Primate Hemoglobins Research Articles

Molecular Basis for the Evolution of Species-Specific Hemoglobin Capture by Staphylococcus aureus

Metals are a limiting resource for pathogenic bacteria and must be scavenged from host proteins. Hemoglobin provides the most abundant source of iron in the human body and is required by several pathogens to cause invasive disease. However, the consequences of hemoglobin evolution for bacterial nutrient acquisition remain unclear. Here we show that the α- and β-globin genes exhibit strikingly parallel signatures of adaptive evolution across simian primates. Rapidly evolving sites in hemoglobin correspond to binding interfaces of IsdB, a bacterial hemoglobin receptor harbored by pathogenic Staphylococcus aureus Using an evolution-guided experimental approach, we demonstrate that the divergence between primates and staphylococcal isolates governs hemoglobin recognition and bacterial growth. The reintroduction of putative adaptive mutations in α- or β-globin proteins was sufficient to impair S. aureus binding, providing a mechanism for the evolution of disease resistance. These findings suggest that bacterial hemoprotein capture has driven repeated evolutionary conflicts with hemoglobin during primate descent.IMPORTANCE During infection, bacteria must steal metals, including iron, from the host tissue. Therefore, pathogenic bacteria have evolved metal acquisition systems to overcome the elaborate processes mammals use to withhold metal from pathogens. Staphylococcus aureus uses IsdB, a hemoglobin receptor, to thieve iron-containing heme from hemoglobin within human blood. We find evidence that primate hemoglobin has undergone rapid evolution at protein surfaces contacted by IsdB. Additionally, variation in the hemoglobin sequences among primates, or variation in IsdB of related staphylococci, reduces bacterial hemoglobin capture. Together, these data suggest that S. aureus has evolved to recognize human hemoglobin in the face of rapid evolution at the IsdB binding interface, consistent with repeated evolutionary conflicts in the battle for iron during host-pathogen interactions.

Identification of PRE-γ-GLOBIN

A pre-γ-globin species was identified by high performance liquid chromatography in platyrrhine primates. Although pre-γ-globin has not been observed in human hemoglobin, its identification in platyrrhine hemoglobin was facilitated by the functional inactivation of one of the duplicated γ-globin genes in platyrrhines, which simplified the high performance liquid chromatography elution pattern. Part, but not all, of the pre-γ was glutathionyl γ2-globinI and matrix-assisted laser desorption/ionization mass spectrometry was used to demonstrate that the glutathionyl residue was located on cysteine 93. As this residue is invariant in primates, it is predicted that the formation of glutathionyl γ-globin will be seen in all primate hemoglobins under appropriate conditions.

A discrete Fourier analysis for evolutionary trees.

Discrete Fourier transformations have recently been developed to model the evolution of two-state characters (the Cavender/Farris model). We report here the extension of these transformations to provide invertible relationships between a phylogenetic tree T (with three probability parameters of nucleotide substitution on each edge corresponding to Kimura's 3ST model) and the expected frequencies of the nucleotide patterns in the sequences. We refer to these relationships as spectral analysis. In either model with independent and identically distributed site substitutions, spectral analysis allows a global correction for all multiple substitutions (second- and higher-order interactions), independent of any particular tree. From these corrected data we use a least-squares selection procedure, the closest tree algorithm, to infer an evolutionary tree. Other selection criteria such as parsimony or compatibility analysis could also be used; each of these criteria will be statistically consistent for these models. The closest tree algorithm selects a unique best-fit phylogenetic tree together with independent edge length parameters for each edge. The method is illustrated with an analysis of some primate hemoglobin sequences.

A Comparative Study on the Structural Differences of Primate Hemoglobins by Spin Labeling Technique

The hemoglobins of human and five non-human primates were spin-labeled with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide, and the ESR spectra of their deoxy, oxy, and carbonmonoxy forms were measured. The analyses of the spectra indicated that the local protein conformation in the vicinity of the spin-labeled cysteine residue at position 93(F9) in the beta-chain is slightly but significantly different among species, and that each hemoglobin shows a similar change in conformation upon conversion from the oxy form to the carbonmonoxy one except for human hemoglobin. Human hemoglobin was suggested to undergo a significantly different conformational change upon this conversion, indicating that it has unique characteristics among the primate hemoglobins.

Monoclonal antibodies recognizing single amino acid substitutions in hemoglobin.

Four monoclonal antibodies (mAb) to non-human primate hemoglobin referred to as Cap-4, Cap-5, Rh-2, and Rh-4, and two mAb to human hemoglobin, referred to as H-1 and H-3 were isolated and were partially characterized. Binding studies with these mAb on a panel of hemoglobins and isolated alpha and beta globin chains revealed a unique reactivity pattern for each mAb. Amino acid sequence analysis of the antigens used to generate the binding data suggests that the specific recognition of certain hemoglobin antigens by each mAb is controlled by the presence of a particular amino acid at a specific position within the epitope. The use of synthetic peptides as antigens confirmed this observation for five of the mAb. No synthetic peptides were tested with the sixth mAb, Rh-2. The amino acids required for binding of mAb Cap-4, Cap-5, Rh-4, and Rh-2 to hemoglobin are alanine at beta 5, threonine at beta 13, glutamine at beta 125, and leucine at alpha 68. The non-human primate hemoglobin antibodies require a specific amino acid that is not present in human hemoglobin. The amino acid required for binding of Cap-4, Cap-5, and Rh-4 could arise by a single base change in the beta globin gene, whereas the amino acid required for Rh-2 binding would only occur if two base changes occurred. Thus these mAb are candidate probes for a somatic cell mutation assay on the basis of the detection of peripheral blood red cells that possess single amino acid substituted hemoglobin as a result of single base substitutions in the globin genes of precursor cells.

The Rate of Molecular Evolution of Primate Hemoglobin is Becoming Slower

The Rate of Molecular Evolution of Primate Hemoglobin is Becoming Slower

Primary structures of adult hemoglobins of silvery marmoset, Callithrix argentatus, and cotton-headed tamarin, Saguinus oedipus.

Tryptic peptides of the alpha and beta chains from silvery marmoset (Callithrix argentatus) and cotton-headed tamarin (Saguinus oedipus) hemoglobins were isolated and sequenced, respectively, by conventional methods. The ordering of the peptides in each chain was deduced from the homology of their sequences with those of human adult hemoglobin. The primary structures thus deduced are compared with those of other primate hemoglobins, and the rate of evolution in New World monkey hemoglobins is discussed.

In Vitro Stimulation of Primate Hemoglobin Synthesis by L-Thyroxine

Bone marrow cells from adult and abortus primates (marmosets) were incubated in vitro to determine their responsiveness to L-thyroxine. 3H-leucine incorporation into purified globin chains was the parameter assayed to determine responsiveness. Bone marrow from spontaneously aborted animals consistently was stimulated by the presence of physiologic levels of L-thyroxine. Bone marrow cells from adult animals were unaffected by the hormone.

In vitro stimulation of primate hemoglobin synthesis by L-thyroxine

Bone marrow cells from adult and abortus primates (marmosets) were incubated in vitro to determine their responsiveness to L-thyroxine. 3H- leucine incorporation into purified globin chains was the parameter assayed to determine responsiveness. Bone marrow from spontaneously aborted animals consistently was stimulated by the presence of physiologic levels of L-thyroxine. Bone marrow cells from adult animals were unaffected by the hormone.

The inhibition of the primary interaction between 1 2 5I-labeled human hemoglobin and rabbit anti-human hemoglobin sera: A sensitive radioimmunoassay technique

A rapid and sensitive radioimmunoassay technique for hemoglobin based upon a modification of the Farr test is described. Since hemoglobin is soluble in ammonium sulfate whereas antibody is precipitated, the amount of free and antibody-bound radiolabeled human hemoglobin can be quantitatively measured after ammonium sulfate precpitation. Nanogram quantities of hemoglobin can be detected by the addition of unlabeled human hemoglobin to the antiserum before the labeled hemoglobin, thus establshing a competition reaction between labeled and unlabeled antigen for antibody. If sufficient quantities of unlabeled human hemoglobin are present, all of the antibody combining sites become saturated and the primary interaction between 1 2 5I-labeled hemoglobin and antibody is completely inhibited. The method can be employed to discriminate various normal human hemoglobins, non-human primate hemoglobins as well as sickle-cell hemoglobin (HbS). Other possible applications of the technique are discussed.

Variation in protein structure and function: Primate hemoglobins

Variation in structure among primate hemoglobins is associated with variation in function. This supports the hypothesis that most substitutions observed among homologous proteins in different species have been fixed by natural selection because they contribute to the fitness of the genotype. It does not support the concept that most substitutions result from the fixation of neutral alleles by genetic drift.

Primate hemoglobins: Polymorphisms and evolutionary patterns

Abstract In this rrport we illustrate the apparently great incidence in the number of different kinds of electrophoretically undetectable intraspecies variants found in the course of characterizing primate hemoglobin α, β and δ sequences. Our primary interest is to assess the significance of such findings in a setting provided by fixed amino acid differences that have accumulated between species during primate evolution. Observed non-randomness in fixed mutations, with respect to both linear distribution and certain kinds of change, is interpreted as due to the restrictive effects of natural selection rather than to either differential mutability at particular positions or disturbed permutations of the genetic code in general. Thus, in the micropatterns of accumulated mutation, we adduce strong evidence for Darwinism. However, we also find that the δ chain of minor hemoglobin A2 (α2δ2)—while apparently visible to selection—seems less restricted than the β chain of the major component, hemoglobin A (α2β2). Such findings are used to support our earlier presumption that comparative changes in β and δ can serve as a model for evaluating mechanisms of amino acid replacement during evolution. We believe that equivalence between β and δ in the numbers of recognizable fixed mutations is more easily explained by non-adaptive mechanisms than by adaptive ones. The frequencies of seven different kinds of electrically neutral variants found within-species during detailed analysis of ∼70 allele products are reported here. Most, if not all, are common. We propose that the overall incidence of different kinds of electrically neutral polymorphisms is probably at least fivefold greater than the kinds of electrophoretically detectable polymorphism. In their electrical neutrality and location the electrophoretically silent polymorphisms strongly resemble the fixed mutations accumulated during evolution of primate hemoglobin chains and are thus candidates for the transient phase of non-adaptive evolution.

Structure, function and evolution of primate hemoglobins—II. A survey of the oxygen-binding properties

1.1. The oxygen-binding properties of hemoglobins from more than twenty species and subspecies of nonhuman primates were compared at 15 and 20°C in the pH range 5·0–8·5.2.2. Most species of primates have hemoglobins with distinct oxygen-binding properties3.3. Prosimian hemoglobins have a lower affinity for oxygen near physiological pH values than do monkey and ape hemoglobins.4.4. Human hemoglobin has oxygen-binding properties intermediate to those of other primate hemoglobins.5.5. Functional variation apparently parallels structural variation.6.6. As predicted from considerations of allosterism, variation in one oxygen-binding parameter is often associated with variation in additional parameters.7.7. This prohibits rapid evolutionary changes, but does allow for multiple and precise control of function.

Primate hemoglobins: Some sequences and some proposals concerning the character of evolution and mutation.

During a multipurpose survey we examined electrophoretic mobilities of major (A, i.e., α2β2) and minor (A2, i.e., α2δ2) adult hemoglobins from populations of nine primate genera representing a total of 440 New World monkeys and apes. Sequences of hemoglobin chains were inferred from differences in amino acid composition between homologous tryptic peptides supplemented by detailed placement of more than 270 residues. Beta sequences were thus analyzed in five genera (Aotus, Ateles, Hylobates, Saimiri, and Saguinus) and δ sequences in seven (foregoing plus Gorilla and Pan). In most genera, sequences from several individuals, often from several species, were delineated. Fifteen kinds of intraspecies mutants were detected; 10 of these were precisely characterized. Five of the 15 mutants form electrophoretically detected genetic polymorphisms of δ; none such occur in β. Six electrophoretically detected mutants, four in α and two in δ, are uncommon. One of these represents the complete absence of minor component. Three kinds of variants, two in δ and one in β, are electrophoretically neutral and chance findings during sequence analysis of the equivalent of 38 allele products. Two of the neutral variants are not especially common; one may have polymorphic frequency. Several general conclusions stem from these and supplementary findings. First, comparisons of sequences suggest that δ and β genes in all primates either arose from a single event in a common ancestor or from two approximately coincident events. Either assumption allows reconstruction of a reasonably accurate archetype sequence that is effectively common to all descendants. Second, there is a pancellular quantitative disproportion between major and minor hemoglobins ranging from 16:1 to 220:1 in species studied. Delta is consequently presumed to be functionally and adaptively less vital than β. When these premises are adopted, δ is expected to be relatively invisible to natural selection, and, where darwinism is the principal arbiter of evolution and polymorphism, δ is expected to show fewer fixed changes and fewer genetic polymorphisms than β. The opposite is observed. Delta exhibits as many or more changes from archetype than β. This finding and the comparative abundance of δ polymorphism are attributed to nonadaptive factors which are thus considered the source of much evolutionary change. Third, particular sequence positions in various species are the site of recurrent mutations in both β and δ. One such area is occupied by the majority of genetic polymorphisms found in man and other primates. The overall distribution of mutations arising in evolution is remarkably nonrandom in β, δ, and a pool of both. These results are quite unlike most other observations in higher organisms. The sources of such nonrandomness are either selection and/or differential mutability. We rely on our prior assumption of relative selective invisibility for δ and, in part, ascribe the nonrandom distribution of changes to microzones of enhanced mutability. Fourth, the six uncommon electrophoretically detected mutants provide an estimate of heterozygosity (1/73) at hemoglobin loci that is tenfold greater than observed in man. Fifth, the unprecedented chance detection of three kinds of electrophoretically neutral intraspecies mutants among the equivalent of 38 characterized allele products suggests that neutral changes are as common as electrostatically active ones and at least tenfold more common than expected in extrapolation from human variant surveys. Sixth, β analyses from three kinds of gibbon (Hylobates) hemoglobin suggest that one of these is a potentially unchanged relict of the ancient archetype and, further, indicate a degree of homozygous diversity within a species that nearly equals the difference between gibbon and man.

Primate hemoglobins: Their structure, function and evolution—I. Amino acid compositions of the tryptic peptides from the beta chain of Cebus albifrons

1. 1. Two fragments were isolated after cyanogen bromide cleavage of the aminoethylated β chain from the major hemoglobin of a New World monkey ( Cebus albifrons). Each fragment was digested with trypsin and the resulting peptides were isolated. The peptides were placed in sequence by homology with the corresponding peptides from the human β chain. The locations of residues that differ from those in the human β chain were deduced from analyses of the thermolysin peptides produced by subfragmentation of selected tryptic peptides. The β chain of C. albifrons differs from that of man at a minimum of seven positions; in addition, two residues (threonine and alanine) were found at position 13. 2. 2. Comparison with β chains from other New World monkeys, Old World monkeys and apes indicates that the ceboid β chains comprise a related group of polypeptide chains which are more similar to those of cercopithecoids than they are to those of hominoids.

MOLECULES AND MONKEYS.

MOLECULES AND MONKEYS.