Sickle hemoglobin instability: a mechanism for malarial protection

Abstract

Heterozygosity for the mutant sickle hemoglobin confers protection from severe Plasmodium falciparum malaria. It is here proposed that this protection derives from the instability of sickle hemoglobin, which clusters red cell membrane protein band 3 and triggers accelerated removal by phagocytic cells. This explanation requires that sickle trait cells manifest greater hemoglobin instability than normal red cells, something that could derive from their content of sickle hemoglobin. The mechanism also implicates splenic function as a determinant of the protective effect.     

Sickle hemoglobin fibers: mechanisms of depolymerization

We examined the depolymerization of hemoglobin (Hb) S fibers in the presence of CO by using photolysis of COHbS to create and isolate individual fibers, then removing photolysis to induce depolymerization. Depolymerization occurs at two sites, fiber ends and fiber sides, with different kinetics and by different mechanisms. At low partial pressure of CO (pCO), end-depolymerization is dominant, proceeding at approximately 1 microm s(-1), whereas at high pCO fibers vanish very rapidly, in much less than one second, by side-depolymerization. Each kind of depolymerization could occur by a ligand-independent path, in which deoxyHb depolymerizes and then is prevented from returning to the polymer by liganding with CO, or by a ligand-dependent path in which CO binds to the polymer inducing dissociation of the newly liganded molecules from it. We find that ligand-independent depolymerization is the dominant path for end-depolymerization and ligand-dependent depolymerization dominates, at least at high pCO, for side-depolymerization. On the basis of our kinetic results and electron micrographs of depolymerizing fibers, we propose a model for side-depolymerization in which a hole is nucleated by cooperative loss of a few molecules from fiber sides, followed by rapid depolymerization from the newly created fiber ends abutting the hole. Potential significance of these results for the pathophysiology of sickle cell disease is discussed.     

Mitotic catastrophe: a mechanism for avoiding genomic instability

The improper distribution of chromosomes during mitosis compromises cellular functions and can reduce cellular fitness or contribute to malignant transformation. As a countermeasure, higher eukaryotes have developed strategies for eliminating mitosis-incompetent cells, one of which is mitotic catastrophe. Mitotic catastrophe is driven by a complex and poorly understood signalling cascade but, from a functional perspective, it can be defined as an oncosuppressive mechanism that precedes (and is distinct from) apoptosis, necrosis or senescence. Accordingly, the disruption of mitotic catastrophe precipitates tumorigenesis and cancer progression, and its induction constitutes a therapeutic endpoint.     

Sickle hemoglobin polymer melting in high concentration phosphate buffer

Sickle cell hemoglobin (HbS) prepared in argon-saturated 1.8 M phosphate buffer was rapidly mixed with carbon monoxide (CO)-saturated buffer. The binding of CO to the sickle hemoglobin and the simultaneous melting of the hemoglobin polymers were monitored by transmission spectroscopy (optical absorption and turbidity). Changes in the absorption profile were interpreted as resulting from CO binding to deoxy-HbS while reduced scattering (turbidity) was attributed to melting (depolymerization) of the HbS polymer phase. Analysis of the data provides insight into the mechanism and kinetics of sickle hemoglobin polymer melting. Conversion of normal deoxygenated, adult hemoglobin (HbA) in high concentration phosphate buffer to the HbA-CO adduct was characterized by an average rate of 83 s-1. Under the same conditions, conversion of deoxy-HbS in the polymer phase to the HbS-CO adduct in the solution phase is characterized by an average rate of 5.8 s-1 via an intermediate species that grows in with a 36 s-1 rate. Spectral analysis of the intermediate species suggests that a significant amount of CO may bind to the polymer phase before the polymer melts.     

Structural analysis of polymers of sickle cell hemoglobin. II. Sickle hemoglobin macrofibers

Sickle cell hemoglobin macrofibers are an important intermediate in the low pH crystallization pathway of deoxygenated hemoglobin S that link the fiber to the crystal. Macrofibers are a class of helical particles differing primarily in their diameters but are related by a common packing of their constituent subunits. We have performed three-dimensional reconstructions of three types of macrofibers. These reconstructions show that macrofibers are composed of rows of Wishner-Love double strands in an arrangement similar to that in the crystal. We have measured the orientation and co-ordinates of double strands in macrofibers using cross-correlation techniques. In this approach, the electron density projections of double strands calculated from the known high-resolution crystal structure are compared with regions along the length of the particles in which the distinct pattern of double strands in c-axis projection may be observed. Contrary to assertions by Makinen & Sigountos (1984), our results unambigously demonstrate that adjacent rows of double strands in macrofibers are oriented in an antiparallel manner, as in the Wishner-Love crystal. Adjacent rows of antiparallel double strands are displaced along the helical axis relative to their co-ordinates in the crystal. Electron density models of macrofibers based on the crystallographic structure of the sickle hemoglobin double strand are in good agreement with the projections of macrofibers observed in electron micrographs. We have studied the structure of a closely related crystallization intermediate, the sickle hemoglobin paracrystal. The arrangement of double strands in paracrystals is similar to that in Wishner-Love crystals, except that they are displaced along the a-axis of the crystal. Measurements of the double strand co-ordinates reveal that the distribution of strand positions is bimodal. These results further establish the close structural relationship between macrofibers and paracrystals as intermediates in the crystallization of deoxygenated sickle hemoglobin.     

Ligand binding to the dimeric hemoglobin from Scapharca inaequivalvis, a hemoglobin with a novel mechanism for cooperativity

The homodimeric hemoglobin from Scapharca inaequivalvis has an unusual spatial arrangement of the subunits (Royer, W.E., Jr., Love, W.E., and Fenderson, F.F. (1985) Nature 316, 277-280). The time course of oxygen and nitric oxide rebinding to this protein following flash photolysis has been measured on a nanosecond time scale. A large amplitude is observed with a half-time of 20 ns (NO). With oxygen the half-time decreases from 70 ns at low fractional photolysis to 30 ns at large breakdown. The second order rate of NO binding is 1.6 x 10(7)/MS, and is the same as that for oxygen. Analysis of the geminate data suggests that oxygen and nitric oxide react more rapidly with the heme than in myoglobin, but also escape much more rapidly from its vicinity.     

Purification and characterization of a hemoglobin degrading aspartic protease from the malarial parasite Plasmodium vivax

Aspartic proteases of human malarial parasites are thought to play key roles in essential pathways of merozoite release, invasion and host cell hemoglobin degradation during the intraerythrocytic stages of their life cycle. Therefore, we have purified and characterized Plasmodium vivax aspartic protease, to determine if this enzyme can be used as potential drug target/immunogen, and its inhibitors as potential antimalarial drug. The P. vivax aspartic protease has been purified by a combination of ion exchange and size exclusion chromatographies and HPLC. Its properties were examined in order to define a role in the hemoglobin degradation process. The purified enzyme migrated as a single band on native PAGE and SDS/PAGE with a molecular mass of 40 kDa. Gelatin zymogram analyses revealed a clear zone of proteolytic activity corresponding to the band obtained on native PAGE and SDS/PAGE. The enzyme has an optimal pH of 4.0 and exhibits its highest activity at 37 degrees C. The enzyme is inhibited by pepstatin, but not by other inhibitors including o-phenanthroline, EDTA, PMSF or E-64, supporting its designation as an aspartic protease; its IC50 value was found to be 3.0 microM. A Lineweaver Burk double reciprocal plot with pepstatin shows that the inhibition is competitive with respect to the substrate. Ca2+ and Mg2+ ions enhance the protease activity, whereas Cu2+ and Hg2+ ions were found to be inhibitory. The pivotal role of aspartic protease in initiating hemoglobin degradation in P. vivax malaria parasite is also demonstrated.     

Sickle hemoglobin gelation. Reaction order and critical nucleus size.

Sickle hemoglobin (Hb S) gelation displays kinetics consistent with a rate-limiting nucleation step. The approximate size of the critical nucleus can be inferred from the order of the reaction with respect to Hb S activity, but determination of the reaction order is complicated by the fact that Hb S activity is substantially different from Hb S concentration at the high protein concentrations required for gelation. Equilibrium and kinetic experiments on Hb S gelation were designed to evaluate the relative activity coefficient of Hb S as a function of concentration. These experiments used non-Hb S proteins to mimic, and thus evaluate, the effect on activity coefficients of increasing Hb S concentration. At Hb S concentrations near 20% the change in Hb S activity coefficient generates two-thirds of the apparent dependence of nucleation rate on Hb S concentration. When this effect is explicitly accounted for, the nucleation reaction is seen to be approximately 10th-order with respect to effective number concentration of Hb S. The closeness of the reaction order to the number of strands in models of Hb S fibers suggests a nucleus close to the size of one turn of the Hb S fiber. These experiments introduce a new approach to the study of Hb S gelation, the equal activity isotherm, used here also to show that Hb S.Hb A (normal adult hemoglobin) hybrids do incorporate into growing nuclei and stable microtubules but that A.S hybridization is neutral with respect to promotion of Hb S nucleation and the sol-gel equilibrium.     

Extended linkage disequilibrium surrounding the hemoglobin E variant due to malarial selection

The hemoglobin E variant (HbE; ( beta )26Glu-->Lys) is concentrated in parts of Southeast Asia where malaria is endemic, and HbE carrier status has been shown to confer some protection against Plasmodium falciparum malaria. To examine the effect of natural selection on the pattern of linkage disequilibrium (LD) and to infer the evolutionary history of the HbE variant, we analyzed biallelic markers surrounding the HbE variant in a Thai population. Pairwise LD analysis of HbE and 43 surrounding biallelic markers revealed LD of HbE extending beyond 100 kb, whereas no LD was observed between non-HbE variants and the same markers. The inferred haplotype network suggests a single origin of the HbE variant in the Thai population. Forward-in-time computer simulations under a variety of selection models indicate that the HbE variant arose 1,240-4,440 years ago. These results support the conjecture that the HbE mutation occurred recently, and the allele frequency has increased rapidly. Our study provides another clear demonstration that a high-resolution LD map across the human genome can detect recent variants that have been subjected to positive selection.     

Heterogeneous nucleation in sickle hemoglobin: experimental validation of a structural mechanism

Sickle hemoglobin polymerizes by two types of nucleation: homogeneous nucleation of aggregates in solution, and heterogeneous nucleation on preexisting polymers. It has been proposed that the same contact that is made in the interior of the polymer between the mutant site beta6 and its receptor pocket on an adjacent molecule is the primary contact site for the heterogeneous nucleus. We have constructed cross-linked hybrid molecules in which one beta-subunit is from HbA with Glu at beta6, and the other is from HbS with a Val at beta6. We measured solubility (using sedimentation) and polymerization kinetics (using laser photolysis) on cross-linked hybrids, and cross-linked HbS as controls. We find approximately 4000 times less heterogeneous nucleation in the cross-linked AS molecules than in cross-linked HbS, in strong confirmation of the proposal. In addition, changes in stability of the nucleus support a further proposal that more than one beta6 contact is involved in the homogeneous nucleus.     

Sickle hemoglobin polymer stability probed by triple and quadruple mutant hybrids.

As part of an effort to understand the interactions in HbS polymerization, we have produced and studied a recombinant triple mutant, D6A(alpha)/D75Y(alpha)/E121R(beta), and a quadruple mutant comprising the preceding mutation plus the natural genetic mutation of sickle hemoglobin, E6V(beta). These recombinant hemoglobins expressed in yeast were extensively characterized, and their structure and oxygen binding cooperativity were found to be normal. Their tetramer-dimer dissociation constants were within a factor of 2 of HbA and HbS. Polymerization of these mutants mixed with HbS was investigated by a micromethod based on volume exclusion by dextran. The elevated solubility of mixtures of HbS with HbA and HbF in dextran could be accurately predicted without any variable parameters. Relative to HbS, the copolymerization probability of the quadruple mutant/HbS hybrid was found to be 6.2, and the copolymerization probability for the triple mutant/HbS hybrid was 0.52. The pure quadruple mutant had a solubility slightly above that of its hybrid with HbS. One way to explain these results is to require significant cis-trans differences in the polymer and that HbA assemble above 42.5 g/dl. A second way to explain these data is by the modification of motional freedom, thereby changing vibrational entropy in the polymer.     

Introduction of a new regulatory mechanism into human hemoglobin

Previous studies on bovine hemoglobin (HbBv) have suggested amino acid substitutions, which might introduce into human hemoglobin (HbA) functional characteristics of HbBv, namely a low intrinsic oxygen affinity regulated by Cl(-). Accordingly, we have constructed and characterized a multiple mutant, PB5, [beta(V1M + H2 Delta + T4I + P5A + A76K)] replacing four amino acid residues of HbA with those present at structurally analogous positions in HbBv, plus an additional substitution, beta T4I, which does not occur in either HbBv or HbA. This 'pseudobovine' hemoglobin has oxygen binding properties very similar to those of HbBv: the P(50) of HbA, PB5 and HbBv in the absence of Cl(-) are 1.6, 4.6 and 4.8 torr, respectively, and in 100 mM Cl(-) are 3.7, 10.5 and 12 torr, respectively. Moreover, PB5 has 3-fold slower autoxidation rate compared to HbA and HbBv. These are desirable characteristics for a human hemoglobin to be considered for use as a clinical artificial oxygen carrier. Although the functional properties of PB5 and HbBv are similar, van't Hoff plots indicate that the two hemoglobins interact differently with water, suggesting that factors regulating the R to T equilibrium are not the same in the two proteins. A further indication that PB5 is not a functional mimic of HbBv derives from PB5(control), a human hemoglobin with the same substitutions as PB5, except the beta T4I replacement. PB5(control) has a high oxygen affinity (P(50)=2.3 torr) in the absence of Cl(-), but retains the Cl(-) effect of PB5. The Cl(-) regulation of oxygen affinity in PB5 involves lysine residues at beta 8 and beta 76. PB4, which has the same substitutions as PB5 except beta A76K, and PB6, which has all the substitutions of PB5 plus beta K8Q, both have a low intrinsic oxygen affinity, like HbBv and PB5, but exhibit a decreased sensitivity to Cl(-). Since HbBv has lysine residues at both beta 8 and beta 76, these results imply that Cl(-) regulation in HbBv likewise involves these two residues. The mechanism responsible for the low intrinsic oxygen affinity of HbBv remains unclear. It is suggested that residues peculiar to HbBv at the alpha(1)beta(1) interface may play a role.     

A chemical instability mechanism for asymmetric cell differentiation

We propose a mechanism of asymmetric mitosis applicable for cells even without inherent architectural asymmetry and in the presence of symmetric conditions such as a homogeneous environment. The theory is based on the instability of the symmetric development in time of the prospective daughters of a cell in mitosis. The macroscopic equations of chemical reaction, diffusion, and permeation of the various chemical species in the cell are given formal expression, and are then linearized about the symmetric development in order to test the stability to asymmetric perturbations. Instability to such perturbations indicates the deterministic onset of asymmetric division (differentiation). Only small external gradients of concentration, temperature, light, etc. are necessary to polarize the asymmetry for the purpose of a particular morphology. The theory is compared qualitatively with experiments on melanocytogenesis, is used to suggest new experiments, and is proposed as a possible alternative to other mechanisms. Possible application of the theory to the experiments on the first division in the egg of Fucus is considered. Finally, a simple model of a product-enhanced reaction mechanism is developed in detail which shows that the history of the initial preparation of the cell prior to mitosis may play a role in determining the possibility of asymmetric division.     

Adaptive amplification: an inducible chromosomal instability mechanism

Adaptive mutation is an induced response to environmental stress in which mutation rates rise, producing permanent genetic changes that can adapt cells to stress. This contrasts with neo-Darwinian views of genetic change rates blind to environmental conditions. DNA amplification is a flexible, reversible genomic change that has long been postulated to be adaptive. We report the discovery of adaptive amplification at the lac operon in Escherichia coli. Additionally, we find that adaptive amplification is separate from, and does not lead to, adaptive point mutation. This contradicts a prevailing alternative hypothesis whereby adaptive mutation is normal mutability in amplified DNA. Instead, adaptive mutation and amplification are parallel routes of inducible genetic instability allowing rapid evolution under stress, and escape from growth inhibition.