Cellular and functional characterization of three recombinant antithrombin mutants that caused pleiotropic effect-type deficiency.
Inherited antithrombin deficiency is associated with a predisposition for familial venous thromboembolic disease. Pleiotropic effect-type mutants of antithrombin that have an amino acid replacement in a distal hinge region including strands 1C, 4B, and 5B of the polypeptide chain are known to exhibit impaired interactions with both thrombin and heparin, coupled with a secretion defect. To examine the mechanism of pleiotropic effect-type antithrombin deficiency, we expressed three mutants, Oslo (Ala404-->Thr), Kyoto (Arg406-->Met), and Utah (Pro407-->Leu), in baby hamster kidney (BHK) cells, and compared their secretion rates, affinities for heparin and abilities to form thrombin-antithrombin (TAT) complexes with those of wild-type (Wt) antithrombin. Pulse-chase experiments showed that the Oslo- and Kyoto-mutants were secreted at rates similar to Wt antithrombin. In contrast, the Utah-mutant underwent partial intracellular degradation. The intracellular degradation of the Utah-mutant was not inhibited by lysosomotropic inhibitors, but by proteasome inhibitors such as carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (LLL) and lactacystin, indicating that a part of the Utah-mutant was degraded by proteasome through quality control in the endoplasmic reticulum (ER). Crossed immunoelectrophoresis in the presence of heparin showed that only the Oslo-mutant lacks heparin-binding ability. Incubation with thrombin showed that the Kyoto- and Utah-mutants, but not the Oslo-mutant, formed a weak but detectable TAT complex. Furthermore, heparin enhanced the TAT complex formation by the Kyoto- and Utah-mutants, suggesting heparin cofactor activities of these mutants. These results show that each of the Oslo-, Kyoto-, and Utah-mutants exhibits different properties as to secretion, intracellular degradation and functional activity, although they are grouped as pleiotropic effect-type mutants.
Shirotani
H
H
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Garden City, Hyogo, 678-1297, Japan.
Tokunaga
F
F
Koide
T
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000990:Antithrombin III / Q000378:metabolism; D000991:Antithrombins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases / Q000378:metabolism; D014162:Transfection