Expression of the beta subunit of spectrin in nonerythroid cells.

Antibodies raised against electrophoretically purified chicken erythrocyte beta subunit of spectrin, called "beta-spectrin," have been used to demonstrate the presence of an immunoreactive form of this polypeptide in nonerythroid tissues. Immunoautoradiography shows that, in chicken erythrocytes, this antiserum reacts with beta-spectrin (Mr 220,000) and another polypeptide (Mr 230,000) that, by two-dimensional tryptic peptide analysis, shows extensive homology with beta-spectrin but not with the alpha subunit of spectrin, called "alpha-spectrin." Immunoautoradiography and immunoprecipitation of various chicken tissues with this antiserum shows that either one variant or both variants of beta-spectrin are expressed. Indirect immunofluorescence reveals that the antiserum reacts with a plasma membrane-associated component of erythroid and some nonerythroid cells. Particularly strong fluorescence is observed in skeletal and cardiac muscle cells where beta-spectrin appears to form a grid-like network along the inner surface of the sarcolemma. The noncoordinated distribution of alpha- and beta-spectrin variants indicates that their expression may be tailored to the functional requirements of the plasma membrane in different cells.     

Erythrocyte ankyrin: immunoreactive analogues are associated with mitotic structures in cultured cells and with microtubules in brain.

Human erythrocyte ankyrin, the membrane attachment protein for spectrin, has been detected by radioimmunoassay in a variety of cells and tissues. This report identifies polypeptides crossreacting with ankyrin in brain and HeLa cells and demonstrates that one function of these ankyrin analogues involves association with microtubules. Ankyrin immunoreactivity was localized by indirect immunofluorescence in a colchicine- and detergent-sensitive cytoplasmic meshwork in interphase cells. There also was specific nuclear staining, localized in a bright spots, which was displaced entirely by ankyrin or by high molecular weight microtubule-associated proteins (MAPs) from brain. In dividing cells, the punctate nuclear staining and the meshwork disappeared. Fluorescence was localized at the spindle pole during metaphase and was redistributed to the cleavage furrow in later stages of mitosis. An immunoreactive Mr 370,000 polypeptide comigrating with MAP1 was identified in brain extracts and copolymerized with microtubules through repeated cycles of polymerization and depolymerization. Finally, erythrocyte ankyrin associated with microtubules prepared from pure tubulin, and this binding was displaced by brain MAPs.     

Ankyrin is fatty acid acylated in erythrocytes.

Ankyrin is a peripheral membrane protein that mediates the attachment of the erythrocyte membrane skeleton to the plasma membrane. We show that [3H]palmitic acid is incorporated into ankyrin in vivo. The majority of the 3H-labeled fatty acid is covalently bound to the polypeptide, as it cannot be removed by strong detergents or by chloroform/methanol extraction but is labile to alkaline hydrolysis. The binding of fatty acid occurs predominantly after the assembly of ankyrin onto the membrane skeleton, since it continues when protein synthesis is inhibited with emetine. Fatty acid acylation of ankyrin is constitutive in erythroid cells throughout chicken embryo development. It also occurs in mature avian and mammalian erythrocytes suggesting that the fatty acid bound to ankyrin turns over more rapidly than the polypeptide. Fatty acid acylation of assembled ankyrin may modulate the interaction of ankyrin with the plasma membrane. It may also provide a mechanism by which the membrane skeleton influences the organization of the lipid bilayer.     

Monoclonal antibodies detect a spectrin-like protein in normal and dystrophic human skeletal muscle.

Spectrin is the major protein of the erythrocyte membrane skeleton, which is bound to the cytoplasmic surface of the membrane's lipid bilayer and is responsible for cell shape and membrane elasticity. Inability to identify spectrin in other cell types led to the assumption that this protein was unique to erythrocytes. However, spectrin-like proteins have been demonstrated recently in a variety of cell types, including skeletal and cardiac muscle, in several species. We used monoclonal antibodies against human erythrocyte spectrin subunits in an immunocytochemical study to detect related proteins in normal and diseased human skeletal muscle. Six of seven monoclonal antibodies against beta-spectrin determinants were bound at the cytoplasmic surface of muscle fiber plasma membranes, whereas none of six monoclonal antibodies against alpha-spectrin determinants was bound. Muscle fibers of patients with neuromuscular diseases showed similar distribution and specificity of antibody binding to those of normal subjects, but the intensity of binding was increased. In contrast, probable regenerating fibers in muscle of patients with muscular dystrophies showed reduced binding of antibodies, but reduced binding was not seen in fetal muscle fibers nor in those of a patient with a myotubular myopathy. We conclude that human skeletal muscle fibers possess a spectrin-related protein associated with their plasma membrane that shows extensive beta-chain similarities to erythrocyte spectrin but differs significantly with respect to the alpha-subunit. Its function may be associated with the maintenance of membrane and myofibril integrity during contraction, and the increased antibody binding in diseased muscle may reflect a structural rearrangement of spectrin or a compensatory increase in spectrin abundance in response to increased stress on these systems.     

Hereditary elliptocytosis, spherocytosis and related disorders: consequences of a deficiency or a mutation of membrane skeletal proteins

The membrane skeleton, a protein lattice that laminates the internal side of the red cell membrane, contains four major proteins: spectrin, actin, protein 4.1 and ankyrin. By mass, the most abundant of these proteins is spectrin, a fibre-like protein composed of two chains, alpha and beta, which are twisted along each other into a heterodimer. At their head region, spectrin heterodimers are assembled into tetramers. At their distal end, these tetramers are interconnected into a two dimensional network by their linkage to oligomers of actin. This interaction is greatly strengthened by protein 4.1. The skeleton is attached to the membrane by ankyrin, a protein that connects the spectrin beta chain to the major transmembrane protein band 3, the anion channel protein. Additional attachment sites are those of protein 4.1 with several glycoproteins, namely glycophorin A and C, as well as direct interactions between spectrin, protein 4.1 and the negatively charged lipids of the inner membrane lipid bilayer. Hereditary spherocytosis, elliptocytosis and pyropoikilocytosis represent a group of disorders that are due to deficiency or dysfunction of one of the membrane skeletal proteins (Fig. 1). Known deficiency states include that of spectrin, ankyrin and protein 4.1. Severe spectrin and ankyrin deficiencies (with decrease in spectrin and ankyrin contents to about 50% of the normal amount) are both rare disorders associated with severe autosomal recessive hereditary spherocytosis. On the other hand, mild spectrin deficiency is found in the majority of patients with autosomal dominant spherocytosis in which the degree of spectrin deficiency correlates with the clinical severity of the disease. Protein 4.1 deficiency, in contrast, is associated with hereditary elliptocytosis, which in certain populations constitutes about 20% of all such patients. Known skeletal protein dysfunctions include mutants of both alpha and beta spectrin that involve the spectrin heterodimer self-association site. These are clinically expressed as hereditary elliptocytosis (HE) and a closely related disorder, hereditary pyropoikilocytosis (HPP). At the level of protein function, this defect can be detected by analysis of the content of spectrin dimers and tetramers in 0 degrees C low ionic strength extracts of red cell membranes. Their structural identification is accomplished by limited proteolytic digestion of spectrin followed by two-dimensional tryptic peptide mapping.(ABSTRACT TRUNCATED AT 400 WORDS)     

Involvement of spectrin in cell-surface receptor capping in lymphocytes.

Human and mouse lymphocytes of T- and B-cell lineages express a protein (Mr, 240,000) that crossreacts with antibodies raised against chicken erythrocyte alpha-spectrin as judged by immunofluorescence, immunoprecipitation, and immunoautoradiography; by the same criteria, antibodies raised against chicken erythrocyte beta-spectrin do not react with any lymphocyte polypeptide. In all T and B cells analyzed, before surface-directed ligand challenge with concanavalin A and surface immunoglobulins the polypeptide antigenically related to erythrocyte alpha-spectrin is distributed diffusely at the plasma membrane. Upon challenge, the redistribution of this polypeptide is concurrent with that of the cell-surface receptors initially in patches and then in a cap. Immunoprecipitation of NaDodSO4-solubilized lymphocytes with erythrocyte alpha-spectrin antiserum shows that in all cases a polypeptide with the same apparent molecular weight as erythrocyte alpha-spectrin is precipitated. Variable amounts of another polypeptide (Mr, 235,000) are also coimmunoprecipitated. Immunoprecipitations and subsequent immunoautoradiography show that the lymphocyte polypeptide doublet has a composition similar to that of (brain) fodrin, a polypeptide doublet that previously has been found mainly in the cells of nervous tissue.     

Immunofluorescence localization at the mammalian neuromuscular junction of the Mr 43,000 protein of Torpedo postsynaptic membranes.

Highly purified cholinergic postsynaptic membranes from Torpedo electric tissue contain, in addition to the acetylcholine receptor (AcChoR), major proteins of Mr 43,000 and Mr approximately 90,000 and minor proteins that can be removed from the membranes by alkaline treatment. We have prepared an antiserum to these alkaline-extractable proteins that reacts with the Mr 43,000 protein but not with any of the other major membrane proteins, including the AcChoR subunits. Immunofluorescent staining of sections of Torpedo electric tissue shows that this antiserum binds to the innervated but not the uninnervated surface of the electrocytes. In rat diaphragm muscle, the antigens recognized by this antiserum are highly concentrated at the synapse. Synaptic staining of muscle is eliminated by prior incubation of the antiserum with the Mr 43,000 protein but not by incubation with affinity-purified AcChoR. This antiserum stains end plates of muscles denervated for 7 days. Antiserum to AcChoR binds to the subsynaptic membranes of electrocytes and muscle but does not react with the Mr 43,000 protein. Purified AcChoR blocks staining of synapses by anti-AcChoR but the Mr 43,000 protein does not. These results indicate that the Mr 43,000 protein is located in the innervated membrane of Torpedo electrocytes and that an immunologically similar component is highly concentrated in the postsynaptic membrane of mammalian muscle.     

Association state of human red blood cell band 3 and its interaction with ankyrin

We have studied the association state of band 3, the anion channel and predominant transmembrane protein of the human red blood cell, and the anomalous stoichiometry and dynamics of its interaction with ankyrin, which acts as a link to the spectrin of the membrane skeletal network. Band 3 exists in benign nonionic detergent solutions as a dimer. Tetramer is formed irreversibly in the course of manipulations, particularly in ion-exchange chromatography. The dimer in solution binds ankyrin without self-associating. In ankyrin-free inside-out membrane vesicles and when incorporated into phosphatidylcholine liposomes, only some 10% to 15% of band 3 chains bind ankyrin at saturation. Moreover, in liposomes this was independent of protein:lipid ratio between 1:2 and 1:40. The bound fraction of band 3 remains with the detergent-extracted membrane cytoskeleton, but is released if the ankyrin has been cleaved with chymotrypsin before detergent treatment; thus, the attachment to the membrane cytoskeleton is entirely through ankyrin and not through other constituents such as protein 4.1. The ratio of band 3 to ankyrin in this complex implies that it consists of two chains of band 3 and one chain of ankyrin, at least after detergent extraction. The bound and free populations of band 3 exchange freely in the membrane. In the artificial liposome membrane binding of ankyrin to band 3 dimers cause association of the band 3 into higher aggregates, as seen in freeze-fracture electron microscopy. Successive manipulations of the red blood cell membrane, which are involved in the preparation of ghosts, of inside-out vesicles, and of inside-out vesicles stripped of peripheral proteins are accompanied by progressive aggregation of intramembrane particles, as judged by freeze-fracture electron microscopy. Thus the intramembrane particles are evidently stabilized in the intact cell by the peripheral protein network and the cytosolic milieu. Aggregation may be expected to limit the number of functional ankyrin binding sites. However, although extraneous ankyrin binds to the unoccupied binding site on the spectrin tetramers in intact ghost membranes, little or no ankyrin can bind to the unoccupied band 3 dimers in situ, perhaps by reason of occlusion of binding sites by the membrane skeletal network.     

Increased concentration of spectrin is observed in avian dystrophic muscle.

A significant increase in the concentration of spectrin has been observed in dystrophic chicken pectoralis major muscle when compared to normal fast-twitch muscle. In normal muscle, alpha-spectrin-specific immunofluorescence delineates each myofiber with a network pattern of staining at the sarcolemma with little staining within the cytoplasm. In dystrophic fibers, numerous intensely stained areas occur within the cytoplasm and staining at the sarcolemma is increased, thereby obscuring or eliminating the highly regular network arrangement of spectrin usually seen in this region. When immunofluorescence experiments are performed on microsomal vesicles isolated from normal and dystrophic tissues, only a small fraction of normal vesicles are stained, whereas most of the dystrophic vesicles are associated with spectrin. An increase in spectrin concentration is observed using immunoautoradiography of whole muscle and isolated microsomes, thus supporting the immunofluorescent observations described above. The early-age post-hatching when increases in spectrin concentration can be detected and the simplicity of the immunofluorescent technique make this observation useful as a new diagnostic parameter. This observation also shows that the distribution of spectrin and its concentration within nonerythroid cells can be modified by abnormal physiological states; this modification may contribute to subsequent symptoms, such as increased rigidity and abnormal calcium metabolism, that are observed in dystrophy.     

Ankyrin binds to two distinct cytoplasmic domains of Na,K-ATPase alpha subunit.

Ankyrin has emerged as a ubiquitous protein linking integral membrane transport proteins such as Na,K-ATPase to an underlying spectrin cytoskeleton. This interaction is mediated by the alpha subunit of Na,K-ATPase; however, the nature of the ankyrin binding site in Na,K-ATPase is unknown. As a step to determine the mechanism of this interaction, the ankyrin binding region of human erythrocyte spectrin and each of five putative cytoplasmic domains of the Na,K-ATPase alpha subunit have been prepared as recombinant fusion proteins in bacteria and analyzed for their interaction with erythrocyte and kidney ankyrin (Ank1 and Ank3, respectively) in vitro. Spectrin binds both Ank1 and Ank3 avidly, as expected. Two of the Na,K-ATPase domains, immobilized on a bioaffinity column, also interact specifically with both of these ankyrins. These ATPase domains are encoded by codons 140-290 (domain II) and 345-784 (domain III), with domain II displaying the greatest apparent affinity. Sequences in domain II are highly conserved between species and isoforms of Na,K-ATPase and are homologous to a cytoplasmic domain in H,K-ATPase and to a limited region of sequence in Ca-ATPase. Conversely, domain II shares no significant homology with other ankyrin binding proteins such as band 3 and Na(+)-channel proteins. These results identify a clear function for a conserved but previously not understood region of the alpha subunit of Na,K-ATPase and suggest that the interaction of ankyrin with membrane transport proteins may involve complex tertiary structural determinants not easily deduced from the primary sequence.     

Erythrocyte membrane protein alterations underlying clinical heterogeneity in hereditary spherocytosis

Summary. Hereditary spherocytosis (HS) is a very heterogenous condition both at clinical and biochemical level. To establish the relationship between these aspects we performed a clinical and biochemical study in 87 Italian HS subjcts. Patients were divided into three groups based on clinical severity (mild, typical and severe) and into five subgroups based on specific membrane abnormalities identified by polyacrylamide gel electrophoresis (isolated spectrin deficiency, spectrin deficiency combined with mild ankyrin reduction, spectin deficiency combined with severe ankyrin reduction, band 3 reduction and isolated protein 4.2 reduction). We were not able to assess any alteration in six HS patients. A good correlation between clinical HS forms and memberane protein defects is shown. We conclude that erythrocyte memberane analysis should be carried out after diagnosis of HS in order to predict the clinical course of the disease.     

Fodrin and band 4.1 in a plasma membrane-associated fraction of human neutrophils

Erythrocytes possess a well-characterized submembranous filamentous network which interacts with transmembrane glycoproteins and is composed primarily of spectrin, ankyrin, band 4.1, and short actin filaments. An analogous structure was recently described in platelets. Human polymorphonuclear leukocytes (PMNs) were examined for the presence and plasma membrane association of similar proteins. Isolated PMNs, free of contamination with erythrocytes or platelets, were disrupted by nitrogen cavitation and separated into subcellular organelles on a discontinuous Percoll gradient. Detergent lysates of plasma membrane vesicles, but not azurophilic or specific granules, contained insoluble actin filaments and associated proteins. Immunoblots of detergent-insoluble plasma membrane fractions contained proteins recognized by antibodies to brain fodrin and erythrocyte band 4.1, whereas blots probed with antibodies to erythrocyte spectrin and ankyrin were negative. Fodrin and band 4.1 were not detected in granule fractions, but some fodrin was present in the cytosol. The association of proteins related to fodrin and band 4.1 with the plasma membrane suggests that PMNs contain a submembranous skeleton structurally analogous to that of erythrocytes and platelets. The specific function of these proteins and their structural organization in human PMNs await further study.     

Detection and ultrastructural localization of human smooth muscle myosin-like molecules in human non-muscle cells by specific antibodies.

Spectrin, a protein complex which is peripherally attached to the cytoplasmic surface of the human erythrocyte membrane, cannot be detected (by complement fixation with anti-spectrin antibodies) in homogenates of several different human non-muscle cells studied. On the other hand, a protein antigenically identical or similar to human smooth muscle myosin was detected (by complement fixation with antibodies to uterine smooth muscle myosin) in these cells. In the case of human fibroblast line WI38, this smooth muscle myosin like component was shown (by ferritin-antibody experiments in electron microscopy) to be at least partly associated with cytoplasmic surface of the plasma membrane of the cell. It is proposed that the spectrin complex of the erythrocyte membrane and the smooth muscle myosin-like component of the fibroblast membrane play similar roles in regulating the translational mobilities of integral proteins in their respective membranes.     

Involvement of spectrin in membrane fusion: induction of fusion in human erythrocyte ghosts by proteolytic enzymes and its inhibition by antispectrin antibody.

In contrast to intact human erythrocytes, human erythrocyte ghosts can be agglutinated but not fused by Sendai virus. Membrane fusion can, however, be induced in virus-agglutinated erythrocyte ghosts by addition of proteolytic enzymes such as trypsin, papain, or Pronase. When erythrocyte ghosts were reacted with antispectrin antiserum, the antiserum inhibited both the induction of fusion and the proteolysis of the membrane spectrin. The correlation between the membrane fusion process and the membrane cytoskeleton is discussed.     

An 11-amino acid beta-hairpin loop in the cytoplasmic domain of band 3 is responsible for ankyrin binding in mouse erythrocytes

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 beta-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 beta-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.     

Chromosomal location of three spectrin genes: relationship to the inherited hemolytic anemias of mouse and man.

Three genetic loci in the mouse affect the synthesis and assembly of the erythrocyte membrane skeleton. The spherocytosis and jaundiced loci affect the membrane skeletal protein known as spectrin. The normoblastosis locus affects the spectrin binding protein called ankyrin. We have obtained genetic data that define the linkage relationships among three spectrin genes and the spherocytosis and jaundiced loci. The erythroid alpha-spectrin gene is tightly linked to the spherocytosis locus on chromosome 1 and the jaundiced locus is on chromosome 12, tightly linked to the erythroid beta-spectrin gene. The brain alpha-spectrin (alpha-fodrin) gene is located on the centromeric end of chromosome 2 and is not closely linked to any previously mapped erythroid or neurological mutation. These results are consistent with the hypothesis that defects in the alpha- and beta-spectrin genes cause the spherocytosis and jaundiced hemolytic anemias in mice. All five loci studied are located within chromosomal segments that are conserved between mouse and man. Analysis of the data from the chromosome 12 study defines a new order for the genes on that chromosome and delineates the largest mouse/human conserved chromosomal segment yet known.     

Visualization of the protein associations in the erythrocyte membrane skeleton.

We have obtained clear images of the erythrocyte membrane skeleton from negatively stained preparations that originate directly from the intact cell but in which the spectrin meshwork is artificially spread to allow close inspection. Our procedure requires less than 2 min at 5 degrees C in phosphate buffers. We find 200-nm-long spectrin tetramers crosslinked by junctional complexes. Each junction contains a regular 37-nm rod, probably an actin oligomer of approximately 13 monomers. Densities appear at variable places in the meshwork but distinct globules occur with great frequency 78 nm from the spectrin tetramer's junctional insertion end, very close to the known binding site for ankyrin. Most frequently, five or six spectrin tetramers insert into each junction, producing a meshwork that displays remarkably regular long range order.