scholarly journals Impaired secretion of mutant alpha 2-plasmin inhibitor (alpha 2 PI- Nara) from COS-7 and HepG2 cells: molecular and cellular basis for hereditary deficiency of alpha 2-plasmin inhibitor

Blood ◽  
1990 ◽  
Vol 75 (5) ◽  
pp. 1092-1096
Author(s):  
O Miura ◽  
N Aoki
Keyword(s):  
Cell Line ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Hepatoma Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Secretory Process ◽  
Hepatoma Cell Line ◽  
Pi Deficiency ◽  
Plasmin Inhibitor

The elongated mutant of alpha 2-plasmin inhibitor (alpha 2 PI) designated as alpha 2 PI-Nara is caused by a frameshift mutation found near the 3′ end of the coding region of the alpha 2 PI gene. To elucidate the mechanism by which this molecular abnormality leads to alpha 2 PI deficiency in plasma, we transfected an expression plasmid for alpha 2 PI-Nara into a monkey kidney cell line COS-7 or human hepatoma cell line HepG2 synthesizing alpha 2 PI, and analyzed the secretory process of the expressed alpha 2 PI-Nara by radioimmunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography. The results obtained showed that the recombinant alpha 2 PI-Nara was retained within the cells for prolonged periods as an endoglycosidase H- sensitive precursor form, and only a small portion of the recombinant protein was secreted into the medium as a neuraminidase-sensitive mature form. These results suggest that instead of being secreted from the cells, most of the alpha 2 PI-Nara undergoes degradation within the cells while its transport is retarded in the intracellular secretory pathway; thus, alpha 2 PI-Nara should lead to the alpha 2 PI deficiency primarily by causing a block in the intracellular transport from the endoplasmic reticulum to the Golgi complex.

scholarly journals Impaired secretion of mutant alpha 2-plasmin inhibitor (alpha 2 PI- Nara) from COS-7 and HepG2 cells: molecular and cellular basis for hereditary deficiency of alpha 2-plasmin inhibitor

Blood ◽  
1990 ◽  
Vol 75 (5) ◽  
pp. 1092-1096 ◽  
Author(s):  
O Miura ◽  
N Aoki
Keyword(s):  
Cell Line ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Hepatoma Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Secretory Process ◽  
Hepatoma Cell Line ◽  
Pi Deficiency ◽  
Plasmin Inhibitor

Abstract The elongated mutant of alpha 2-plasmin inhibitor (alpha 2 PI) designated as alpha 2 PI-Nara is caused by a frameshift mutation found near the 3′ end of the coding region of the alpha 2 PI gene. To elucidate the mechanism by which this molecular abnormality leads to alpha 2 PI deficiency in plasma, we transfected an expression plasmid for alpha 2 PI-Nara into a monkey kidney cell line COS-7 or human hepatoma cell line HepG2 synthesizing alpha 2 PI, and analyzed the secretory process of the expressed alpha 2 PI-Nara by radioimmunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography. The results obtained showed that the recombinant alpha 2 PI-Nara was retained within the cells for prolonged periods as an endoglycosidase H- sensitive precursor form, and only a small portion of the recombinant protein was secreted into the medium as a neuraminidase-sensitive mature form. These results suggest that instead of being secreted from the cells, most of the alpha 2 PI-Nara undergoes degradation within the cells while its transport is retarded in the intracellular secretory pathway; thus, alpha 2 PI-Nara should lead to the alpha 2 PI deficiency primarily by causing a block in the intracellular transport from the endoplasmic reticulum to the Golgi complex.

scholarly journals The route of secretion of procollagen. The influence of αα′-bipyridyl, colchicine and antimycin A on the secretory process in embryonic-chick tendon and cartilage cells

1976 ◽  
Vol 156 (1) ◽  
pp. 81-90 ◽  
Author(s):  
R Harwood ◽  
M E Grant ◽  
D S Jackson
Keyword(s):  
Intracellular Transport ◽  
Secretory Pathway ◽  
Smooth Endoplasmic Reticulum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Extracellular Proteins ◽  
Secretory Process ◽  
Antimycin A ◽  
Embryonic Chick ◽  
Extracellular Medium ◽  
Cartilage Cells

I. Embryonic-chick tendon cells were pulse-labelled for 4 min with [14C]proline and the 14C-labelled polypeptides were chased with unlabelled proline for up to 30 min. Isolation of subcellular fractions during the chase period and their subsequent analysis for bacterial collagenase-susceptible 14C-labelled peptides demonstrated the transfer of procollagen polypeptides from rough to smooth microsomal fractions and thence to the extracellular medium. Parallel analyses of Golgi-enriched fractions indicated the involvement of this organelle in the secretory pathway of procollagen. Sodium dodecylsulphate/polyacrylamide-gel electrophoresis of the 14C-labelled polypeptides present in the Golgi-enriched fractions demonstrated that the procollagen polypeptides were all present as disulphide-linked pro-gamma components. 2. When similar kinetic studies of the intracellular transport of procollagen were conducted with embryonic-chick cartilage cells almost identical results were obtained, but the rate of translocation of cartilage procollagen was significantly slower than that observed for tendon procollagen. 3. When hydroxylation of procollagen polypeptides was inhibited by alphaalpha′-bipyridyl, the nascent polypeptides accumulated in the rough microsomal fraction. 4. When cells were pulse-labelled for 4min with [14C)proline and the label was chased in the presence of colchicine, secretion of procollagen was inhibited and an intracellular accumulation of procollagen 14C-labelled polypeptides was observed in the Golgi-enriched fractions. 5. The energy-dependence of the intracellular transport of procollagen was demonstrated in experiments in which antimycin A was found to inhibit the transfer of procollagen polypeptides from rough to smooth endoplasmic reticulum. 6. It is concluded that procollagen follows the classical route of secretion taken by other extracellular proteins.

scholarly journals Effect of warfarin on prothrombin synthesis and secretion in human Hep G2 cells

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 773-778 ◽  
Author(s):  
S Karpatkin ◽  
TH Finlay ◽  
AL Ballesteros ◽  
M Karpatkin
Keyword(s):  
Hepatoma Cell ◽  
Carbohydrate Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hepatoma Cell Line ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Human Hepatoma Cell ◽  
Sds Page ◽  
The Media

Abstract Prothrombin synthesis and secretion were studied in a human hepatoma cell line (Hep G2) incubated with 35S-methionine for 2 to 24 hours at 37 degrees C. Extracellular and intracellular prothrombin were detected by immunoprecipitation with affinity-purified antiprothrombin antibody. Incorporation of 35S-methionine into prothrombin was monitored by counting specific bands excised from 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). Prothrombin represented 0.3% to 0.7% of total newly synthesized protein secreted into the media. Warfarin had no effect on total prothrombin synthesis (extracellular plus intracellular). However, warfarin inhibited secretion of newly synthesized prothrombin by 58% to 73% over a 2 to 4 hour period. This was accompanied by the intracellular accumulation of an immunoprecipitable species of prothrombin of 78 kd, 6 kd less than extracellular prothrombin. At the end of the 4-hour incubation with warfarin, intracellular prothrombin increased from 44% to 82% (twofold) of total prothrombin, whereas extracellular prothrombin decreased from 56% to 19% (threefold) of total prothrombin. After 24-hour incubation with warfarin, intracellular and extracellular immunoprecipitable prothrombin approached control values. Deglycosylation of extracellular and intracellular prothrombin with hydrofluoric acid (HF) resulted in a decrease in mol wt for both species to 66 kd. Endoglycosidase-H treatment, which digests “early mannosyl” residues, resulted in a decrease in the mol wt of the intracellular species of 8 kd with no effect on the extracellular species. Thus, the lower mol wt intracellular species that accumulates following early warfarin treatment is due to the presence of incompletely processed carbohydrate chain. The data are compatible with the hypothesis that optimum glycosylation and secretion require Vitamin K-dependent carboxylation.

scholarly journals Effect of lysosomotropic amines on the secretory pathway and on the recycling of the asialoglycoprotein receptor in human hepatoma cells.

1985 ◽  
Vol 101 (2) ◽  
pp. 531-539 ◽  
Author(s):  
G J Strous ◽  
A Du Maine ◽  
J E Zijderhand-Bleekemolen ◽  
J W Slot ◽  
A L Schwartz
Keyword(s):  
Golgi Complex ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Human Hepatoma ◽  
Receptor Recycling ◽  
Human Hepatoma Cells ◽  
Human Hepatoma Cell ◽  
Response Characteristics

We studied the intracellular transport of secretory and membrane proteins in the human hepatoma cell line HepG-2 infected with vesicular stomatitis virus. Cells were pulse-labeled in the presence of [35S]methionine and chased in the presence of the lysosomotropic agent primaquine. At a concentration of 0.3 mM primaquine effectively inhibited the secretion of albumin and, to a lesser extent, that of orosomucoid and transferrin. The drug also prevented the budding of virus particles at the cell surface. The intracellular transport to the Golgi complex of the membrane protein VSV-G was not affected by primaquine as it acquires resistance to endo-beta-N-acetylglucosaminidase H at the same rate as in control cells. Addition of primaquine at various times after the initiation of the chase period indicates that the effect of primaquine occurs just before secretion. In confirmation of the biochemical data, immunocytochemical localization of albumin in cells treated with NH4Cl demonstrated that albumin accumulated in vesicles at the trans side of the Golgi complex. The effect of primaquine on secretion was also compared with its effect on receptor recycling. The dose-response characteristics of the effect of primaquine on receptor recycling are identical to those of the effects on protein secretion and virus budding. These results indicate that both processes involve the same transport mechanism, and/or that they occur via at least one identical intracellular compartment.

scholarly journals Effects of cyclosporin A on erythropoietin production by the human Hep3B hepatoma cell line

Blood ◽  
1993 ◽  
Vol 82 (3) ◽  
pp. 978-984
Author(s):  
AM Vannucchi ◽  
A Grossi ◽  
A Bosi ◽  
D Rafanelli ◽  
M Statello ◽  
...  
Keyword(s):  
Cyclosporin A ◽  
Cell Line ◽  
Secretory Pathway ◽  
Hepatoma Cell ◽  
Allogeneic Bone Marrow Transplantation ◽  
Inhibitory Effect ◽  
Hepatoma Cell Line ◽  
Allogeneic Bone ◽  
Hep3b Cells

There is evidence that the inadequate erythropoietin (Epo) production observed in patients undergoing allogeneic bone marrow transplantation (BMT) might be ascribed to an inhibitory effect caused by the immunosuppressive drug cyclosporin A (CsA). In this in vitro study, we have evaluated the effects of CsA on the release of Epo in the culture medium by the human Hep3B hepatoma cell line. In cultures incubated with both CsA and the nonimmunosuppressive CsA analog MeAla-6, but not with the CsA-unrelated immunosuppressive agent FK-506, the levels of Epo in the medium were significantly reduced in comparison with controls, at concentrations (0.01 to 1.6 mumol/L) not affecting total protein synthetic rate nor the constitutive secretion of alpha- fetoprotein. Hep3B cells were found to contain a CsA-binding molecule, with an M(r) of 18 Kd, as assessed by high performance liquid chromatography (HPLC) and ligand-blotting analysis. CsA did not affect the expression of the Epo gene, as judged by Northern blot analysis, but caused a significant amount of Epo to remain unsecreted within the cells; almost all (97% of total) of the intracellular Epo was associated with the plasma membrane subcellular fraction. We conclude that: (1) CsA is able to inhibit Epo release in vitro by Hep3B cells, further supporting the hypothesis that the drug might have a role in the inappropriately low Epo levels observed in BMT patients; (2) the inhibitory effect appears to be specific and not caused by a general impairment of protein synthesis and/or secretion; and (3) the reduced Epo levels found in the medium of CsA-treated Hep3B cultures are supposed to be the consequence of an inability of the cells to correctly process Epo molecules for the secretory pathway.

scholarly journals Effects of cyclosporin A on erythropoietin production by the human Hep3B hepatoma cell line

Blood ◽  
1993 ◽  
Vol 82 (3) ◽  
pp. 978-984 ◽  
Author(s):  
AM Vannucchi ◽  
A Grossi ◽  
A Bosi ◽  
D Rafanelli ◽  
M Statello ◽  
...  
Keyword(s):  
Cyclosporin A ◽  
Cell Line ◽  
Secretory Pathway ◽  
Hepatoma Cell ◽  
Allogeneic Bone Marrow Transplantation ◽  
Inhibitory Effect ◽  
Hepatoma Cell Line ◽  
Allogeneic Bone ◽  
Hep3b Cells

Abstract There is evidence that the inadequate erythropoietin (Epo) production observed in patients undergoing allogeneic bone marrow transplantation (BMT) might be ascribed to an inhibitory effect caused by the immunosuppressive drug cyclosporin A (CsA). In this in vitro study, we have evaluated the effects of CsA on the release of Epo in the culture medium by the human Hep3B hepatoma cell line. In cultures incubated with both CsA and the nonimmunosuppressive CsA analog MeAla-6, but not with the CsA-unrelated immunosuppressive agent FK-506, the levels of Epo in the medium were significantly reduced in comparison with controls, at concentrations (0.01 to 1.6 mumol/L) not affecting total protein synthetic rate nor the constitutive secretion of alpha- fetoprotein. Hep3B cells were found to contain a CsA-binding molecule, with an M(r) of 18 Kd, as assessed by high performance liquid chromatography (HPLC) and ligand-blotting analysis. CsA did not affect the expression of the Epo gene, as judged by Northern blot analysis, but caused a significant amount of Epo to remain unsecreted within the cells; almost all (97% of total) of the intracellular Epo was associated with the plasma membrane subcellular fraction. We conclude that: (1) CsA is able to inhibit Epo release in vitro by Hep3B cells, further supporting the hypothesis that the drug might have a role in the inappropriately low Epo levels observed in BMT patients; (2) the inhibitory effect appears to be specific and not caused by a general impairment of protein synthesis and/or secretion; and (3) the reduced Epo levels found in the medium of CsA-treated Hep3B cultures are supposed to be the consequence of an inability of the cells to correctly process Epo molecules for the secretory pathway.

scholarly journals Biosynthesis, transport and processing of myeloperoxidase in the human leukaemic promyelocytic cell line HL-60 and normal marrow cells

1984 ◽  
Vol 223 (3) ◽  
pp. 911-920 ◽  
Author(s):  
I Olsson ◽  
A M Persson ◽  
K Strömberg
Keyword(s):  
Cell Line ◽  
Intracellular Transport ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Cytosol Fraction ◽  
Leukaemia Cell Line ◽  
A Chain ◽  
Intermediate Density ◽  
Marrow Cells

The processing and intracellular transport of myeloperoxidase were studied in the human promyelocytic leukaemia cell line HL-60 and in normal marrow cells labelled with [35S]methionine or [14C]leucine. Myeloperoxidase was precipitated with antimyeloperoxidase serum; the immunoprecipitates were subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and radiolabelled myeloperoxidase visualized by fluorography. During a 1 h pulse, myeloperoxidase was labelled in a chain of apparent Mr 90 000. With a subsequent chase, the Mr 90 000 polypeptide disappeared and was replaced by chains of Mr 62 000 and 12 400 corresponding roughly to the size of neutrophil myeloperoxidase subunits. The identification of the radioactive polypeptides as different forms of myeloperoxidase was established also by the similarity in patterns generated by partial proteolysis with V8 proteinase from Staphylococcus aureus. Processing of myeloperoxidase in HL-60 was slow; mature polypeptides were significantly increased only after 6 h. Another myeloperoxidase chain of apparent Mr 82 000 was an intermediate precursor or degradation form. Pulse-chase experiments in combination with sucrose-density-gradient separations of homogenates showed that the Mr 90 000 precursor was located in light density organelles only and not in granule fractions, whereas the Mr 82 000 precursor was located only in intermediate density organelles, suggesting that the latter is a product of the former. Processed mature myeloperoxidase was concentrated in the granule fraction, but some occurred in lower density organelles, which may indicate processing during intracellular transport. Only the Mr 90 000 polypeptide was secreted into the culture medium; this was also the only form found in the cytosol fraction.

INHIBITOR OF THE FACTOR VIIA-TISSUE FACTOR COMPLEX IS REDUCED IN PATIENTS WITH DISSEMINATED INTRAVASCULAR COAGULATION BUT NOT IN PATIENTS WITH SEVERE HEPATOCELLULAR DISEASE

1987 ◽  
Author(s):  
M S Bajaj ◽  
S V Rana ◽  
R B Wysolmerski ◽  
S P Bajaj
Keyword(s):  
Endothelial Cells ◽  
Cell Line ◽  
Tissue Factor ◽  
Disseminated Intravascular Coagulation ◽  
Factor Viia ◽  
Hepatoma Cell ◽  
Sodium Dodecyl ◽  
Hepatoma Cell Line ◽  
Intravascular Coagulation ◽  
Hepatocellular Disease

Recently, inhibition of factor VIIa-tissue factor activity by a plasma component(s) which requires factor Xa has been described. In this communication, we have developed a specific radiometric assay (which utilizes 3H-factor IX and is sensitive to <1% of plasma level) for this inhibitor and have measured its activity in various disease states. Strikingly, the levels of this inhibitor were found to be normal in patients with advanced chronic hepatocellular disease but low in patients with disseminated intravascular coagulation (DIC). When endotoxin was used to induce DIC in rabbits, the levels of this inhibitor fell by 30 to 90%. Human umbilical vein endothelial cells (HUVE), bovine pulmonary artery endothelial cells, and a human hepatoma cell line (HepG2) all synthesized and secreted this inhibitor whereas a promyelocytic cell line (HL-60) did not and a monocytic cell line (U937) appears to synthesize only small amounts. When ammonium sulfate fractionated human plasma, and serum-free conditioned media from both HUVE and HepG2 cells were electrophoresed on sodium dodecyl sulfate acrylamide gels, two activity peaks corresponding to Mr ≃45,000 and Mr =33,000 were eluted in each case. These observations suggest that (a) the inhibitor is consumed in DIC and that (b) endothelial cells (or other cells) synthesize sufficient amounts of this inhibitor in vivo to compensate for any decreased production by liver cells. Furthermore, the inhibitor levels were found to be normal in patients on chronic warfarin therapy suggesting that the inhibitor is not a vitamin K-dependent protein.

scholarly journals Effect of warfarin on prothrombin synthesis and secretion in human Hep G2 cells

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 773-778
Author(s):  
S Karpatkin ◽  
TH Finlay ◽  
AL Ballesteros ◽  
M Karpatkin
Keyword(s):  
Hepatoma Cell ◽  
Carbohydrate Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hepatoma Cell Line ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Human Hepatoma Cell ◽  
Sds Page ◽  
The Media

Prothrombin synthesis and secretion were studied in a human hepatoma cell line (Hep G2) incubated with 35S-methionine for 2 to 24 hours at 37 degrees C. Extracellular and intracellular prothrombin were detected by immunoprecipitation with affinity-purified antiprothrombin antibody. Incorporation of 35S-methionine into prothrombin was monitored by counting specific bands excised from 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). Prothrombin represented 0.3% to 0.7% of total newly synthesized protein secreted into the media. Warfarin had no effect on total prothrombin synthesis (extracellular plus intracellular). However, warfarin inhibited secretion of newly synthesized prothrombin by 58% to 73% over a 2 to 4 hour period. This was accompanied by the intracellular accumulation of an immunoprecipitable species of prothrombin of 78 kd, 6 kd less than extracellular prothrombin. At the end of the 4-hour incubation with warfarin, intracellular prothrombin increased from 44% to 82% (twofold) of total prothrombin, whereas extracellular prothrombin decreased from 56% to 19% (threefold) of total prothrombin. After 24-hour incubation with warfarin, intracellular and extracellular immunoprecipitable prothrombin approached control values. Deglycosylation of extracellular and intracellular prothrombin with hydrofluoric acid (HF) resulted in a decrease in mol wt for both species to 66 kd. Endoglycosidase-H treatment, which digests “early mannosyl” residues, resulted in a decrease in the mol wt of the intracellular species of 8 kd with no effect on the extracellular species. Thus, the lower mol wt intracellular species that accumulates following early warfarin treatment is due to the presence of incompletely processed carbohydrate chain. The data are compatible with the hypothesis that optimum glycosylation and secretion require Vitamin K-dependent carboxylation.

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