The oxygenation state of erythrocytes is known to impact several cellular processes. As the only known O2-binding protein in red blood cells, haemoglobin has been implicated in the oxygenation-mediated control of cell pathways and properties. Band 3, an integral membrane protein linked to the spectrin/actin cytoskeleton, preferentially binds deoxygenated haemoglobin at its N-terminus, and has been postulated to participate in the mechanism by which oxygenation controls cellular processes. Because the ankyrin-binding site on band 3 is located near the deoxyHb (deoxygenated haemoglobin)-binding site, we hypothesized that deoxyHb might impact the association between band 3 and the underlying erythrocyte cytoskeleton, a link that is primarily established through band 3–ankyrin bridging. In the present paper we show that deoxygenation of human erythrocytes results in displacement of ankyrin from band 3, leading to release of the spectrin/actin cytoskeleton from the membrane. This weakening of membrane–cytoskeletal interactions during brief periods of deoxygenation could prove beneficial to blood flow, but during episodes of prolonged deoxygenation, such as during sickle cell occlusive crises, could promote unwanted membrane vesiculation.
The best-studied cytoskeletal system is the inner surface of the erythrocyte membrane, which provides an erythrocyte with the structural support needed to be stable yet flexible as it passes through the circulation. Current structural models predict that the spectrin-actin-based cytoskeletal network is attached to the plasma membrane through interactions of the protein ankyrin, which binds to both spectrin and the cytoplasmic domain of the transmembrane protein band 3. The crystal structure of the cytoplasmic domain of band 3 predicted that the ankyrin binding site was located on a -hairpin loop in the cytoplasmic domain. In vitro, deletion of this loop eliminated ankyrin affinity for band 3 without affecting any other protein-band 3 interaction. To evaluate the importance of the ankyrin-band 3 linkage to membrane properties in vivo, we generated mice with the nucleotides encoding the 11-aa -hairpin loop in the mouse Slc4a1 gene replaced with sequence encoding a diglycine bridge. Mice homozygous for the loop deletion were viable with mildly spherocytic and osmotically fragile erythrocytes. In vitro, homozygous ld/ld erythrocytes were incapable of binding ankyrin, but contrary to all previous predictions, abolishing the ankyrin-band 3 linkage destabilized the erythrocyte membrane to a lesser degree than complete deficiencies of either band 3 or ankyrin. Our data indicate that as yet uncharacterized interactions between other membrane proteins must significantly contribute to linkage of the spectrin-actin-based membrane cytoskeleton to the plasma membrane.cytoskeleton ͉ membrane ͉ transgenic ͉ spherocytosis
The anion exchange protein (band 3) is an integral erythrocyte membrane protein that is associated with the spectrin-actin cytoskeleton through ankyrin, which binds to both band 3 and spectrin. Improper band 3/ankyrin interactions result in erythrocyte instability leading to spherocytosis and hemolytic anemia. The crystal structures of ankyrin and band 3 predict that the ankyrin amino-terminus, containing comb-like ANKYRIN repeat subdomains, binds to a β-hairpin loop (residues 175-185) in the band 3 cytoplasmic domain. We have previously shown that replacement of human band 3 residues 175–185 with a di-glycine bridge eliminates the binding of an ankyrin fragment containing domains 3 and 4 to the cytoplasmic domain of band 3 in vitro. To test this model for ankyrin/band 3 interactions in vivo, we used homologous recombination in embryonic stem cells to replace the corresponding mouse band 3 sequences, residues 189–199 in exon 7, with a di-glycine bridge to create a knock-in transgenic mouse. Analysis of mRNA expression in transgenic mice carrying the targeted locus demonstrated that the level of mRNA from the mutant allele (which contains the neomycin resistance gene used for positive selection of embryonic stem cells) was 50-70 fold lower than the level of mRNA from the wild type allele. Heterozygous transgenic mice were mated to mice expressing the Cre recombinase protein in the germ line to remove the neomycin resistance gene. After removal of the neomycin resistance gene from the band 3 locus, mice homozygous for the 189–199 deletion/di-glycine insertion were born in a normal Mendelian ratio and had normal numbers of red blood cells, white blood cells and platelets. Quantitative RT-PCR analysis showed that the level of band 3 mRNA from the wild type and mutant alleles were identical. SDS poly acrylamide gel electrophoresis and Western Blot analysis demonstrated normal levels of band 3 and other red cell membrane proteins in wild type, heterozygous and homozygous mutant erythrocytes. Homozygous mutant mice showed a modest but significant increase in osmotic fragility (p<0.01) compared to wild type or heterozygous mice. Ankyrin binding to inside out vesicles (IOV) from wild type, heterozygous and homozygous mutant erythrocytes was measured using an 125I labeled peptide containing domains 3 and 4 of mouse ankyrin. Ankyrin binding to homozygous mutant IOVs was 50% or less than the level of ankyrin binding to WT IOVs. We conclude from these studies that the 11 amino acid β-hairpin loop encoded by residues 189–199 of the band 3 gene is responsible for at least 50% of the ankyrin binding in mouse erythrocytes. We predict that mutations in this region of human band 3 would lead to a phenotype of Hereditary Spherocytosis similar to that seen with ankyrin deficiency. Space filling models of the cytoplasmic domain of band 3 have identified a second homologous β-hairpin loop encoded by residues 63-75, which is the target of our future investigation.
Csenge Kolozsvari, "Bodylandscapes I." (10:58). A proposition for remembering the ecological ways of belonging, a feeling into other ways of knowing, connecting into the vastness that surrounds us and moves across us, becoming-environment once again. // Anja Plonka, Marko Stefanovic, and Rasmus Nordholt-Frieling, "Breathing Gaia: Searching for Kinship Around Walensee" (8:28). The video essay creates a speculative-utopian body and existence of human and non-human. The body as an archive of traumatic inscriptions practices transformation as a being in resonance with Gaia. // Jessica Marion Barr, Jenn Cole, and LA Alfonso, "Our Bodies, These Lands: Practising Reciprocity" (6:03). As artist-researchers with embodied practices and relationships with lands and waters, we explore a unique part of Michi Saagig Nishnaabeg territory wherein “rockmills” or “kettles” offer spaces for our human selves to be held and surrounded by massive ancient rock beings. // Alessandro Guglielmo, "Wisdom and Trouble: Notes on Blood, Care, and Death in Multispecies Settings" (9:30). In this video essay, I employ my emplacement as a vegetarian anthropologist witnessing the killing of a non-human being to produce an understanding of more-than-human ecologies. I reflect on narratives of death, and the trouble of care and killing in multispecies settings.
Reduction of α‐santonin (I) with DIBAL yields the hemiacetals (II) and (III).
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.