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Papers In Press, published online ahead of print October 23, 2002
Department of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama, Toyama 930-0194
Corresponding Author: imanaka{at}ms.toyama-mpu.ac.jp
Antithrombin (AT) is a major plasma protease inhibitor with three intramolecular disulfide bonds and its deficiency is associated with increased venous thrombosis. Recently, we found a novel missense mutation named AT Morioka (C95R), which causes the loss of one of the three disulfide bonds. In this study, we prepared CHO cells stably overexpressing wild type or mutant AT and examined the intracellular fate of the ATs. In pulse-chase experiments, newly synthesized wild type AT was secreted into the medium with a half- life of ~1.5 h. In contrast, most of the mutant type AT was not secreted during the chase period of 9 h and surprisingly, was not degraded in the cells. The kinetics of the secretion suggests that the mutant was secreted about 50 times more slowly into the medium. Most of the mutant AT in the cells had high-mannose type oligosaccharides, suggesting that it was retained in the endoplasmic reticulum (ER). In addition, half of the mutant AT existed in a dimeric form with an intermolecular disulfide bond. On immunoelectron microscopy, the mutant AT was found to have accumulated in variously sized structures surrounded by a single membrane in the cytoplasm. Immuno-gold particles exhibiting calnexin-immunoreactivity were detected on the membranes. Further, we prepared CHO cells stably overexpressing another mutant AT in which two cysteine residues at 21 and 95, responsible for disulfide bond formation, were substituted for arginines. In pulse-chase experiments, the mutant AT (C21, 95R) was secreted faster than that of AT Morioka (C95R) into the medium. These results suggest that AT Morioka remained for a long time in ER without being degraded and accumulated in newly formed membrane structures derived from the ER. The dimerization of AT Morioka (C95R) through C21 seems to be critical for its intracellular accumulation.
J. Biol. Chem, 10.1074/jbc.M210231200
Submitted on October 7, 2002
Revised on October 23, 2002
Accepted on October 23, 2002
Intracellular accumulation of antithrombin morioka (Cys95Arg), A novel mutation causing type I antithrombin deficiency
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