Henan International Joint Laboratory of Thrombosis and Hemostasis, Henan University of Science and Technology, Luoyang, Henan, People's Republic of China; Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, USA.
College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, Jiangsu, People's Republic of China.
J Thromb Haemost. 2023 Nov;21(11):3124-3137. doi: 10.1016/j.jtha.2023.06.021. Epub 2023 Jun 29.
Inherited protein C deficiency (PCD) caused by mutations in protein C (PC) gene (PROC) increases the risk of thrombosis. Missense mutations in PC's signal peptide and propeptide have been reported in patients with PCD, but their pathogenic mechanisms, except mutations in R42 residue, remain unclear.
To investigate the pathogenic mechanisms of inherited PCD caused by 11 naturally occurring missense mutations in PC's signal peptide and propeptide.
Using cell-based assays, we evaluated the impact of these mutations on various aspects such as activities and antigens of secreted PC, intracellular PC expression, subcellular localization of a reporter protein, and propeptide cleavage. Additionally, we investigated their effect on pre-messenger RNA (pre-mRNA) splicing using a minigene splicing assay.
Our data revealed that certain missense mutations (L9P, R32C, R40C, R38W, and R42C) disrupted PC secretion by impeding cotranslational translocation to the endoplasmic reticulum or causing endoplasmic reticulum retention. Additionally, some mutations (R38W and R42L/H/S) resulted in abnormal propeptide cleavage. However, a few missense mutations (Q3P, W14G, and V26M) did not account for PCD. Using a minigene splicing assay, we observed that several variations (c.8A>C, c.76G>A, c.94C>T, and c.112C>T) increased the incidence of aberrant pre-mRNA splicing.
Our findings suggest that variations in PC's signal peptide and propeptide have varying effects on the biological process of PC, including posttranscriptional pre-mRNA splicing, translation, and posttranslational processing. Additionally, a variation could affect the biological process of PC at multiple levels. Except for W14G, our results provide a clear understanding of the relationship between PROC genotype and inherited PCD.
由于蛋白 C（PROC）基因突变导致的遗传性蛋白 C 缺乏症（PCD）会增加血栓形成的风险。已有研究报道，PC 的信号肽和前肽中的错义突变可导致 PCD，但除 R42 残基突变外，其致病机制仍不清楚。
研究 11 种天然存在的 PC 信号肽和前肽错义突变导致遗传性 PCD 的致病机制。
采用基于细胞的检测方法，我们评估了这些突变对分泌型 PC 的活性和抗原、细胞内 PC 表达、报告蛋白的亚细胞定位以及前肽切割等多个方面的影响。此外，我们还通过小基因剪接试验研究了它们对前信使 RNA（pre-mRNA）剪接的影响。
我们的数据显示，某些错义突变（L9P、R32C、R40C、R38W 和 R42C）通过阻碍共翻译易位到内质网或导致内质网滞留，从而破坏 PC 的分泌。此外，一些突变（R38W 和 R42L/H/S）导致异常的前肽切割。然而，少数错义突变（Q3P、W14G 和 V26M）不能解释 PCD。通过小基因剪接试验，我们发现几种变异（c.8A>C、c.76G>A、c.94C>T 和 c.112C>T）增加了异常 pre-mRNA 剪接的发生率。
我们的研究结果表明，PC 的信号肽和前肽的变异对 PC 的生物学过程（包括转录后 pre-mRNA 剪接、翻译和翻译后加工）有不同的影响。此外，一种变异可以在多个水平上影响 PC 的生物学过程。除了 W14G，我们的结果还明确了 PROC 基因型与遗传性 PCD 之间的关系。
