scholarly journals Biosynthesis of complement C1 inhibitor by Hep G2 cells. Reactivity of different glycosylated forms of the inhibitor with C1s

1986 ◽  
Vol 237 (1) ◽  
pp. 93-98 ◽  
Author(s):  
M H Prandini ◽  
A Reboul ◽  
M G Colomb
Keyword(s):  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
C1 Inhibitor ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Complex Type ◽  
Human Hepatoma Cell ◽  
Minor Form ◽  
A Minor ◽  
Endoglycosidase H

The biosynthesis of C1 Inh (C1 inhibitor) was studied in a human hepatoma cell line (Hep G2) by metabolic labelling, immunoprecipitation with anti-(C1 Inh) serum, analysis on SDS/polyacrylamide gel slabs and fluorography. Two forms of C1 Inh are secreted by Hep G2: a minor form of Mr 90,000 and a major form of Mr approximately 100,000. The latter form is also found in small amounts intracellularly in co-existence with an 80,000-Mr form. Accumulation of the 80,000-Mr C1 Inh is favoured when the cells are labelled at 23 degrees C instead of 37 degrees C or when they are treated with monensin. In the presence of tunicamycin, a compound that blocks the formation of N-asparagine-linked oligosaccharide chains, a decrease in Mr of both secreted and intracellular major forms is observed, indicating that secreted and intracellular C1 Inh contain N-linked oligosaccharide units. The 100,000 Mr secreted C1 Inh is sensitive to endoglycosidase F but resistant to endoglycosidase H, and it incorporates [3H]galactose, [3H]glucosamine and [3H]galactosamine, indicating the presence of both N-linked oligosaccharides of the complex type and O-linked oligosaccharides. The intracellular C1 Inh contains N-linked oligosaccharide units of the high-mannose type as demonstrated by endoglycosidase H-sensitivity. The functional activity of C1 Inh during its biosynthesis was tested by studying its reactivity towards C1s. Both secreted and intracellular C1 Inh form covalent-like complexes with purified plasma C1s. The underglycosylated C1 Inh secreted in presence of tunicamycin is still reactive with purified C1s. These results clearly show that sugars are not essential for this inhibitory activity of C1 Inh.

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 The metabolism in vitro of human low-density lipoprotein by the human hepatoma cell line Hep G2

1983 ◽  
Vol 214 (3) ◽  
pp. 951-958 ◽  
Author(s):  
L Havekes ◽  
V van Hinsbergh ◽  
H J Kempen ◽  
J Emeis
Keyword(s):  
Cell Line ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Human Hepatoma ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Human Hepatoma Cell ◽  
Human Hepatoma Cell Line ◽  
High Affinity ◽  
G2 Cells

The human hepatoma cell line Hep G2 was studied with respect to metabolism of human low-density lipoprotein (LDL). The Hep G2 cells bind, take up and degrade human LDL with a high-affinity saturable and with a low-affinity non-saturable component. The high-affinity binding possesses a KD of 25 nM-LDL and a maximal amount of binding of about 70 ng of LDL-apoprotein/mg of cell protein. The high-affinity binding, uptake and degradation of LDL by Hep G2 cells is dependent on the extracellular Ca2+ concentration and is down-regulated by the presence of fairly high concentrations of extracellular LDL. Incubation of the Hep G2 cells with LDL results in suppression of the intracellular cholesterol synthesis. It is concluded that the human hepatoma cell line Hep G2 possesses specific LDL receptors similar to the LDL receptors demonstrated on extrahepatic tissue cells.

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.

scholarly journals Steady state levels of factor X mRNA in liver and Hep G2 cells

Blood ◽  
1987 ◽  
Vol 69 (1) ◽  
pp. 224-230 ◽  
Author(s):  
BR Bahnak ◽  
R Howk ◽  
JH Morrissey ◽  
GA Ricca ◽  
TS Edgington ◽  
...  
Keyword(s):  
Steady State ◽  
Human Liver ◽  
Hepatoma Cell ◽  
Specific Factor ◽  
Hepatoma Cell Line ◽  
Factor X ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Steady State Levels ◽  
G2 Cells

Abstract In order to examine the control of human factor X biosynthesis we have molecularly cloned the cDNA and investigated the expression of the Factor X gene. A recombinant clone of approximately 1100 base pairs in length containing the sequence of factor X was identified in a lambda gt11 human liver cDNA library by screening with polyclonal antibodies. One plaque was selected and confirmed for specificity with a mixture of five factor X specific monoclonal antibodies (MoAbs). A partial nucleic acid sequence of the 5′ end of the cDNA corresponded to the described amino acid sequence between residues 41 and 56 of the light chain of factor X. Northern blot analysis of RNA from human liver and the hepatoma cell line, Hep G2, identified the factor X mRNA as a single molecular species of approximately 1700 bases. Cell lines which do not secrete factor X did not contain factor X mRNA indicating restriction of transcription to hepatocytes. Slot-blot hybridization analysis of factor X and actin mRNA demonstrated no change in the levels of total or specific factor X mRNA in Hep G2 cells following treatment with warfarin or vitamin K. We conclude that modulation of factor X production by these drugs, known to influence gamma-carboxylation and total factor X secretion by these cells, is mediated by changes in posttranscriptional events rather than by effects on the steady state levels of factor X mRNA.

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