Specific Alterations in Membrane Lipid Levels Correlate with the Degree of Sickling in a Humanized Mouse Model

Sickle cell Disease (SCD) is caused by a mutation at position 6 of the beta- globin gene. The consequences of this altered hemoglobin (HbS) include anemia as well as injuries to multiple organs including the brain, lungs and vasculature. The design of therapies to treat and cure SCD has been slow. Hydroxyurea, the only FDA-approved drug to treat SCD, was approved in 1998. In addition, the greatest number of SCD patients reside in sub-Saharan Africa where there is an acute need for low-cost, systemic therapies. Our goal is to discover novel anti-sickling targets for which affordable and easy to administer drugs can be developed.

Band 3 is the most abundant protein in the mammalian erythrocyte membrane. Band 3 is a transmembrane protein that functions both as an anion exchange channel and as the junction where ankyrin links the spectrin/actin cytoskeleton to the membrane. We have previously shown that Band 3 acts as an "oxygen sensor” in red cells, through the reversible binding of deoxygenated hemoglobin (deoxy Hb) and the enzymes involved in glycolysis to the cytoplasmic domain of Band 3 (CdB3). We generated transgenic mice in which the sequence encoding the first 35 amino acids of the wild-type human CdB3 replaced the corresponding mouse Band 3 sequences in the Slc4a1 gene (WT). We created a second mutant line in which human amino acids 12-21 were deleted, removing the deoxy Hb binding site (-Hb), and a third line in which amino acids 1-11 were deleted, creating a high affinity binding site for deoxy Hb (++Hb). Erythrocytes from the mutant lines were insensitive to oxygen concentration resulting in changes in oxygen dependent deformability and other physical properties compared to the WT line (Chu et al. Blood 128, 2016, Zheng et al. JBC 294, 2019, Zhou et al. Sci. Adv. 5, 2019).

We crossed our three humanized Band 3 strains to the Townes SCD mouse model, generating human Hb-AA, -AS and -SS mice homozygous for each of the humanized CdB3 genes. We measured the percentage of sickled cells and the rate of sickling of SS red cells in each strain (Dunkelberger et al., J. Phys. Chem. B 122, 2018). We observed that -Hb-SS mice showed an accelerated rate of sickle cell formation and a higher percent of sickled cells than WT-SS mice (p<0.01). Conversely, ++Hb-SS mice showed an inhibition of both the rate of sickling and the precent of sickled cells compared to WT-SS mice (p<0.05).

We hypothesized that the Hb binding mutations alter erythrocyte physiology in a way that either promotes or inhibits sickle cell formation. To test this hypothesis, we performed a lipidomic profile of erythrocytes from Hb-AA, -AS and -SS mice of the WT-, -Hb- and ++Hb- strains as well as WT-AA cells from animals that were phlebotomized to create an anemic state similar to that seen in HbSS animals (anemic control). A panel of 75 membrane lipids were quantified by LC-MS/MS using a Waters C18 column (Xbridge BEH, 2.1 mm x 150 mm, 2.5 uM particle size) and an API 4500 triple quadrupole mass spectrometer (Sciex). Stable isotope controls of deuterated polar lipid and fatty acid standards were added during sample extraction and used for peak normalization. The data were collected in MRM mode and normalized peak areas were analyzed using Partial Least Squares-Dimensional Analysis (PLS-DA). PLS-DA identified lipids present at different levels in SS cells compared to AA, AS and anemic control cells. We then used the PLS-DA plots to identify the lipids with the greatest Variable Importance in the Projection (VIP score) among SS-specific lipids in WT-, -Hb- and ++Hb- SS cells. A VIP score of <1 is considered important. We identified 8 lipids with scores of 1.5 or greater that correlated with the percentage and rate of sickling. The levels of 1-decanoyl-2-tetracosanoyl-sn-glycero-3-phosphocholine (PC 34:0; VIP 2.6), PC 38:2 (1.7), PC 36:1 (1.6) and PC 36:0 (1.5) levels were highest in -Hb-SS cells, intermediate in WT-SS cells and lowest in ++Hb-SS cells. The levels of 1-Arachidonyl-glycero-3-posphocholine (Lyso PC 20:0; 2.3), Oleic acid (1.9), Arachidonic acid (1.9) and PC 40:1 (1.5) were highest in ++Hb-SS cells, intermediate in WT-SS cells and lowest in -Hb-SS cells. We conclude that elevated or reduced levels of lipids such as PC 34:0 or Lyso PC 20:0 could be useful biomarkers for determining the severity of SCD. It is also possible that pharmacological or dietary manipulation of the levels of PC 34:0 and/or Lyso PC 20:0 could be a novel method to treat SCD.

No relevant conflicts of interest to declare.