Protein 4.2 is a major red blood cell (RBC) protein that interacts with the band 3 protein and with ankyrin. Inherited deficiencies of this protein are associated with spherocytic hemolytic anemia, but the molecular basis of this defect is unknown. We have studied the underlying defect in a patient with spherocytic hemolytic anemia whose RBCs had a partial (29% +/- 5%) deficiency of protein 4.2. We have first studied the binding of normal ankyrin and protein 4.2 to patient inside-out vesicles (IOVs) stripped of peripheral proteins. While the binding of ankyrin was normal, the predicted maximal binding capacity of patient IOVs for band 4.2 was 20% to 33% lower than that of control IOVs, suggesting a defect in the cytoplasmic domain of band 3 (cdb3). An additional line of evidence pointing to a possible abnormality of band 3 was an abnormal proteolytic digest of cdb3. To elucidate the underlying molecular defect, we have cloned and sequenced the cDNA coding for cdb3 from the patient. One band 3 allele was found to be normal, while clones corresponding to the other allele contained two mutations: substitution A----G in nucleotide 166, changing codon 56 from AAG to GAG (Lys----Glu), and substitution C----G in nucleotide 980, changing codon 327 from CCC to CGC (Pro----Arg). Since the Lys56--- -Glu56 substitution is found in a common asymptomatic variant of the band 3 protein designated band 3 Memphis, we conclude that either the Pro327----Arg327 substitution itself, or in combination with the band 3 Memphis polymorphism, underlies the abnormal binding of protein 4.2 to cdb3 and results in the spherocytic.
Human platelets were tested for the presence of proteins immunologically cross-reactive with red cell spectrin and protein 4.1. As assessed by indirect immunofluorescence microscopy, platelets were specifically reactive with affinity-purified rabbit antisera against red cell spectrin and protein 4.1. The immunoreactive platelet constituents were further analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, followed by electrophoretic transfer to nitrocellulose paper and immunoperoxidase staining. We found that whole platelets, membranes, and cytoskeletal preparations isolated by Triton X-100 extraction contain small amounts of proteins reacting with anti-spectrin or anti-protein 4.1 antiserum. The immunoreactive spectrin-like platelet protein has an apparent molecular weight of 240,000 and comigrates with the alpha-subunit of red cell spectrin. The major immunoreactive protein 4.1-like constituent has an apparent molecular weight of 78,000, which is slightly less than that of red cell protein 4.1. We conclude that platelets contain a spectrin- like protein which, by analogy with red cell spectrin, may have a role in membrane-cytoskeletal attachment. The properties and function of the platelet protein 4.1-like constituent are not yet known.
We investigated the role of glycophorins C and D in the association of band 4.1 with the erythrocyte membrane by measuring the binding of band 4.1 to erythrocyte inside-out vesicles stripped of endogenous band 4.1. Vesicles were prepared from either normal erythrocytes or erythrocytes completely lacking glycophorins C and D (Leach phenotype). Band 4.1 binding to vesicles from normal erythrocytes gave rise to a nonlinear Scatchard plot, indicative of two classes of binding sites: a low-capacity, high-affinity class of sites (about 10% of the total) and a high-capacity, low-affinity class of sites. Vesicles prepared from Leach erythrocytes had a binding capacity for band 4.1 that was, on average, 32% lower than that of vesicles from normal erythrocytes. This difference was caused by the complete absence of the high-affinity binding sites as well as by a decrease in the number of low-affinity binding sites. Reduction of membrane phosphatidylinositol 4,5-biphosphate (PIP2) content by adenosine triphosphate depletion or activation of phosphoinositidase C resulted in a decrease in band 4.1 binding capacity to a similar extent in both control and Leach vesicles. The principal effect of PIP2 depletion was a reduction in the number of low-affinity band 4.1 binding sites in control and Leach vesicles. The fact that PIP2 depletion induced a decrease in band 4.1 binding to Leach vesicles shows that glycophorin C or D is not required for the formation of PIP2-sensitive band 4.1 binding sites, and may not be involved in PIP2-sensitive band 4.1 binding sites even when they are present. Our studies give new insights into the involvement of glycophorins and of PIP2 in modulating cytoskeletal-membrane interactions.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.