Comparative Profiling of Red Blood Cells from the Beta-Adducin Knockout Mouse Model of Hemolytic Anemia Using Label Free Differential Proteomics

Abstract Inherited hemolytic anemia (spherocytosis or elliptocytosis) is one of the most common inherited diseases. While mild to severe inherited hemolytic anemias can arise from defects in the red blood cell (RBC) membrane skeleton, fundamental questions remain unanswered surrounding the clinical variability of known red blood cell mutations. To identify candidate proteins involved in hemolytic anemia pathophysiology, we utilized a label-free comparative approach to detect differences in RBCs from normal and beta-adducin (ADD2KO) knock-out mice. For each genotype, whole blood was taken from independent biological replicates and RBCs were purified using cellulose acetate chromatography. The isolated RBCs were lysed to generate RBC ghosts whose protein complements were digested with trypsin. For each biological replicate, three replicate runs utilizing 0.5 μg digested protein were performed via microcapillary liquid chromatography coupled with tandem mass spectrometry. Using the recently developed software package CRAWDAD, we detected 7 proteins that were decreased and 31 proteins with a greater abundance in the beta-adducin knock-out RBC ghosts. Differences were detected for previously known membrane skeleton components, including the predicted absence of beta-adducin and decrease in alpha-adducin. The actin binding protein capping protein (actin filament) muscle Z-line alpha 1 was increased along with ankyrin 1, spectrin alpha, tropomyosin, and glycophorin C. Interestingly, differences were also detected for the protein oxidative stress-responsive 1 and the uncharacterized protein C10orf58 which contains a thioredoxin motif. While the protein differences span a broad range of cellular processes, some of the proteins are attractive candidates for modifiers of disease severity. This label free approach is the first demonstration of comprehensively analyzing wild type versus kockout mice that circumvents the need for metabolic or chemical labeling with isotopes. This new approach adds a valuable tool to the list of standard assays for analysis of RBCs in cases of hereditary hemolytic anemia.

Download Full-text

Direct Measurement of the Area Expansion and Shear Moduli of the Human Red Blood Cell Membrane Skeleton

Biophysical Journal ◽

10.1016/s0006-3495(01)75678-0 ◽

2001 ◽

Vol 81 (1) ◽

pp. 43-56 ◽

Cited By ~ 116

Author(s):

Guillaume Lenormand ◽

Sylvie Hénon ◽

Alain Richert ◽

Jacqueline Siméon ◽

François Gallet

Keyword(s):

Cell Membrane ◽

Blood Cell ◽

Direct Measurement ◽

Red Blood Cell ◽

Membrane Skeleton ◽

Shear Moduli ◽

Red Blood Cell Membrane ◽

Area Expansion

Download Full-text

Label-Free Transient Absorption Microscopy for Red Blood Cell Flow Velocity Measurement in Vivo

Analytical Chemistry ◽

10.1021/acs.analchem.7b01952 ◽

2017 ◽

Vol 89 (19) ◽

pp. 10120-10123 ◽

Cited By ~ 6

Author(s):

Tao Chen ◽

Yanyi Huang

Keyword(s):

Blood Cell ◽

Flow Velocity ◽

Red Blood Cell ◽

Velocity Measurement ◽

Transient Absorption ◽

Label Free ◽

Flow Velocity Measurement

Download Full-text

Sickle Cell Disease Model Mice Lacking 2,3-Dpg Show Reduced RBC Sickling and Improvements in Markers of Hemolytic Anemia

Blood ◽

10.1182/blood-2020-142052 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 27-28

Author(s):

Kelly M. Knee ◽

Amey Barakat ◽

Lindsay Tomlinson ◽

Lila Ramaiah ◽

Zane Wenzel ◽

...

Keyword(s):

Sickle Cell Disease ◽

Blood Cell ◽

Hemolytic Anemia ◽

Sickle Cell ◽

Red Blood Cell ◽

Organ Damage ◽

Complete Elimination ◽

Cell Disease ◽

The Impact ◽

2,3 Dpg

Sickle cell disease (SCD) is a severe genetic disorder caused by a mutation in hemoglobin (b6Glu-Val), which allows the mutant hemoglobin to assemble into long polymers when deoxygenated. Over time, these polymers build up and deform red blood cells, leading to hemolytic anemia, vaso-occlusion, and end organ damage. A number of recent therapies for SCD have focused on modulating the mutant hemoglobin directly, however, reduction or elimination of 2,3-DPG to reduce Hb S polymerization and RBC sickling has recently been proposed as a therapeutic strategy for SCD. Current clinical studies focus on activation of pyruvate kinase to reduce 2,3-DPG, however, direct targeting of the enzyme which produces 2,3-DPG; Bisphosphoglycerate Mutase (BPGM) may also be possible. In this study we evaluate the impact of elimination of 2,3-DPG on SCD pathology by complete knockout of BPGM in Townes model mice. Animals with complete knockout of BPGM (BPGM -/-) have no detectable 2,3-DPG, while animals that are heterozygous for BPGM (BPGM -/+) have 2,3-DPG levels comparable to Townes mice. Western Blot analysis confirms that BPGM -/- animals completely lack BPGM, while BPGM -/+ animals have BPGM levels that are nearly equivalent to Townes mice. As expected from the lack of 2,3-DPG, BPGM -/- animals have increased oxygen affinity, observed as a 39% decrease in p50 relative to Townes mice. Complete elimination of 2,3-DPG has significant effects on markers of hemolytic anemia in BPGM -/- mice. Mice lacking 2,3-DPG have a 60% increase in hemoglobin (3.7 g/dL), a 53% increase in red blood cell count, and a 29% increase in hematocrit relative to Townes mice. The BPGM -/- mice also have a 57% decrease in reticulocytes, and a 61% decrease in spleen weight relative to Townes animals, consistent with decreased extramedullary hematopoiesis. Consistent with the reduction in hemolysis, BPGM -/- animals had a 59% reduction in red blood cell sickling under robust hypoxic conditions. BPGM -/+ animals had hemoglobin, RBC, and hematocrit levels that were similar to Townes animals, and a similar degree of RBC sickling to Townes mice. Liver phenotype was similar across all variants, with areas of random necrosis observed in BPGM -/-, BPGM -/+ and Townes mice. Higher percentages of microcytic and/or hyperchromic RBCs were observed in BPGM -/- animals relative to BPGM -/+ or Townes animals. These results suggest that modulation of 2,3-DPG has a positive effect on RBC sickling and hemolytic anemia, which may have therapeutic benefits for SCD patients. However, the lack of improvement in organ damage suggests that modulation of 2,3-DPG alone may not be sufficient for complete elimination of SCD phenotypes, and further investigation of this therapeutic avenue may be necessary. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Interleukin-10–mediated regulatory T-cell responses to epitopes on a human red blood cell autoantigen

Blood ◽

10.1182/blood-2002-05-1383 ◽

2002 ◽

Vol 100 (13) ◽

pp. 4529-4536 ◽

Cited By ~ 54

Author(s):

Andrew M. Hall ◽

Frank J. Ward ◽

Mark A. Vickers ◽

Lisa-Marie Stott ◽

Stanislaw J. Urbaniak ◽

...

Keyword(s):

T Cell ◽

Blood Cell ◽

Hemolytic Anemia ◽

Red Blood Cell ◽

Regulatory T Cell ◽

Autoimmune Hemolytic Anemia ◽

T Cell Responses ◽

Interleukin 10 ◽

Cell Responses ◽

Immune Mediated

Regulatory T cells have been shown to control animal models of immune-mediated pathology by inhibitory cytokine production, but little is known about such cells in human disease. Here we characterize regulatory T-cell responses specific for a human red blood cell autoantigen in patients with warm-type autoimmune hemolytic anemia. Peripheral blood mononuclear cells from patients with autoimmune hemolytic anemia were found either to proliferate and produce interferon-γ or to secrete the regulatory cytokine interleukin 10 when stimulated in vitro with a major red blood cell autoantigen, the RhD protein. Flow cytometric analysis confirmed that the majority of the responding cells were of the CD4+phenotype. Serial results from individual patients demonstrated that this bias toward proliferative or interleukin-10 responses was unstable over time and could reverse in subsequent samples. Epitope mapping studies identified peptides from the sequence of the autoantigen that preferentially induced interleukin-10 production, rather than proliferation, and demonstrated that many contain naturally processed epitopes. Responses to such peptides suppressed T-cell proliferation against the RhD protein, an inhibition that was mediated largely by interleukin 10 and dependent on cytotonic T lymphocyte–associated antigen (CTLA-4) costimulation. Antigenic peptides with the ability to stimulate specific regulatory cells may represent a new class of therapeutic agents for immune-mediated disease.

Download Full-text

Three cases of hereditary nonspherocytic hemolytic anemia associated with red blood cell glutathione deficiency

Blood ◽

10.1182/blood.v87.5.2071.2071 ◽

1996 ◽

Vol 87 (5) ◽

pp. 2071-2074 ◽

Cited By ~ 24

Author(s):

A Hirono ◽

H Iyori ◽

I Sekine ◽

J Ueyama ◽

H Chiba ◽

...

Keyword(s):

Blood Cell ◽

Hemolytic Anemia ◽

Red Blood Cell ◽

Japanese Patients ◽

Glutathione Synthetase ◽

Synthetase Activity ◽

Glutathione S Transferase ◽

Mild Deficiency ◽

Glutathione Deficiency ◽

Generalized Type

Abstract Three unrelated Japanese patients with chronic nonspherocytic hemolytic anemia wer found to have marked deficiency of red blood cell (RBC) reduced glutathoine (GSH) (4.4%, 13.1%, and 6.9% of normal, respectively). A panel of RBC enzyme assays showed that one patient had decreased glutathione synthetase activity and the other two were moderately deficient in gamma-glutamylcystine synthetase. Some family members of each patient showed mild deficiency of the respective enzymes. RBCs of these patients also showed a decreased level of glutathione-S-transferase as in previously described GSH-deficient cases. Hemolytic anemia was their only manifestation, and neither 5- oxoprolinemia nor 5-oxoprolinuria, which are usually associated with to generalized type of glutathione synthetase deficiency, was noted in our patients.

Download Full-text

Molecular Modelling of Human Red Blood Cell Pyruvate Kinase: Structural Implications of a Novel G1091 to A Mutation Causing Severe Nonspherocytic Hemolytic Anemia

Blood ◽

10.1182/blood.v90.12.4987 ◽

1997 ◽

Vol 90 (12) ◽

pp. 4987-4995 ◽

Cited By ~ 20

Author(s):

Wouter W. van Solinge ◽

Rob J. Kraaijenhagen ◽

Gert Rijksen ◽

Richard van Wijk ◽

Bjarne B. Stoffer ◽

...

Keyword(s):

Blood Cell ◽

Pyruvate Kinase ◽

Hemolytic Anemia ◽

Red Blood Cell ◽

Molecular Modelling ◽

Human Liver ◽

The Other ◽

Salt Bridges ◽

Compound Heterozygotes ◽

Allosteric Properties

Abstract We present a novel G1091 to A mutation in the human liver and red blood cell (RBC) pyruvate kinase (PK) gene causing severe hemolytic anemia. In two families, three children were severely PK-deficient compound heterozygotes exhibiting the G1091 to A mutation and a common G1529 to A mutation on the other allele. In one family, the mother, a G1091 to A heterozygote, later had a second baby with a new husband, also a G1091 to A carrier. The baby was homozygous for the G1091 to A mutation and died 6 weeks after birth from severe hemolysis. Both mutant alleles were expressed at the RNA level. The G1091 to A mutation results in the substitution of a conserved glycine by an aspartate in domain A of RBC PK, whereas the G1529 to A mutation leads to the substitution of a conserved arginine residue with glutamine in the C-domain. Molecular modelling of human RBC PK, based on the crystal structure of cat muscle PK, shows that both mutations are located outside the catalytic site at the interface of domains A and C. The mutations are likely to disrupt the critical conformation of the interface by introducing alternative salt bridges. In this way the Gly364 to Asp and Arg510 to Gln substitutions may cause PK deficiency by influencing the allosteric properties of the enzyme.

Download Full-text

Contribution of the band 3-ankyrin interaction to erythrocyte membrane mechanical stability

Blood ◽

10.1182/blood.v77.7.1581.1581 ◽

1991 ◽

Vol 77 (7) ◽

pp. 1581-1586 ◽

Cited By ~ 71

Author(s):

PS Low ◽

BM Willardson ◽

N Mohandas ◽

M Rossi ◽

S Shohet

Keyword(s):

Blood Cell ◽

Red Blood Cell ◽

Mechanical Stability ◽

Membrane Integrity ◽

Membrane Skeleton ◽

Band 3 ◽

Cell Membrane Integrity ◽

The Face ◽

Cell Stability

Abstract In an effort to evaluate the role of the band 3-ankyrin linkage in maintenance of red blood cell membrane integrity, solution conditions were sought that would selectively dissociate the band 3-ankyrin linkage, leaving other membrane skeletal interactions intact. For this purpose erythrocytes were equilibrated overnight in nutrient-containing buffers at a range of elevated pHs and then examined for changes in mechanical stability and membrane skeletal composition. Band 3 was found to be released from interaction with the membrane skeleton over a pH range (8.4 to 9.5) that was observed to dissociate the band 3- ankyrin interaction in vitro. In contrast, all other membrane skeletal associations appeared to remain intact up to pH 9.3, after which they were also seen to dissociate. Whereas hemolysis of mechanically unstressed cells did not begin until approximately pH 9.3, where the membrane skeletons began to disintegrate, enhanced fragmentation of shear stressed membranes was seen to begin near pH 8, where band 3 dissociation was first observed. Furthermore, the shear-induced fragmentation rate was found to reach a maximum at pH 9.4, ie, where band 3 dissociation was essentially complete. Based on these correlations, we hypothesize that the band 3-ankyrin linkage of the membrane skeleton to the lipid bilayer is essential for red blood cell stability in the face of mechanical distortion but not for cellular integrity in the absence of mechanical stress.

Download Full-text