Biogenesis of the Red Blood Cell Membrane in Health and Disease
Tim Satchwell Postdoctoral Research Associate No conflicts of interest to declare Biogenesis of the Red Blood Cell Membrane in Health and Disease
CD47 ICAM4
GPA GPB RhAG/ Rh30
band 3
band 3 MEMBRANE
protein 4.2
ankyrin
Tim Satchwell The Erythrocyte Membrane
Rigid yet highly deformable Cytoskeleton – hexagonal spectrin lattice Vertical linkages to major multiprotein complexes in the membrane
Liu et al 1987
CD47 ICAM4
GPA GPB RhAG/ Rh30 band 3
band 3 MEMBRANE
protein 4.2
ankyrin
Mankelow et al, BCMD, 2012 Erythropoiesis
Takes place in the bone marrow in vivo – 2 million erythrocytes per second
Erythroblasts mature over 7 days – decrease in cell volume, haemoglobinise, nuclear condensation
Enucleation and loss of organelles
Synthesise, deliver and assemble membrane complexes, remodel cytoskeleton
basophilic polychromatic orthochromatic enucleating nascent reticulocyte erythrocyte proerythroblast erythroblast erythroblast erythroblast normoblast reticulocyte An in vitro system of erythropoiesis
Erythroid Progenitor Expansion Curve 10000 Epo, SCF, Dex, IGF1, cholesterol mix 1000 PERCOLL DIFFERENTIATION
100
Cells (x10^6) Cells EXPANSION High Epo, insulin,
10 transferrin,
+ IL3 human serum, thyroid hormone 1 0 2 4 6 8 10 12 14 16 18 20 22 Days
van den Akker et al, Haematologica 2010; Satchwell et al Blood 2011 Monitoring Protein Expression, Localisation and Interactions
Band 3 GPA Rh RhAG CD47 GPC CD71 c-kit 0 24 48 72 96 120 144 168 Median fluorescence (a.u.) intensity fluorescence Median Time in differentiation (hours)
0 24 48 72 120 144 anti-band 3
anti-protein 4.2
Band 3 Immunoprecipitation Disrupted Complex Assembly in Hereditary Haemolytic Anaemias CD47 ICAM4 GPA GPB RhAG/ Rh30 band 3 band 3 MEMBRANE
protein 4.2 Mutations/absences in band 3, ankyrin and ankyrin protein 4.2 result in hereditary spherocytosis Studies of secondary protein deficiencies in patient erythrocytes have informed models of the RBC membrane ? Difficulties associated with studies of erythropoiesis have hampered mechanistic understanding of protein loss Uncovering the mechanisms of protein loss in disrupted erythroid complex assembly: a band 3 null tale
Unique band 3 null patient – homozygous V488M band 3 Coimbra Multiple secondary protein deficiencies Patient completely transfusion dependent
CD47 ICAM4
GPA GPB RhAG/ Rh30 band 3
band 3 ? X X MEMBRANE
protein 4.2
ankyrin Bruce et al, Blood 2003 B3Coimbra Key questions… How does loss of band 3 impact upon expression and stability of proteins known to associate in erythrocytes as the membrane develops during erythropoiesis? When does band 3 dependency establish? How and where are associated proteins lost? What are the properties of band 3 that dictate its role in complex assembly and stability? CD47 ICAM4
GPA GPB RhAG/ Rh30 band 3
band 3 ? X X MEMBRANE
protein 4.2
ankyrin B3Coimbra How does absence of band 3 influence surface expression of associated proteins during erythropoiesis?
Protein Antibody B3Coimbra Mean Surface Expression (% of control) Gross expected reductions in reticulocyte band 3 BRIC71 0.3 ± 0.1 expression of band 3 associated proteins band 3 BRIC200 0.2 ± 0.2
Wrb BRIC13 95 3.1± 0.3 anti- band 3 (N-terminus) GPA BRIC256 39.5 ± 3.6 Discrepancy between reticulocytes and CD47 BRIC32 34.2 ± 2.7 95 anti- band 3 (C-terminus) Rh BRIC69 40.1 ± 2.0 erythrocytes remains RhAG LA1818 67.5 ± 2.5 anti-protein 4.2 CD44 BRIC222 72 132.0 ± 6.7 GPC BRIC4 94.9 ± 6.2
band 3 (BRIC71) band 3 (BRIC200) GPA (BRIC256) GPC (BRIC4) 60 40 90 30 35 80 50 25 30 70 40 60 20 25 50 30 20 15 40 15
arbitrary units) arbitrary 20 30 10
3 10 20 10 5 5 10 0 0 0 0 0 24 48 72 96 120 144 168 0 24 48 72 96 120 144 168 0 24 48 72 96 120 144 168 0 24 48 72 96 120 144 168
CD47 (BRIC32) Rh (BRIC69) CD44 (BRIC222) 25 18 16 control 16 14 20 14 12 B3Coimbra 12 15 10 reticulocytes 10 8 8 10 6 6
Mean fluorescent intensity (x10 intensity fluorescent Mean 4 5 4 2 2 0 0 0 0 24 48 72 96 120 144 168 0 24 48 72 96 120 144 168 0 24 48 72 96 120 144 168
Differentiation timepoint (hours) What about enucleation?
reticulocytes nuclei erythroblasts
erythroblasts reticulocytescontrol B3Coimbra band 3
FSC Integral membrane proteins band 3
reticulocytes Wrb nuclei (pyrenocytes) nuclei GPA nucleus erythroblasts BRIC4 (GPC) BRIC4
BRIC256 (GPA) BRIC256 Reticulocyte Nucleus
APC - reticulocyte
CD47 APC - Hoechst Rh Hoechst Hoechst RhAG Integral membrane proteins Spectrin cytoskeleton CD44
GPC
0% 20% 40% 60% 80% 100% 0% 20% 40% 60% 80% 100% Reticulocyte Nucleus
Spectrin cytoskeleton Griffiths et al, Blood 2012 Identifying temporal and mechanistic details of protein loss in hereditary spherocytosis
Protein Partitioning Disrupted Complex during Enucleation Assembly RhAG LAMP-1 DAPI merge
Disrupted assembly of band 3 control tetrameric complex
ANK KD 1048
720 Unstable 480 Tetrameric band 3 RhAG 242 complexes absent internalised 146 ANK KD
ER anti-ankyrin ankyrin band 3 Lysosomal RhAG anti-RhAG degradation
anti-GAPDH Rapid degradation of protein 4.2 Manipulating red blood cell protein expression: a challenging proposition
Day 0 Day 3 Day 5 Further expansion Day 9 Differentiation Day 16
Lentiviral Selection CD34+ isolation Differentiation Enucleation transduction e.g. FACS/drugs
Express fluorescently tagged wild type/mutant Lentivirus proteins production in HEK293T cells Deplete protein expression using shRNAs Introduce enhanced or novel functionality Band 3 to the rescue?
2. GFP-B3mem (missing cytoskeleton binding sites)
1. GFP-B3 3. GFP-kB3 (tagged wt) (missing ankyrin binding site, retains protein 4.2 binding site) Expression of GFP-band 3 in patient erythroblasts rescues secondary protein deficiencies
GFP band 3 (BRIC200)
91% +ve control B3Coimbra B3Coimbra + GFPB3
GFPB3 +ve T72 erythroblasts
band 3 (BRIC200) CD47 (BRIC32) Rh (BRIC69) GFP
130 anti-band 3 95
72 anti-protein 4.2 APC-BRIC200 APC-BRIC32 APC-BRIC69 Band 3 membrane domain alone is not sufficient to rescue reduced reticulocyte expression of associated proteins
150
125 control
100 B3Coimbra B3Coimbra + GFPB3 B3Coimbra + GFPB3mem 130 75 anti-band 3 95 B3Coimbra + GFPkB3 50 72 anti-protein 4.2 25 MFI percentageof control (%) 0 BRIC71 BRIC200 BRIC13 BRIC256 BRIC32 BRIC69 LA1818 BRIC222 BRIC4 band 3 band 3 Wrb GPA CD47 Rh RhAG CD44 GPC
band 3 (BRIC200) GPA (BRIC256) Rh (BRIC69) RhAG (LA1818) GPC (BRIC4)
control B3C
GFP B3C + GFPB3 B3C + GFPB3mem
BRIC200 BRIC256 BRIC69 LA1818 BRIC4 The cytoskeletal binding domain of band 3 is required for efficient retention of proteins during enucleation
control Integral membrane proteins
B3Coimbra
B3Coimbra + GFPB3 nucleus Reticulocyte Nucleus reticulocyte
B3Coimbra + GFPB3mem
Spectrin B3Coimbra + GFPkB3 cytoskeleton
0% 20% 40% 60% 80% 100% The cytoskeletal binding domain of band 3 is required for efficient retention of proteins during enucleation band 3 GPC Integral membrane proteins control
B3Coimbra
B3C + GFPB3 Reticulocyte Nucleus
B3C + GFPB3mem
B3C + GFPkB3
0% 20% 40% 60% 80% 100% Spectrin 0% 20% 40% 60% 80% 100% cytoskeleton
GPA Rh CD47 RhAG
control
B3Coimbra
B3C + GFPB3
B3C + GFPB3mem
B3C + GFPkB3
0% 20% 40% 60% 80% 100% 0% 20% 40% 60% 80% 100% 0% 20% 40% 60% 80% 100% 0% 20% 40% 60% 80% 100% Summary (Health)
Complex Assembly Erythroblast Enucleation
band 3/GPA
protein 4.2 ankyrin ER
RhAG Rh CD47
ankyrin
Preliminary protein association of key proteins including band 3/protein 4.2 and Rh/RhAG is evident from the onset of expression in basophilic erythroblasts Formation of an ankyrin mediated band 3 complex in the early stages of differentiation stabilises Rh complex components within the plasma membrane Ability to associate with the cytoskeleton is important for retention of band 3 and associated proteins but not the sole determinant Summary (Disease)
Complex Assembly Erythroblast Enucleation
Unstable band 3/GPA Reduced band 3 and associated Tetrameric band 3 RhAG protein reticulocyte retention complexes absent internalised protein 4.2 ankyrin
ER RhAG Rh CD47
Lysosomal RhAG degradation Reticulocyte Maturation
Gross disruption of cytoskeletal Rapid architecture/cytoskletal connectivity degradation of protein 4.2
Secondary protein loss in erythrocyte membrane biogenesis of hereditary spherocytosis patients occurs via multiple mechanisms The absence of core complex components results in membrane destabilisation and degradation of dependent proteins at an early stage Impaired direct or indirect cytoskeletal association results in protein loss due to aberrant protein partitioning during erythroblast enucleation Additional loss of protein occurs in post enucleation reticulocyte maturation Acknowledgements
Ash Toye Emile van den Akker
The Lab Bristol Institute of Transfusion Science Steph Pellegrin Dave Anstee Beth Hawley Lee Bruce Mandy Bell Rosey Mushens Patient Samples …Lisa Satchwell Kat Mordue Paola Bianchi Charlie Severn FACS Richard van Wijk Roz Williamson Andy Herman Jean Delaunay Marjolein Meinders Leticia Ribeiro Joe Hawksworth Molecular Models Lea Hampton O’Neil Nick Burton