Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na,KATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we started from the fact that a closely related enzyme, the gastric H,K-ATPase, does not bind glycosides like ouabain. Previously, we showed that a chimera of these two enzymes, in which only the M3-M4 and M5-M6 hairpins were of Na,K-ATPase, bound ouabain with high affinity (
Pfs48/45, a member of a Plasmodium-specific protein family, displays conformation-dependent epitopes and is an important target for malaria transmission-blocking (TB) immunity. To design a recombinant Pfs48/45-based TB vaccine, we analyzed the conformational TB epitopes of Pfs48/45. The Pfs48/45 protein was found to consist of a C-terminal six-cysteine module recognized by anti-epitope I antibodies, a middle four-cysteine module recognized by anti-epitopes IIb and III, and an N-terminal module recognized by anti-epitope V antibodies. Refolding assays identified that a fragment of 10 cysteines (10C), comprising the middle four-cysteine and the C-terminal six-cysteine modules, possesses superior refolding capacity. The refolded and partially purified 10C conformer elicited antibodies in mice that targeted at least two of the TB epitopes (I and III). The induced antibodies could block the fertilization of Plasmodium falciparum gametes in vivo in a concentration-dependent manner. Our results provide important insight into the structural organization of the Pfs48/45 protein and experimental support for a Pfs48/45-based subunit vaccine.Malaria parasites are spread in the human population by Plasmodium-infected Anopheles mosquitoes. After a blood meal on infected humans, mosquitoes become infected by ingesting a sexual form of the malaria parasite called gametocytes. Subsequent sporogonic development in the mosquito can be prevented by the presence of anti-malaria, transmissionblocking (TB) 3 antibodies in the ingested blood meal (1-3). Pfs48/45 is a TB vaccine candidate that belongs to a family of malaria-specific proteins that contain conserved motifs with four-or six-cysteine residues (4). Pfs48/45 plays a key role in parasite fertilization (5), and monoclonal antibodies (mAbs) against Pfs48/45 prevent fertilization (6). Anti-Pfs48/45 antibodies are present in human sera from endemic areas, and there is evidence that the presence of anti-Pfs48/45 antibodies in natural sera correlates with TB activity (7). The induction of antibodies after natural infection, as observed in the field, creates the highly beneficial potential of vaccine boosting in the endemic setting. At least four different epitopes (I, IIb, III, and V) on Pfs48/45 are targeted by monoclonal antibodies that block or reduce malaria transmission (8, 9). Apart from epitope V, the TB epitopes are sensitive to reducing agents, which indicates the importance of the disulfide bridges and hence the conformational dependence of these epitopes (8, 10).To design a Pfs48/45-based TB vaccine, we aimed to delineate and characterize the TB epitopes of Pfs48/45. Protease digestion of the native protein, expression in Escherichia coli of truncations, cysteine mutations, and refolding assays were evaluated by immunological analysis of the TB epitopes. An N-terminally truncated protein with higher refolding efficiency was identified, partially purified, and tested in mice immunization experiments. The elicited antibodies recognized the native Pfs48/45 protein, competed ...
The Na,K-ATPase is an ion-translocating transmembrane protein that actively maintains the electrochemical gradients for Na+ and K+ across the plasma membrane. The functional protein is a heterodimer comprising a catalytic alpha-subunit (four isoforms) and an ancillary beta-subunit (three isoforms). Mutations in the alpha2-subunit have recently been implicated in familial hemiplegic migraine type 2, but almost no thorough studies of the functional consequences of these mutations have been provided. We investigated the functional properties of the mutations L764P and W887R in the human Na,K-ATPase alpha2-subunit upon heterologous expression in Xenopus oocytes. No Na,K-ATPase-specific pump currents could be detected in cells expressing these mutants. The binding of radiolabelled [3H]ouabain to intact cells suggested that this could be due to a lack of plasma membrane expression. However, plasma membrane isolation showed that the mutated pumps are well expressed at the plasma membrane. 86Rb+-flux and ATPase activity measurements demonstrated that the mutants are inactive. Therefore, the primary disease-causing mechanism is loss-of-function of the Na,K-ATPase alpha2-isoform.
Ouabain is a glycoside that binds to and inhibits the action of Na ؉ ,K ؉ -ATPase. Little is known, however, about the specific requirements of the protein surface for glycoside binding. Using chimeras of gastric H ؉ ,K ؉ -ATPase and Na ؉ ,K ؉ -ATPase, we demonstrated previously that the combined presence of transmembrane hairpins M3-M4 and M5-M6 of Na ؉ ,K ؉ -ATPase in a backbone of H ؉ ,K ؉ -ATPase (HN34/56) is both required and sufficient for high affinity ouabain binding. Since replacement of transmembrane hairpin M3-M4 by the N terminus up to transmembrane segment 3 (HNN3/56) resulted in a low affinity ouabain binding, hairpin M5-M6 seems to be essential for ouabain binding. To assess which residues of M5-M6 are required for ouabain action, we divided this transmembrane hairpin in seven parts and individually replaced these parts by the corresponding sequences of H ؉ ,K ؉ -ATPase in chimera HN34/56. Three of these chimeras failed to bind ouabain following expression in Xenopus laevis oocytes. Altogether, these three chimeras contained 7 amino acids that were specific for Na ؉ ,K ؉ -ATPase. Individual replacement of these 7 amino acids by the corresponding amino acids in H ؉ ,K ؉ -ATPase revealed a dramatic loss of ouabain binding for F783Y, T797C, and D804E. As a proof of principle, the Na ؉ ,K ؉ -ATPase equivalents of these 3 amino acids were introduced in different combinations in chimera HN34. The presence of all 3 amino acids appeared to be required for ouabain action. Docking of ouabain onto a three-dimensional-model of Na ؉ ,K ؉ -ATPase suggests that Asp 804 , in contrast to Phe 783 and Thr 797 , does not actually form part of the ouabain-binding pocket. Most likely, the presence of this amino acid is required for adopting of the proper conformation for ouabain binding.
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