Degradation of band3 and PRDX2 in erythrocytes during severe acute GVHD

Abstract We investigated the proteins of erythrocytes from stem cell transplantation patients and found decreased expression of band3 and C‐terminal‐truncated peroxiredoxin 2 (PRDX2) only during severe graft‐versus‐host disease (GVHD), using time‐of‐flight mass spectrometry (TOF‐MS) analysis and Western blotting. During the same period, PRDX2 dimerization and calpain‐1 activation were observed, indicating severe oxidative stress. We also found a putative cleavage site for calpain‐1 in the C‐terminal‐truncated site of PRDX2. Decreased band3 expression impairs the plasticity and stability of erythrocytes, and C‐terminal‐truncated PRDX2 induces irreversible dysfunction of antioxidant activity. These effects may exacerbate microcirculation disorders and the progression of organ dysfunction.


INTRODUCTION
Reactive oxygen species (ROS) are not only produced in a basic electron transport system of the life activity, but also from various external stimulations and inflammation. Each organism has various mechanisms to remove ROS because they are harmful to the living body. However, excessive production of ROS beyond the permissible level causes serious damage to the living body. It has been reported that ROS are involved and play crucial roles during the progression of diabetes and arteriosclerotic diseases.
Erythrocytes are the most powerful buffer against ROS-induced in vivo oxidative stress. Erythrocytes carry oxygen from the lungs to the entire body and are consequently exposed to oxidative stress.
The most abundant and powerful substance against oxidative stress in erythrocytes is peroxiredoxin 2 (PRDX2), which removes ROS via This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2023 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd. the glutathione system [1]. PRDX2 has two cysteine residues, and the thiol-dependent hydrogen peroxide removal activity is regulated by the thioredoxin/NADPH reduction mechanism. PRDX2 is located in the cytoplasm; it forms a dimer when oxidized in the presence of reactive oxygen and is then translocated to bind to the cell membrane [2,3].
Examination of the erythrocyte membrane proteins of 16 recipients of allogeneic hematopoietic cells showed that these changes were detected only in six cases with GVHD3 or 4, but not in cases without GVHD or with GVHD1 or 2 (Table S1). Long-term analysis showed that reduced expression of band3 recovered and truncated PRDX2 disappeared after the resolution of GVHD ( Figure S2).
However, it has been reported that the erythrocyte protease calpain is activated through autoproteolysis induced by oxidative stress via calcium, and moves to the cell membrane from the cytoplasm [7]. Additionally, anoxia has been reported to activate calpain [8]. Furthermore, activated calpain degrades band3 [9], and the analysis of calpain-1knockout mice has revealed that activated calpain degrades band3 in vivo [10]. According to these reports, we examined calpain autoproteolysis in erythrocytes and found that calpain activation was detected in accordance with the decreased band3 expression and 20 kDa band appearance ( Figure 2). PRDX2 consists of 198 amino acids and recognizes several putative cleavage sites for calpain [11], out of which the 157th and 182nd amino acid residue sites are determined at the C-terminus of PRDX2.
It might be possible that degradation of band3 or PRDX2 is induced by physical impact, such as hemolysis or fragmentation in thrombotic microangiopathy. However, it is difficult to explain the constant cutting site of PRDX2 in this context. Furthermore, none of the patients we examined presented clinically determined thrombotic microangiopathy or hemolytic disorder before the onset of degradation of band3 and PRDX2.
The decreased expression of band3 affects the plasticity and stability of erythrocytes [12], and the C-terminal-truncated PRDX2 presents irreversible dysfunction [13,14],

AUTHOR CONTRIBUTIONS
Masayuki Nagasawa conceptualized and designed the study, analyzed the data, and wrote the manuscript.

CONFLICT OF INTEREST STATEMENT
The author declares no conflicts of interest.

DATA AVAILABILITY STATEMENT
Data are available on request from the author.

ETHICS STATEMENT
This study was performed in compliance with the ethical treatment policy of human and animal research participants and the Declaration of Helsinki. This study was approved by institutional review board of Tokyo Medical and Dental University.

PATIENT CONSENT STATEMENT
Written informed consent was obtained from the guardians of each SCT patient.

SUPPORTING INFORMATION
Additional supporting information can be found online in the Supporting Information section at the end of this article.