Immunoreactive forms of human erythrocyte ankyrin are present in diverse cells and tissues.

Ankyrin is a polypeptide of molecular weight (MW) 200,000 which is tightly bound to the cytoplasmic surface of the human erythrocyte membrane and has been identified as the high-affinity membrane attachment protein for spectrin. This protein has also been shown to be associated with band 3 (ref. 4), the major transmembrane protein which links a cytoplasmic structural protein to an integral membrane protein. A water-soluble, 72,000-MW, proteolytic fragment of ankyrin has been purified which retains the ability to bind to spectrin, and competitively inhibits reassociation of spectrin with membranes. Monospecific antibodies directed against this fragment have been prepared and demonstrated to cross-react only with ankyrin among the erythrocyte membrane proteins. The present study reports the use of these antibodies to develop a radioimmunoassay capable of detecting femtomolar quantities of ankyrin, and demonstrates the presence of small but significant amounts of immunoreactivity in a variety of types of cells and tissues.     

Partial deficiency of erythrocyte spectrin in hereditary spherocytosis

Hereditary spherocytosis (HS) is a common, clinically heterogeneous haemolytic anaemia in which the primary erythrocyte defect is believed to be some abnormality in the spectrin-actin membrane skeleton, leading to loss of surface membrane. Recessively inherited spectrin deficiency with extreme erythrocyte fragility and spherocytosis has been identified in certain mutant mice and two severely anaemic humans. Although suspected, deficiency of spectrin has not been demonstrated in less severe forms of human HS. We not report the quantitation of erythrocytes spectrin by radioimmunoassay. We found that normal erythrocytes contained 240,000 copies of spectrin heterodimer, whereas erythrocytes from 14 patients with a variety of types of HS were all partially deficient in spectrin (range 74,000-200,000 copies), the magnitude of the deficiency correlating with the severity of the disease. Spectrin deficiency of varying degrees is common in HS and probably represents the principal structural defect leading to loss of surface membrane.     

Modulation of spectrin-actin assembly by erythrocyte adducin

The spectrin-based membrane skeleton, an assembly of proteins tightly associated with the plasma membrane, determines the shape and mechanical properties of erythrocytes. Spectrin, the most abundant component of this assembly, is an elongated and flexible molecule that, with potentiation by protein 4.1, is cross-linked at its ends by short actin filaments to form a lattice beneath the membrane. These and other proteins stabilize the plasma membrane, organize integral membrane proteins and maintain specialized regions of the cell surface. A membrane-skeleton-associated calmodulin-binding protein of erythrocytes is a major substrate for Ca2+- and phospholipid-dependent protein kinase C (ref. 5), and thus is a target for Ca2+ by two regulatory pathways. Here we demonstrate that this protein, called adducin: (1) binds tightly in vitro to spectrin-actin complexes but with much less affinity either to spectrin or to actin alone; (2) promotes assembly of additional spectrin molecules onto actin filaments; and (3) is inhibited in its ability to induce the binding of additional spectrin molecules to actin by micromolar concentrations of calmodulin and Ca2+. Adducin may be involved in the action of Ca2+ on erythrocyte membrane skeleton and in the assembly of spectrin-actin complexes.     

Spectrin assembly in avian erythroid development is determined by competing reactions of subunit homo- and hetero-oligomerization

Erythroid differentiation entails the biogenesis of a membrane skeleton, a network of proteins underlying and interacting with the plasma membrane, whose major constituent is the heterodimeric protein spectrin, composed of two structurally similar but distinct subunits, alpha (relative molecular mass (Mr) 240,000) and beta (Mr 220,000), which interact side-on with each other to form a long rod-like molecule. Interaction of this network with the membrane is mediated by the binding of the beta subunit to ankyrin, which in turn binds to the cytoplasmic domain of the transmembrane anion transporter (also referred to as band 3). Purified alpha and beta subunits of spectrin from the membrane of mature red blood cells will spontaneously heterodimerize, suggesting that assembly of the spectrin-actin skeleton is a simple self-assembly process, but in vivo studies with developing chicken embryo erythroid cells have indicated that assembly in vivo is more complex. We now present evidence that newly synthesized spectrin subunits in vivo or in vitro rapidly adopt one of two competing conformations, a heterodimer or a homo-oligomer. These competing reactions seem to determine the overall extent of spectrin assembled during erythroid development by determining which conformation will assemble onto the membrane-skeleton (the heterodimer) and which conformations are targeted for degradation (the homo-oligomers).     

Ankyrin binding to (Na+ + K+)ATPase and implications for the organization of membrane domains in polarized cells

The interaction between membrane proteins and cytoplasmic structural proteins is thought to be one mechanism for maintaining the spatial order of proteins within functional domains on the plasma membrane. Such interactions have been characterized extensively in the human erythrocyte, where a dense, cytoplasmic matrix of proteins comprised mainly of spectrin and actin, is attached through a linker protein, ankyrin, to the anion transporter (Band 3). In several nonerythroid cell types, including neurons, exocrine cells and polarized epithelial cells homologues of ankyrin and spectrin (fodrin) are localized in specific membrane domains. Although these results suggest a functional linkage between ankyrin and fodrin and integral membrane proteins in the maintenance of membrane domains in nonerythroid cells, there has been little direct evidence of specific molecular interactions. Using a direct biological and chemical approach, we show here that ankyrin binds to the ubiquitous (Na+ + K+)ATPase, which has an asymmetrical distribution in polarized cells.     

Hereditary spherocytosis associated with deletion of human erythrocyte ankyrin gene on chromosome 8

Hereditary spherocytosis (HS) is one of the most common hereditary haemolytic anaemias. HS red cells from both autosound dominant and recessive variants are spectrin-deficient, which correlates with the severity of the disease. Some patients with recessive HS have a mutation in the spectrin alpha-2 domain (S.L.M. et al., unpublished observations), and a few dominant HS patients have an unstable beta-spectrin that is easily oxidized, which damages the protein 4.1 binding site and weakens spectrin-actin interactions. In most patients, however, the cause of spectrin deficiency is unknown. The alpha- and beta-spectrin loci are on chromosomes 1 and 14 respectively. The only other genetic locus for HS is SPH2, on the short arm of chromosome 8 (8p11). This does not correspond to any of the known loci of genes for red cell membrane proteins including protein 4.1 (1p36.2-p34), the anion exchange protein (AE1, band 3; 17q21-qter), glycophorin C (2q14-q21), and beta-actin (7pter-q22). Human erythrocyte ankyrin, which links beta-spectrin to the anion exchange protein, has recently been cloned. We now show that the ankyrin gene maps to chromosome 8p11.2, and that one copy is missing from DNA of two unrelated children with severe HS and heterozygous deletions of chromosome 8 (del(8)(p11-p21.1)). Affected red cells are also ankyrin-deficient. The data suggest that defects or deficiency or ankyrin are responsible for HS at the SPH2 locus.     

再生障碍性贫血病人红细胞膜骨架蛋白的研究

十二烷基磺酸钠—聚丙烯酰胺凝胶电泳(简称为SDS-PAGE)方法分离12例再生障碍性贫血(简称再障)病人红细胞膜蛋白,发现其中有6例红细胞骨架蛋白带1、带2和带4有不同程度减少或缺损。同时用扫描电子显微镜观察其红细胞形态,发现亦有不同程度异常,表现为棘状、刺状及波浪状。提示红细胞骨架蛋白的含量与红细胞的形态有密切关系。     

Regulation of the association of membrane skeletal protein 4.1 with glycophorin by a polyphosphoinositide

Many of the physical properties of the erythrocyte membrane appear to depend on the membrane skeleton, which is attached to the membrane through associations with transmembrane proteins. A membrane skeletal protein, protein 4.1, is pivotal in the assembly of the membrane skeleton because of its ability to promote associations between spectrin and actin. Protein 4.1 also binds to the membrane through at least two sites: a high-affinity site on the glycophorins and a site of lower affinity associated with band 3 (ref. 11). The glycophorin-protein 4.1 association has been proposed to be involved in maintenance of cell shape. Here we show that the association between glycophorin and protein 4.1 is regulated by a polyphosphoinositide cofactor. This observation suggests a mechanism which may explain the recently reported dependence of red cell shape on the level of polyphosphoinositides in the membrane.     

Appearance of new variants of membrane skeletal protein 4.1 during terminal differentiation of avian erythroid and lenticular cells

The erythrocyte plasma membrane is lined with a network of extrinsic proteins, mainly spectrin and actin, which constitute a reticulum tethered to the intrinsic anion transport protein of the lipid bilayer through a linker protein, ankyrin. Protein 4.1 forms a stable ternary complex with spectrin and actin, thereby strengthening the reticulum and anchoring it directly to the lipid bilayer or to another intrinsic protein, glycophorin. It has been found recently that spectrin, ankyrin and protein 4.1 are not erythrocyte-specific; this has elucidated further the mechanisms of plasma membrane assembly and modelling during the differentiation of diverse tissues. We have shown previously that protein 4.1 in chickens is most abundant in erythrocytes and lens cells, but is scarce or absent from other spectrin-rich cell types. In addition, it exists as a family of related polypeptides showing differential expression in these two tissues, suggesting variant-specific functions. Here we show that the pattern of protein 4.1 variants changes during the terminal differentiation of erythroid and lenticular cells, with novel variants appearing in postmitotic cells. The accumulation of these variants may lead to the final stabilization of the plasma membrane skeletons of these cells.     

Regulation of glutamate receptor binding by the cytoskeletal protein fodrin

The erythrocyte cytoskeleton, which consists primarily of a meshwork of spectrin and actin, controls cell shape and the disposition of proteins within the membrane. Proteins similar to spectrin have recently been found in diverse cells and tissues, and it is possible that they mediate the capping of cell-surface receptors, although this has not been demonstrated directly. In neurones, the spectrin-like protein fodrin lines the cortical cytoplasm and may link actin filaments to the membrane. Fodrin has been hypothesized to regulate the number of receptor binding sites on neuronal membranes for the putative neurotransmitter L-glutamate. Micromolar calcium concentrations activate the thiol protease calpain I, induce fodrin degradation and more than double the density of glutamate binding sites; these effects are all blocked by thiol protease inhibitors. We have now used specific antibodies to examine further the role of fodrin proteolysis in regulating glutamate receptors. We report that fodrin antibodies block the fodrin degradation and increase in glutamate binding normally induced by calcium, and so provide direct evidence for control of membrane receptors by a non-erythroid spectrin.     

Synapsin I is a spectrin-binding protein immunologically related to erythrocyte protein 4.1

The membrane-associated cytoskeleton is considered to be the apparatus by which cells regulate the properties of their plasma membranes, although recent evidence has indicated additional roles for the proteins of this structure, including an involvement in intracellular transport and exocytosis (see refs 1-3 for review). Of the membrane skeletal proteins, to date only spectrin (fodrin) and ankyrin have been purified and characterized from non-erythroid sources. Protein 4.1 in the red cell is a spectrin-binding protein that enhances the binding of spectrin to actin and can apparently bind to at least one transmembrane protein Immunoreactive forms of 4.1 have been detected in several cell types, including brain. Here we report the purification of brain 4.1 on the basis of its cross-reactivity with erythrocyte 4.1 and spectrin-binding activity. We further show that brain 4.1 is identical to the synaptic vesicle protein, synapsin I, one of the brain's major substrates for cyclic AMP and Ca2+-calmodulin-dependent kinases. Spectrin and synapsin are present in brain homogenates in an approximately 1:1 molar ratio. Although synapsin I has been implicated in synaptic transmission, no activity has been previously ascribed to it.     

Effect on membrane transport in the erythrocytes by band 3 cross-linking

Band 3 and glucose transport protein (GluT1) are two kinds of important proteins in the human erythrocyte membranes. Bis(sulfosuccinimidyl)suberate (BS³), an impermeable cross-linker of band 3, inhibited NO₂ ⁻ transport, showing that anion exchange is affected by the association state of band 3 in the intact erythrocyte membranes. At the same time, the rates of glucose transport of both exit and entry declined. The amount of monomers of band 3 was decreased after treatment of the erythrocytes with BS³, but there was no change in GluT1 according to the SDS-PAGE patterns. This demonstrates that band 3 and GluT1 would be linkaged together in the erythrocyte membranes for the requirement of rapid and cooperative performance of physiological functions of the membrane proteins.     

遗传球形红细胞增多症红细胞膜力学特性研究

彩和微管吸吮实验技术并结合十二烷基硫酸钠－聚丙烯酰胺凝胶电泳等生化方法研究遗传球形红细胞增多症红细胞膜粘弹性特及膜骨架蛋白的变化。结果表明：ＨＳ红细胞膜的弹性模量和正常对照组比较无明显差异而粘性系数显著高于正常值,细胞粘滞性增大型汽轮发电机组轴系的变形能力降低;     

Primary structure and transmembrane orientation of the murine anion exchange protein

The amino-acid sequence of murine band 3, deduced from the nucleotide sequence of a complementary DNA clone, confirms that this integral membrane glycoprotein is composed of two major structural domains which correlate with its dual functions as the anchor for the erythrocyte cytoskeleton and as a plasma membrane anion antiporter. This latter activity resides within a highly hydrophobic domain that crosses the plasma membrane at least 12 times.     

Expression and characterization of anion exchanger1 (AE1) 55 kD transmembrane region and glycophorin A

A complete transmembrane coding region of human erythrocyte anion exchanger1 (AE1, band 3) and the full length of glycophorin A (GPA) were amplified by PCR amplification. The genes were subcloned into the baculovirus transfer vectors and expressed in Sf9 cells. Western blot showed a cross reaction between band 3 transmembrane domain and GPA. The yeast two-hybrid experiment confirmed the interaction between the two proteins.     

Membrane skeleton spectrin in pollen and pollen tube

Immuno-western blots, immunofluorescence and immuno-gold labeling methods were used to study the existence and distribution of a homologue of the membrane skeleton spectrin in the pollen and pollen tube ofLilium davidii Duch. A spectrin homologue was found to exist in the pollen and pollen tube, distributed on membranes of secretory vesicles near Golgi apparatus.     

用红外光谱法研究Azone对红细胞膜结合水的影响

用傅里叶变换红外光谱法(FT-IR)研究了透皮吸收促进剂Azone对红细胞膜结合水量的影响.结果表明Azone使膜结合水量减少.很可能Azone嵌入细胞膜,改变了脂质头部之间的相互作用,降低了膜结合水.     

硫丹对长爪沙鼠血细胞免疫相关因子表达的影响

目的 探讨有机氯农药硫丹对长爪沙鼠血细胞免疫功能相关因子的影响。方法将50只长爪沙鼠随机分成5组,各组动物所用硫丹灌胃的剂量依次为：0mg／（kg·d）、0．4mg／（kg·d）、0．8mg／（kg·d）、1．6mg／（kg·d）和6．4mg／／（kg·d）。灌胃21d后,取血检测血细胞免疫功能相关因子的表达。结果药物处理后,红细胞表面CD59因子和B淋巴细胞表面CD35因子的表达量在各实验组和对照组间没有明显差异（P〉0．05）。红细胞膜表面CD58因子随硫丹浓度的升高显著下降,0．8mg,／（kg·d）、1．6mg／（kg·d）和6．4／mg／（kg·d）组较对照组有显著降低（P〈0．05）。结论硫丹能抑制长爪沙鼠红细胞膜表面CD58因子表达,但对红细胞膜表面CD59和B细胞表面的CD35的表达没有影响。     

The asymmetric membrane structure of erythrocytes from Crucian carp studied by atomic force microscopy

Cell membranes play a key role in cellular activities. Fish erythrocytes, prototype of the nucleated erythrocytes of lower vertebrates, are predominantly oval, biconvex discs with elliptical nucleus and much larger in size than human erythrocytes. In attempts to disclose the correlation of membrane structure between fish and human erythrocytes, we used in situ atomic force microscope （AFM） combined with single-molecule force spectroscopy to study the membrane structure of Crucian carp erythrocytes under quasi-native conditions. Our results revealed the asymmetric distribution of proteins in Crucian carp erythrocyte membrane： the outer leaflet of membrane is rather smooth without any proteins, whereas the inner leaflet of membrane is very rough with dense proteins. The asymmetry of fish erythrocyte membrane structure fits well with the semi-mosaic model of human erythrocyte membrane structure. This similarity of membrane structure between human and fish erythrocytes extends the semimosaic model of erythrocytes membrane structure to a wider range of species.