Department of Clinical Laboratory Science, Division of Health Sciences, Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa, Japan.
Research Center for Experimental Modeling of Human Disease, Kanazawa University, Kanazawa, Ishikawa, Japan.
Thromb Res. 2023 Oct;230:18-26. doi: 10.1016/j.thromres.2023.08.010. Epub 2023 Aug 15.
Hereditary antithrombin (AT) deficiency type I causes venous thrombosis due to decreased levels of AT antigen in the blood. We identified one novel and one known abnormal variant in two unrelated Japanese families with venous thrombosis. In this study, we analyzed the mechanism by which these abnormal variants cause type I AT deficiency.
Wild-type and variant AT expression vectors were constructed and transiently expressed in human embryonic kidney 293 cells, and AT antigen levels and N-glycosylation of cell lysates and culture medium were evaluated by western blot analysis. Subcellular co-localization of AT was also examined using confocal microscopy, and chase experiments with cycloheximide and MG132 were performed to investigate the degradation pathway of AT variants.
Genetic analysis identified a novel variant, c.613delC (p.Leu205Trpfs79), and the known variant c.283T>C (p.Tyr95His). These AT variants exhibited significantly reduced extracellular secretion compared with the wild-type; N-glycosylation of the AT protein was normal. Co-localization analysis suggested that the transport of these abnormal AT proteins to the Golgi apparatus was impaired. The c.613delC variant was degraded early by the proteasome, suggesting that the c.283T>C variant is stored in the endoplasmic reticulum (ER).
The AT variants identified here synthesize abnormal AT proteins that exhibit suppressed secretion and impaired transport from the ER to the Golgi apparatus. These results provide clues that could help elucidate the mechanism of type I AT deficiency and facilitate therapy development.
遗传性抗凝血酶 (AT) 缺陷型 I 由于血液中 AT 抗原水平降低而导致静脉血栓形成。我们在两个无血缘关系的日本静脉血栓形成家族中发现了一个新的和一个已知的异常变异体。在这项研究中，我们分析了这些异常变异体导致 I 型 AT 缺陷的机制。
构建了野生型和变异型 AT 表达载体，并在人胚肾 293 细胞中转染，通过 Western blot 分析评估细胞裂解物和培养上清液中 AT 抗原水平和 N-糖基化。使用共聚焦显微镜检查 AT 的亚细胞共定位，并用环己酰亚胺和 MG132 进行追踪实验，以研究 AT 变异体的降解途径。
遗传分析鉴定出一种新的变异体 c.613delC（p.Leu205Trpfs79）和已知的变异体 c.283T>C（p.Tyr95His）。与野生型相比，这些 AT 变异体的细胞外分泌明显减少；AT 蛋白的 N-糖基化正常。共定位分析表明，这些异常 AT 蛋白向高尔基体的转运受到损害。c.613delC 变异体被蛋白酶体早期降解，提示 c.283T>C 变异体储存在内质网 (ER) 中。
本研究鉴定的 AT 变异体合成异常 AT 蛋白，表现为分泌抑制和从 ER 到高尔基体的转运受损。这些结果为阐明 I 型 AT 缺陷的机制和促进治疗方法的发展提供了线索。
