Spectrin and other membrane-skeletal components in human red blood cells of different age

Abstract

Background: Old human red blood cells (RBCs) have a reduced surface area with respect to young RBCs. If this decrease occurred through the release of vesicles similar to the spectrinfree vesicles that are shed in vitro under different experimental conditions or during storage, there would be no decrease of membrane-skeleton, but only of lipid bilayer surface area, during RBC ageing in vivo. However, we observed a decrease in spectrin and other membraneskeletal proteins in old RBCs. Because RBCs contain components of the ubiquitin-proteasome system and other hydrolytic systems for protein degradation, we asked whether increased membrane-skeleton fragments could be detected in older RBCs. Methods: Four different anti-spectrin antibodies and an antibody anti-ubiquitin conjugates were used to analyse, by Western blotting, fragments of spectrin and other proteins in RBCs of different age separated in density gradients and characterized for their protein 4.1a/4.1b ratio as a cell age parameter. Results: spectrin fragments do exist in RBCs of all ages, they represent a minute fraction of all spectrin, are membrane-bound and not cytoplasmic and do not increase with cell age. Besides spectrin, other membrane-skeletal components decrease with cell age. Conclusion: Observed results challenge the commonly accepted view that decrease in cell membrane throughout RBC life in vivo occurs via the release of spectrin-free vesicles.