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Transfusion. 2015 Mar;55(3):499-506. doi: 10.1111/trf.12855. Epub 2014 Sep 4.

Thiol-based regulation of glyceraldehyde-3-phosphate dehydrogenase in blood bank-stored red blood cells: a strategy to counteract oxidative stress.

Author information

1
Department of Ecological and Biological Sciences, University of Tuscia, Viterbo, Italy.

Abstract

BACKGROUND:

Red blood cell (RBC) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme normally inhibited upon binding to the membrane-spanning protein Band 3, but active when free in the cytosol. Accumulating evidence in other cells indicates that oxidative thiol modifications in cytosolic GAPDH drive this molecule into functional avenues that deviate from glycolysis. This study aimed to investigate the role of GAPDH in oxidative stress-dependent metabolic modulations occurring in SAGM-stored RBCs, to increase the knowledge of the molecular mechanisms affecting RBC survival and viability under blood banking conditions.

STUDY DESIGN AND METHODS:

Membranes and cytosol from CPD SAGM-stored RBCs were subjected to Western blotting with anti-GAPDH at 0, 7, 14, 21, 28, 35, and 42 days of preservation. Immunoreactive bands were excised, digested with trypsin, and analyzed by mass spectrometry for the presence of oxidative posttranslational modifications. GAPDH enzymatic activity was also measured in the cytosolic fraction during storage.

RESULTS:

At 21 days of storage, we demonstrated that cytosolic GAPDH undergoes temporary inactivation due to the formation of an intramolecular disulfide bond between the active-site Cys-152 and nearby Cys-156, a mechanism to rerouting glucose flux toward the pentose phosphate pathway. In addition, an increase in the membrane-bound GAPDH was detected in long-stored RBCs.

CONCLUSION:

Reversible inhibition or activation of cytosolic GAPDH may represent a protective strategy against oxidative stress to favor NADPH production in stored RBCs.

PMID:
25196942
DOI:
10.1111/trf.12855
[Indexed for MEDLINE]

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