Oxidized and poorly glycosylated band 3 is selectively phosphorylated by Syk kinase to form large membrane clusters in normal and G6PD-deficient red cells

Antonella Pantaleo (1) , Emanuela Ferru , Giuliana Giribaldi , Franca Mannu , Franco Carta , Alessandro Mattè , Lucia de Franceschi , Franco Turrini

1 UNITO - Università degli studi di Torino = University of Turin

Oxidative events involving band 3 (Anion Exchanger 1) have been associated with red blood cells (RBCs) removal through binding of naturally occurring antibodies (NAbs), however, the underlying mechanism has been only partially characterized. In addition to inducing direct membrane protein oxidative modification, oxidative treatment, specifically triggers the phosphorylation of band 3 tyrosine residues. This study reports that diamide, a sulfhydryl group oxidant, induces disulfide cross-linking of poorly-glycosylated band 3 and that the oligomerised band 3 fraction is selectively tyrosine (Tyr-) phosphorylated both in glucose-6-phosphate dehydrogenase (G6PD) deficient and control erythrocytes. This phenomenon is irreversible in G6PD deficient RBCs while it is temporarily limited in control RBCs. Diamide treatment caused p72 Syk phosphorylation and translocation to the membrane. Diamide also induced p72 Syk co-immunoprecipitation with aggregated band 3. Moreover, following size exclusion separation of Triton X-100 extracted membrane proteins, Syk was found only in the high molecular weight (MW) fraction containing oligomerised / phosphorylated band 3. Src family inhibitors efficiently abrogated band 3 Tyr-phosphorylation, band 3 clustering and NAbs binding to the RBCs surface suggesting a causal relationship between these events. Experiments performed with the non-permeant cross-linker BS3 showed that band 3 Tyr-phosphorylation enhances its capability to form large aggregates. Our results suggest that selective Tyr-phosphorylation of oxidised band 3 by Syk may play a role in the recruitment of oxidized band 3 in large membrane aggregates that show high affinity to NAbs, leading to RBCs removal from circulation.

Mots clés: Life Sciences

Antonella Pantaleo, Emanuela Ferru, Giuliana Giribaldi, Franca Mannu, Franco Carta, et al.. Oxidized and poorly glycosylated band 3 is selectively phosphorylated by Syk kinase to form large membrane clusters in normal and G6PD-deficient red cells. Biochemical Journal, 2009, 418 (2), pp.359-367. ⟨10.1042/BJ20081557⟩. ⟨hal-00479079⟩