Mediation of oxygen-dependent erythrocyte properties by band 3 (AE1)
Abstract
Over the years the concept that erythrocytes are “simple” cells has been dispelled by evidence of impressive complexity in structure and function. For example, oxygenation status of red blood cells has been implicated in such diverse functions as volume control, glycolytic enzyme activity and membrane structure. Because band 3 is thought to be involved in all three processes, and since hemoglobin (Hb) binding to band 3 is strongly oxygen-dependent, the possibility arises that the preferential association of deoxyHb with cytoplasmic domain of band3 (cdb3) might constitute an O 2 sensor that mediates these processes. Our hypothesis is that deoxyHb associates in a reversible manner with the NH2-terminus of the cdb3 changing the interaction of band 3 with other red blood cell proteins, resulting in the aforementioned O2 regulation of red cell properties. In order to characterize this interaction, the first goal of my study was to find and optimize a method to evaluate hemoglobin-cdb3 interaction in solution. We found that fluorescence resonance energy transfer (FRET) can be used to study this interaction in vitro under both oxygenated or deoxygenated conditions. The second goal was to find whether the cdb3-Hb interaction in mouse species is O2-dependent and to localize the binding site of deoxyhemoglobin on cdb3 using FRET analysis. This binding site was found to be in the first 23 amino acids from the NH2-terminus of cdb3 in the sequence 12-23. Also, introduction of negatively charged amino acids adjacent to the binding site (12-23) resulted in a high affinity murine cdb3 mutant. These findings will allow us to study the effect of constant binding or no binding between band3-hemoglobin on red blood cell properties. Understanding how and why red blood cells respond to changes in oxygen partial pressure will be beneficial for different reasons. First, understanding how red cells fight oxidative stress might help us to better realize how to improve red cell storage in blood banks. Second, unraveling cell volume regulation is important for the control of some ion transporter abnormalities and also sickle cell anemia.
Degree
Ph.D.
Advisors
Christian, Purdue University.
Subject Area
Biochemistry
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