Allergens of non-biting midges (Diptera: Chironomidae): a systematic survey of chironomid haemoglobins

Various genera of non-biting midges (Diptera: Chironomidae) possess haemoglobins in larvae and adults. For certain species, these haemoglobins have been implicated in human allergic disease in several countries. The present study confirms and extends observations that haemoglobin is present in many species of Chironomidae, establishes that it is retained from the larval to the adult stage and shows that haemoglobin persists both in live and in dead dry flies. Previous suggestions that Chironomidae should be seen as important environmental and occupational allergens are clearly substantiated.     

Expression and evolution of functionally distinct haemoglobin genes in plants

Haemoglobin genes have been found in a number of plant species, but the number of genes known has been too small to allow effective evolutionary inferences. We present nine new non-symbiotic haemoglobin sequences from a range of plants, including class 1 haemoglobins from cotton, Citrus and tomato, class 2 haemoglobins from cotton, tomato, sugar beet and canola and two haemoglobins from the non-vascular plants, Marchantia polymorpha (a liverwort) and Physcomitrella patens (a moss). Our molecular phylogenetic analysis of all currently known non-symbiotic haemoglobin genes and a selection of symbiotic haemoglobins have confirmed the existence of two distinct classes of haemoglobin genes in the dicots. It is likely that all dicots have both class 1 and class 2 non-symbiotic haemoglobin genes whereas in monocots we have detected only class 1 genes. The symbiotic haemoglobins from legumes and Casuarina are related to the class 2 non-symbiotic haemoglobins, whilst the symbiotic haemoglobin from Parasponia groups with the class 1 non-symbiotic genes. Probably, there have been two independent recruitments of symbiotic haemoglobins. Although the functions of the two non-symbiotic haemoglobins remain unknown, their patterns of expression within plants suggest different functions. We examined the expression in transgenic plants of the two non-symbiotic haemoglobins from Arabidopsis using promoter fusions to a GUS reporter gene. The Arabidopsis GLB1 and GLB2 genes are likely to be functionally distinct. The class 2 haemoglobin gene (GLB2) is expressed in the roots, leaves and inflorescence and can be induced in young plants by cytokinin treatment in contrast to the class 1 gene (GLB1) which is active in germinating seedlings and can be induced by hypoxia and increased sucrose supply, but not by cytokinin treatment.     

The haemoglobin system of the mudfish, Labeo capensis: adaptations to temperature and hypoxia

The effect of temperature and hypoxic acclimation on the haemoglobin system and intraerythrocytic organic phosphate concentrations in the South African mudfish, Labeo capensis, have been investigated. Exposure to hypoxia or increased temperature raised haemoglobin concentration and decreased NTP/Hb ratio. Temperature acclimation did not effect the oxygenation characteristics of the haemolysate or haemoglobin multiplicity, as evident from isoelectric focussing experiments that showed one cathodic (Hb I) and three anodic haemoglobins (Hb II, III and IV). Oxygen equilibria of the isolated components showed a smaller Bohr effect and lower temperature and organic phosphate sensitivities in the cathodic than in the anodic haemoglobins. Unlike the trout and eel haemoglobin systems, both the anodic and cathodic haemoglobins from L. capensis exhibited sensitivity to organic phosphates but the effect was smaller in the latter. The results indicate that oxygen transport in mudfish blood is supported by variations in the red cell organic phosphate\haemoglobin ratio and the functional differentiation between anodic and cathodic haemoglobins.     

A comparative approach to protein-and ligand-dependence of the Root effect for fish haemoglobins

Ligand-binding equilibria, kinetics and (13)C n.m.r. spectra of bound (13)CO, of the haemoglobins from two fishes that are very distant on the evolutionary scale, i.e. the fourth haemoglobin component from Salmo irideus and the single component from Osteoglossum bicirrhosum, were studied. The C-terminal sequence was also determined for the haemoglobin from Osteoglossum. The results show that (i) the C-terminal residues of both chains are not directly responsible for the characteristic heterotropic effect known as Root effect, since for both fish haemoglobins these residues are identical with those of human haemoglobins. (ii) In all haemoglobins characterized by the Root effect a dependence of the (13)CO n.m.r. resonances on pH is observed. However, the extent of the shift(s) depends on the particular protein, and is probably the result of a combination of both tertiary and quaternary conformational changes. (iii) Both haemoglobins from trout and Osteoglossum manifest a functional heterogeneity between the two types of chains in the tetramer, which increases with proton activity. For CO, the effect is very small for trout haemoglobin IV, and very marked for Osteoglossum haemoglobin; for O(2) strongly heterogeneous binding curves were obtained at approx. pH6.2 with both haemoglobins. (iv) Estimations of the relative values of the affinity constants for the alpha and beta chains in the tetramer were obtained for both haemoglobins from (13)CO n.m.r. spectra at low fractional saturation. On the basis of these findings the molecular mechanism underlying the Root effect is discussed.     

A comparative study of haemoglobins from the Artiodactyla by isoelectric focusing.

1. Isoelectric focusing on 7% acrylamide gels in 2% ampholyte of pH range 6-8 was used to fractionate the haemoglobins of 81 animals, representing 30 species from 4 families of the Order Artiodactyla. 2. Isoelectric points of the major haemoglobins were determined and the relative distribution of minor haemoglobins compared. 3. Marked haemoglobin heterogeneity was observed, with polymorphic and multiple haemoglobins. No one haemoglobin was common to all species.     

Low-temperature sensitivity and enhanced Bohr effect in red deer (Cervus elaphus) haemoglobin: a molecular adaptive strategy to life at high altitude and low temperature.

A study of the functional properties of haemoglobin from red deer (Cervus elaphus) whose habitat varies over a wide range of latitude, was performed. The oxygen-binding properties of the most common haemoglobin phenotype from the species living in Sardinia were examined with particular attention to the effect of pH, chloride, 2, 3-bisphosphoglycerate and temperature. Results indicate that red deer haemoglobin, like all haemoglobins from ruminants so far examined, is characterized by a low intrinsic oxygen affinity, with chloride being its main physiological modulator in vivo. The functional results and the low temperature sensitivity of the oxygen affinity are discussed in the light of the amino acid sequence of closely related ruminant haemoglobins.     

Biochemische und cytogenetische Untersuchungen zur Natur des Hämoglobin-Polymorphismus bei Chironomus tentans und Chironomus pallidivittatus

The haemoglobin of chironomids is dissolved in the body fluid of the larvae and can be separated electrophoretically in Chironomus tentans into 10, and in C. pallidivittatus into 8, different bands. The molecular weight determined under the electrophoretic conditions was 15,000±1,000 for each haemoglobin band. This means that each haemoglobin band represented a single protein chain. In each species 7 haemoglobins could be characterised as species specific according to their different electrophoretic mobilities, developmental characteristics and the fact that one haemoglobin could be correlated genetically with a specific chromosome inversion. The inheritance of all these species specific haemoglobins was found to be co-dominant. With cytogenetic methods it was possible to define the loci of the species specific haemoglobin genes as being restricted to certain parts of chromosome 3. This finding suggests gene duplication as the most likely mecanism of the evolution of haemoglobins in Chironomus.     

Koelliker haemoglobins in developing chick embryo

1. Three Koelliker haemoglobins, HbKE, HbKA and HbKH, derived from a post-translational loss of alpha-Arg-141, were isolated from red cells of chicken embryos. HbKE is typical of embryos up to 7 days of incubation, HbKA and HbKH are found in mature embryos. 2. All the precursor haemoglobins contain alpha A chains. HbKA derives from adult haemoglobin A whose globin composition is alpha A2 beta 2, HbKH from embryonic haemoglobin H with a globin composition alpha A2 beta H2 and HbKE from embryonic haemoglobin E with globin composition alpha A2 epsilon 2. 3. No Koelliker derivatives of haemoglobins with alpha-like chains other than alpha A were observed.     

Biophysical characterization of Artemia salina (L.) extracellular haemoglobins.

Sedimentation coefficients (s0 20,w) of 11.57 +/- 0.10 S and 11.52 +/- 0.09 S were assigned for Artemia salina (L.) extracellular haemoglobins II and III respectively. These values are not significantly different. The molecular weights, M0w and M0z, of the native haemoglobins as determined by the high-speed sedimentation-equilibrium method were for haemoglobin II 239 400 +/- 7200 and 240 400 +/- 2600 respectively, and for haemoglobin III 216 300 +/- 6500 and 219 300 +/- 4500 respectively. The observed increase of Mapp. with concentration suggested that association was occurring over the concentration range investigated. Exposure of haemoglobin II to either 6 M-guanidinium chloride or to low pH (pH 4) resulted in dissociation to units of approximately half the size of the native protein, with molecular weights approx. 115 000. Electron-microscopic observations indicated a molecular structure composed of two stacked lobed discs. These results strongly support the dimeric model for Artemia haemoglobins proposed by Moens & Kondo [(1978) Eur. J. Biochem. 82, 65-72].     

Molecular modeling and small angle X-ray scattering studies of Hoplosternum littorale cathodic haemoglobin

Considerable interest is currently focused on fish haemoglobins in order to identify the structural basis for their diversity of functional behavior. Hoplosternum littorale is a catfish that presents bimodal gill (water)/gut (air)-breathing, which allows this species to survive in waters with low oxygen content. The hemolysate of this fish showed the presence of two main haemoglobins, cathodic and anodic. This work describes structural features analyzed here by integration of molecular modeling with small angle X-ray scattering. Here is described a molecular model for the cathodic haemoglobin in the unliganded and liganded states. The models were determined by molecular modeling based on the high-resolution crystal structure of fish haemoglobins. The structural models for both forms of H. littorale haemoglobin were compared to human haemoglobin.     

Two Transitions of Haemoglobin Expression in Xenopus: from Embryonic to Larval and from Larval to Adult

Abstract. Electrophoretic analyses of haemoglobin and globin phenotypes in families of Xenopus borealis and Xenopus l. laevis revealed two developmental haemoglobin transitions during ontogeny. The first transition occurs at the developmental stage when tadpoles begin to feed. It is characterized by the decline of embryonic-specific globins in favour of novel, tadpole-specific globins ( X. borealis ) correlated to changes in the haemoglobin pattern. We suppose that this switch results from the replacement of a primitive, ventral blood island-dependent erythrocyte population by tadpole erythrocytes from other erythropoietic sites. Several other globin chains and haemoglobins are present in both young tadpoles and throughout larval life. The second, well-known transition occurs during metamorphosis, where all tadpole haemoglobins are replaced by adult haemoglobins composed of entirely different globin chains.     

Two transitions of haemoglobin expression in Xenopus: from embryonic to larval and from larval to adult

Electrophoretic analyses of haemoglobin and globin phenotypes in families of Xenopus borealis and Xenopus l. laevis revealed two developmental haemoglobin transitions during ontogeny. The first transition occurs at the developmental stage when tadpoles begin to feed. It is characterized by the decline of embryonic-specific globins in favour of novel, tadpole-specific globins (X. borealis) correlated to changes in the haemoglobin pattern. We suppose that this switch results from the replacement of a primitive, ventral blood island-dependent erythrocyte population by tadpole erythrocytes from other erythropoietic sites. Several other globin chains and haemoglobins are present in both young tadpoles and throughout larval life. The second, well-known transition occurs during metamorphosis, where all tadpole haemoglobins are replaced by adult haemoglobins composed of entirely different globin chains.     

Two new haemoglobins: haemoglobin Perspolis (alpha 64 (E13) Asp leads to Tyr) and haemoglobin J-Kurosh (alpha 19 (AB) Ala leads to Asp).

Two new haemoglobins are described which were found during a regular survey on voluntary blood donors in Iran. They are haemoglobin Perspolis [alpha 64 (E13) Asp leads to Tyr] and haemoglobin J-Kurosh [alpha 19 (AB) Ala leads to Asp]. The amino acid substitution in these two variants was determined by fingerprinting and amino acid analysis of the tryptic peptides and thermolytic peptides derived from abnormal tryptic peptides. Neither haemoglobin was associated with clinical symptoms.     

Rat haemoglobin heterogeneity. Two structurally distinct alpha chains and functional behaviour of selected components.

Six haemoglobins were separated analytically from haemolysates of adult Wistar rats (Rattus norvegicus) by cellulose acetate electrophoresis and preparatively by DEAE-cellulose chromatography. The globin chains were separated from unfractionated haemolysates by CM-cellulose chromatography by using a non-linear formic acid-pyridine gradient followed by CM-cellulose chromatography in 8M-urea by using a gradient of increasing Na+ concentration in phosphate buffer, pH 6.7. Two alpha chains and three non-alpha chains were identified. Chains isolated from purified haemoglobins were correlated with chains isolated from unfractionated haemolysates by electrophoresis on urea-starch gels to make presumptive assignments of the subunit composition of the six haemoglobin tetramers. Partial amino acid sequences were determined for the major and minor alpha chains. The oxygen equilibria of two of the major haemoglobin components and of the unfractionated haemolysate were examined at pH 7.5 and 8.0. The two purified haemoglobins exhibited similar oxygen affinities; the haemolysate, however, had a lower oxygen affinity than either of the two purified haemoglobins. Both the haemolysate and the two haemoglobins showed an alkaline Bohr effect larger than that of human haemoglobin A.     

Oxygen binding properties of non-mammalian nerve globins

Oxygen-binding globins occur in the nervous systems of both invertebrates and vertebrates. While the function of invertebrate nerve haemoglobins as oxygen stores that extend neural excitability under hypoxia has been convincingly demonstrated, the physiological role of vertebrate neuroglobins is less well understood. Here we provide a detailed analysis of the oxygenation characteristics of nerve haemoglobins from an annelid (Aphrodite aculeata), a nemertean (Cerebratulus lacteus) and a bivalve (Spisula solidissima) and of neuroglobin from zebrafish (Danio rerio). The functional differences have been related to haem coordination: the haem is pentacoordinate (as in human haemoglobin and myoglobin) in A. aculeata and C. lacteus nerve haemoglobins and hexacoordinate in S. solidissima nerve haemoglobin and D. rerio neuroglobin. Whereas pentacoordinate nerve globins lacked Bohr effects at all temperatures investigated and exhibited large enthalpies of oxygenation, the hexacoordinate globins showed reverse Bohr effects (at least at low temperature) and approximately twofold lower oxygenation enthalpies. Only S. solidissima nerve haemoglobin showed apparent cooperativity in oxygen binding, suggesting deoxygenation-linked self-association of the monomeric proteins. These results demonstrate a remarkable diversity in oxygenation characteristics of vertebrate and invertebrate nerve haemoglobins that clearly reflect distinct physiological roles.     

Evolutionary trace analysis of plant haemoglobins: implications for site-directed mutagenesis

Haemoglobins are found ubiquitously in eukaryotes and many bacteria. In plants, haemoglobins were first identified in species, which can fix nitrogen via symbiosis with bacteria. Recent findings suggest that another class of haemoglobins termed as nonsymbiotic haemoglobins are present through out the plant kingdom and are expressed differentially during plant development. Limited data available suggests that non-symbiotic haemoglobins are involved in hypoxic stress and oversupply of nutrients. Due to lack of information on structurally conserved, functionally important residues in non-symbiotic haemoglobins, further studies to elucidate the molecular mechanisms underlying the biological role are hampered. To determine functionally important residues in non-symbiotic haemoglobins, I have analyzed a number of sequences from plant haemoglobin family, in the context of the known crystal structures of plant by evolutionary trace method. Results indicate that the, evolutionary trace method like conventional phylogentic analysis, could resolve phylogentic relationships between plant haemoglobin family. Evolutionary trace analysis has identified candidate functional (trace) residues that uniquely characterize the heme-binding pocket, dimer interface and possible novel functional surfaces. Such residues from specific three-dimensional clusters might be of functional importance in nonsymbiotic haemoglobins. These data, together with our improved knowledge of possible functional residues, can be used in future structure-function analysis experiments.     

The haemoglobins of the fish Sarotherodon mossambicus (Peters): functional significance and ontogenetic changes

The haemoglobin patterns of adult and young larval stages of the fish species Sarotherodon mossambicus were determined by polyacrylamide–gel electrophoresis. Oxygen affinity determinations of the adult and larval haemolysates and of each haemoglobin component of the adult indicates that the affinity changes with the pH, temperature and osmotic pressure. The possible evolutionary significance of the multiple haemoglobins is discussed. The electrophoretic pattern of adult S. leucostictus , a species closely related to S. mossambicus was also determined and used as a comparative study.     

Organ regulated expression of the Parasponia andersonii haemoglobin gene in transgenic tobacco plants

Summary Plant haemoglobin genes are known to occur in legume and non-legume families and in both nodulating (e.g. Parasponia andersonii) and non-nodulating species (e.g. Trema tomentosa). Their presence in non-nodulating plants raises the possibility that haemoglobins might serve a function in non-symbiotic tissues distinct from their role in the nitrogen-fixing root nodules induced by micro-organisms. We report here that a P. andersonii haemoglobin promoter can regulate expression of either the P. andersonii haemoglobin gene, or a hybrid construct with the bacterial chloramphenicol acetyltransferase gene (cat), in the nonsymbiotic plant, Nicotiana tabacum. Expression is predominantly in the roots, implying that haemoglobins might have a function in roots of non-nodulated plants. We have also observed a low level of haemoglobin protein in non-nodulated P. andersonii roots, but not leaves, supporting this assertion. The expression in transgenic plants will allow further characterization of the promoter sequences essential for the organ-specific expression of haemoglobins in nonsymbiotic tissues.     

Functionally distinct haemoglobins of the cryopelagic Antarctic teleost Pagothenia borchgrevinki

Pagothenia borchgrevinki , has a higher haemoglobin concentration than other Antarctic notothenioids and the high oxygen capacity may correlate with the relatively active mode of life of this fish. The fish has five haemoglobins (Hb C, Hb 0, Hb 1, Hb 2 and Hb 3) with Hb 1 accounting for 70–80% of the total, and Hb C being present in trace amounts. Hb 1 and Hb 2 are functionally similar in terms of Bohr and Root effects. Hb 3 has a weaker Bohr effect than Hb 1 and Hb 2, and the Root effect is similar to that of Hb 1. Hb 0 has a strong Bohr effect and the Root effect is enhanced to a larger extent by the physiological effectors chlorides and phosphates than that of the other components with the exception of Hb C. The heats of oxygenation are lower than those of temperate fish haemoglobins. Temperature variations may have a different effect on the functional properties of each haemoglobin, and chloride and phosphates may play an important role in the conformational change between the oxy and deoxy structures. The complete amino acid sequences of Hb 1 and Hb 0, as well as partial N-terminal or internal sequences of the other haemoglobins, have been established. The high multiplicity of functionally distinct haemoglobins indicates that P. borchgrevinki , has a specialized haemoglobin system.