Interaction of Deoxyhemoglobin with the Cytoplasmic Domain of Murine Erythrocyte Band 3

Abstract

The partial pressure of oxygen constitutes an important factor in the regulation of human erythrocyte physiology, including control of cell volume, membrane structure, and glucose metabolism. Because band 3 is thought to be involved in all three processes and because binding of hemoglobin (Hb) to the cytoplasmic domain of band 3 (cdb3) is strongly oxygen-dependent, the possibility that the reversible association of deoxyhemoglobin (deoxyHb) with cdb3 might constitute an O 2 -dependent sensor that mediates O 2 -regulated changes in erythrocyte properties arises. While several lines of evidence support this hypothesis, a major opposing argument lies in the fact that the deoxyHb binding sequence on human cdb3 is not conserved. Moreover, no effect of O 2 pressure on Hb− band 3 interactions has ever been demonstrated in another species. To explore whether band 3−Hb interactions might be widely involved in O 2 -dependent regulation of erythrocyte physiology, we undertook characterization of the effect of O 2 on band 3−Hb interactions in the mouse. We report here that murine band 3 binds deoxyHb with significantly greater affinity than oxyHb, despite the lack of significant homology within the deoxyHb binding sequence. We further map the deoxyHb binding site on murine band 3 and show that deletion of the site eliminates deoxyHb binding. Finally, we identify mutations in murine cdb3 that either enhance or eliminate its affinity for murine deoxyHb. These data demonstrate that despite a lack of homology in the sequences of both murine band 3 and murine Hb, a strong oxygen-dependent association of the two proteins has been conserved.

Deoxygenation Affects Composition of Membrane-Bound Proteins in Human Erythrocytes

“…the major integral protein of erythrocytes membrane, plays a key role in the organization of membrane cytoskeleton as well as in anion transport (anion exchanger 1, AE1) [37]. It was shown that both in humans and mice the affinity of band 3 fragment with M r of 43 kDa (CDB3) for T-Hb is much higher than for oxygenated hemoglobin [38,39]. These data allowed researcher to assume that T-Hb/band3 interaction is crucial for triggering the downstream pO 2 -dependent signals including altered cytoskeleton assembly which role in the maintenance of erythrocyte membrane integrity is well-documented [40][41][42][43].…”

Understanding Red Blood Cell Rheology in Sepsis and its Role in Clinical Practice. From Biomolecular Aspects to Possible Therapeutic Interventions

“…Band 3 phosphorylation then determines increased cytosol concentration of glyceraldehyde dehydrogenase, phosphofructokinase and aldolase (glycolitic enzymes anteriorly inactive and attached to cdb3) and enhanced glycolysis [75]. Band 3 phosphorylation is also determined by deoxyhemoglobin which through binding to cdb3 enhances glycolisis via the same mechanism described above [77]. Sepsis hypoxic status reduces drastically the hexose monophosphate pathway and further, NADPH concentration, which is vital in producing reductants in order to protect RBCs from oxidative damage [78,79].…”

“…Band 3 phosphorylation and dephosphorylation depends on several intraerytrocytic alterations: calcium concentration [75], oxidative damage on RBC membrane [76], low oxygen partial pressure leading to increased deoxyhemoglobin levels [77]. These alterations are already described in sepsis and can, in part, explain this phenomenon.…”

Regulation of erythrocyte Na+/K+/2Cl− cotransport by an oxygen-switched kinase cascade

“…In a second transgenic mouse, the same human sequence was inserted into murine band 3, except the amino acids responsible for deoxyHb binding (residues 12-23) were deleted, allowing us to determine how the lack of a deoxyHb-band 3 affects O 2 regulation of RBC properties. In the present study, we introduced a third mutation into murine band 3 (deletion of residues 1-11) that endows band 3 with a significantly higher affinity for deoxyHb (33,35) (Fig. 1).…”