Naoki Kimata

Membrane Peroxidation and Methemoglobin Formation are Both Necessary for Band 3 Clustering: Mechanistic Insights into Human Erythrocyte Senescence

Oxidative damage and clustering of band 3 in the membrane have been implicated in the removal of senescent human erythrocytes from the circulation at the end of their 120 day life span. However, the biochemical and mechanistic events leading to band 3 cluster formation have yet to be fully defined. Here we show that while neither membrane peroxidation nor methemoglobin (MetHb) formation on their own can induce band 3 clustering in the human erythrocytes, they can do so when acting in combination. We further show that binding of MetHb to the cytoplasmic domain of band 3 in peroxidized, but not in untreated, erythrocyte membranes induces cluster formation. Age-fractionated populations of erythrocytes from normal human blood, obtained by a density gradient procedure, have allowed us to examine a subpopulation, highly enriched in senescent cells. We have found that band 3 clustering is a feature of only this small fraction, amounting to ∼0.1% of total circulating erythrocytes. These senescent cells are characterized by an increased proportion of MetHb as a result of reduced nicotinamide adenine dinucleotide-dependent reductase activity and accumulated oxidative membrane damage. These findings have allowed us to establish that the combined effects of membrane peroxidation and MetHb formation are necessary for band 3 clustering, and this is a very late event in erythrocyte life. A plausible mechanism for the combined effects of membrane peroxidation and MetHb is proposed, involving high-affinity cooperative binding of MetHb to the cytoplasmic domain of oxidized band 3, probably because of its carbonylation, rather than other forms of oxidative damage. This modification leads to dissociation of ankyrin from band 3, allowing the tetrameric MetHb to cross-link the resulting freely diffusible band 3 dimers, with formation of clusters.

G protein subtype specificity of rhodopsin intermediates metarhodopsin Ib and metarhodopsin II.

Rhodopsin is one of the members of the G protein‐coupled receptor family that can catalyze a GDP–GTP exchange reaction on the retinal G protein transducin (Gt) upon photon absorption. There are at least two intermediate states, meta‐Ib and meta‐II, which exhibit direct interaction with Gt. Meta‐Ib binds to GDP‐bound Gt, while meta‐II forms a complex with Gt having no nucleotide, suggesting that meta‐Ib is a state that initially interacts with Gt. Here we investigated whether or not meta‐Ib exhibits specific interaction with G protein similar to meta‐II, by examining the binding efficiencies of meta‐Ib and meta‐II to Giα and its mutants whose C‐terminal 11 amino acids were replaced with those of Goα, Gqα and Gsα. The affinity of meta‐Ib to the C‐terminal 11 amino acids of Gtα was similar to those of Giα and its mutant with Goα’s C‐terminal 11 amino acids, whereas meta‐II exhibited affinity to the C‐terminal 11 amino acids of Giα mutant with Goα’s C‐terminal 11 amino acids about half of what was seen for Gtα and Giα. Both intermediates exhibited no affinity to the Giα mutants containing the C‐terminal 11 amino acids of Gqα and Gsα. These results suggested that meta‐Ib is the state that exhibits specific interaction with G protein as meta‐II does, although meta‐Ib exhibits a slightly lenient binding selectivity compared to that of meta‐II.

The C-Terminus of the G Protein α Subunit Controls the Affinity of Nucleotides

The C-terminus of the G protein α subunit has a well-known role in determining the selective coupling with the cognate G protein-coupled receptor (GPCR). In fact, rhodopsin, a prototypical GPCR, exhibits active state [metarhodopsin II (MII)] stabilization by interacting with G protein [extra formation of MII (eMII)], and the extent of stabilization is affected by the C-terminal sequence of Gα. Here we examine the relationship between the amount of eMII and the activation efficiency of Gi mutants whose Giα forms have different lengths of the C-terminal sequence of Goα. The results show that both the activation efficiencies of Gi and the amounts of eMII were affected by mutations; however, there was no correlation between them. This finding suggested that the C-terminal region of Gα not only stabilizes MII (active state) but also affects the nucleotide-binding site of Gα. Therefore, we measured the activation efficiency of these mutants by MII at several concentrations of GDP and GTP and calculated the rate constants of GDP release, GDP uptake, and GTP uptake. These rate constants of the Gi mutants were substantially different from those of the wild type, indicating that the replacement of the amino acid residues in the C-terminus alters the affinity of nucleotides. The rate constants of GDP uptake and GTP uptake showed a strong correlation, suggesting that the C-terminus of Giα controls the accessibility of the nucleotide-binding site. Therefore, our results strongly suggest that there is a long-range interlink between the C-terminus of Giα and its nucleotide-binding site.