Headpiece domain of dematin is required for the stability of the erythrocyte membrane

Abstract

Dematin is an actin-binding and bundling protein of the erythrocyte membrane skeleton. Dematin is localized to the spectrin-actin junctions, and its actin-bundling activity is regulated by phosphorylation of cAMP-dependent protein kinase. The carboxyl terminus of dematin is homologous to the ''headpiece'' domain of villin, an actin-bundling protein of the microvillus cytoskeleton. The headpiece domain contains an actin-binding site, a cAMP-kinase phosphorylation site, plays an essential role in dematin self-assembly, and bundles F-actin in vitro. By using homologous recombination in mouse embryonic stem cells, the headpiece domain of dematin was deleted to evaluate its function in vivo. Dematin headpiece null mice were viable and born at the expected Mendelian ratio. Hematological evaluation revealed evidence of compensated anemia and spherocytosis in the dematin headpiece null mice. The headpiece null erythrocytes were osmotically fragile, and ektacytometry͞micropore filtration measurements demonstrated reduced deformability and filterability. In vitro membrane stability measurements indicated significantly greater membrane fragmentation of the dematin headpiece null erythrocytes. Finally, biochemical characterization, including the vesicle͞cytoskel-eton dissociation, spectrin self-association, and chemical crosslinking measurements, revealed a weakened membrane skeleton evidenced by reduced association of spectrin and actin to the plasma membrane. Together, these results provide evidence for the physiological significance of dematin and demonstrate a role for the headpiece domain in the maintenance of structural integrity and mechanical properties of erythrocytes in vivo.T he membrane bilayer and the network of membrane-associated proteins together regulate the characteristic shape and elastic properties of red blood cells (1, 2). When membrane skeletons are prepared in the presence of a high concentration of monovalent salt, the core of the membrane skeleton consists of spectrin, actin, protein 4.1, and dematin (3). Although the functions of spectrin, actin, and protein 4.1 have been extensively characterized (4, 5), virtually nothing is known about the physiological function in mature erythrocytes of dematin, a three-subunit protein that migrates in the protein 4.9 region during electrophoresis. The earliest evidence suggesting a membrane stabilizing role for dematin came from Holdstock and Ralston (6). They demonstrated that charged sulfhydryl compounds such as p-chloromercuribenzene sulfonateS preferentially attach to dematin and cause the disruption of erythrocyte cytoskeletons. Dematin is a substrate for multiple protein kinases, and phosphorylation of dematin by the cAMP-dependent protein kinase is known to regulate dematin's actin-bundling activity in vitro (7-9). The major phosphorylation site of the cAMPdependent protein kinase is located within the headpiece domain of dematin (10), but the physiological significance of dematin phosphorylation is not known.Siegel and Branton (11) conducted the first ...

The NMR Structure of Dematin Headpiece Reveals a Dynamic Loop That Is Conformationally Altered upon Phosphorylation at a Distal Site

“…The dematin headpiece is particularly intriguing, since knockout mice with a truncated dematin protein missing only the headpiece domain have a fragile red cell phenotype (7). In addition, dematin is the only headpiece that is phosphorylated in vivo and contains the longest predicted variable loop region of any headpiece sequence yet identified.…”

“…Recently, a dematin headpiece knockout mouse was shown to have a fragile red cell phenotype with spheroidal erythrocytes, indicating an important role for the headpiece of dematin in maintaining the mechanical properties of the red cell membrane (7). Dematin has been mapped to human chromosome 8p21.1 (8), a region often deleted in prostate cancer.…”

UCP2 Modulates Cell Proliferation through the MAPK/ERK Pathway during Erythropoiesis and Has No Effect on Heme Biosynthesis

“…The supernatant was collected, and absorbance was measured at 540 nm with appropriate controls. The lysis percentage of RBCs was calculated from the absorbance, and a fragility curve was generated by plotting varying salt concentrations versus hemolysis (43,44).…”

Osmotic and morphological effects on red blood cell membrane: action of an aqueous extract of Lantana camara

“…Different techniques have been used to evaluate the effects of the interaction between drugs and plasma membrane (Li et al, 1999, Pompei et al, 2005. The osmotic fragility assay is a classical, rapid, useful and easy technique that has permitted to obtain relevant information about the interactions of natural and synthetic drugs with cellular membrane (Khanna et al, 2002). Morphological analysis is another available method that has permitted to evaluate the effects of natural products on membrane of red blood cells (Oliveira et al, 2002, Oliveira et al, 2003Presta et al, 2007;Giani et al, 2007).…”