Hereditary spherocytosis of man. Altered binding of cytoskeletal components to the erythrocyte membrane

Abstract

Human erythrocytes possess a lattice work of extrinsic proteins on the inner face of the membrane (;cytoskeleton') that maintains the shape and deformability of the cell. The major proteins of the cytoskeleton are spectrin and actin, which are attached to the membrane by protein bands 2.1 (;ankyrin') and 4.1. The interactions of spectrin/actin with erythrocyte membranes from normal subjects and from patients with hereditary spherocytosis (HS) have been studied by using an air-driven ultracentrifuge, which can rapidly separate membranes from soluble proteins (150000g for 30s). The total amount of spectrin/actin in HS and normal ghosts is similar. However, the rate of dissociation of spectrin and actin from HS erythrocyte membranes at low ionic strength is significantly lower than that observed for normal membranes. Spectrin and actin isolated from either HS or normal membranes re-associated in a similar manner to spectrin/actin-depleted vesicles prepared from normal cells. Scatchard analysis showed an average binding capacity of 278mug/mg of membrane protein. However, spectrin/actin-depleted vesicles prepared from HS cells bound significantly less spectrin/actin prepared from either the normal or abnormal cells (average binding capacity 158mug/mg of membrane protein). The defect was defined further by studying the cytoskeleton obtained by Triton X-100 extraction of membranes. Under conditions of low ionic strength cytoskeletons prepared from HS membranes dissociated more slowly than those prepared from normal membranes, and only 80% of the protein from HS cytoskeletons could be solubilized after 180min compared with 100% for normal cytoskeletons. The difference between HS and normal membranes, which persists in isolated cytoskeletons, suggests that alterations in either the primary structure or the degree of phosphorylation of protein bands 2.1 or 4.1 may be central to the molecular basis of hereditary spherocytosis.

Diminished Extractable Spectrin in the Erythrocytes of a Patient with ‘Sporadic’ Hereditary Spherocytosis

“…A similar picture was reported in 2 out of 7 kindreds [12] and in 10 out of 11 probands [13]. Defective binding of the skeleton to the red cell mem brane was described in 7 spherocytics [14] and also in 1 proband [15].…”

Hereditary spherocytosis of man. Defective cytoskeletal interactions in the erythrocyte membrane

“…Our previous studies have shown that spectrin and actin dissociate more slowly from HS ghosts than from normal ghosts in low-ionic-strength media (Hill et al, 1981). We have now extended our study to 12 HS families and all have shown this consistent abnormality.…”

“…Selection of HS patients. Patients from five different families were diagnosed as having HS as previously described (Hill et al, 1981). All but one were post-splenectomy and had normal reticulocyte counts; all had increased red cell osmotic fragility.…”

“…Fresh venous blood from normal or HS subjects was collected into heparinized tubes. Erythrocyte ghosts were prepared by hypo-osmotic lysis as described previously (Hill et al, 1981). Spectrin tetramers were prepared by the method of Cohen & Foley (1980), which minimizes contamination of the preparation with band 4.1.…”

“…Dissociation of shells. The time course of the dissociation of shells at low ionic strength was followed using a procedure similar to that described for the dissociation of cytoskeletons (Hill et al, 1981). Shells (approx.…”