Kovács Kitti B, Pataki István, Bárdos Helga, Fekete Attila, Pfliegler György, Haramura Gizella, Gindele Réka, Komáromi István, Balla György, Ádány Róza, Muszbek László, Bereczky Zsuzsanna
Division of Clinical Laboratory Science, University of Debrecen, Debrecen, Hungary.
Department of Pediatrics, University of Debrecen, Debrecen, Hungary.
Thromb Res. 2015 Apr;135(4):718-26. doi: 10.1016/j.thromres.2015.01.011. Epub 2015 Jan 15.
Protein C (PC) is a major anticoagulant and numerous distinct mutations in its coding gene result in quantitative or qualitative PC deficiency with high thrombosis risk. Homozygous or compound heterozygous PC deficiency usually leads to life-threatening thrombosis in neonates.
The molecular consequences of 3 different missense mutations of two patients have been investigated. The first patient suffered from neonatal purpura fulminans and was a compound heterozygote for p.Asp77Gly and p.Ala163Glu mutations. The second patient had severe deep venous thrombosis in young adulthood and carried the p.Ala163Val mutation. The fate of mutant proteins expressed in HEK cells was monitored by ELISA, by Western blotting, by investigation of polyubiquitination and by functional assays. Their intracellular localization was examined by immunostaining and confocal laser scanning microscopy. Molecular modeling and dynamics simulations were also carried out.
The 163Val and 163Glu mutants had undetectable levels in the culture media, showed intracellular co-localization with the 26S proteasome and were polyubiquitinated. The 77Gly mutant was secreted to the media showing similar activity as the wild type. There was no difference among intracellular PC levels of wild type and mutant proteins. The 163Val and 163Glu mutations caused significant changes in the relative positions of the EGF2 domains suggesting misfolding with the consequence of secretion defect. No major structural alteration was observed in case of 77Gly mutant; it might influence the stability of protein complexes in which PC participates and may have an impact on the clearance of PC requiring further research.
蛋白C（PC）是一种主要的抗凝剂，其编码基因中的众多不同突变会导致定量或定性的PC缺乏，从而具有高血栓形成风险。纯合子或复合杂合子PC缺乏通常会导致新生儿出现危及生命的血栓形成。
对两名患者的3种不同错义突变的分子后果进行了研究。第一名患者患有暴发性紫癜新生儿型，是p.Asp77Gly和p.Ala163Glu突变的复合杂合子。第二名患者在青年期患有严重的深静脉血栓形成，携带p.Ala163Val突变。通过酶联免疫吸附测定（ELISA）、蛋白质印迹法、多聚泛素化研究和功能测定来监测在人胚肾（HEK）细胞中表达的突变蛋白的命运。通过免疫染色和共聚焦激光扫描显微镜检查它们的细胞内定位。还进行了分子建模和动力学模拟。
163Val和163Glu突变体在培养基中的水平无法检测到，显示与26S蛋白酶体在细胞内共定位并且被多聚泛素化。77Gly突变体分泌到培养基中，显示出与野生型相似的活性。野生型和突变蛋白的细胞内PC水平没有差异。163Val和163Glu突变导致表皮生长因子2（EGF2）结构域的相对位置发生显著变化，提示错误折叠并导致分泌缺陷。在77Gly突变体中未观察到主要的结构改变；它可能影响PC参与的蛋白质复合物的稳定性，并且可能对PC的清除有影响，这需要进一步研究。
