Alterations of spectrin assembly in the red cell membrane: Functional consequences

Abstract

The internal side of the red cell membrane is laminated by fibres of spectrin tetramers linked into a two-dimensional cytoskeleton by actin oligomers and polypeptide 4.1. We have investigated abnormalities in associations among the skeletal and integral membrane proteins and their functional consequences. We employed spectrin extraction from the membrane and analysis of its oligomeric states by gel filtration, oxidative cross-linking (XL) of membrane protein neighbours and we examined shape and structural stability of red cells and membrane skeletons.

We describe three cytoskeletal perturbations and their functional consequences. (1) Defective association of membrane-bound spectrin dimers (Sp-D) into tetramers (Sp-T). This is found in fresh red cells from patients with hereditary pyropoikilocytosis (HPP) and some patients with hereditary ellip-tocytosis (HE). In normal red cell ghosts Sp-T are transformed to Sp-D by incubation (37°C) at low ionic strength. Cytoskeletons prepared from all of the above ghosts with increased Sp-D Sp-T ratio (e.g. fresh HE, HPP ghosts or normal ghosts incubated at low ionic strength) are markedly unstable in vitro. We suggest that the skeletal instability may underlie the decreased stability of intact HPP, HE red cells both in vitro and in vivo. (2) Spectrin aggregation in the membrane as revealed by membrane protein XL. Such aggregation is induced by exposure of normal red cells and HPP red cells (which are more susceptible than normal to thermal damage in vitro) to temperatures >49°C and 45°C respectively. At these temperatures, normal and HPP erythrocytes and their membrane cytoskeletons exhibit a marked spontaneous fragmentation. (3) Spontaneous oxidative cross-linking (XL). Such XL takes place in aerobically ATP depleted erythrocytes and results in a stabilization (‘fixation’) of their echinocytic shape. The propensity of such cells to recover the discocytic shape after repletion of intracellular ATP stores is restored by reducing agents, which cleave the intermolecular disulphide cross-links.

These data demonstrate that cytoskeletal protein associations are of critical significance in a control of structural stability of the membrane and reversible shape transformations.

Key Words:

• membrane fragmentation
• protein cross-linking
• poikilocytosis
• cytoskeleton
• spectrin aggregation
• spectrin dimers and tetramers.