Oral Presentation 22nd Annual Lorne Proteomics Symposium 2017

Signalling networks in the regulation of red blood cell integrity and survival. (#15)

Alison Louw 1 2 , Nicole Kucera 1 2 , Neli S Slavova-Azmanova 1 2 , Cindy Le 1 2 , Jiulia Satiaputra 1 2 , Wendy Erber 3 , Lucia DeFranceschi 4 , Narla Mohandas 5 , Margaret L Hibbs 6 , Evan Ingley 1 2
  1. Harry Perkins Institute of Medical Research and Centre for Medical Research, The University of Western Australia, Nedlands, WA, Australia
  2. Centre for Medical Research, The University of Western Australia, Crawley, WA, Australia
  3. Pathology and Laboratory Medicine, University of Western Australia, Crawley, WA, Australia
  4. Department of Medicine, University of Verona, Verona, Italy
  5. Lindsley F. Kimball Research Institute, New York Blood Center, New York, USA
  6. Immunology, Monash University, Melbourne, VIC, Australia

Red blood cells (RBC) are essential for transport and exchange of O2/CO2 around the body. Two major multi-protein complexes are critical for RBC integrity as they bridge the lipid bilayer and connect integral membrane proteins to the cytoskeleton (composed of α/β-spectrin and actin), designated as the ankyrin complex and the 4.1R complex. Both complexes encompass the anion exchanger Band3, which is essential for CO2 removal by exchanging chloride for bicarbonate. Its exchange activity is mediated by its C-terminal transmembrane domain, while its N-terminal cytosolic region anchors many proteins including ankyrin (linking directly to α/β-spectrin), glycophorin C, adducin, Band4.1, deoxy-haemoglobin (deoxy-Hb) and Duffy. Lyn and Syk tyrosine kinases are responsible for phosphorylating Band3 in response to stress stimulation (oxidation, osmotic and physical). However, apart from this aspect of RBC biochemistry, not much is known of signalling networks in RBCs. We have recently shown that excessive Lyn activity (Lynup/up mice) causes formation of RBC acanthocytes (RBC with thorn-like protrusions). Acanthocytes arise in several conditions when there are alterations to membrane structural proteins. Indeed, patients with chorea acanthocytosis (ChAc) display circulating acanthocytes and possess abnormally high Lyn activity in their RBCs, which is the cause of their acanthocytic morphology. Proteomic analysis of ChAc RBCs showed altered phosphorylation of Band3, ankyrin and β-spectrin, which affects cross-talk with adducin. Significantly, altering Lyn levels and activity has important effects on RBC biology in response to stress stimulation including in vivo half-life, in vitro viability and integrity, and membrane protein association. We have used several proteomic approaches to investigate the intricacies of signaling networks controlling the RBC membrane/cytoskeletal. Using quantitative iTRAQ as well as phospho-tyrosine-peptide mass-spectrometry we have identified specific pY sites in Band3, Band4.1, α/β-spectrin, and ankyrin mediated by Lyn/Src kinases as well as quantitative changes in membrane/cytoskeletal protein associations regulated by Lyn.