Mechanism of oxygen regulation of erythrocyte properties

Haiyan Chu, Purdue University

Abstract

Band 3, the major protein of the human erythrocyte membrane, associates with multiple metabolic, ion transport, and structural proteins. Functional studies demonstrate that the oxygenation state of the erythrocyte regulates cellular properties performed by these and/or related proteins. Because deoxyhemoglobin, but not oxyhemoglobin, binds band 3 reversibly with high affinity, these observations raise the hypothesis that hemoglobin might regulate erythrocyte properties through its reversible, oxygenation-dependent association with band 3. (Chapter 1) To explore this hypothesis, the interactions of band 3 with other erythrocyte proteins were studied. First, I have precisely characterized the association of band 3 with glycolytic enzymes and report that band 3 provides a nucleation site for the glycolytic enzyme complex on the erythrocyte membrane. I have determined that the binding sites of aldolase, GAPDH and PFK on band 3 are in NH2-terminal residues 1-23. (Chapter 2) Assembly of the glycolytic enzyme complex on the erythrocyte membrane protein band 3 can be beneficial to generate ATP for transmembrane ion pumps use. Therefore, I have characterized the compartmentalization of ATP on the erythrocyte membrane that directly fuels both the Na+/K +- and Ca2+-pumps, and report that band 3, GAPDH, β-spectrin, and ankyrin form a compartment to entrap ATP. The specific peptides from these proteins corralling the entrapped ATP were also identified by Mass Spectrometry. (Chapter 3) I have also characterized the binding site of deoxyHb on human erythrocyte band 3 and report that: i) deoxyHb binds to residues 12-23 of band 3, which is proximal to binding sites for glycolytic enzymes, protein 4.1, and ankyrin, suggesting possible mechanisms through which multifarious erythrocyte properties might be regulated by the oxygenation state of the cell; ii) mutation of residues on either side of this binding sequence greatly enhance affinity of deoxyHb for band 3, suggesting that evolution of a higher affinity interaction would have been possible had it been beneficial for survival. This carefully tuned intermediate affinity of band 3 for deoxyHb binding might have evolved so that the band 3-Hb interaction can serve as a "molecular switch" for O 2-dependent regulation of erythrocyte physiology. (Chapter 4) To explore whether this "molecular switch" truly functions in vivo, two transgenic mice with altered band 3 sequences were prepared. I have characterized the oxygen dependence of erythrocyte properties in the transgenic mice and report that oxygen can regulate erythrocyte properties in the mice containing the deoxyHb binding site on band 3, but not in the transgenic mice without the deoxyHb binding site on band 3. These findings demonstrate that the oxygen-dependent reversible association of deoxyHb with band 3 constitutes a molecular switch for oxygen regulation of RBC properties in vivo. (Chapter 5)

Degree

Ph.D.

Advisors

Low, Purdue University.

Subject Area

Molecular biology|Analytical chemistry|Biochemistry

Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server
.

Share

COinS