Quality control of protein C: protein C synthesized in the presence of warfarin is selectively degraded in the endoplasmic reticulum.
Warfarin is known to disrupt the microsomal vitamin K cycle, which results in a decrease of the plasma level of protein C, an anticoagulant factor, as well as some other vitamin K-dependent coagulation factors. We examined the effect of warfarin on secretion of recombinant protein C expressed in human kidney 293 or BHK cells. In transient expression, warfarin caused a two- to four-fold decrease in the quantity of protein C secreted, compared to findings with vitamin K-treated cells. Pulse-chase experiments using stable cells showed that, although recombinant protein C was secreted in the presence of vitamin K, the decrease in total amount of the radioactivity in the warfarin-treated cells suggested intracellular degradation. This degradation depended on the concentration of warfarin and was not inhibited by an endoplasmic reticulum (ER)-Golgi transport inhibitor or by lysosomotropic inhibitors. Thus, protein C synthesized in the presence of warfarin is selectively degraded and the degradation occurs in a pre-Golgi, nonlysosomal compartment. Among the protease inhibitors tested, N-acetyl-Leu-Leu-methioninal and N-acetyl-Leu-Leu-norleucinal blocked the degradation of protein C synthesized in the presence of warfarin and the protein C accumulated intracellularly, in a dose-dependent manner. Both inhibitors, however, did not disturb the secretion of protein C in the vitamin K-treated cells. Thus, a cysteine protease(s) appeared to be responsible for the degradation. These results suggest that protein C synthesized in the presence of warfarin was selectively degraded by a cysteine protease(g) in the ER through a "quality control" mechanism.
Koide
T
T
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Hyogo, Japan.
Tokunaga
F
F
Wakabayashi
S
S
D001673:Biodegradation, Environmental; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D011233:Precipitin Tests; D011480:Protease Inhibitors / Q000494:pharmacology; D011486:Protein C / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014812:Vitamin K / Q000494:pharmacology; D014859:Warfarin / Q000494:pharmacology*
D018076:DNA, Complementary; D011480:Protease Inhibitors; D011486:Protein C; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D014812:Vitamin K; D014859:Warfarin