Persistence of external chloride and DIDS binding after chemical modification of Glu-681 in human band 3. (17/661)

Although its primary function is monovalent anion exchange, the band 3 protein also cotransports divalent anions together with protons at low pH. The putative proton binding site, Glu-681 in human erythrocyte band 3, is conserved throughout the anion exchanger family (AE family). To determine whether or not the monovalent anion binding site is located near Glu-681, we modified this residue with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate; WRK). Measurements of Cl(-) binding by (35)Cl-NMR show that external Cl(-) binds to band 3 even when Cl(-) transport is inhibited approximately 95% by WRK modification of Glu-681. This indicates that the external Cl(-) binding site is not located near Glu-681 and thus presumably is distant from the proton binding site. DIDS inhibits Cl(-) binding even when WRK is bound to Glu-681, indicating that the DIDS binding site is also distant from Glu-681. Our data suggest that the DIDS site and probably also the externally facing Cl(-) transport site are located nearer to the external surface of the membrane than Glu-681.  (+info)

Topology studies with biosynthetic fragments identify interacting transmembrane regions of the human red-cell anion exchanger (band 3; AE1). (18/661)

The red-cell anion exchanger (band 3; AE1) is a multispanning membrane protein that traverses the bilayer up to 14 times and is N-glycosylated at Asn-642. We have shown that the integrity of six different loops are not essential for stilbene disulphonate-sensitive chloride uptake in Xenopus oocytes. We used an N-glycosylation mutagenesis approach to examine the orientation of the N-terminus and the endogenous glycosylation site of each C-terminal fragment by cell-free translation. The fragments initiating in the loops preceding spans 2, 9 and 11 did not insert into the membrane with the expected orientation. Furthermore, N-glycosylation of Asn-642 might facilitate the membrane integration of span 7. The correct integration of spans 2-3 required the presence of the region containing span 4 and that the luminal exposure of the C-terminus of span 7 is increased in the presence of the region including span 6 or span 8. The results suggest the span 8 region is required for the correct folding of spans 9-10, at least in the presence of the span 11-12 region. Our results suggest that there are intramolecular interactions between the regions of transmembrane spans 1 and 2, 2 and 4, 4 and 5, 7 and 8, 8 and 9-10, and 9-10 and 11-12. Spans 1, 4, 5, 6 and 8 might act as a scaffold for the assembly of spans 2-3, 7 and 9-10. This approach might provide a general method for dissecting the interactions between membrane-spanning regions of polytopic membrane proteins.  (+info)

Structural model for the organization of the transmembrane spans of the human red-cell anion exchanger (band 3; AE1). (19/661)

We have examined the functional co-assembly of non-complementary pairs of N- and C-terminal polypeptide fragments of the anion transport domain (b3mem) of human red-cell band 3. cDNA clones encoding non-contiguous pairs of fragments with one transmembrane (TM) region omitted, or overlapping pairs of fragments with between one and ten TM regions duplicated, were co-expressed in Xenopus oocytes and a cell-free translation system. Stilbene disulphonate-sensitive chloride uptake assays in oocytes revealed that the omission of any single TM region of b3mem except spans 6 and 7 caused a complete loss of functional expression. In contrast, co-expressed pairs of fragments overlapping a single TM region 5, 6, 7, 8, 9-10 or 11-12 retained a high level of functionality, whereas fragments overlapping the clusters of TM regions 2-5, 4-5, 5-8 and 8-10 also mediated some stilbene disulphonate-sensitive uptake. The co-assembly of N- or C-terminal fragments with intact band 3, b3mem or other fragments was examined by co-immunoprecipitation in non-denaturing detergent solutions by using monoclonal antibodies against the termini of b3mem. All the fragments, except for TM spans 13-14, co-immunoprecipitated with b3mem. The medium-sized N-terminal fragments comprising spans 1-6, 1-7 or 1-8 co-immunoprecipitated particularly strongly with the C-terminal fragments containing spans 8-14 or 9-14. The fragments comprising spans 1-4 or 1-12 co-immunoprecipitated less extensively than the other N-terminal fragments with either b3mem or C-terminal fragments. There is sufficient flexibility in the structure of b3mem to allow the inclusion of at least one duplicated TM span without a loss of function. We propose a working model for the organization of TM spans of dimeric band 3 based on current evidence.  (+info)

Mechanism of band 3 dimer dissociation during incubation of erythrocyte membranes at 37 degrees C. (20/661)

The mechanism of dissociation of the stable dimer of band 3 was investigated during the incubation of isolated erythrocyte membranes or resealed ghosts at 37 degrees C. The kinetics of changes in the structural and functional integrity of the membrane domain of band 3 (MDB3) were measured and correlated with the change in the Stokes radius of band 3. MDB3 integrity was determined as follows: (1) by measuring the fluorescence emission spectrum of 4, 4'-di-isothiocyanostilbene-2,2'-disulphonate (DIDS) bound covalently to MDB3; (2) by measuring the number of DIDS covalent binding sites present after incubation of unlabelled resealed ghosts; and (3) by measuring the anion transport V(max) by using the same resealed ghosts. Incubation of membranes at 37 degrees C caused the dissociation of band 3 dimers to monomers but only after a lag period lasting approx. 50 h. The observation of such a lag implies that dissociation involves a sequence of molecular events beginning with some type of initial process. We have discovered that this initial process involves a conformation change in MDB3. There was a shift in the fluorescence spectrum for DIDS-labelled band 3 and a decrease in the DIDS binding capacity and transport activity of the unlabelled protein. Incubation of membranes at 4 degrees C inhibited the conformational change in MDB3 and the dissociation of dimers. Furthermore, no conformational change in MDB3 was observed when erythrocytes were incubated at 37 degrees C. We suggest that MDB3 unfolding is the molecular event responsible for the subsequent dissociation of stable dimers of band 3 to monomers during the incubation of erythrocyte membranes at 37 degrees C. The monomers so generated are either not functional in anion exchange or they have an attenuated functionality. The absence of a conformational change for band 3 in erythrocytes might imply that haemolysis perturbs the membrane structure and somehow predisposes band 3 to the conformational change that occurs during incubation at 37 degrees C.  (+info)

Heavy transfusions and presence of an anti-protein 4.2 antibody in 4. 2(-) hereditary spherocytosis (949delG). (21/661)

BACKGROUND AND OBJECTIVE: A patient with hereditary spherocytosis (HS) was found not to have red cell membrane protein 4.2. This rare form of HS, or 4.2 (-) HS, stems from mutations within the ELB42 or the EPB3 genes. The patient had long suffered from a gastric ulcer and impaired liver function. He had had several dramatic episodes of gastrointestinal tract bleeding and had received numerous transfusions. An antibody against a high frequency, undefined antigen was found, creating a transfusional deadlock. We elucidated the responsible mutation and searched for an anti-protein 4.2 antibody. DESIGN AND METHODS: Red cell membranes were analyzed by SDS-PAGE and by Western blotting. Nucleotide sequencing was performed after reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR. RESULTS: The not previously described mutation was a single base deletion: 949delG (CGCAECC, exon 7, codon 317) in the homozygous state. It was called protein 4.2 Nancy. The deletion placed a non-sense codon shortly downstream so that no viable polypeptide could be synthesized. The patient carried a strong antibody against protein 4.2 as shown by Western blotting. INTERPRETATION AND CONCLUSIONS: The manifestations resulting from the mutation described were compared with the picture of HS stemming from other ELB42 gene mutations. We discuss the mechanism through which the anti-protein 4.2 antibody developed. There was no way to establish or to rule out whether the antibody participated in the transfusional deadlock found in our patient.  (+info)

The noncompetitive inhibitor WW781 senses changes in erythrocyte anion exchanger (AE1) transport site conformation and substrate binding. (22/661)

WW781 binds reversibly to red blood cell AE1 and inhibits anion exchange by a two-step mechanism, in which an initial complex (complex 1) is rapidly formed, and then there is a slower equilibration to form a second complex (complex 2) with a lower free energy. According to the ping-pong kinetic model, AE1 can exist in forms with the anion transport site facing either inward or outward, and the transition between these forms is greatly facilitated by binding of a transportable substrate such as Cl(-). Both the rapid initial binding of WW781 and the formation of complex 2 are strongly affected by the conformation of AE1, such that the forms with the transport site facing outward have higher affinity than those with the transport site facing inward. In addition, binding of Cl(-) seems to raise the free energy of complex 2 relative to complex 1, thereby reducing the equilibrium binding affinity, but Cl(-) does not compete directly with WW781. The WW781 binding site, therefore, reveals a part of the AE1 structure that is sensitive to Cl(-) binding and to transport site orientation, in addition to the disulfonic stilbene binding site. The relationship of the inhibitory potency of WW781 under different conditions to the affinities for the different forms of AE1 provides information on the possible asymmetric distributions of unloaded and Cl(-)-loaded transport sites that are consistent with the ping-pong model, and supports the conclusion from flux and nuclear magnetic resonance data that both the unloaded and Cl(-)-loaded sites are very asymmetrically distributed, with far more sites facing the cytoplasm than the outside medium. This asymmetry, together with the ability of WW781 to recruit toward the forms with outward-facing sites, implies that WW781 may be useful for changing the conformation of AE1 in studies of structure-function relationships.  (+info)

Adherence of phosphatidylserine-exposing erythrocytes to endothelial matrix thrombospondin. (23/661)

Phospholipid asymmetry is well maintained in erythrocyte (RBC) membranes with phosphatidylserine (PS) exclusively present in the inner leaflet. The appearance of PS on the surface of the cell can have major physiologic consequences, including increased cell-cell interactions. Because increased adherence of PS-exposing RBCs to endothelial cells (ECs) may be pathologically important in hemoglobinopathies such as sickle cell disease and thalassemia, we studied the role of PS exposure in calcium ionophore-treated normal RBC adherence to human umbilical vein endothelial cell (HUVEC) monolayers. When HUVEC monolayers were incubated with these PS-exposing RBCs, the ECs retracted and the RBCs adhered primarily in the gaps opened between the ECs. A linear correlation was found between the number of PS-exposing RBCs in the population and the number of adhering RBCs to the monolayer. Pretreatment of RBCs with annexin V significantly decreased adherence by shielding PS on the RBCs. Similarly, PS-containing lipid vesicles decreased RBC binding by competing for the PS binding sites in the monolayer. PS-exposing RBCs and PS-containing lipid vesicles adhered to immobilized thrombospondin (TSP) and matrix TSP, respectively, and adherence of PS-exposing RBCs to EC monolayers was reduced by antibodies to TSP and to its EC receptor, alpha(v)beta(3). Together, these results indicate a role for PS and matrix TSP in the adherence of PS-exposing RBCs to EC monolayers, and suggest an important contribution of PS-exposing RBCs in pathologies with reported vascular damage, such as sickle cell anemia. (Blood. 2000;95:1293-1300)  (+info)

Ca(2+)-dependent and Ca(2+)-independent calmodulin binding sites in erythrocyte protein 4.1. Implications for regulation of protein 4.1 interactions with transmembrane proteins. (24/661)

In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH(2)-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca(2+) and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca(2+)/CaM to down-regulate 4. 1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca(2+) and CaM requires binding of CaM to both Ca(2+)-independent and Ca(2+)-dependent sites in protein 4.1.  (+info)