The elongated proteins of the spectrin family (dystrophin, alpha-actinin, and spectrin) contain tandemly repeated segments and form resilient cellular meshworks by cross-linking actin filaments. The structure of one of the repetitive segments of alpha-spectrin was determined at a 1.8 angstrom resolution. A segment consists of a three-helix bundle. A model of the interface between two tandem segments suggests that hydrophobic interactions between segments may constrain intersegment flexibility. The helix side chain interactions explain how mutations that are known to produce hemolytic anemias disrupt spectrin associations that sustain the integrity of the erythrocyte membrane.

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Many proteins contain a repetitive sequence motif, which implies that they contain a repetitive structural motif. Spectrin and the related proteins dystrophin and a-actinin consist largely of repeated motifs of 100-120 residues. But the repeating motif is degenerate and it has been difficult to derme the boundaries of the repeating sequence unit or its corresponding structural unit. We have determined at which residues the structural units that correspond to spectrin's repeating 106lOaano acid motifs begin and end. Drosophila a-spectrin BdNAs were expressed in bacteria to show that single (106 amino acids) and pairs of segments encoded by selected regions of spectrin cDNA can fold into stable conformations whose biophysical and biochemical properties are similar to those of native spectrin. Because such folig was critically dependent on the phasing ofthe expressed sequence with respect to the apparent boundaries of the repeating motifs, our data provide experimental evidence that relates the boundaries of the folded, conformational unit to the chemical sequence of repeating motifs.Spectrins, some of which are also called fodrin, are elongated, flexible molecules whose primary structure is dominated by tandem, homologous, 106-residue motifs (1) that have been referred to as repeating units or segments (2). Dystrophin and a-actinin, other members of the spectrin superfamily (3, 4), contain comparable repetitive sequences. Although secondary-structure predictions suggest that each of the repetitive motifs of spectrin folds as a closely packed, triple a-helical unit, neither spectrin itself, nor proteolytically derived domains of spectrin, have been tractable samples for direct structure determination. Because the 106-residue motifs are generally repeated without interruptions, the residue that corresponds to the beginning (or end) of a structural unit is not self-evident. Consider, for example, the repetitive sequences of letters ABCDABCDABCD .... To produce a 4-letter segment of this sequence that can form a structural unit, it is essential to know whether the structure is ABCD, BCDA, CDAB, or DABC. Similarly, to express a 106-residue segment of spectrin that will fold into a structural unit, one must know at which position in any of the repetitive motifs that are evident in the primary sequence the segment should start and end. Speicher and Marchesi (1) stated clearly that the convention they adopted to illustrate the repetitive sequences was not intended to correlate with the conformational unit of the folded protein.Using cDNA constructs derived from Drosophila a-spectrin and expressed in bacteria, we show that single repetitive motifs (106 amino acids) and pairs of motifs can fold into stable conformations similar to that of native spectrin when their N-terminal ends are 26 residues downstream of the residue that, according to the convention of Speicher and Marchesi (1), has usually been designated to be in the first position of each repetitive motif. Similarly sized polypeptides with other phasing do ...

Abstract. Infections with the human malaria Plasmodium falciparum are characterized by the retention of parasitized erythrocytes in tissue capillaries and venules. Erythrocytes containing trophozoites and schizonts attach to the endothelial cells that line these vessels by means of structurally identifiable excrescences present on the surface of the infected cell. Such excrescences, commonly called knobs, are visible by means of scanning or transmission electron microscopy. The biochemical mechanisms responsible for erythrocyte adherence to the endothelial cell are still undefined. In an attempt to identify the cytoadhesive molecule on the surface of the infected cell, we have prepared monoclonal antibodies to knob-bearing erythrocytes infected with the FCR-3 strain of P.falciparum. One of these monoclonal antibodies, designed 4A3, is an IgM that reacts (by means of immunofluorescence) with the surface of unfixed erythrocytes bearing mature parasites of the knobby line; it does not react with knobless lines or uninfected erythrocytes. By immunoelectron microscopy the monoclonal antibody 4A3 was localized to the knob region. In an in vitro cytoadherence assay, the monoclonal antibody partially blocked the binding of knob-bearing cells (FCR-3 strain) to formalin-fixed amelanotic melanoma cells. The monoclonal antibody was used to immunoprecipitate a protein from extracts of knobby erythrocytes that had been previously surface iodinated. By a two-dimensional peptide mapping technique, the antigen recognized by the monoclonal antibody was found to be structurally related to band 3 protein, the human erythrocyte anion transporter.

The a and P chains of spectrin are homologous, yet they have acquired different structural features that work in synergy to give the multimer its overall properties. The primary amino acid sequence of each spectrin subunit is dominated by tandemly repeated 106-residue motifs. By comparing the complete Drosophila f-spectrin sequence with other spectrins we have discovered evidence that a higher-order, 848-amino acid supra-motif is tandemly repeated in both aand .3-spectrin. These data argue that a-and (3-spectrin, rather than evolving independently from sequences encoding the ancestral 106-residue motifs, must have arisen after the establishment of a large supra-motif composed of eight of the 106-residue motifs. Our data suggest the segment structure of a progenitor gene that gave rise to both a-and 13-spectrin as well as dystrophin. The structural differences that evolved after the split between the a-and /3-spectrin genes confer the independent functions that exist in their products today.Spectrin is an important component of the membrane skeleton in most, if not all, cell types. The a and /3 subunits of spectrin are large, elongated polypeptides that have common evolutionary origins but have since acquired many distinct structural and functional attributes. For example, although the known a subunits are very similar to each other in molecular mass (-280 kDa), the molecular mass of known 13-spectrins range from 246 kDa to 430 kDa (1, 2). In addition, diversity in the /8 subunits is thought to account for most of the distinctive structural and functional attributes of different spectrin isoforms (3, 4).As first described by Speicher and Marchesi (5), a structural feature common to the a-and ,B-spectrin subunits is the presence of tandemly repeated 106-residue motifs that are also referred to as repeating units or segments (6). The repeating segments typically display a limited sequence similarity with one another and are defined by the presence of a consensus profile of conserved residues that occur at characteristic positions in each segment. Dystrophin and a-actinin, other members of the spectrin superfamily (7, 8), also contain homologous repetitive motifs. It has been postulated that a-spectrin arose by the serial duplication of an ancestral sequence that encoded the 106-residue segments, followed by the duplication of a larger block of DNA encoding several of these 106-residue segments (9-11). Here we present data showing that, rather than a simple duplication in a-spectrin alone, the repetitive regions of both present-day a-and P-spectrin genes were derived from a tandemly repeated supra-motif that encoded eight of the 106-amino acid motifs.tWe also find that the primary sequences of Drosophila and human ,B-spectrin are quite similar and coextensive except at their carboxyl-terminal ends. MATERIALS AND METHODSNested deletions of two cDNA clones, B15 and B32, that contain the complete coding region of Drosophila f8-spectrin were generated by exonuclease digestion essentially as described (12). Nucle...

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Erythrocytes infected with a knobby variant of Plasmodium falciparum selectively bind IgG autoantibodies in normal human serum. Quantification of membrane-bound IgG, by use of '251-labeled protein A, revealed that erythrocytes infected with the knobby variant bound 30 times more protein A than did noninfected erythrocytes; infection with a knobless variant resulted in less than a 2-fold difference compared with noninfected erythrocytes. IgG binding to knobby erythrocytes appeared to be related to parasite development, since binding of 1251-labeled protein A to cells bearing young trophozoites (less than 20 hr after parasite invasion) was similar to binding to uninfected erythrocytes. By immunoelectron microscopy, the membrane-bound IgG on erythrocytes infected with the knobby variant was found to be preferentially associated with the protuberances (knobs) of the plasma membrane. The removal of aged or senescent erythrocytes from the peripheral circulation is reported to involve the binding of specific antibodies to an antigen (senescent antigen) related to the major erythrocyte membrane protein band 3. Since affinity-purified autoantibodies against band 3 specifically bound to the plasma membrane of erythrocytes infected with the knobby variant of P. fakiparum, it is clear that the malaria parasite induces expression of senescent antigen.Erythrocytes infected with the human malaria parasite Plasmodium falciparum become immunologically altered as a result ofparasite development. These changes, which occur primarily toward the end of the parasite's 48-hr developmental cycle, have been identified by techniques capable of detecting membrane-bound immunoglobulins (1-3). It has been proposed (4) that the newly exposed antigens on the surface of infected erythrocytes are the result of parasitesynthesized proteins that are inserted into the host cell membrane; however, in spite of intense efforts by several laboratories such parasite proteins have been neither fully characterized nor specifically localized, and the manner of transport from the parasite to the erythrocyte surface is yet to be convincingly demonstrated.It is entirely possible that some of the antigenic changes observed for malaria-infected erythrocytes result from parasiteprovoked exposure or modification of host cell antigens. For example, Kay (5) showed that immunoglobulins were specifically bound to the surface of old erythrocytes, and as a result in vitro phagocytosis by macrophages occurred. These autoantibodies, found in the sera ofnormal individuals, bound to the major erythrocyte integral membrane protein, band 3 (6, 7 Elution of membrane-bound IgG was performed according to the procedure of Rekvig and Hannesta (13), except that the incubation time in isotonic glycine buffer was increased to 5 min at 0WC. Cells were layered over a 10% (wt/vol) dextran cushion and centrifuged for 2 min in a Beckman Microfuge. The pelleted cells were washed five to seven times in PBS and then incubated with antibody (70 ,ug/ml) purified by band 3-Sepharose ...

Little is known about the molecular mechanism underlying the release of merozoites from malaria-infected erythrocytes. In the present study, video microscopy was carried out, and images throughout the process of merozoite release from Plasmodium falciparum-infected erythrocytes were digitized and analyzed. Merozoites were shown to escape from the infected host cell in about 1 s through a single site of the infected erythrocyte membrane, whose dimension was estimated to be 2.5 microm. Merozoites were released together with the residual body containing hemozoin, leaving behind a membranous structure that persisted even after an extended period of observation. Densitometric measurements showed that the cytoplasmic content of the infected erythrocyte did not diffuse out as parasites were released, but was gradually lost thereafter. This would indicate that the release of merozoites from infected erythrocytes is not mediated by an explosive event.