Investigating citrullinated proteins in tumour cell lines

Abstract

Background: The conversion of arginine into citrulline, termed citrullination, has important consequences for the structure and function of proteins. Studies have found PADI4, an enzyme performing citrullination, to be highly expressed in a variety of malignant tumours and have shown that PADI4 participates in the process of tumorigenesis. However, as citrullinated proteins have not been systematically investigated in tumours, the present study aimed to identify novel citrullinated proteins in tumours by 2-D western blotting (2-D WB).

Methods: Two identical two-dimensional electrophoresis (2-DE) gels were prepared using extracts from ECA, H292, HeLa, HEPG2, Lovo, MCF-7, PANC-1, SGC, and SKOV3 tumour cell lines. The expression profiles on a 2-DE gel were trans-blotted to PVDF membranes, and the blots were then probed with an anti-citrulline antibody. By comparing the 2-DE profile with the parallel 2-D WB profile at a global level, protein spots with immuno-signals were collected from the second 2-DE gel and identified using mass spectrometry. Immunoprecipitation was used to verify the expression and citrullination of the targeted proteins in tumour cell lines.

Results: 2-D WB and mass spectrometry identified citrullinated α-enolase (ENO1), heat shock protein 60 (HSP60), keratin 8 (KRT8), tubulin beta (TUBB), T cell receptor chain and vimentin in these cell lines. Immunoprecipitation analyses verified the expression and citrullination of ENO1, HSP60, KRT8, and TUBB in the total protein lysates of the tumour cell lines.

Conclusions: The citrullination of these proteins suggests a new mechanism in the tumorigenic process.

Figure 1

Two-dimensional polyacrylamide gel electrophoresis and the parallel 2-D western blotting of total proteins from various tumour cell lines. The total proteins extracted from ECA, H292, HeLa, HEPG2, Lovo, MCF-7, PANC-1, SGC, and SKOV3 cells were separated using two identical 2-DE gels. The tumour cell proteins on one gel were trans-blotted onto a PVDF membrane and probed with an anti-citrulline antibody. The protein spots in the same position as those recognised by the parallel 2-D WB were selected and identified by MALDI-TOF MS.

Figure 2

Immunoprecipitation of ENO1, HSP60, KRT8, PDI, TUBB, and VIME in ECA, HEPG2, SKOV3, HeLa, PANC-1, and SGC cell lines. The immunoprecipitation was performed with anti-ENO1, -HSP60, -KRT8, -PDI, -TUBB, or -VIME antibodies to extract the corresponding proteins. Western blotting was performed with anti-ENO1, -HSP60, -KRT8, -PDI, -TUBB, -VIME, and anti-citrulline antibodies to determine the citrullination of these candidate proteins. The thick arrow indicates the candidate proteins. The thin arrow indicates the molecular weights of the protein marker.