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(Circulation Research. 2003;92:865.)
© 2003 American Heart Association, Inc.

Cellular Biology

Defective Sorting to Secretory Vesicles in Trans-Golgi Network Is Partly Responsible for Protein C Deficiency

Molecular Mechanisms of Impaired Secretion of Abnormal Protein C R169W, R352W, and G376D

Masao Naito, Jun Mimuro, Hitoshi Endo, Seiji Madoiwa, Kyo-ichi Ogata, Jiro Kikuchi, Teruko Sugo, Takanori Yasu, Yusei Kariya, Yuichi Hoshino, Yoichi Sakata

From the Research Division of Cell and Molecular Medicine (M.N., J.M., S.M., K.O., J.K., T.S., Y.S.), Department of Biochemistry (H.E.), Cardiovascular Division of Ohmiya Medical Center (T.Y.), and Department of Orthopedics (Y.K., Y.H.), Jichi Medical School, Tochigi-Ken, Japan.

Correspondence to Jun Mimuro, MD, PhD, Research Division of Cell and Molecular Medicine, Jichi Medical School, Tochigi-Ken 329-0498, Japan. E-mail mimuro-j{at}jichi.ac.jp

Three thrombophilic patients with protein C (PC) deficiency were found to have independent mutations in the PC gene. These mutations resulted in single amino acid substitutions of R169W, R352W, and G376D in the affected PC molecules. These abnormal PC molecules were expressed in CHO-K1 cells in the presence or absence of vitamin K, and their synthesis, posttranslational modification, and secretion were studied. PC G376D was not secreted from the cells and was gradually degraded inside the cells. There was partial secretion of PC R169W and PC R352W, but most of these molecules were not secreted but were degraded intracellularly. On the basis of pulse-chase, immunofluorescence, and endo-ß-N-acetylglucosaminidase H digestion experiments, the majority of wild-type PC molecules localize not in the Golgi apparatus but in the rough endoplasmic reticulum inside the cells. This suggests that wild-type PC molecules are secreted immediately after -carboxylation and modification at the Golgi apparatus. In contrast, the mutant PC molecules were retained inside the cells even after modification of oligosaccharides at the trans-Golgi apparatus, which was probably due to impaired conformation of the abnormal molecules. Data suggest that these abnormal PC molecules were not sorted to secretory vesicles in the trans-Golgi network because of conformational defects in addition to the transport defect from the rough endoplasmic reticulum to the Golgi apparatus and were degraded inside the cells, thereby resulting in a PC deficiency in the affected patients.

Key Words: thrombosis • protein C deficiency • intracellular protein transport

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