Spectrin's E2/E3 ubiquitin conjugating/ligating activity is diminished in sickle cells

Abstract

Erythrocyte spectrin contains E2/E3 ubiquitin conjugating/ligating activity in its a subunit. Ankyrin is a target of spectrin's E2/E3 ubiquitin conjugating/ligating activity in vitro and in vivo. We compare the ubiquitination levels of ankyrin mediated by control and sickle cell spectrin using a biotinylated ubiquitin cell-free assay. Sickle cell spectrin has diminished ability to transfer ubiquitin from an intermediate spectrin-ubiquitin thioester adduct (a 0 spectrin) to ankyrin, which may be due to glutathiolation of spectrin's E2 and/ or E3 active site cysteines. There is also a diminished ability of the sickle cell ankyrin to serve as target of spectrin's E2/E3 activity, probably due to oxidative damage to ankyrin. A direct correlation exists between the a 0 /a spectrin ratio and spectrin's ability to ubiquitinate ankyrin. There is also an inverse correlation between severity of the disease and the a 0 /a spectrin ratio in SS erythrocytes. These results suggest that reduced spectrin E2/ E3 activity is an important determinant of sickle cell severity. Am. J. Hematol. 79:89-96, 2005.

Alteration of α-spectrin ubiquitination after hemorrhagic shock

“…Since recent studies in sickle cell disease have implicated a decrease in alpha-spectrin ubiquitination in the pathogenesis of red cell rigidification [16], we compared the ratio of ubiquinated to non-ubiquinated alpha-spectrin in the T/HS red cells to that of T/SS red cells. These studies revealed a decrease in alpha-spectrin ubiquitination in the erythrocytes of the T/HS animals as compared to the T/SS animals resulting in a decrease in the ratio of ubiquitinated α-spectrin to total α-spectrin (Figure 6).…”

“…Since alpha-spectrin has ubiquitin conjugating (E2) and ligating (E3) activity and the ability to ubiquinate beta-spectrin, ankyrin and Band 3 [12,20,21], plus recent studies on sickle cells have correlated decreased spectrin E2/E3 activity with decreased RBC deformability [16], we tested whether T/HS caused a decrease in the amount of ubiquinated alpha-spectin. Our results documented that there was a decrease in the amount of ubiquitinated alpha-spectrin resulting in a decreased ratio of non-ubiquitinated to total alpha-spectrin.…”

“…That is, although ubiquitin has been thought of as primarily functioning in the proteosomal degredation of proteins, it is now recognized that ubiquitin plays an important role in many cellular events ranging from protein-protein interactions to transcriptional activation and even apoptosis [22]. The importance of the ubiquinating activity of alpha-spectin in maintaining normal RBC deformability has been recently established in sickle cells with abnormal deformability, where the a decrease in the alpha-spectrin ubiquitin to non-ubiquitin ratio correlated tightly with sickle cell severity and deformability [16]. The proposed mechanism for how decreased alpha-spectrin ubiquitin levels and activity leads to decreased RBC deformability is based on the observation that reduced spectrin ubiquitination leads to tightened spectrin-Protein 4.1-actin and spectrin-adductin-actin complexes that dissociate slowly [16,23,24].…”

Spectrin’s chimeric E2/E3 enzymatic activity

“…43 SCA RBC spectrin demonstrates diminished ubiquitination (50-90% reduced) when compared to control RBC spectrin. 28,45,47 This is caused by diminished spectrin E2/ E3 activity, which we believe to be caused by the conversion of active site cysteine thiolates (C2071 and C2100) into cysteine oxiforms, which cause loss of function. 47 As nonubiquitinated spectrin participates in a more tightly associated spectrin-4.1-actin and spectrin-adducin-actin ternary complex than ubiquitinated spectrin, 45,46 the rate of SCA ternary complex dissociation is far slower than the control ternary complex dissociation rate.…”

“…28,45,47 This is caused by diminished spectrin E2/ E3 activity, which we believe to be caused by the conversion of active site cysteine thiolates (C2071 and C2100) into cysteine oxiforms, which cause loss of function. 47 As nonubiquitinated spectrin participates in a more tightly associated spectrin-4.1-actin and spectrin-adducin-actin ternary complex than ubiquitinated spectrin, 45,46 the rate of SCA ternary complex dissociation is far slower than the control ternary complex dissociation rate. 40 Therefore, the molecular basis of the formation of the ISC is a membrane skeleton that disassembles and reassembles slowly leading to a cell ''locked'' into the sickled shape.…”