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

Biochem J. 2009 Mar 1;418(2):359-67. doi: 10.1042/BJ20081557.


Oxidative events involving band 3 (Anion Exchanger 1) have been associated with RBC (red blood cell) removal through binding of NAbs (naturally occurring antibodies); 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. The present study reports that diamide, a thiol group oxidant, induces disulfide cross-linking of poorly glycosylated band 3 and that the oligomerized band 3 fraction is selectively tyrosine phosphorylated both in G6PD (glucose-6-phosphate dehydrogenase)-deficient and control RBCs. This phenomenon is irreversible in G6PD-deficient RBCs, whereas 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-mass fraction containing oligomerized/phosphorylated band 3. Src family inhibitors efficiently abrogated band 3 tyrosine phosphorylation, band 3 clustering and NAbs binding to the RBC surface, suggesting a causal relationship between these events. Experiments performed with the non-permeant cross-linker BS(3) (bis-sulfosuccinimidyl-suberate) showed that band 3 tyrosine phosphorylation enhances its capability to form large aggregates. The results of the present study suggest that selective tyrosine phosphorylation of oxidized band 3 by Syk may play a role in the recruitment of oxidized band 3 in large membrane aggregates that show a high affinity to NAbs, leading to RBC removal from the circulation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anion Exchange Protein 1, Erythrocyte / metabolism*
  • Antibodies / metabolism
  • Antibodies / physiology
  • Diamide / pharmacology
  • Erythrocytes / metabolism
  • Erythrocytes / pathology*
  • Glucosephosphate Dehydrogenase / genetics
  • Glucosephosphate Dehydrogenase / metabolism
  • Glucosephosphate Dehydrogenase Deficiency / blood
  • Glucosephosphate Dehydrogenase Deficiency / metabolism
  • Glucosephosphate Dehydrogenase Deficiency / pathology*
  • Glycosylation
  • Humans
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Membrane Proteins / metabolism
  • Models, Biological
  • Oxidation-Reduction
  • Phosphorylation
  • Protein Binding
  • Protein Multimerization* / genetics
  • Protein Multimerization* / physiology
  • Protein-Tyrosine Kinases / metabolism*
  • Substrate Specificity
  • Sulfhydryl Reagents / pharmacology
  • Syk Kinase
  • Tyrosine / metabolism


  • Anion Exchange Protein 1, Erythrocyte
  • Antibodies
  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • Sulfhydryl Reagents
  • Diamide
  • Tyrosine
  • Glucosephosphate Dehydrogenase
  • Protein-Tyrosine Kinases
  • SYK protein, human
  • Syk Kinase