Presence and localization of proteins immunologically related to erythrocyte protein 4.1 in human skin

Summary Analogues of human erythrocyte protein 4.1 have been examined in the human skin by immunochemical techniques using anti-human erythrocyte protein 4.1 antibodies. Immunoblot analysis revealed that human epidermis contains 4.1-like proteins of 80 kDa and 78 kDa that cross react with anti-protein 4.1 antibodies.Analysis with immunofluorescence microscopy revealed that the plasma membrane of the human epidermal keratinocyte was stained intensively in the basal cells, whereas spinous cells were moderately stained. It is noted that eccrine sweat gland cells and ductal cells were also stained in the peripheral cytoplasma. Taken together, these results demonstrate that 4.1-like proteins are present in human epidermal keratinocytes, eccrine sweat gland cells and ductal cells. The present findings enable us to suggest that a membrane skeletal protein lattice might exist in these cells.     

Presence and localization of proteins immunologically related to erythrocyte protein 4.1 in human skin

Analogues of human erythrocyte protein 4.1 have been examined in the human skin by immunochemical techniques using anti-human erythrocyte protein 4.1 antibodies. Immunoblot analysis revealed that human epidermis contains 4.1-like proteins of 80 kDa and 78 kDa that cross react with anti-protein 4.1 antibodies. Analysis with immunofluorescence microscopy revealed that the plasma membrane of the human epidermal keratinocyte was stained intensively in the basal cells, whereas spinous cells were moderately stained. It is noted that eccrine sweat gland cells and ductal cells were also stained in the peripheral cytoplasma. Taken together, these results demonstrate that 4.1-like proteins are present in human epidermal keratinocytes, eccrine sweat gland cells and ductal cells. The present findings enable us to suggest that a membrane skeletal protein lattice might exist in these cells.     

Correlation between protein 4.1a/4.1b ratio and erythrocyte life span

Erythrocyte membranes from various healthy mammals contained a doublet of protein 4.1a and 4.1b, which appeared to differ by 2-3 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The ratio of protein 4.1a/4.1b showed much variety among animal species, and the 4.1a/4.1b ratio correlated to the mean erythrocyte life span, that is, the mean cell age in circulating blood. We also found that the 4.1b is the predominant form in the immature erythroid cells such as reticulocytes and K562 cells. In addition, the 4.1b but not 4.1a protein was metabolically labeled with [35S]methionine in the erythropoietic cells from anemic mouse. Immunological detection showed that there is a doublet of minor variants of protein 4.1 with apparent molecular masses slightly more than those of 4.1a and 4.1b. The ratio of these minor isoforms designated as 4.1a + and 4.1b + revealed the alteration during erythrocyte senescence as observed in 4.1a/4.1b ratio. These results show that protein 4.1 may be synthesized as 4.1b and 4.1b + and intercalated into membrane skeletons at an early stage of erythroidal differentiation, and that the posttranslational modification into 4.1a and 4.1a + appears to occur by a common mechanism in many mammalian species. Feline erythrocytes, however, appeared to lack such a postsynthetic processing of protein 4.1, and exhibited one major component of 4.1b with the other minor variant of 4.1b +.     

Interaction of a peripheral protein of the erythrocyte membrane, band 4.1, with phosphatidylserine-containing liposomes and erythrocyte inside-out vesicles

Interactions of band 4.1 with mixed phospholipid membranes [phosphatidylserine (PtdSer), phosphatidylethanolamine, phosphatidylcholine, etc.] and erythrocyte inside-out vesicles were studied. Band 4.1 showed a higher affinity to PtdSer-containing membranes. The amount of binding to PtdSer-containing liposomes was larger than that to PtdSer-lacking liposomes. The amount of binding to inside-out vesicles did not change significantly on a protease treatment of the vesicles. The amount of band 4.1 bound on inside-out vesicles decreased on PtdSer-decarboxylase treatment of the vesicles. Ca2+ acted inhibitory to the binding of band 4.1. Band 4.1 together with PtdSer-containing vesicles but not with PtdSer-lacking vesicles induced gelation of spectrin-actin copolymer solution. Ca2+ inhibited the gelation. Fluorescence energy transfer from PtdSer-containing vesicles to band 4.1 was larger than that from PtdSer-lacking vesicles. Band 4.1 caused a marked release of tempocholine from preloaded PtdSer-containing liposomes but not from PtdSer-lacking liposomes. The release was larger from liposomes containing more PtdSer. Ca2+ was inhibitory to the tempocholine release. We suggest from these results that band 4.1 provides another anchoring site for the cytoskeletal spectrin-actin network to PtdSer domains in the inner layer of erythrocyte membrane. This anchoring may be involved in functional regulation since the interaction causes the membrane permeability change that is dependent on Ca2+.     

Identification of a calcium-dependent calmodulin-binding domain in Xenopus membrane skeleton protein 4.1

Xenopus membrane skeleton protein 4.1 is expressed constitutively during embryonic development and accumulates to high levels within the retina during normal morphogenesis. There exists a high degree of amino acid identity between Xenopus protein 4.1 and human protein 4.1, suggesting that the mechanisms known to modulate the function(s) of human protein 4.1 may also serve to regulate Xenopus protein 4.1. Calmodulin (CaM) is one regulatory protein known to affect membrane-cytoskeletal interactions. An in vitro binding assay was used to test the ability of Xenopus protein 4.1 to bind CaM. Two independent approaches, involving protein 4.1 synthesized in vitro from synthetic RNA or a partial length protein 4.1 fusion protein expressed in Escherichia coli, demonstrate calcium-dependent, CaM binding. Both approaches demonstrate that the CaM-binding site is within the amino-terminal region of Xenopus protein 4.1. Results of this calmodulin binding activity suggest a possible regulatory mechanism by which calcium and calmodulin may affect the function of protein 4.1 during development.     

High molecular weight microtubule-associated proteins from pig brain are immunologically related to human erythrocyte membrane proteins spectrin, ankyrin, proteins 4.1 and 4.2

The microtubule-associated proteins MAPs 1 and 2 from pig brain have been found to react with antibodies directed against human ankyrin and spectrin, respectively (Bennett and Davis, 1981; Davis and Bennett, 1982). In a complementary approach we have prepared antibodies against MAP1 alpha. MAP1 gamma and MAP2 purified from pig brain and tested their reactivity with human erythrocyte membrane proteins. Anti-MAP1 alpha was shown to react with alpha and beta-spectrin and with protein 4.1; anti-MAP1 gamma reacted with alpha-spectrin and ankyrin and with a 60 K peptide which copurified with human spectrin. Finally anti-MAP2 was specific for beta-spectrin and protein 4.2. The biological function of protein 4.2 is still unknown but details on the interactions between ankyrin, spectrin and protein 4.1 and their role in mediating the linkage of oligomeric actin on the erythrocyte membrane are well documented. The present results, which demonstrate extended immunological analogies between pig brain high molecular weight MAPs and human erythrocyte membrane proteins, may reflect the presence, in the two families of proteins, of similar functionally important epitopes.     

Phosphatidylinositol-4,5-biphosphate (PIP2) differentially regulates the interaction of human erythrocyte protein 4.1 (4.1R) with membrane proteins

Human erythrocyte protein 4.1 (4.1R) participates in organizing the plasma membrane by linking several surface-exposed transmembrane proteins to the internal cytoskeleton. In the present study, we characterized the interaction of 4.1R with phosphatidylinositol-4,5-bisphosphate (PIP2) and assessed the effect of PIP2 on the interaction of 4.1R with membrane proteins. We found that 4.1R bound to PIP2-containing liposomes through its N-terminal 30 kDa membrane-binding domain and PIP2 binding induced a conformational change in this domain. Phosphatidylinositol-4-phosphate (PIP) was a less effective inducer of this conformational change, and phosphatidylinositol (PI) and inositol-1,4,5-phosphate (IP3) induced no change. Replacement of amino acids K63,64 and K265,266 by alanine abolished the interaction of the membrane-binding domain with PIP2. Importantly, binding of PIP2 to 4.1R selectively modulated the ability of 4.1R to interact with its different binding partners. While PIP2 significantly enhanced the binding of 4.1R to glycophorin C (GPC), it inhibited the binding of 4.1R to band 3 in vitro. PIP2 had no effect on 4.1R binding to p55. Furthermore, GPC was more readily extracted by Triton X-100 from adenosine triphosphate (ATP)-depleted erythrocytes, implying that the GPC-4.1R interaction may be regulated by PIP2 in situ. These findings define an important role for PIP2 in regulating the function of 4.1R. Because 4.1R and its family members (4.1R, 4.1B, 4.1G, and 4.1N) are widely expressed and the PIP2-binding motifs are highly conserved, it is likely that the functions of other 4.1 proteins are similarly regulated by PIP2 in many different cell types.     

Interactions of Plasmodium falciparum erythrocyte membrane protein 3 with the red blood cell membrane skeleton

Plasmodium falciparum parasites express and traffick numerous proteins into the red blood cell (RBC), where some associate specifically with the membrane skeleton. Importantly, these interactions underlie the major alterations to the modified structural and functional properties of the parasite-infected RBC. P. falciparum Erythrocyte Membrane Protein 3 (PfEMP3) is one such parasite protein that is found in association with the membrane skeleton. Using recombinant PfEMP3 proteins in vitro, we have identified the region of PfEMP3 that binds to the RBC membrane skeleton, specifically to spectrin and actin. Kinetic studies revealed that residues 38-97 of PfEMP3 bound to purified spectrin with moderately high affinity (K_D(kin) = 8.5 × 10^− 8 M). Subsequent deletion mapping analysis further defined the binding domain to a 14-residue sequence (IFEIRLKRSLAQVL; K_D(kin) = 3.8 × 10^− 7 M). Interestingly, this same domain also bound to F-actin in a specific and saturable manner. These interactions are of physiological relevance as evidenced by the binding of this region to the membrane skeleton of inside-out RBCs and when introduced into resealed RBCs. Identification of a 14-residue region of PfEMP3 that binds to both spectrin and actin provides insight into the potential function of PfEMP3 in P. falciparum-infected RBCs.     

A FERM-adjacent (FA) region defines a subset of the 4.1 superfamily and is a potential regulator of FERM domain function

Background

It is customary, in population genetics studies, to consider Basques as the direct descendants of the Paleolithic Europeans. However, until now there has been no irrefutable genetic proof to support this supposition. Even studies based on mitochondrial DNA (mtDNA), an ideal molecule for constructing datable maternal genealogies, have failed to achieve this. It could be that incoming gene flow has replaced the Basque ancient lineages but it could also be that these lineages have not been detected due to a lack of resolution of the Basque mtDNA genealogies. To assess this possibility we analyzed here the mtDNA of a large sample of autochthonous Basques using mtDNA genomic sequencing for those lineages that could not be unequivocally classified by diagnostic RFLP analysis and control region (HVSI and HVSII) sequencing.

Results

We show that Basques have the most ancestral phylogeny in Europe for the rare mitochondrial subhaplogroup U8a. Divergence times situate the Basque origin of this lineage in the Upper Palaeolithic. Most probably, their primitive founders came from West Asia. The lack of U8a lineages in Africa points to an European and not a North African route of entrance. Phylogeographic analysis suggest that U8a had two expansion periods in Europe, the first, from a south-western area including the Iberian peninsula and Mediterranean France before 30,000 years ago, and the second, from Central Europe around 15,000–10,000 years ago.

Conclusion

It has been demonstrated, for the first time, that Basques show the oldest lineages in Europe for subhaplogroup U8a. Coalescence times for these lineages suggest their presence in the Basque country since the Upper Paleolithic. The European U8 phylogeography is congruent with the supposition that Basques could have participated in demographic re-expansions to repopulate central Europe in the last interglacial periods.     

Alternatively spliced exon 5 of the FERM domain of protein 4.1R encodes a novel binding site for erythrocyte p55 and is critical for membrane targeting in epithelial cells

Direct physical linkage of MAGUKs to the actin cytoskeleton was first established by the interaction of erythrocyte p55 with the FERM domain of protein 4.1R. Subsequently, it was reported that p55 binds to a 51-amino acid peptide, encoded by exon 10, located within the FERM domain of protein 4.1R. In this study, we investigated the nature of the p55-FERM domain binding interface and show that p55 binds to a second 35-amino acid peptide, encoded by an alternatively spliced exon 5, located within the FERM domain of protein 4.1R. Competition and Surface Plasmon Resonance-binding measurements suggest that the peptides encoded by exons 5 and 10 bind to independent sites within the D5 domain of p55. Interestingly, the full length 135 kDa isoform of protein 4.1R containing both exons 5 and 10 was targeted exclusively to the plasma membrane of epithelial cells whereas the same isoform without exon 5 completely lost its membrane localization capacity. Together, these results indicate that p55 binds to two distinct sites within the FERM domain, and the alternatively spliced exon 5 is necessary for the membrane targeting of protein 4.1R in epithelial cells. Since sequences similar to the exon 5-peptide of protein 4.1R and D5 domain of p55 are conserved in many proteins, our findings suggest that a similar mechanism may govern the membrane targeting of other FERM domain containing proteins.     

Associations of erythrocyte membrane proteins. Binding of purified bands 2.1 and 4.1 to spectrin

Specific associations of spectrin with Bands 2.1 and 4.1 have been examined by measuring the binding of purified 125I-Band 2.1 and 125I-Band 4.1 to [32P]spectrin in solution. Binding of Bands 2.1 and 4.1 to spectrin was measured as 125I radioactivity precipitated by an anti-spectrin.Staphylococcus aureus complex. The association between spectrin and Band 2.1 is characterized by relatively high affinity (Kd congruent to 10(-7) M at pH 7.6) and saturation of available binding sites at a molar ratio of 1:1 (Band 2.1/spectrin heterodimer). Band 4.1 binding to spectrin is characterized by a similar affinity (Kd congruent to 10(-7) M at pH 7.6) with saturation of available sites occurring at a stoichiometric ration of 2:1 (Band 4.1/spectrin heterodimer). Scatchard plots of Band 4.1 binding to spectrin are curvilinear and consistent with a positively cooperative interation. Bands 2.1 and 4.1 bind to different sites on the spectrin molecule: unlabeled Band 4.1 does not competitively displace 125 I-Band 2.1 from spectrin in solution, and low angle rotary-shadowed platinum-carbon replicas of these polypeptides reveal two discrete binding sites.     

Interactions of Plasmodium falciparum erythrocyte membrane protein 3 with the red blood cell membrane skeleton

Plasmodium falciparum parasites express and traffick numerous proteins into the red blood cell (RBC), where some associate specifically with the membrane skeleton. Importantly, these interactions underlie the major alterations to the modified structural and functional properties of the parasite-infected RBC. P. falciparum Erythrocyte Membrane Protein 3 (PfEMP3) is one such parasite protein that is found in association with the membrane skeleton. Using recombinant PfEMP3 proteins in vitro, we have identified the region of PfEMP3 that binds to the RBC membrane skeleton, specifically to spectrin and actin. Kinetic studies revealed that residues 38-97 of PfEMP3 bound to purified spectrin with moderately high affinity (K(D(kin))=8.5 x 10(-8) M). Subsequent deletion mapping analysis further defined the binding domain to a 14-residue sequence (IFEIRLKRSLAQVL; K(D(kin))=3.8 x 10(-7) M). Interestingly, this same domain also bound to F-actin in a specific and saturable manner. These interactions are of physiological relevance as evidenced by the binding of this region to the membrane skeleton of inside-out RBCs and when introduced into resealed RBCs. Identification of a 14-residue region of PfEMP3 that binds to both spectrin and actin provides insight into the potential function of PfEMP3 in P. falciparum-infected RBCs.     

Regulation of protein 4.1R, p55, and glycophorin C ternary complex in human erythrocyte membrane

Three binary protein-protein interactions, glycophorin C (GPC)-4.1R, GPC-p55, and p55-4.1R, constitute the GPC-4.1R-p55 ternary complex in the erythrocyte membrane. Little is known regarding the molecular basis for the interaction of 4.1R with either GPC or p55 and regarding the role of 4.1R in regulating the various protein-protein interactions that constitute the GPC-4.1R-p55 ternary complex. In the present study, we present evidence that sequences in the 30-kDa domain encoded by exon 8 and exon 10 of 4.1R constitute the binding interfaces for GPC and p55, respectively. We further show that 4.1R increases the affinity of p55 binding to GPC by an order of magnitude, implying that 4.1R modulates the interaction between p55 and GPC. Finally, we document that binding of calmodulin to 4.1R decreases the affinity of 4.1R interactions with both p55 and GPC in a Ca(2+)-dependent manner, implying that the GPC-4.1R-p55 ternary protein complex can undergo dynamic regulation in the erythrocyte membrane. Taken together, these findings have enabled us to identify an important role for 4.1R in regulating the GPC-4.1R-p55 ternary complex in the erythrocyte membrane.     

The 4.1-like proteins of the bovine lens: spectrin-binding proteins closely related in structure to red blood cell protein 4.1

The superficial cortical fiber cells of the bovine lens contain membrane-associated proteins of 150,000, 80,000, and 78,000 D that cross-react with antisera prepared against red blood cell (RBC) protein 4.1 (Aster, J. C., G. J. Brewer, S. M. Hanash, and H. Maisel, 1984, Biochem. J., 224:609-616). To further study their relationship to protein 4.1, these proteins were immunoprecipitated from detergent extracts of crude lens membranes with purified polyclonal and monoclonal anti-4.1 antibodies and resolved by SDS PAGE. The electrophoretic mobilities of the lens proteins of 80,000 and 78,000 D were found to be identical to bovine RBC protein 4.1a and protein 4.1b, respectively. One- and two-dimensional peptide mapping revealed that a high degree of structural homology exists among all three of the lens 4.1-like proteins and RBC protein 4.1a and protein 4.1b. Despite the large difference in apparent molecular mass, the 150,000-D lens protein showed only minor peptide map differences. A nitrocellulose filter overlay assay showed that all three of the lens 4.1-like proteins bind to RBC and lens spectrins. We conclude that the bovine lens contains proteins of 80,000 and 78,000 D that are highly similar to protein 4.1 in structure and functional capacity. Additionally, the lens also contains a 4.1 isomorph of 150 kD. Analogous to RBC protein 4.1, these proteins may function in the lens by promoting association of spectrin with actin and by playing a role in the coupling of lens cytoskeleton to plasma membrane.     

A monoclonal antibody recognizes a phosphorylated epitope shared by proteins of the cell nucleus and the erythrocyte membrane skeleton

Monoclonal antibody 3C5 recognizes a family of proteins in the nuclei of cultured cells [(1985) Eur. J. Cell Biol. 38, 344]. This antibody has now been shown to recognize equivalent proteins in liver nuclei and in the Triton-insoluble fraction of tissue extracts. In human erythrocytes the antibody recognized a single protein, present in the membrane skeleton fraction and with the molecular mass and extraction properties of beta-spectrin. The epitope recognized by 3C5 was destroyed by alkaline phosphatase. We conclude that this antibody recognizes a phosphorylation site shared by nuclear proteins and a protein of the erythrocyte membrane skeleton, probably beta-spectrin.     

Dematin, a component of the erythrocyte membrane skeleton, is internalized by the malaria parasite and associates with Plasmodium 14-3-3

The malaria parasite invades the terminally differentiated erythrocytes, where it grows and multiplies surrounded by a parasitophorous vacuole. Plasmodium blood stages translocate newly synthesized proteins outside the parasitophorous vacuole and direct them to various erythrocyte compartments, including the cytoskeleton and the plasma membrane. Here, we show that the remodeling of the host cell directed by the parasite also includes the recruitment of dematin, an actin-binding protein of the erythrocyte membrane skeleton and its repositioning to the parasite. Internalized dematin was found associated with Plasmodium 14-3-3, which belongs to a family of conserved multitask molecules. We also show that, in vitro, the dematin-14-3-3 interaction is strictly dependent on phosphorylation of dematin at Ser(124) and Ser(333), belonging to two 14-3-3 putative binding motifs. This study is the first report showing that a component of the erythrocyte spectrin-based membrane skeleton is recruited by the malaria parasite following erythrocyte infection.