Alterations in the expression pattern of RBC membrane associated proteins (RMAPs) in whole body γ-irradiated Sprague Dawley rats

Abstract

Purpose

Peripheral blood serum/plasma proteins are frequently studied for their potential use as radiation exposure biomarkers. Here we report RBC membrane associated proteins (RMAPs), which show alterations in expression level following whole-body γ-irradiation of rats at sub-lethal/lethal doses.

Materials and Methods

RBCs from peripheral blood of Sprague Dawley rats were segregated using the Ficoll-Hypaque method, and membrane fractions were hypotonically isolated at various time points (6 h, 24 h, 48 h) after γ-irradiation at 2 Gy, 5 Gy, and 7.5 Gy doses. Following purification of proteins from these fractions, two-dimensional electrophoresis (2-DE) was carried out. Treatment induced differentially expressed protein spots (≥2 fold increase/decrease) were picked up, trypsinized, and identified using LC-MS/MS analysis. Western immunoblots using protein specific antibodies were used to confirm the results. Gene ontology and interactions of these proteins were also studied.

Results

From a number of differentially expressed radiation-responsive 2-DE protein spots detected, eight were identified unequivocally using LC-MS/MS. Out of these, actin, cytoplasmic 1 (ACTB) showed detectable yet insignificant variation (<50%) in expression. In contrast, peroxiredoxin-2 (PRDX2) and 26S proteasome regulatory subunit RPN11 (PSMD14) were the two most prominently over-expressed proteins. Five more proteins, namely tropomyosin alpha-3 chain (TPM3), exosome component 6 (EXOSC6), isoform 4 of tropomyosin alpha-1 chain (TPM1), serum albumin (ALB), and the 55 kDa erythrocyte membrane protein (P55) showed distinct alteration in their expression at different time-points and doses. ALB, EXOSC6, and PSMD14 were the most responsive at 2 Gy, albeit at different time-points. While EXOSC6 and PSMD14 showed maximum over-expression (5-12 fold) at 6 h post-irradiation, ALB expression increased progressively (4 up to 7 fold) from 6 h to 48 h. TPM1 showed over-expression (2-3 fold) at all doses and time-points tested. TPM3 showed a dose-dependent response at all time-points studied; with no variation at 2 Gy, ∼2 fold increase at 5 Gy, and 3-6 fold at the highest dose used (7.5 Gy). The p55 protein was over-expressed (∼2.5 fold) only transiently at 24 h following the lethal (7.5 Gy) dose.

Conclusion

This is the first study to report γ-radiation induced alterations in the RBC membrane associated proteins. We are further evaluating the potential of these proteins as radiation biomarkers. Due to the abundance and easy use of RBCs, this approach can prove very useful for detecting ionizing radiation exposure.

Keywords:

• γ-radiation
• RBC
• membrane proteins
• radiation biomarkers
• rat

Disclosure statement

No potential conflict of interest was reported by the author(s). The authors alone are responsible for the content and writing the paper.

Additional information

Funding

This work was supported by the Defence Research and Development Organisation (DRDO), Ministry of Defence, India (DRDO Project No. TD-15/INM-313).

Notes on contributors

Prabuddho Mukherjee

Prabuddho Mukherjee received his MSc in Biotechnology from the University of Mumbai and has worked as a doctoral research fellow at INMAS, Delhi in the area of proteomics and miRNA as pertaining to radiation biology and biomarkers.

Kamendra Kumar

Kamendra Kumar received his PhD in Neuroscience from Jiwaji University, Gwalior in 2014 and worked as a postdoctoral fellow at INMAS (DRDO), Delhi, India. Presently he is working as a postdoctoral fellow at Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington-DC, USA. His research interests include radiation biology, proteomics and carcinogenesis.

Bincy Babu

Bincy Babu received her MTech in Genetic Engineering from SRM University, Chennai and has worked as a doctoral research fellow at INMAS, Delhi in the area of miRNA-based biomarkers in disease and molecular radiation biology.

Jubilee Purkayastha

Jubilee Purkayastha holds a PhD in Life Sciences from NEIST, CSIR, Jorhat, and worked as an Associate Project Scientist (Post PhD) at IIT, Guwahati. Currently she is working as a Scientist in INMAS, Delhi. Her research interests include radiation biology and biomarker studies.

Sudhir Chandna

Sudhir Chandna holds a PhD in Biomedicine from University of Delhi. He has long experience in radiation biology and is presently heading the Division of Molecular & Radiation Biosciences at INMAS, Delhi. His research interests include radioresistance mechanisms, low-dose hyper-radiosensitivity, radiation carcinogenesis, and molecular biomarkers. He served as the PI for this work.