Protein 4.1R-dependent multiprotein complex: New insights into the structural organization of the red blood cell membrane Marcela Salomao*, Xihui Zhang*, Yang Yang*, Soohee Lee†, John H. Hartwig‡, Joel Anne Chasis§, Narla Mohandas*, and Xiuli An*¶ *Red Cell Physiology Laboratory and †Membrane Biochemistry Laboratory, New York Blood Center, New York, NY 10065; ‡Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; and §Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720 Communicated by Joseph F. Hoffman, Yale University School of Medicine, New Haven, CT, April 3, 2008 (received for review January 4, 2008) Protein 4.1R (4.1R) is a multifunctional component of the red cell The membrane-skeletal network is coupled to the lipid bilayer membrane. It forms a ternary complex with actin and spectrin, which through transmembrane proteins. One such linkage is generated by defines the nodal junctions of the membrane-skeletal network, and ankyrin, which forms a bridge between spectrin and band 3 tet- its attachment to the transmembrane protein glycophorin C creates a ramers (14–17). Band 3 also contains a binding site for carbonic bridge between the protein network and the membrane bilayer. We anhydrase II at its C-terminal cytoplasmic domain (18) and binding now show that deletion of 4.1R in mouse red cells leads to a large sites for glycolytic enzymes, hemoglobin, and protein 4.2 at its diminution of actin accompanied by extensive loss of cytoskeletal N-terminal cytoplasmic domain (19). In addition, there is a clear lattice structure, with formation of bare areas of membrane. Whereas interaction between glycophorin A (GPA) and band 3 (20–23). The band 3, the preponderant transmembrane constituent, and proteins association of these proteins with band 3 forms the band-3-based known to be associated with it are present in normal or increased complex. In addition to the band 3 complex, studies using human amounts, glycophorin C is missing and XK, Duffy, and Rh are much Rh-null erythrocytes suggested the existence of the Rh protein reduced in the 4.1R-deficient cells. The inference that these are complex comprising RhAG, Rh, CD47, LW, and GPB (24, 25). associated with 4.1R was borne out by the results of in vitro pull- More recently, the finding that components of both the band 3 down assays. Furthermore, whereas Western blot analysis showed complex and the Rh complex are absent or reduced in band-3- normal levels of band 3 and Kell, flow cytometric analysis using an deficient erythrocytes led to the concept of a band-3-based mac- antibody against the extracellular region of band 3 or Kell revealed romolecular complex (26). reduction of these two proteins, suggesting a conformational change A second membrane skeleton–bilayer link, consisting of a nexus of band 3 and Kell epitopes. Taken together, we suggest that 4.1R among 4.1R, p55, and the transmembrane glycophorin C (GPC), is organizes a macromolecular complex of skeletal and transmembrane located at the network junctions (27–29). GPC and p55 are missing proteins at the junctional node and that perturbation of this macro- from 4.1RϪ/Ϫ mouse red cells (30) and are much reduced in human molecular complex not only is responsible for the well characterized 4.1R-deficient red cells (31, 32). These proteins, as well as some membrane instability but may also remodel the red cell surface. transmembrane blood group proteins, Duffy, Lu, and CD44, and the glucose transporter GLUT1 are found in normal or elevated macromolecular complex ͉ cytoskeleton amounts in band-3-deficient red cells (26). The work described here was undertaken to examine whether and to what extent 4.1R plays n essential attribute of the red cell is its ability to undergo a part in the formation of membrane structures other than the Ϫ/Ϫ Aextensive and repeated deformations while maintaining struc- network junctions. The results, based on the study of 4.1R mouse tural integrity. The cell owes this mechanical resilience to the red cells, have allowed us to identify a 4.1R-based macromolecular membrane-associated protein skeleton (1, 2). This has the form of complex and to develop a more refined model of red cell membrane a lattice, made up of spectrin tetramers, formed by self-association organization. ␣␤ of spectrin heterodimers (3). The tetramers are attached at their Results ends to predominantly sixfold junctions consisting of short F-actin filaments (protofilaments) and several actin-binding proteins, in- Specificity of Various Anti-Mouse Antibodies. To compare the ex- pression of red cell membrane proteins between wild-type and cluding 4.1R, protein 4.9 (dematin), adducin, tropomyosin, and Ϫ/Ϫ tropomodulin (4). Defects or deficiency of components of the 4.1R cells, we first needed to generate a panel of various junctional complexes, and especially of 4.1R, lead to instability of antibodies against mouse transmembrane and cytoskeletal pro- the network and consequently of the cell. This reveals itself in teins. For transmembrane proteins we usually generate two anti- progressive fragmentation in vivo (5). bodies, one against the extracellular region and one against the In addition to the above-mentioned cytoskeletal proteins, a cytoplasmic part. The antigens used for antibody production are number of transmembrane proteins that specify blood group anti- listed in supporting information (SI) Table S1. All of the antibodies gens have also been purified and characterized biochemically. are raised in rabbit with the exception of monoclonal anti-Kell These include band 3, glycophorin A, glycophorin B, glycophorin C, antibody, which was generated in mice using red cell as antigen. The RhAG, Rh, Duffy, Lu, LW, CD44, CD47, Kell, and XK (6). These specificity of our antibodies was confirmed by Western blot analysis transmembrane proteins exhibit diverse functions. For example, band 3 functions as an anion exchanger. Rh/RhAG are probably gas Author contributions: S.L., J.A.C., N.M., and X.A. designed research; M.S., X.Z., Y.Y., S.L., and transporters although there is some controversy regarding whether J.H.H. performed research; M.S., S.L., J.H.H., J.A.C., N.M., and X.A. analyzed data; and M.S., they transport ammonia or carbon dioxide (7, 8). Duffy serves as a N.M., and X.A. wrote the paper. chemokine receptor and is also a receptor for the malarial parasite The authors declare no conflict of interest. Plasmodium vivax (9, 10). Lu, LW, and CD44 are proteins that are ¶To whom correspondence should be addressed at: Red Cell Physiology Laboratory, 310 involved in adhesive interactions (11). CD47 can function as a East 67th Street, New York, NY 10065. E-mail: [email protected]. marker of self on erythrocytes by binding to the inhibitory receptor This article contains supporting information online at www.pnas.org/cgi/content/full/ SIRP␣ (12). Kell possess endothin-3-converting enzyme activity 0803225105/DCSupplemental. (13), but the function of XK remains to be defined. © 2008 by The National Academy of Sciences of the USA 8026–8031 ͉ PNAS ͉ June 10, 2008 ͉ vol. 105 ͉ no. 23 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0803225105 Downloaded by guest on September 26, 2021 and thalassemia (data not shown). Another major skeleton protein, ankyrin, was unchanged. Structural Consequences of 4.1R Deletion. Having discovered the significant reduction of actin in 4.1R-deficient red cells, we then examined the structural consequences of 4.1R deletion. Staining of fixed red cells with fluorescent phalloidin demonstrates that F-actin is indeed much sparser on the 4.1RϪ/Ϫ cell membranes (Fig. S2). To determine whether F-actin is reduced or redistributed, we quanti- tated the fluorescence levels (pixel intensity/unit area) and found an Ϸ30% reduction in phalloidin levels in 4.1R-null red cells when compared with wild-type erythrocytes [knockout, 17,958 Ϯ 1,783; wild type, 25,479 Ϯ 1,858 (n ϭ 8, P ϭ 0.0000004)]. To establish how Fig. 1. Immunoblots of membrane skeletal proteins in red cells of 4.1Rϩ/ϩ Ϫ/Ϫ the actin deficiency affects the structure of the network, we and 4.1R mice. Blots of SDS/PAGE of total membrane protein were probed examined the membranes by electron microscopy under negative with antibodies against the indicated proteins. Note the absence of 4.1R, as well as p55 in the 4.1R-deficient cells, the reduced actin concentration, and the stain. Fig. 2 demonstrates that the regularity of the lattice is grossly elevated tropomyosin and adducin. disrupted, with large bare regions. It thus appears that many of the junctions that are normally present are missing in the mutant membranes. using corresponding knockout mice as negative controls. All anti- bodies generated recognize the corresponding mouse proteins, and Integral Membrane Proteins in 4.1R؊/؊ Cells. It has been shown that some also recognize the cognate human proteins. Fig. S1 demon- in both mouse and human band-3-deficient red cells the known or strates the specificity of a representative set of antibodies against surmised band-3-associated proteins, namely GPA, GPB, RhAG, mouse red cell proteins. Rh, CD47, and LW, are missing or greatly reduced. GPC, 4.1R, and p55, which are confined to the network junctions, are present in Analysis of Cytoskeletal Protein Components of 4.1R؊/؊ Red Cells by normal amounts, and so also are other transmembrane proteins Western Blot. We have previously shown that the membranes of (Duffy, Lu, GLUT1, LFA-3, and CD44) with no known cytoskel- mouse red cells lacking 4.1R have greatly impaired shear resistance eton interactions (26). We have used Western blots to compare the (30). The same is true of human 4.1R-deficient cells (5).