast, the comparable fragment from glycosylated fibronectin was resistant to protease digestion. Heparin-binding domains that normally lack carbohydrate are equally susceptible to proteases in glycosylated and nonglycosylated fibronectin. We conclude that the carbohydrate component of fibronectin plays an important role in the stabilization of a specific domain of the protein against proteolytic degradation; however, the carbohydrate does not alter overall proteolytic specificity.

Animals↗
PubMed1981

Inhibition of fusion of embryonic muscle cells in culture by tunicamycin is prevented by leupeptin.

The carbohydrate requirement for alignment and fusion of embryonic quail muscle cells has been examined in tissue culture by use of tunicamycin (TM). The mononucleated, spindle-shaped proliferating myoblasts were treated with TM at various times before fusion and differentiation into multinucleated muscle fibers capable of spontaneous contraction. Tm blocked protein glycosylation and expression of glycoproteins on the cell surface, and strongly inhibited fusion when added to cultures of differentiating muscle cells before the fusion "burst," but had no apparent effect on cell alignment. The inhibition of fusion was partially prevented when TM was administered in the presence of protease inhibitors such as leupeptin and pepstatin, but the inhibition of glycosylation was not prevented. Both glycosylation and fusion were completely restored to normal by the removal of the antibiotic from the medium. These studies provide strong support for the idea that myoblast fusion is partially mediated by glycoproteins with asparagine-linked oligosaccharides. However, the requirement for the carbohydrate portion of the glycoprotein appears to be indirect in that it acts to stabilize the protein moiety against proteolytic degradation. Our findings do not rule out the possibility that oligosaccharide units of surface glycolipids have some role in myoblast fusion.

Animals↗
PubMed1980

Biosynthetic processing of the oligosaccharide chains of cellular fibronectin.

We have examined the maturation or processing of the oligosaccharides of cellular fibronectin in cultured chick embryo fibroblasts. Fibronectin was pulse-labeled with [2-3H]mannose of [35S]methionine, and the turnover rates of carbohydrate and polypeptide portions of immunoprecipitated fibronectin were compared. The oligosaccharides on fibronectin were analyzed by gel electrophoresis for alterations in sensitivity to the enzyme endo-beta-N-acetylgluosaminidase H, which specifically cleaves the 'high-mannose' class of asparagine-linked oligosaccharide. Incorporated mannose was removed only at early time points, suggesting that the structure of fibronectin oligosaccharides was altered due to processing. This possibility was confirmed by the analysis of glycopeptides generated by exhaustive pronase digestion. Two major glycopeptide structures were detected; their properties correspond to a 'high-mannose' oligosaccharide precursor and a 'complex' carbohydrate product. The precursor-product relationship of these two forms of oligosaccharide chains was demonstrated by pulse-chase labeling experiments. The precursor glycopeptide had an apparent size (Mr 2100) comparable to (Man)9GlcNAc (Mr 2080), and was sensitive to endo-beta-N-acetylglucosaminidase H; nearly all of the labeled mannose incorporated in a 10 min pulse was released from fibronectin glycopeptides by this enzyme. During a 90 min chase period, the glycopeptides became larger and increasingly resistant to endo-beta-N-acetylglucosaminidase H cleavage. The final 'complex' or processed oligosaccharide structure contained approximately two-thirds less [3H]mannose, was insensitive to endo-beta-N-acetylglucosaminidase H and had an apparent Mr of 2300 as estimated by gel filtration. We conclude that the carbohydrate portion of fibronectin is synthesized as a 'high-mannose' intermediate and is subsequently processed to give the characteristic 'complex' oligosaccharide chains of fibronectin.

Acetylglucosaminidase↗
PubMed1980

Interferon treatment inhibits glycosylation of a viral protein.

Earlier, we reported a 30-200-fold reduction in the yield of infectious vesicular stomatitis virus (VSV) released from L cells treated with 10-30 reference units ml-1 of interferon (IFN); however, in these cultures virus particle production, as measured by VSV particle-associated viral RNA, virus nucleocapsid protein and viral transcriptase, was inhibited less than 10-fold. There was biochemical and morphological evidence of a significant reduction in glycoprotein (G) and membrane protein (M) of VSV particles released from IFN-treated cells. We compare here the effects of tunicamycin (TM) and IFN in L cells. Treatment with TM or IFN reduced the production of infectious VSV particles, decreased the amount of G and M proteins in VSV released from treated cells, and inhibited an early step in the formation of asparagine-linked oligosaccharide chains, the incorporation by membrane preparations from treated cells of N-acetylglucosamine into glycolipids with the properties of dolichol derivatives.

Dolichol Phosphates↗
PubMed1980

Carbohydrate requirement for expression and stability of acetylcholine receptor on the surface of embryonic muscle cells in culture.

We have investigated the significance of protein glycosylation for metabolism of acetylcholine receptors (AcChoR) in primary cultures of embryonic chicken muscle cells. Tunicamycin, a specific inhibitor of the glycosylation of asparagine residues on glycoproteins, decreased AcChoR accumulation and accelerated its degradation. In contrast, there was no evidence that tunicamycin treatment affected AcChoR biosynthesis, intracellular transport, or incorporation into surface membranes. Leupeptin, an inhibitor of intracellular proteases, markedly increased accumulation of AcChoR on the external surface of muscle cells treated with tunicamycin. Our findings indicate that impairment of protein glycosylation prevents accumulation of AcChoR by increasing its susceptibility to degradation by cellular proteases.

Animals↗
PubMed1979

Evidence for role of glycoprotein carbohydrates in membrane transport: specific inhibition by tunicamycin.

Using tunicamycin, we have investigated the role of glycoproteins in membrane transport. Tunicamycin is a glucosamine-containing antibiotic that specifically inhibits dolichol pyrophosphate-mediated glycosylation of asparaginyl residues of glycoproteins. Inhibition of protein glycosylation in chick embryo fibroblasts by tunicamycin or other inhibitors of glycosylation resulted in defective transport of glucose, uridine, and amino acid analogs (alpha-aminoisobutyrate and cycloleucine). The defect in glucose transport is accompanied by decreased glucose metabolism, as determined by rates of CO2 and lactate production. In contrast, tunicamycin treatment did not affect other membrane-associated processes, such as secretion of fibronectin and procollagen, uptake of glucose by passive diffusion, Na+/K+ ATPase and adenylate cyclase activities, or stimulation of adenylate cyclase by prostaglandin and cholera toxin. Two glucose/glycosylation-regulated membrane proteins with apparent subunit molecular weights of 95,000 and 75,000 were induced by tunicamycin treatment. Our results indicate that glycoprotein glycosylation is required for membrane transport.

Amino Acids↗
PubMed1979

Role of carbohydrate in biological function of the adhesive glycoprotein fibronectin.

We have investigated the role of the carbohydrate moiety in the biological activity of fibronectin in vitro by using tunicamycin to inhibit the glycosylation of this glycoprotein. Tunicamycin is a glucosamine-containing antibiotic that specifically inhibits glycosylation of protein asparaginyl residues mediated by dolichol pyrophosphate. Fibronectin synthesized in the presence of 0.5 microgram of tunicamycin per ml was not glycosylated, as determined by amino sugar analysis, lack of incorporation of [14C]glucosamine and [3H]mannose, and concanavalin A binding studies. Nonglycosylated fibronectin that was isolated from chicken embryo fibroblasts and added to transformed cells in vitro was as effective as the glycosylated protein in promoting a more normal fibroblastic phenotype, including cell flattening, elongation of cell processes, and parallel alignment of cells. The nonglycosylated protein was also as effective as the glycosylated species in mediating cell attachment to collagen and spreading on plastic, as well as in agglutination of formalin-fixed sheep erythrocytes. The nonglycosylated protein was twice as sensitive as the glycosylated protein to proteolytic hydrolysis in vitro as had been suggested by previous studies with intact cells [Olden, K., Pratt, R.M. & Yamada, K.M. (1978) Cell 13, 461-473]. We conclude that the carbohydrate moiety of fibronectin is not required for the mediation of a number of biological activities characteristic of this glycoprotein.

Animals↗
PubMed1978

Fibronectins--adhesive glycoproteins of cell surface and blood.

A recently characterised class of adhesive, high molecular weight glycoproteins is present on the surfaces of cells, in connective tissue matrices, and in extracellular fluids. These proteins may have important roles in cellular adhesion, malignant transformation, reticuloendothelial system function, and embryonic differentiation.

Actins↗
PubMed1978

Transformation-sensitive cell surface protein: isolation, characterization, and role in cellular morphology and adhesion.

Cell surface protein is the major cell surface glycoprotein of chick embryo fibroblasts. We have isolated and purified this glycoprotein and find that it is an adhesive protein that increases cell-cell and cell-substratum adhesiveness in a variety cellular adhesion assays. Transformation of chick fibroblasts results in decreased quantities of CSP due primarily to a fivefold reduction in CSP biosynthesis, although increased proteolytic degradation and shedding from the cell surface also contribute. The decreased biosynthesis is apparently due to a fivefold reduction in translatable mRNA for CSP. Reconstitution of isolated purified CSP on 14 transformed cell lines from several species results in reversion to a more normal fibroblastic morphology, adhesiveness, cell surface architecture, microfilament bundle organization, motility, and alignment at confluence. Cell surface protein does not restore growth control. The effects of CSP appear to be due to at least two actions, increased cell-substratum adhesion plus altered cell-cell interactions. Untransformed chick cells treated with affinity-purified antibodies to CSP develop the rounded morphology characteristic of many transformed cells that are deficient in CSP (LETS protein). Cell surface protein is found primarily in fibrillar aggregates on the cell surface. These CSP fibrils are relatively immobile and do not affect the mobility of other cell surface components. However, CSP can be eventually redistributed to caplike structures with anti-CSP. Isolated CSP consists of highly asymmetric disulfide-linked dimers and multimers. The interchain disulfide bridges are confined to a short terminal fragment that is readily removed by trypsin. Cell surface protein and cold-insoluble globulin have similar compositions but differ in solubility and amino termini. Cell surface protein contains primarily asparagine-linked oligosaccharides that appear to be responsible for CSP's concanavalin A receptor activity. Inhibition of CSP's glycosylation by treatment with tunicamycin results in decreased CSP due to marked increases in its degradation rate, without inhibition of synthesis or secretion. Studies of this major cell surface glycoprotein have provided insight into the biochemical mechanisms of cellular adhesion, morphology, and social interaction and provide an approach to analyze the dynamics and regulation of protein synthesis, glycosylation, secretion, and turnover.

Animals↗
PubMed1978

Role of carbohydrates in protein secretion and turnover: effects of tunicamycin on the major cell surface glycoprotein of chick embryo fibroblasts.

Using tunicamycin, we have investigated the role of glycosylation in the biosynthesis, processing and turnover of CSP, the major cell surface glycoprotein of chick embryo fibroblasts (CEF). This antibiotic specifically inhibits glycosylation mediated by dolichol pyrophosphate and consequently inhibits the glycosylation of asparaginyl residues of glycoproteins. Tunicamycin inhibited the incorporation of 3H-mannose into CSP by 92--98% and 14C-glucosamine by 84--96%, whereas total protein synthesis was decreased by only 15--45%. Tunicamycin treatment decreased total amounts of CSP by approximately 50--65%, with equal decreases in CSP occurring on the cell surface and in culture medium, whereas intracellular pools of CSP were not substantially affected. In contrast to CSP, three other membrane-associated proteins of apparent molecular weights 75,000, 95,000 and 150,000 daltons were found in increased amounts. Procollagen secretion was not inhibited by tunicamycin. Both procollagen and CSP secretion into culture medium were also not increased in AD6, a glycosylation-deficient, mutant mouse 3T3 cell line compared to wild-type cells. We examined the mechanism of the decrease in CSP after tunicamycin treatment. The rate of CSP biosynthesis as measured by pulse-labeling with 14C-leucine was not altered. Tunicamycin had only a slight effect on the initial times and rates of CSP appearance on the cell surface; some apparent intracellular redistribution of CSP was detected by immunofluorescence. The major effect of tunicamycin treatment was to accelerate the rate of degradation of CSP 2--3 fold. This increase is sufficient to account for the observed decreases after tunicamycin treatment. Our results suggest that carbohydrates may not be essential for CSP or procollagen synthesis, intracellular processing and secretion, but that carbohydrates may help stabilize CSP against proteolytic degradation.

Anti-Bacterial Agents↗