Mutation Study of Antithrombin: The Roles of Disulfide Bonds in Intracellular Accumulation and Formation of Russell Body-Like Structures
| Autor: | Isao Kitajima, Yu-ki Tanaka, Shoji Okamura, Tsuneo Imanaka, Masashi Morita, Kazue Ueda, Sadaki Yokota, Tetsuo Ozawa |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Arginine
Immunoblotting Mutant Mutation Missense Russell bodies CHO Cells Endoplasmic Reticulum medicine.disease_cause Biochemistry Antithrombins Microscopy Electron Transmission Cricetinae Centrifugation Density Gradient medicine Animals Secretion Antithrombin Proteins Cysteine Disulfides Microscopy Immunoelectron Molecular Biology Inclusion Bodies Mutation Chemistry Endoplasmic reticulum Wild type General Medicine Molecular Weight |
| Zdroj: | Journal of Biochemistry. 137:273-285 |
| ISSN: | 0021-924X |
| DOI: | 10.1093/jb/mvi040 |
| Popis: | Antithrombin (AT) is a major plasma protease inhibitor with three intramolecular disulfide bonds and a deficiency of it is associated with venous thrombosis. Recently, we prepared CHO cells overexpressing a novel mutant, AT(C95R), with a disulfide bond removed, and revealed that this mutant remained for a long time in the endoplasmic reticulum (ER) without being degraded and also accumulated in newly formed membrane structures that resembled Russell bodies (RB) [Tanaka, Y. et al. (2002) J. Biol. Chem. 277, 51058-51067]. In this study, we replaced each of the individual cysteine residues of AT with an arginine and also two paired cysteine residues with arginines. We stably expressed these mutant ATs in CHO cells, and examined the roles of each cysteine residue or disulfide bond in the accumulation of mutant ATs and the formation of RB-like structures. In pulse-chase experiments, the secretion of mutant ATs with single mutations decreased approximately 1/5-1/50 times compared to that of the wild type AT. All of the mutant ATs were retained in the ER and were also found to accumulate in the RB-like structures. On the other hand, the fates of mutant ATs with double mutations fell into two categories. Secretion of mutant AT(C8R,C128R) decreased only approximately 1/2 times and no RB-like structures appeared. Mutants AT(C21R,C95R) and AT(C247R,C430R) exhibited similar secretion kinetics to the mutant ATs with the single mutations and were found in RB-like structures. On a sucrose gradient, all of the mutant ATs that induced RB-like structures migrated as oligomeric structures, whereas wild type AT and AT(C8R,C128R) migrated as monomers. Further, to clarify the morphological pathway through which RB-like structures are formed, we prepared CHO cells in which the expression of AT(C95R) was controlled by the Tet-On system. During expression of AT(C95R), RB-like structures formed through expansion of the ER. These results suggest that the correct folding with each disulfide bond is essential for the secretion of AT and oligomerization of mutant ATs in the ER is involved in the formation of RB-like structures. |
| Databáze: | OpenAIRE |
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