Istituto Oncologico Veneto IOV-IRCCS, Padova, Italy.
Exp Biol Med (Maywood). 2011 Mar;236(3):281-90. doi: 10.1258/ebm.2011.010229. Epub 2011 Mar 7.
SERPINB3 has been found in chronic liver damage and hepatocellular carcinoma, but not in normal liver. By direct mRNA sequencing, a new SERPINB3 polymorphism (SCCA-PD) has been identified, presenting the substitution Gly351Ala in the reactive center loop of the protein. The prevalence of the SCCA-PD isoform has been found to be significantly higher in patients with cirrhosis than in patients with chronic liver disease and in normal subjects. The aim of this study was to investigate the biological and functional activity of SERPINB3 isoforms using in vitro models. HepG2 and Huh7 cells lines were transfected with plasmid vectors containing wild-type SERPINB3 or its polymorphic variant SCCA-PD and their expression at transcriptional and protein level was determined. To assess the functional activity, both recombinant proteins were produced and kinetic analysis was carried out using papain and cathepsin-L as target proteases. In addition, the inhibition of JNK kinase activity by SERPINB3 isoforms was assessed. The crystal structure of wild-type SERPINB3 at 2.7 Å resolution was used for preparation of refined 3D models of the two isoforms. The results showed that transcriptional activity and protein expression of the two isoforms were similar in both transfected cell lines. Both SERPINB3 preparations exerted a dose-dependent protease inhibitory activity, but the effect of SCCA-PD was higher than that of the wild-type isoform. This result was supported by 3D modelling, where increased hydrophobic profile of the SCCA-PD isoform, introduced by the G351A mutation, was detected. In addition, at high protein concentration, SCCA-PD revealed a 16% higher inhibitory effect on c-Jun phosphorylation by JNK1, compared with wild-type SERPINB3. In conclusion, the single amino acid substitution in the SERPINB3 reactive site loop improves the functional activity of SCCA-PD isoform. This different antiprotease activity might favor disease progression in patients carrying this polymorphism.
丝氨酸蛋白酶抑制剂 B3（SERPINB3）已在慢性肝损伤和肝细胞癌中被发现，但在正常肝脏中未被发现。通过直接 mRNA 测序，发现了一种新的 SERPINB3 多态性（SCCA-PD），其蛋白质的反应中心环中的甘氨酸 351 被丙氨酸取代。研究发现，SCCA-PD 同工型在肝硬化患者中的患病率明显高于慢性肝病患者和正常受试者。本研究旨在使用体外模型研究 SERPINB3 同工型的生物学和功能活性。用含有野生型 SERPINB3 或其多态变体 SCCA-PD 的质粒载体转染 HepG2 和 Huh7 细胞系，并测定其转录和蛋白水平的表达。为了评估功能活性，分别制备了两种重组蛋白，并使用木瓜蛋白酶和组织蛋白酶-L 作为靶蛋白酶进行了动力学分析。此外，还评估了 SERPINB3 同工型对 JNK 激酶活性的抑制作用。使用分辨率为 2.7 Å 的野生型 SERPINB3 的晶体结构，为两种同工型制备了经过优化的 3D 模型。结果表明，两种同工型在转染的细胞系中的转录活性和蛋白表达相似。两种 SERPINB3 制剂均表现出剂量依赖性的蛋白酶抑制活性，但 SCCA-PD 的作用高于野生型同工型。这一结果得到了 3D 建模的支持，其中检测到由 G351A 突变引入的 SCCA-PD 同工型增加的疏水性轮廓。此外，在高蛋白浓度下，与野生型 SERPINB3 相比，SCCA-PD 对 JNK1 磷酸化 c-Jun 的抑制作用提高了 16%。总之，SERPINB3 反应中心环中的单个氨基酸取代提高了 SCCA-PD 同工型的功能活性。这种不同的抗蛋白酶活性可能有利于携带这种多态性的患者疾病的进展。
