Aggregation of band 3 in hereditary ovalocytic red blood cell membranes Electron microscopy and protein rotational diffusion studies

Abstract

Microaggregation of band 3 proteins in hereditary ovalocytic membranes was investigated by rotational diffusion measurements and by electron microscopy. It was previously shown that band 3 in ovalocytic membranes has decreased rotational mobility compared with band 3 in normal cells (Tilley, L., Nash, G.B., Jones, G.L. and Sawyer, W.L. (1991) J. Membr. Biol. 121, 59–66). This result could arise from either altered interactions with cytoskeletal proteins or from band 3 microaggregation. In the present study it was found that removal of spectrin and actin from the membrane had no effect on the rotational mobility of ovalocytic band 3. Additional removal of ankyrin and band 4.1, as well as cleavage of the cytoplasmic domain of band 3 with trypsin, did enhance band 3 mobility, as is the case in the membranes from normal cells. However, the rotational mobility of ovalocytic band 3 was always considerably less than that of normal band 3 under the same conditions. Scanning electron microscopy and low power electron micrographs of freeze-fracture replicas revealed that the surfaces of ovalocytes were more irregular than those of normal erythrocytes. At higher magnification, numerous linearly arranged intramembranous particles were observed on the P-faces of freeze-fractured ovalocytes but not on normal cells. These clusters consist of straight or slightly curved lines of 10–15 particles in single rows. From these results it is deduced that the reduced rotational mobility of band 3 in ovalocytes is a consequence of the formation of microaggregates, which are very probably induced by the mutation in the membrane-bound domain of ovalocytic band 3.

Molecular Basis and Functional Consequences of the Dominant Effects of the Mutant Band 3 on the Structure of Normal Band 3 in Southeast Asian Ovalocytosis

“…Other evidence also suggests the band 3 in SAO membranes has a greater tendency to aggregate than normal band 3 and greater propensity to form tetramers (16,18). Freeze-fracture electron micrographs of SAO red cells also show the presence of unusual linear microaggregates of intramembranous particles that most likely contain band 3 (32). Table 2 and Figure 4 show that the normal band 3 sites not cleaved in band 3 of SAO membranes are distributed over the entire band 3 molecule.…”

Regulation of Band 3 Rotational Mobility by Ankyrin in Intact Human Red Cells

“…Band 3 rotational mobility is constrained in normal RBC membranes by low-affinity and high-affinity binding interactions (24,31,33,34,(41)(42)(43)(44)(45)(58)(59)(60). The rotationally immobile band 3 fraction apparently represents individual band 3 molecules bound with high affinity to ankyrin.…”

“…The rapidly rotating band 3 fraction is presumed to consist of free band 3 dimers, tetramers, and possibly higher order oligomers (34,36). In some pathologic RBCs, the rotationally immobile band 3 fraction contains clustered or linearly aggregated band 3 molecules (31,(41)(42)(43), but in normal cells this fraction is likely to represent band 3 molecules bound with high affinity to ankyrin. The slowly rotating band 3 fraction is less well characterized, however.…”

The in vitro red blood cell microvesiculation exerts procoagulant activity of blood cell storage in Southeast Asian ovalocytosis

“…The decrease in ATP levels during in vitro storage was hypothesized to be an initial step in membrane vesiculation and band 3 protein rearrangement [ 19 ]. Previous data showed that clustering and microaggregation of the Band 3 protein were observed during cold storage [ 40 ]. Karon et al reported that storage at cold temperatures altered Band 3 oligomericity in the RBC membrane [ 16 ].…”