Human embryonic, fetal, and adult hemoglobins have different subunit interface strengths. Correlation with lifespan in the red cell

The different types of naturally occurring, normal human hemoglobins vary in their tetramer-dimer subunit interface strengths (stabilities) by three orders of magnitude in the liganded (CO or oxy) state. The presence of embryonic zeta-subunits leads to an average 20-fold weakening of tetramer-dimer interfaces compared to corresponding hemoglobins containing adult alpha-subunits. The dimer-monomer interfaces of these hemoglobins differ by at least 500-fold in their strengths; such interfaces are weak if they contain zeta-subunits and exchange with added beta-subunits in the form of beta(4) (HbH) significantly faster than do those with alpha-subunits. Subunit exchange occurs at the level of the dimer, although tetramer formation reciprocally influences the amount of dimer available for exchange. Competition between subunit types occurs so that pairs of weak embryonic hemoglobins can exchange subunits to form the stronger fetal and adult hemoglobins. The dimer strengths increase in the order Hb Portland-2 (zeta(2)beta(2)) < Hb Portland-1 (zeta(2)gamma(2)) approximately equal Hb Gower-1 (zeta(2)epsilon(2)) < Hb Gower-2 (alpha(2)epsilon(2)) < HbF(1) < HbF (alpha(2)gamma(2)) < HbA(2) (alpha(2)delta(2)), i.e., from embryonic to fetal to adult types, representing maturation from weaker to stronger monomer-monomer subunit contacts. This increasing order recapitulates the developmental order in which globins are expressed (embryonic --> fetal --> adult), suggesting that the intrinsic binding properties of the subunits themselves regarding the strengths of interfaces they form with competing subunits play an important role in the dynamics of protein assemblies and networks.     

Gelation of sickle cell hemoglobin in mixtures with normal adult and fetal hemoglobins.

We report the results of thermodynamic and kinetic studies on the gelation of mixtures of sickle cell (S) deoxyhemoglobin with normal human adult (A) and fetal (F) deoxyhemoglobins. The delay time of thermally induced gelation was monitored by the increase in turbidity. At the completion of gelation the solubility was determined by sedimenting the polymers and measuring the supernatant concentration spectrophotometrically. Addition of hemoglobins A or F, at mole fractions from 0 to 0.6, resulted in large increases in both the solubility and the delay time. For a 50:50 mixture of deoxyhemoglobin F with deoxyhemoglobin S, the solubility increased by a factor of 1.8 and the delay time by a factor of 10⁷ relative to pure deoxyhemoglobin S at the same total concentration, while for a 50:50 mixture of deoxyhemoglobins A and S the solubility increased by a factor of 1.4 and the delay time by a factor of 10⁴. The relative delay times were independent of both temperature and total hemoglobin concentration. The data have been analyzed according to theoretical models which treat the effects of temperature, concentration, non-ideality and solution composition on the thermodynamics and kinetics of gelation. The increased solubility in mixtures with deoxyhemoglobin F is fully explained by a model in which only deoxyhemoglobin S molecules polymerize. The effect of fetal hemoglobin (α₂γ₂) and hybrid α₂γβ^S molecules is to increase the solution non-ideality through the contribution of their excluded volume. The smaller increase in the solubility observed in comparable mixtures with deoxyhemoglobin A requires that the hybrid α₂β^Aβ^S molecules copolymerize with the deoxyhemoglobin S. The kinetic results for the mixtures can be quantitatively accounted for using a nucleation model in which the equilibrium properties of the polymer are used to describe the critical nucleus. The very large increases in delay time observed for the $\text{S}\text{F}$ mixtures can be explained by assuming that only α₂β₂^S molecules participate in the formation of a nucleus containing about 25 monomers. As in the thermodynamic analysis, the smaller effect of adding deoxyhemoglobin A can be attributed to the contribution of the hybrid molecules in forming the critical nucleus. Thus the difference between the polymerization properties of mixtures of deoxyhemoglobin S with deoxyhemoglobins A and F can be attributed solely to the copolymerization of the α₂β^Aβ^S hybrid molecule and the absence of any significant copolymerization of the α₂γβ^S hybrid.     

N-terminal contributions of the γ-subunit of fetal hemoglobin to its tetramer strength: Remote effects at subunit contacts

The greatly increased tetramer strength of liganded fetal hemoglobin compared with adult hemoglobin is shown by its 70-fold smaller tetramer-dimer dissociation constant. This property has been shown previously to be only partially caused by the 5-amino-acid differences at both types of interfaces in each hemoglobin. A major contributor to tetramer strengthening is the 18-amino-acid N-terminal A helix of the gamma-subunit of fetal hemoglobin, which differs from the beta-subunit of adult hemoglobin at eight amino acid residues. This long-distance communication between the A helix and the distant C helix and FG helical corner comprising the subunit contacts at the allosteric interface represents internal signaling. Physiologically, its greater tetramer strength endows fetal hemoglobin with the capacity to abstract oxygen from maternal adult hemoglobin. It also leads to resistance of fetal red cells to the malaria parasite because the HbF tetramer does not dissociate to dimers as readily as HbA; dimers are digested by malaria proteases but tetramers are not. In this communication, we report which sites on the A helix of the gamma-subunit are important for tetramer strengthening in HbF by substituting certain amino acids in the beta-subunit by the corresponding residues in the gamma-subunit. The recombinant hemoglobins containing up to five replacements together have been extensively characterized. Mass values were within 1 unit of theory. Gly 1 (gamma) of HbF with its high pK(a) of 8.1 compared with a 7.1 value for Val 1 (beta) of HbA creates a highly electropositive N terminus that may couple with the electronegative sequence just after it on the gamma-subunit. The Leu 3 to Phe replacement has no apparent role; however, position 5 is important because replacement of Pro 5 (beta) by Glu 5 (gamma) promotes tetramer strengthening. The Glu --> Asp replacement at position 7 enhances this effect because of the lower pK(a) of Asp but the Val --> Ile substitution at position 11 has no effect. Thus, the three positive/negative sites at positions 1, 5, and 7 account for practically all of the tetramer strength of HbF, as illustrated by an electrostatic surface potential analysis. The pathway by which information is transmitted to the distant allosteric subunit interfaces is currently under study. Oxygen-binding properties of the hemoglobins with charged substitutions more closely resemble those of HbA rather than those of HbF. Thus, whereas the A helix has a major role in controlling the strength of interactions at the tetramer-dimer allosteric interface, oxygen-binding properties of HbA and HbF are influenced by sequences in the C helix and at the FG helical corner constituting the allosteric interface.     

Multiple fetal and adult hemoglobins in Malaysian Macaca nemestrina: consequences of a-chain variation

The hemoglobins of fetal and adult Macaca nemestrina were investigated electrophoretically and chromatographically. Standard electrophoretic techniques failed to reveal variation in either fetal or adult hemoglobins beyond that previously attributed to the presence of the duplicate γ-chain loci characteristic of this species. However, column chromatography of whole hemolysates on CM-Sephadex or of globin chains on CM-cellulose in 8 M urea revealed that some fetuses had four major hemoglobins and some adults had two major hemoglobin components. All animals examined were of Malaysian origin; the variation described is a consequence of variation in α-chain structure in Malaysian populations of this species, and of the interaction of different α chains with the products of the duplicate γ-chain loci.     

Carboxyl-terminal modification alters the subunit interactions and assembly pathways of normal and sickle hemoglobins

Parallel isofocusing studies established that carboxypeptidase A removal of the His-146 (HC3) and Tyr-145 (HC2) residues of heme subunits affected the assembly properties of both Des (A) and Des (S) with heme chains, albeit to differing degrees. Indeed, the rate of Des (A) oligomer dissociation (k ₁), as determined by visible spectroscopy, was 4.3-fold faster than that of its native (A) counterpart. Furthermore, Soret spectral studies have affirmed distinct rates of normal (HbA), sickle (HbS), and Des HbA hemoglobin assembly (k₂) from their and [Des (A)] heme-containing monomers. Matching kinetic analysis of Des (A) and Des (S) chain assembly (with an identical chain) revealed 4.6- and 7.8-fold faster combination rates than those seen for (A) and (S) chains, respectively. This 3-fold disparity in rates strongly supports the critical role of the -6 (A3) residue, and its amino-terminal region, in chain partner recognition and subsequent human hemoglobin assembly.     

Models to study ancient host-pathogen interactions: lessons from Crete

The Aegean Conferences' first International Conference on Model Hosts took place on the picturesque Greek island of Crete. This meeting was the first of its kind and gathered together international experts who are using a vast array of hosts as models of infection, including worms, insects, mice, fish, rats, humans, squids, pigs, monkeys, protozoa, amoebae and ticks.     

Protein--carbohydrate interactions: learning lessons from nature

Protein--carbohydrate interactions are at the heart of many important biological processes including signalling, recognition and catalysis. A deeper understanding of these interactions at the molecular level will enable the development of novel, effective and highly selective therapeutics. Glycosyltransferases and glycosidases, carbohydrate-processing enzymes responsible for the synthesis and breakdown of oligosaccharides, have emerged as important targets in the fight against bacterial and fungal pathogenesis, cancer and AIDS. Binding and recognition phenomena are essential processes by which the body exerts control over complex biological functions. In this regard, heparin has retained ongoing interest reflecting its importance as a major pharmaceutical. Recent studies on heparin have shed light onto the mechanisms of cross-reactivity that cause life-threatening side effects and have provided impetus for the development of more selective anti-clotting agents. Important targets for therapeutic intervention are the binding processes mediated through multivalent protein--carbohydrate interactions, such as the interactions of bacterial toxins with cell-surface receptors.     

The multifaceted contributions of leukocyte subsets to atherosclerosis: lessons from mouse models

Chronic inflammation drives the development of atherosclerosis, and details regarding the involvement of different leukocyte subpopulations in the pathology of this disease have recently emerged. This Review highlights the surprising contribution of granulocyte subsets and mast cells to early atherogenesis and subsequent plaque instability, and describes the complex, double-edged role of monocyte, macrophage and dendritic-cell subsets through crosstalk with T cells and vascular progenitor cells. Improved understanding of the selective contributions of specific cell types to atherogenesis will pave the way for new targeted approaches to therapy.     

Study of normal human fetal and adult hemoglobins by M?ssbauer spectroscopy

Human normal fetal and adult hemoglobins were investigated by M?ssbauer spectroscopy. The differences discovered in quadrupole splitting are connected with distinctions in protein molecular structures. An approximation of M?ssbauer spectra of hemoglobins in supposition of nonequivalence of Fe(II) electronic structure in alpha-, beta- and gamma-subunits in tetramers was proposed. Qualitative analysis of the relationship of Fe(II) electronic structure, molecular structure of the active site and functionality of hemoglobin was carried out.     

Functional characterization of fetal and adult yak hemoglobins: an oxygen binding cascade and its molecular basis

In contrast to most other mammals, the yak, which is native to high altitudes, has two major fetal and two or four major adult hemoglobin (Hb) components. We report the oxygen affinities and sensitivities to pH and 2,3-diphosphoglycerate of the two fetal and two adult Hbs commonly found in calves, compared to those of adult cow Hb A, and relate these findings to their primary structures and to placental maternal-fetal oxygen transfer at altitude. Arranged in order of decreasing oxygen affinity the Hbs are F1 (alpha I2 gamma 2), F2 (alpha II2 gamma 2), A1 (alpha II2 beta II2), and cow Hb A. The higher affinity of the fetal than the adult yak Hbs correlates with the beta 15Trp----Phe substitution, whereas the higher affinity in yak than in cow Hb correlates with the beta 135Ala----Val substitution. The difference in oxygen affinities between yak Hbs A1 and A2, which have identical beta chains, suggests the existence of yet unknown mechanisms determining oxygen affinity. The larger Bohr effects of F2 than F1 and of A2 than A1 are attributable to alpha-chain differences, most probably the alpha I50Glu----alpha II50His substitution.     

Conjoined hemoglobins. Loss of cooperativity and protein-protein interactions

Hemoglobin cross-linked as a bis(isophthalamide) of the epsilon-amino groups of lysine 82 of each beta-subunit binds and releases oxygen with a Hill coefficient indicative of cooperative oxygen binding (typically approximately 2.0). However, connecting two such cross-linked tetramers with a relatively short covalent linkage produces cross-linked bis-tetramers that bind oxygen with Hill coefficients near unity. To separate the effect of the linkages from the effects of protein-protein interactions in the conjoined proteins, reagents (1 and 2) were used to produce bis-tetramers (A and B). These have a considerably greater distance between cross-linked tetramers than earlier examples. Yet, the bis-tetramers (A and B) bind oxygen with minimal cooperativity (n(50) = 1.4, 1.2). To assess the effect of the linkage itself, cross-linked tetramers (Cand D) were prepared from reactions with the same reagents. These bind oxygen with cooperativity similar to that of cross-linked tetramers without the extended chain (C, n(50) = 2.0; D, n(50) = 1.8). Other tetramers (E and F) with flexible, saturated hydrocarbon appendages were also prepared. These also showed cooperativity in oxygen binding (E, n(50) = 1.7; F, n(50) = 1.8) despite their high degree of hydrophobicity. Thus, the intertetrameric linkages themselves do not induce the loss of cooperativity, leading to the conclusion that solution effects of the tetramers upon one another are the source of the decline in cooperativity: protein-protein interactions are most significant in disrupting the cooperativity of the bis-tetramers, regardless of the span or composition of the linker. This suggests that effects of oligomerization of hemoglobin within red cell substitutes should be considered in terms of such interactions.     

Epigenetic interactions between transposons and genes: lessons from plants

Transposons replicate, increase in copy number and persist in nature by moving, but insertion into genes is generally mutagenic. There is thus a strong selection for transposons that can achieve a balance between their own replication and minimal damage to their host. Epigenetic regulation proves to be a widespread way to achieve this balance, quieting transposition on the one hand, yet reversible on the other. As our understanding of epigenetics improves, the subtleties and the scope of how transposons can affect gene expression, both directly and indirectly, are becoming clearer.     

Temporal kinetics of fluoride accumulation: from fetal to adult deer

In June 2011, a volcano deposited about 100 million tons of tephra over parts of Chile and over 36 million ha of Argentina. Initially, fluoride was considered irrelevant; however, recently wild deer exhibited strong fluorosis, with fluoride level increasing 38-fold among severely affected deer. Whereas mothers averaged 2,151 ppm, their late-term fetuses had only 19.8 ppm, indicating a barrier to fluoride transport in utero. Levels among four age classes increased significantly, at a rate of about 1,000 ppm/year. The temporal kinetics of accumulation suggests that sources of available fluoride are highly effective. Thus, compared to prior background levels (63 ppm in adults) and to fetuses starting at about 20 ppm, 1-year-old calves averaged 1,035 ppm (maximum 1,830 ppm), 2-year olds averaged 2,151 ppm (maximum 2,513 ppm), and older deer averaged 2,806 ppm (maximum 5,175 ppm). As osteofluorosis occurs in deer with >4,000 ppm, accumulation of 1,000 ppm/year would result in adults reaching levels causing osteopathology in 1–2 years. Importantly, impacts may be further exacerbated by regional iodine and selenium deficiencies. Iodine deficiency may increase incidences of dental fluorosis and severity of damages, while selenium deficiency impacts iodine metabolism. Fluorosis will affect population dynamics, morbidity, predation susceptibility, and other ecosystem components like scavenger and plant communities.