Abstract
Red blood cell (RBC) deformability is altered in inherited RBC disorders but the mechanism behind this is poorly understood. Here, we explored the molecular, biophysical, morphological, and functional consequences of α-spectrin mutations in a patient with hereditary elliptocytosis (pEl) almost exclusively expressing the Pro260 variant of SPTA1 and her mother (pElm), heterozygous for this mutation. At the molecular level, the pEI RBC proteome was globally preserved but spectrin density at cell edges was increased. Decreased phosphatidylserine vs. increased lysophosphatidylserine species, and enhanced lipid peroxidation, methemoglobin, and plasma acid sphingomyelinase (aSMase) activity were observed. At the biophysical level, although membrane transversal asymmetry was preserved, curvature at RBC edges and rigidity were increased. Lipid domains were altered for membrane:cytoskeleton anchorage, cholesterol content and response to Ca2+ exchange stimulation. At the morphological and functional levels, pEl RBCs exhibited reduced size and circularity, increased fragility and impaired membrane Ca2+ exchanges. The contribution of increased membrane curvature to the pEl phenotype was shown by mechanistic experiments in healthy RBCs upon lysophosphatidylserine membrane insertion. The role of lipid domain defects was proved by cholesterol depletion and aSMase inhibition in pEl. The data indicate that aberrant membrane content and biophysical properties alter pEl RBC morphology and functionality.
Highlights
During its lifetime, the red blood cell (RBC) undergoes extensive deformations needed to pass through narrow capillaries to deliver oxygen to tissues
Such exceptional deformability relies on RBC intrinsic features, including a biconcave shape due to excess plasma membrane surface vs. cytoplasmic volume, a finely regulated cytoplasmic viscosity controlled by hemoglobin concentration, and a cytoskeleton composed of a meshwork of spectrin tetramers linked to the membrane by the 4.1R- and ankyrin-based anchorage complexes [1,2]
Since GM1- and sphingomyelin-enriched domains were suggested to contribute to Ca2+ exchange [10], we explored the potential consequences of membrane lipid alterations in patient with hereditary elliptocytosis (pEl) RBCs for Ca2+ exchanges upon RBC deformation in stretchable PDMS chambers [10]
Summary
The red blood cell (RBC) undergoes extensive deformations needed to pass through narrow capillaries to deliver oxygen to tissues Such exceptional deformability relies on RBC intrinsic features, including a biconcave shape due to excess plasma membrane surface vs cytoplasmic volume, a finely regulated cytoplasmic viscosity controlled by hemoglobin concentration, and a cytoskeleton composed of a meshwork of spectrin tetramers linked to the membrane by the 4.1R- and ankyrin-based anchorage complexes [1,2]. We took benefit from our expertise in the healthy RBC membrane analysis using complementary imaging, lipidomic and biophysical approaches to elucidate the mechanism behind morphology and functionality defects of RBCs in elliptocytosis. At the morphological and functional levels, we determined the RBC size and circularity, membrane Ca2+ exchanges using PDMS chambers, intracellular Ca2+ content and RBC fragility through hemoglobin release
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