A 96-bp synthetic oligonucleotide corresponding to an amino acid sequence near the N-terminus of erythroid β-spectrin was used to screen a human genomic library, and two overlapping recombinants were isolated. DNA sequence analysis established that the genomic fragment encoded β-fodrin, the nonerythroid form of β-spectrin, by correlation to a known amino acid sequence of human brain β-fodrin. The genomic DNA contained regions that cross-hybridized with an erythroid β-spectrin cDNA probe, and the DNA sequence of these regions revealed a high degree of identity with that of erythroid β-spectrin and a similar exon/intron organization. A single-copy DNA fragment of the β-fodrin genomic clone was used to screen a lymphoid cell cDNA library and two recombinants were isolated. The composite DNA sequence of these various genomic and cDNA clones encoded almost all of the first twelve 106 amino acid repeat segments of β-fodrin that shared 58% identity and 75.5% similarity with the amino acid sequence of β-spectrin and 66% identity with the nucleotide sequence of β-spectrin cDNA. The chromosomal localization of the gene was determined to be chromosome 2 by hybridization of a single-copy probe derived from the cloned genomic DNA to DNA of a panel of somatic hybrid cell lines, and in situ hybridization localized the gene to band 2p21. β-Fodrin was assigned the gene symbol SPTBN1 .

The calcium-dependent proteolysis of fodrin has been implicated in the regulation of secretion, neutrophil and platelet activation, and long-term potentiation in neurons. In vitro studies indicate that calcium-dependent protease I (calpain I) cleaves fodrin in the middle of the alpha subunit and in the COOH-terminal third of the beta subunit. Cleavage at the beta site requires calmodulin, which binds with high affinity to a single site in the alpha subunit. In vitro binding assays, nondenaturing gel electrophoresis, and velocity sedimentation identify a linkage between calcium-dependent protease I proteolysis of fodrin and the ability of calmodulin to regulate the self-association of fodrin and its interaction with actin. Three functional states appear to exist: (i) intact fodrin, which constitutively forms tetramers and binds F-actin; (ii) alpha-cleaved fodrin, which loses its ability to self-associate and bind F-actin in the presence of calmodulin; and (iii) alpha,beta-cleaved fodrin, a form that is incompetent to establish tetramers or bind actin. Because actin binding and fodrin self-association occur at opposite ends of the molecule, whereas calmodulin binds at its center, these results indicate that long-range interactions exist within fodrin. They also offer an example of how two calcium-dependent regulatory processes may act synergistically to reversibly regulate a linkage between the membrane and the cytoskeleton.

The intracellular calcium-dependent proteolysis of fodrin has been postulated to be central to the regulation of plasticity of the cortical cytoskeleton of many eukaryotic cells. The close proximity of the sites of calmodulin (CaM) binding and calcium-dependent protease I (CDP-I) cleavage in mammalian alpha-fodrin suggested that their action may be linked. In hypotonic and isotonic buffers, CDP-I proteolysis of the beta subunit of fodrin was absolutely dependent upon the presence of active CaM. The stimulation by CaM was inhibited by CaM antagonists. The rate of CDP-I proteolysis of both subunits was enhanced by CaM, while the rate of fodrin proteolysis with other proteases was not influenced by CaM. The increase in the susceptibility of fodrin to CDP-I proteolysis was half-maximal at 80 nM CaM, and maximal at 200 nM CaM. The unusual and differential susceptibility of alpha- and beta-fodrin to proteolysis by CDP-I in the absence of CaM was exploited to investigate the quaternary structure of fodrin in which only the alpha subunit was cleaved. Cleavage of the alpha subunit alone did not destroy the tetrameric form of the molecule, whereas CDP-I cleavage of both subunits rendered the molecule incapable of reforming tetramers. These results provide structural and functional evidence that CaM and CDP-I act synergistically in the regulated proteolysis of fodrin.

Fodrin (brain spectrin) binds calmodulin and is susceptible to proteolysis by calcium-dependent protease I (CDP-I, calcium-activated neutral protease I, or calpain I). Both events involve the central region of the alpha-fodrin subunit, and calmodulin binding enhances the sensitivity of fodrin to CDP-I mediated proteolysis. Fragments of fodrin, generated chemically or proteolytically, which retain calmodulin binding activity have been identified and analyzed by two-dimensional peptide mapping and by direct protein sequencing. Both CDP-I and calmodulin interact with the terminal portion of the eleventh repetitive unit in fodrin, which is at the center of the molecule. CDP-I cleavage occurs between Tyr104 and Gly105 and preserves the calmodulin binding activity of the carboxyl-terminal fragment. In contrast, chymotryptic cleavage at Trp120 reduces the ability of this fragment to bind calmodulin, and tryptic cleavage beyond Trp120 completely eliminates calmodulin binding activity. It is concluded that Ser-Lys-Thr-Ala-Ser-Pro-Trp-Lys-Ser-Ala-Arg-Leu-Met-Val-His-Thr-Val-Ala- Thr- Phe-Asn-Ser-Ile-Lys, a 24-residue peptide which bridges repeats 11 and 12 of brain alpha spectrin contains the high affinity calmodulin binding domain.

The processing of brain spectrin (fodrin) by calcium-dependent proteases at the postsynaptic membrane has been postulated to be one of the central molecular mechanisms underlying long-term potentiation (LTP). The effect of such processing on the structure and function of brain spectrin, and on spectrin's ability to organize or otherwise regulate receptor function remains unclear. To address these issues, human and bovine brain spectrin were digested under mild conditions with several proteases, and the resulting cleavage fragments analyzed by 2-dimensional chymotryptic 125I peptide mapping. These studies identify an underlying protease-resistant domain structure reminiscent of, yet distinctly different from, human erythroid spectrin. More importantly, fodrin is unusual for the presence of a single, proteolytically hypersensitive site in the center of the alpha subunit, which is the favored site of action by many proteases, including the calcium-dependent neutral proteases. This proteolytically hypersensitive site is a unique feature of alpha nonerythroid spectrin since it is absent from human erythrocyte spectrin and appears to be the site at which the molecule is processed in vivo. In addition, on the basis of gel overlay techniques, it appears that the hypersensitive site is also the site at which calmodulin binds to the alpha-subunit in a calcium-dependent manner. These studies thus establish at the molecular level 2 calcium-dependent mechanisms by which brain spectrin function might be regulated and provide a conceptual and methodological framework for further investigation into the function of this important molecule.

The ability of protein 4.1 to stimulate the binding of spectrin to F-actin has been compared by cosedimentation analysis for three avian (erythrocyte, brain, and brush border) and two mammalian (erythrocyte and brain) spectrin isoforms. Human erythroid protein 4.1 stimulated actin binding of all spectrins except the brush border isoform (TW 260/240). These results suggested that the beta subunit determined the protein 4.1 sensitivity of the heterodimer, since all avian alpha subunits are encoded by a single gene. Tissue-specific posttranslational modification of the alpha subunit was excluded by examining the properties of hybrid spectrins composed of the purified alpha subunit from avian erythrocyte or brush border spectrin and the beta subunit of human erythrocyte spectrin. A hybrid composed of avian brush border alpha and human erythroid beta spectrin ran on nondenaturing gels as a discrete band, migrating near human erythroid spectrin tetramers. The actin-binding activity of this hybrid was stimulated by protein 4.1, while either chain alone was devoid of activity. Therefore, although both subunits were required for actin binding, the sensitivity of the spectrin-actin interaction to protein 4.1 is a property uniquely bestowed on the heterodimer by the beta subunit. The singular insensitivity of brush border spectrin to stimulation by erythroid protein 4.1 was also consistent with the absence of proteins in avian intestinal epithelial cells which were immunoreactive with polyclonal antisera sensitive to all of the known avian and human erythroid 4.1 isoforms.

A study of human erythrocyte and brain spectrin with particular emphasis on the beta subunits revealed a structural homology but functional dissimilarity between these two molecules. Six monoclonal antibodies raised to human erythrocyte beta spectrin identify three of the four proteolytically defined domains of erythrocyte beta spectrin. Five of these monoclonal antibodies cross-react with human brain spectrin. None of a previously identified set of alpha erythrocyte spectrin monoclonal antibodies [Yurchenco et al: J Biol Chem 257:9102, 1982] reacted with brain spectrin. A domain map generated by limited tryptic digestion shows that brain spectrin is composed of proteolytically resistant domains analogous to erythrocyte spectrin, but the brain protein is more basic. The binding of brain spectrin to erythrocyte ankyrin, both in solution and on erythrocyte IOVs, yielded an association constant approximately 100 time weaker than for erythrocyte spectrin. The binding of azido-calmodulin under native conditions was specific for the erythrocyte beta subunit but was not calcium dependent. In contrast, azido-calmodulin bound only to the alpha subunit of brain spectrin in a calcium-dependent manner. The similarity of structure but modified functional characteristics of the brain and erythrocyte beta spectrins suggest that these proteins serve different cellular roles.

Human erythrocyte and brain spectrin (fodrin, calspectin) have been compared quantitatively with respect to the extent and sites of antigenic and functional similarity. Brain spectrin cross-reacts strongly with approx. 1% of the epitopes in erythrocyte spectrin, but weakly with at least 50%. The distribution of shared determinants is not uniform. Brain spectrin is most deficient in epitopes characteristic of the 80 kDa and 52 kDa domains of the α-subunit (α-I and α-III) and of terminal portions of the 28 kDa and 74 kDa domains of the β-subunit (β-I and β-IV). The functions associated with these domains also differ between the two proteins. Brain spectrin does not undergo extensive polymerization and binds calmodulin at a different site. The unique ability of erythrocyte spectrin to oligomerize beyond the tetramer reflects its role in the membrane skeleton. Non-erythroid spectrins probably function as specific linkers between membrane receptors and the filamentous cytoskeleton. In this sense, they may act as regulated transducers of information flow between the membrane and the cytoplasmic matrix.

Human erythrocytes were observed to undergo a discocyte to echinocyte to spheroechinocyte shape transformation during brief incubation with endotoxic lipopolysaccharide. It was concluded that lipopolysaccharide-membrane interactions alter the curvature of erythrocyte membranes.

Approximately 25 percent of black blood donors have an elevated red blood cell (RBC) sodium (Nai) level compared with white donors. This elevation results in a significant increase in the mean Nai from black (9.00 ± 2.96 mmoles/l RBC) as compared to white blood donors (7.04 ± 1.48 mmoles/l RBC, p less than 0.001). Red blood cells from four black donors with mean Nai levels of 15 ± 2.8 mmoles/l RBC were stored for 35 days in citrate‐phosphate‐dextrose‐adenine and compared to that of four donors with normal levels of Nai. Serial measurements of red blood cell adenosine triphosphate, diphosphoglycerate, glucose‐6‐phosphate dehydrogenase, pyruvic kinase, lactate production rates, and intracellular cations showed no differences between the two donor groups. Furthermore, the mean 24‐hour posttransfusion survival was not significantly different for the high Nai group (83.2 ± 5.6%) as compared with the control group (82.3 ± 6.9%). Based on this study, it is not necessary to eliminate individuals with an elevated red blood cell Nai level as blood donors.