The structure of erythrocyte membranes studied by freeze-etching. II. Localization of receptors for phytohemagglutinin and influenza virus to the intramembranous particles.

The distribution of specific glycoprotein receptors on the external surfaces of red cells was mapped, by the freeze-etching technique, to determine if the receptors coincided with the underlying 75-A intramembranous particles. Phytohemagglutinin, ferritin-conjugated phytohemagglutinin, and influenza virus were used as labeling agents since they can be seen by freeze-etching techniques and each reacts with a different site on the same glycoprotein molecule. The distribution of these labels was studied on intact human red cells, isolated ghost membranes, and trypsin-treated ghost membranes. The results show that the receptors for these labels are distributed uniformly over the surfaces of normal red cell membranes in the same apparent distribution as that of the 75-A particles within the membrane. The association between the external receptors and the underlying particles is especially evident when trypsin-treated ghost membranes are labeled: the labeled receptor sites and the intramembranous particles both form sharply defined, reticulated networks, which overlap. These results provide further support for the idea that membrane-bound glycoproteins are oriented so that their carbohydrate-rich segments, which bear the antigenic sites and receptors, are exposed to the external medium, while hydrophobic segments of the same molecules interact with lipids, and possibly other proteins, to form the intramembranous particles.

Self-assembly of spectrin oligomers in vitro: a basis for a dynamic cytoskeleton.

Purified human erythrocyte spectrin is able to form large oligomeric species without the collaboration of any other proteins. This reversible self-assembly process is both temperature and concentration dependent and seems to be mediated by the same kinds of low affinity noncovalent associations between spectrin monomers that promote tetramer formation. Low ionic strength extracts of erythrocyte membranes also contain these oligomeric species. These results support the idea that spectrin oligomers and the factors that regulate their formation may be responsible for both the stability and the versatility of the erythrocyte membrane cytoskeleton. It is postulated that the high concentrations of spectrin necessary for oligomerization are maintained in vivo by a high-affinity interaction with ankyrin. Such a coupling of high and low affinity interactions in multifunctional proteins may have significant implications for membrane structure and function.

The binding of vimentin to human erythrocyte membranes: a model system for the study of intermediate filament-membrane interactions.

We have characterized the association of the intermediate filament protein, vimentin, with the plasma membrane, using radioiodinated lens vimentin and various preparations of human erythrocyte membrane vesicles. Inside-out membrane vesicles (IOVs), depleted of spectrin and actin, bind I125-vimentin in a saturable manner unlike resealed, right-side-out membranes which bind negligible amounts of vimentin in an unsaturable fashion. The binding of vimentin to IOVs is abolished by trypsin or acid treatment of the vesicles. Extraction of protein 4.1 or reconstitution of the membranes with purified spectrin do not basically affect the association. However, removal of ankyrin (band 2.1) significantly lowers the binding. Upon reconstitution of depleted vesicles with purified ankyrin, the vimentin binding function is restored. If ankyrin is added in excess the binding of vimentin to IOVs is quantitatively inhibited, whereas protein 4.1, the cytoplasmic fragment of band 3, band 6, band 4.5 (catalase), or bovine serum albumin do not influence it. Preincubation of the IOVs with a polyclonal anti-ankyrin antibody blocks 90% of the binding. Preimmune sera and antibodies against spectrin, protein 4.1, glycophorin A, and band 3 exhibit no effect. On the basis of these data, we propose that vimentin is able to associate specifically with the erythrocyte membrane skeleton and that ankyrin constitutes its major attachment site.

The localization of Mg-Na-K-activated adenosine triphosphatase on red cell ghost membranes.

The lead salt method introduced by Wachstein and Meisel (12) for the cytochemical demonstration of ATPase activity was modified and used to determine sites of activity on red cell ghost membranes. Preliminary studies showed that aldehyde fixation and standard concentrations of the capture reagent Pb(NO(3))(2) resulted in marked inhibition of the ATPase activity of these membranes. By lowering the concentration of Pb(2+) and incubating unfixed red cell ghosts, over 50% of the total ATPase activity, which included an ouabain-sensitive, Na-K-activated component, could be demonstrated by quantitative biochemical assay. Cytochemical tests, carried out under the same conditions, gave a reaction product localized exclusively along the inner surfaces of the ghost membranes for both Mg-ATPase and Na-K-ATPase. These findings indicate that the ATPase activity of red cell ghosts results in the release of P(i) on the inside of the ghost membrane at sites scattered over its inner aspect. There were no deposits of reaction product on the outer surface of the ghost membrane, hence no indication that upon ATP hydrolysis P(i) is released outside the ghosts. Nor was there any clear difference in the localization of reaction product of Mg-ATPase as opposed to that of Na-K-ATPase.

Site specificity in vimentin-membrane interactions: intermediate filament subunits associate with the plasma membrane via their head domains.

Fragments of vimentin, generated by chemical or enzymatic cleavages, were analyzed for their capacity to bind to human inverted erythrocyte membrane vesicles. Only peptides comprising the amino-terminal head domain of vimentin molecules were competent in associating with the membranes. In vitro studies also demonstrated that isolated ankyrin (the major vimentin acceptor site on the membrane) binds to an oligomeric species of vimentin and prevents the formation of characteristic 10-nm filaments. These data, taken together with the observation that the NH2-terminal end of vimentin is implicated in the polymerization process (Traub, P., and C. Vorgias, J. Cell Sci., 1983, 63:43-67), imply that intermediate filaments may contact the membrane in an end-on fashion, using the exposed head domains of their terminal subunits.

The localization of spectrin on the inner surface of human red blood cell membranes by ferritin-conjugated antibodies.

Spectrin, a major protein constituent of mammalian red blood cell membrane preparations, has been localized on the inner surface of human red blood cell membranes by techniques that utilized specific ferritin-conjugated antibodies and fixation of membranes shortly after hemolysis so as to allow penetration of the ferritin-antibody labels. The labeling of spectrin was shown to be specific by the following criteria. (a) Nonhomologous ferritin-conjugated antibodies did not specifically bind to either membrane surface. (b) Blocking the membrane-bound spectrin with excess unconjugated antispectrin antibodies prevented ferritin-antibody labeling. (c) Removal of spectrin by treating the membrane preparation with a low ionic strength buffer containing ethylenediaminetetraacetate and beta-mercaptoethanol prevented labeling by specific ferritin-conjugated antibodies.

Heterogeneity of mRNA and protein products arising from the protein 4.1 gene in erythroid and nonerythroid tissues.

Immunologically cross-reactive isoforms of the cytoskeletal element protein 4.1 have been identified in many tissues in which they exhibit heterogeneity of molecular weight, abundance, and intracellular localization. To examine the basis for isoform production in erythroid and nonerythroid tissues, we have compared the structure and expression of cDNAs isolated from human erythroid and nonerythroid sources. We have encountered cDNAs representing many distinct mRNA sequences. These exhibit complete nucleotide sequence homology along most of their lengths. Differences were confined to five sequence blocks designated Motifs I-V, which were present or absent in each mRNA moiety. Motif I was expressed only in erythroid cells; it encodes 21 amino acids in a well-characterized spectrin/actin binding domain. Motif II, located near the COOH terminus of the 80-kD "erythroid" protein 4.1 molecule is present in the vast majority of transcripts from both erythroid and nonerythroid cells. Motifs IV and V alter the 5' untranslated region: simultaneous insertion of Motif IV and deletion of Motif V in the untranslated region inserts a new initiator methionine and establishes a contiguous open reading frame encoding a novel 135-kD protein 4.1 molecule. By immunochemical analysis we have identified the longer isoform in cells. Our results are most consistent with tissue-specific alternative mRNA splicing of transcripts of the protein 4.1 gene to yield numerous isoforms. These isoforms exhibit tissue specificity and alter strategic portions of the molecule. Moreover, we describe a novel high molecular weight form of protein 4.1 that arises by splicing events which allow translation at an upstream site.

A nonerythroid isoform of protein 4.1R interacts with the nuclear mitotic apparatus (NuMA) protein.

Red blood cell protein 4.1 (4.1R) is an 80- kD erythrocyte phosphoprotein that stabilizes the spectrin/actin cytoskeleton. In nonerythroid cells, multiple 4.1R isoforms arise from a single gene by alternative splicing and predominantly code for a 135-kD isoform. This isoform contains a 209 amino acid extension at its NH2 terminus (head piece; HP). Immunoreactive epitopes specific for HP have been detected within the cell nucleus, nuclear matrix, centrosomes, and parts of the mitotic apparatus in dividing cells. Using a yeast two-hybrid system, in vitro binding assays, coimmunolocalization, and coimmunoprecipitation studies, we show that a 135-kD 4.1R isoform specifically interacts with the nuclear mitotic apparatus (NuMA) protein. NuMA and 4.1R partially colocalize in the interphase nucleus of MDCK cells and redistribute to the spindle poles early in mitosis. Protein 4.1R associates with NuMA in the interphase nucleus and forms a complex with spindle pole organizing proteins, NuMA, dynein, and dynactin during cell division. Overexpression of a 135-kD isoform of 4.1R alters the normal distribution of NuMA in the interphase nucleus. The minimal sequence sufficient for this interaction has been mapped to the amino acids encoded by exons 20 and 21 of 4.1R and residues 1788-1810 of NuMA. Our results not only suggest that 4.1R could, possibly, play an important role in organizing the nuclear architecture, mitotic spindle, and spindle poles, but also could define a novel role for its 22-24-kD domain.

Selective solubilization of a protein component of the red cell membrane.

Approximately 20 percent of the membrane-bound protein of erythrocyte ghosts can be solubilized and obtained free of other membrane components by dialysis against adenosine triphosphate and 2-mercaptoethanol. This protein forms one major band on polyacrylamide gels and a single boundary in free-boundary electrophoresis, and it undergoes polymerization in the presence of divalent cations to form coiled filaments visible by electron microscopy. Antibodies to this membrane protein react specifically with red blood cells or their membrane ghosts but do not react with serum, erythrocyte cytoplasm, or other blood cells. The functional role of this protein is unknown, but it appears to be involved in maintaiining the structure of the red cell membrane. We suggest that this protein be called Spectrin since it is obtained from membrane ghosts.

Glycoproteins: isolation from cellmembranes with lithium diiodosalicylate.

A glycoprotein has been extracted in water-soluble form from human red cell membranes with lithium diiodosalicylate. After extraction of the membranes and phosphocellulose chromatography a homogeneous preparation is obtained which was 60 percent carbohydrate and 40 percent protein (by weight). The preparation contains AB and MN blood group antigens, receptors for influenza virus, and various phytohemagglutinins.

Mutant forms of spectrin alpha-subunits in hereditary elliptocytosis.

Two variant spectrins have been described in hereditary elliptocytosis (HE) and pyropoikilocytosis (HPP). Both are characterized by increased susceptibility of the alpha I (N-terminal) 80-kD domain to mild tryptic digestion, yielding peptides of 46-50 or 65-68 kD (T50a and T68 in our terminology). In this report we add a third unstable spectrin alpha I domain found in three kindreds with HE; alpha IT80 in this type of spectrin is cleaved by mild tryptic digestion to a 50-kD peptide (T50b) distinguished from T50a by its more basic isoelectric point. All three spectrins show impaired self-association to form oligomers. Intermediate tryptic peptides of the three unstable alpha I domains from HE spectrins were characterized by monoclonal immunoblotting and I125 limit peptide mapping and affinity purified using polyclonal anti-alpha IT80. Partial amino acid sequences of alpha I domain peptides were obtained from two unrelated patients for each of the three variant spectrins. T50a results from cleavage at arginine 250 or lysine 252 of alpha IT80; a proline replaced the normal leucine or serine at residues 254 and 255, respectively. T50b and a 19-kD peptide result from cleavage at arginine 462 or arginine 464; a proline replaced the normal residue 465 (in T19b) in one of the two patients studied. T68 results from cleavage at arginine 131. In both 68-kD peptides examined, a leucine is inserted at residue 150. The relationship of the sequence changes to the new tryptic cleavages, to the current model of alpha I domain structure, and to defective spectrin self-association is discussed.

Molecular analysis of insertion/deletion mutations in protein 4.1 in elliptocytosis. I. Biochemical identification of rearrangements in the spectrin/actin binding domain and functional characterizations.

Protein 4.1 (80 kD) interacts with spectrin and short actin filaments to form the erythrocyte membrane skeleton. Mutations of spectrin and protein 4.1 are associated with elliptocytosis or spherocytosis and anemia of varying severity. We analyzed two mutant protein 4.1 molecules associated with elliptocytosis: a high molecular weight 4.1 (95 kD) associated with mild elliptocytosis without anemia, and a low molecular weight 4.1 (two species at 68 and 65 kD) associated with moderate elliptocytosis and anemia. 4.1(95) was found to contain a approximately 15-kD insertion adjacent to the spectrin/actin binding domain comprised, at least in part, of repeated sequence. 4.1(68/65) was found to lack the entire spectrin-actin binding domain. The mechanical stability of erythrocyte membranes containing 4.1(95) was identical to that of normal membranes, consistent with the presence of an intact spectrin-actin binding domain in protein 4.1. In contrast, membranes containing 4.1(68/65) have markedly reduced mechanical stability as a result of deleting the spectrin-actin binding domain. The mechanical stability of these membranes was improved following reconstitution with normal 4.1. These studies have thus enabled us to establish the importance of the spectrin-actin binding domain in regulating the mechanical stability of the erythrocyte membrane.

Common structural polymorphisms in human erythrocyte spectrin.

Restricted tryptic digestion of erythrocyte spectrin at 4 degrees C followed by two-dimensional (isoelectric-focusing/sodium dodecyl sulfate) polyacrylamide electrophoresis yields highly reproducible maps of approximately 50 peptides with molecular weights between 80,000 and 12,000. Based on molecular weight and isoelectric point (pI), each unique alpha- and beta-subunit domain can be identified and compared with spectrin peptides from other individuals. The alpha-subunit of spectrin from 60 Caucasian donors contains a 46,000-mol-wt tryptic domain, called alpha II-T46, Type 1; more extensive tryptic digestion of this domain generates peptides with molecular weights of 35,000, 30,000, 25,000, and 16,000. Spectrin from 29 of 37 black donors representing 14 kindreds shows variation in the molecular weight and/or pI of peptides from the alpha II domain. In the most common form, Type 2, alpha II tryptic peptides are increased in molecular weight by 4,000, and the pI becomes more basic. Other alpha II variants are characterized by either the 4,000 increase in molecular weight (Type 3) or by the basic shift in pI (Type 4). When limit peptide maps of intermediate-sized tryptic and CNBr peptides from the alpha II-domain Types 1 and 2 are compared, a consistent alteration in the chromatographic mobility of one limit peptide is observed. Polymorphism in the alpha II subunit of spectrin did not itself produce anemia, nor did it appear to alter the expression of an underlying hereditary spherocytosis or elliptocytosis. In six family studies, the alpha II 46,000-mol-wt variations observed were consistent with Mendelian inheritance.

Sequence and exon-intron organization of the DNA encoding the alpha I domain of human spectrin. Application to the study of mutations causing hereditary elliptocytosis.

We have determined the exon-intron organization and the nucleotide sequence of the exons and their flanking intronic DNA in cloned genomic DNA that encodes the first 526 amino acids of the alpha I domain of the human red cell spectrin polypeptide chain. From the gene sequence we designed oligonucleotide primers to use in the polymerase chain reaction technique to amplify the appropriate exons in DNA from individuals with three variants of hereditary elliptocytosis characterized by the presence of abnormal alpha I spectrin peptides, 46-50 and 65-68 kD in size, in partial tryptic digests of spectrin. The alpha I/68-kD abnormality resulted from a duplication of leucine codon 148 in exon 4: TTG-CTG to TTG-TTG-CTG. The alpha I/50a defect was associated in different individuals with two separate single base changes in exon 6: CTG to CCG (leucine to proline) encoding residue 254, and TCC to CCC (serine to proline) encoding residue 255. In another individual with the alpha I/50a polypeptide defect, the nucleotide sequence encoding amino acid residues 221 through 264 was normal. The alpha I/50b abnormality resulted from a single base change of CAG (glutamine) to CCG (proline) encoding residue 465 in exon 11 in two unrelated individuals. In a third individual with alpha I/50b-kD hereditary elliptocytosis, the entire exon encoding residues 445 through 490 was normal. The relationship of the alpha I domain polypeptide structure to these mutations and the organization of the gene is discussed.

Molecular and functional changes in spectrin from patients with hereditary pyropoikilocytosis.

The structural and functional properties of spectrin from normal and hereditary pyropoikilocytosis (HPP) donors from the two unrelated families were studied. The structural domains of the spectrin molecule were generated by mild tryptic digestion and analyzed by two-dimensional electrophoresis (isoelectric focusing; sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The alpha I-T80 peptide (Mr 80,000) is not detectable in two related HPP donors; instead, two new peptides (Mr 50,000 and 21,000) are generated and have been identified as fragments of the normal alpha I-T80. A third sibling has reduced levels of both the normal alpha I-T80 and the two new peptides. A similar analysis of spectrin from another HPP family indicates that their spectrins contain reduced amounts of the alpha I-T80 and the 50,000 and 21,000 fragments of the alpha I domain. The HPP donor also has other structural variations in the alpha I, alpha II, and alpha III domains. The alpha I-T80 domain of normal spectrin has been shown to be an important site for spectrin oligomerization (J. Morrow and V.T. Marchesi. 1981. J. Cell Biol. 88: 463-468), and in vitro assays indicate that HPP spectrin has an impaired ability to oligomerize. Ghost membranes from HPP donors are also more fragile than membranes from normal erythrocytes when measured by ektacytometry. In both the oligomerization and fragility assays, the degree of impairment is correlated with the amount of normal alpha I-T80 present in the spectrin molecule. We believe that a structural alteration in the alpha I-T80 domain perturbs normal in vivo oligomerization of spectrin, producing a marked decrease in erythrocyte stability.

Comparison of nonerythroid alpha-spectrin genes reveals strict homology among diverse species.

The spectrins are a family of widely distributed filamentous proteins. In association with actin, spectrins form a supporting and organizing scaffold for cell membranes. Using antibodies specific for human brain alpha-spectrin (alpha-fodrin), we have cloned a rat brain alpha-spectrin cDNA from an expression library. Several closely related human clones were also isolated by hybridization. Comparison of sequences of these and other overlapping nonerythroid and erythroid alpha-spectrin genes demonstrated that the nonerythroid genes are strictly conserved across species, while the mammalian erythroid genes have diverged rapidly. Peptide sequences deduced from these cDNAs revealed that the nonerythroid alpha-spectrin chain, like the erythroid spectrin, is composed of multiple 106-amino-acid repeating units, with the characteristic invariant tryptophan as well as other charged and hydrophobic residues in conserved locations. However, the carboxy-terminal sequence varies markedly from this internal repeat pattern and may represent a specialized functional site. The nonerythroid alpha-spectrin gene was mapped to human chromosome 9, in contrast to the erythroid alpha-spectrin gene, which has previously been assigned to a locus on chromosome 1.

Self-association of human erythrocyte glycophorin A. Appearance of low mobility bands on sodium dodecyl sulfate gels.

We have examined the self-association of glycophorin A, the major sialoglycoprotein of the human erythrocyte membrane, using sodium dodecyl sulfate (SDS) polyacrylamide gels and circular dichroism. Pure glycophorin A has a tendency to form multiple bands on SDS gels at positions of higher apparent molecular weight than the PAS 1 and PAS 2 bands previously seen. These high molecular weight bands do not have mobilities corresponding to integral polymers of PAS 1 and PAS 2. Circular dichroism spectra of solutions giving rise to these bands or to PAS 1 and PAS 2 bands alone, indicate that these species all have essentially the same peptide conformation.

Hollow cylinder protein in the cytoplasm of human erythrocytes.

A "Hollow Cylinder Protein' (HCP) similar to the protein originally isolated by Harris from human erythrocyte membranes (Harris, J.R. (1968) Biochim. Biophys. Acta 150, 534--537) is present in the cytosol of erythrocytes at a concentration of more than 15 micrograms/ml packed erythrocytes. When negatively stained and examined in the electron microscope, cytosol HCP is similar in morphology to the HCP associated with erythrocyte ghost membranes. Cytosol HCP can be purified by isoelectric precipitation at pH 5.2 followed by repeated sucrose gradient centrifugation at alkaline pH. Negatively stained purified cytosol HCP appears as a hollow cylinder with apparent dimensions of 18.0 nm in length by 11.8 nm in diameter and contains a hollow core. Purified cytosol HCP migrates as a single band by non-denaturing polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis shows that it is composed of five peptides having apparent molecular weights 21 500, 23 500, 26 000, 27 500 and 29 000. Chymotryptic peptide maps of each of these bands indicate that each is a unique polypeptide chain. These results indicate that erythrocyte cytosol HCP is a macromolecular complex composed of multiple copies of five non-identical subunits arranged as a hollow cylinder.

Interaction between the subunits of human erythrocyte spectrin using a fluorescence probe.

Fluorescence labeling of spectrin subunits was performed with N-(1-anilinonaphthyl-4)maleimide (ANM) to study the interaction between alpha and beta subunits. The fluorescence anisotropy of both ANM alpha and ANM beta increased linearly with the addition of nonfluorescent beta or alpha subunit, and saturated at a protein ratio about 1, indicating that 1 mol alpha subunit binds to 1 mol beta subunit with high affinity in vitro. Furthermore, this binding seemed to be reversible, because the anisotropy value decreased when an excess fo nonfluorescent alpha was added to the ANM alpha/beta mixture. The anisotropy of ANM alpha attained a maximum level within l min after addition of the same quantity of nonfluorescent beta at 12 degrees C, and the anisotropy of this mixture decreased rapidly when an excess of nonfluorescent alpha was added. These findings suggested that both the binding process of beta to ANM alpha and the dissociation step of ANM alpha from the ANM alpha-beta complex were quite rapid. The results obtained here imply that dynamic interaction between alpha and beta subunits of spectrin should be taken into account in understanding the role of the spectrin molecule in the cytoskeletal mesh.

Purification and characterization of acid proteinase from human erythrocyte membranes.

Two forms of an acid proteinase have been purified from human erythrocyte membranes by a simple method involving selective extraction with 0.5% Brij 35, affinity chromatography on pepstatin A-Sepharose 4B, and chromatography on Sephacryl S-200 and DEAE-Sephadex G-100. One species has an apparent molecular weight of 73 000 as measured by gel filtration and has been purified to apparent homogeneity. This form can be resolved by two-dimensional polyacrylamide gel electrophoresis into two bands with isoelectric points of about 4.5 and 5.0 and with molecular weights of 44 000 and 29 000, respectively. The second form of the enzyme has a molecular weight of about 47 000 and has also been purified to apparent homogeneity. In SDS-polyacrylamide gel electrophoresis the second form showed a single protein band corresponding to a molecular weight of 44 000. Two-dimensional polyacrylamide gel electrophoresis revealed a single band with an isoelectric point of about 4.5 and a molecular weight of 44 000. The high-molecular-weight form represented about a 17 500-fold purification over the erythrocyte membranes and about a 24% recovery, while the low-molecular-weight form represented about a 25 000-fold purification and about a 25% recovery. The two species had similar pH optimum and showed equal sensitivity to a number of ions and proteinase inhibitors. Chymotryptic maps of the 44 000 peptide of high-molecular-weight form and the purified low-molecular-weight form revealed no difference.