A posttranslational modification of β-actin contributes to the slow dissociation of the spectrin-protein 4.1-actin complex of irreversibly sickled cells

Abstract

Irreversibly sickled cells (ISCs) remain sickled even under conditions where they are well oxygenated and hemoglobin is depolymerized. In our studies we demonstrate that triton extracted ISC core skeletons containing only spectrin, protein 4.1, and actin also retain their sickled shape; while reversibly sickled cell (RSC) skeletons remodel to a round or biconcave shape. We also demonstrate that these triton extracted ISC core skeletons dissociate more slowly upon incubation at 37°C than do RSC or control (AA) core skeletons. This observation may supply the basis for the inability of the ISC core skeleton to remodel its shape. Using an in vitro ternary complex dissociation assay, we demonstrate that a modification in β-actin is the major determinant of the slow dissociation of the spectrin-protein 4.1-actin complex isolated from the ISC core skeleton. We demonstrate that the difference between ISC and control β-actin is the inaccessibility of two cysteine residues in ISC β-actin to labeling by thiol reactive reagents; due to the formation of a disulfide bridge between cysteine284 and cysteine373 in ISC β-actin, or alternatively another modification of cysteine284 and cysteine373 which is reversible with DTT and adds less than 100 D to the molecular weight of β-actin.