Increased antiprotease activity of the SERPINB3 polymorphic variant SCCA-PD

Abstract

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.

SerpinB3 and Yap Interplay Increases Myc Oncogenic Activity

“…The inhibitory effect of SerpinB3 on calpain was examined using the commercially available Calpain activity assay Kit (Calbiochem, Merk Millipore, Darmstadt, Germany) that utilizes the Suc-LLVY-AMC as synthetic substrate. Inhibition experiments were performed using different amounts of human recombinant SerpinB3 protein 52 (range 0–20 nmol/L). 60μl of human calpain 1 (100 nmol/L) was mixed with increasing amounts of inhibitor (SerpinB3) in presence of 60μl of substrate at 1:100 dilution.…”

SERPINB3 (Serpin Peptidase Inhibitor, Clade B (Ovalbumin), Member 3)

“…The prevalence of SCCA-PD was 24% in the normal population, while in patients with cirrhosis it was 45%, supporting the hypothesis of a higher contribution of this isoform to liver disease progression. In addition, the specific amino acid change detected in the reactive center of this SNP might confer a different biological behavior to the serpin improving the antiprotease activity of SERPINB3 [47]. …”

SerpinB3 and Yap interplay increases Myc oncogenic activity

“…The inhibitory effect of SerpinB3 on calpain was examined using the commercially available Calpain activity assay Kit (Calbiochem, Merk Millipore, Darmstadt, Germany) that utilizes the Suc-LLVY-AMC as synthetic substrate. Inhibition experiments were performed using different amounts of human recombinant SerpinB3 protein 52 (range 0-20 nmol/L). 60μ l of human calpain 1 (100 nmol/L) was mixed with increasing amounts of inhibitor (SerpinB3) in presence of 60μ l of substrate at 1:100 dilution.…”