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Aggregation of band 3 in hereditary ovalocytic red blood cell membranes. Electron microscopy and protein rotational
A Che1, R J Cherry, L H Bannister
1Department of Chemistry and Biological Chemistry, University of Essex, Wivenhoe Park, Colchester, UK.
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.
Insights
Hereditary ovalocytosis involves band 3 protein microaggregation, reducing its membrane mobility. This study confirms microaggregation, not altered cytoskeletal interactions, causes reduced band 3 protein movement in ovalocytic cells.
Area of Science:
- Biochemistry
- Cell Biology
- Membrane Biophysics
Background:
- Band 3 protein in red blood cells is crucial for membrane structure and function.
- Hereditary ovalocytosis is characterized by altered red blood cell shape and membrane properties.
- Previous studies indicated reduced rotational mobility of band 3 in ovalocytic membranes.
Purpose of the Study:
- To investigate the cause of reduced band 3 protein rotational mobility in hereditary ovalocytosis.
- To differentiate between altered protein interactions and band 3 microaggregation as the underlying mechanism.
Main Methods:
- Rotational diffusion measurements of band 3 protein.
- Electron microscopy, including scanning electron microscopy and freeze-fracture electron microscopy.
- Biochemical extraction of membrane proteins (spectrin, actin, ankyrin, band 4.1) and enzymatic cleavage of band 3.
Main Results:
- Removal of spectrin and actin did not affect ovalocytic band 3 mobility.
- Removal of ankyrin and band 4.1, and trypsin cleavage, partially increased mobility but remained lower than normal.
- Electron microscopy revealed irregular ovalocyte surfaces and linear arrangements of intramembranous particles, indicative of aggregation.
Conclusions:
- Reduced band 3 rotational mobility in ovalocytes is caused by microaggregation.
- These microaggregates are likely induced by mutations in the membrane-bound domain of band 3 protein.
- Band 3 microaggregation is a key factor in the membrane abnormalities of hereditary ovalocytosis.