Rice hemoglobins. Gene cloning, analysis, and O2-binding kinetics of a recombinant protein synthesized in Escherichia coli.

Although nonsymbiotic hemoglobins (Hbs) are found in different tissues of dicots and monocots, very little is known about hb genes in monocots and the function of Hbs in nonsymbiotic tissues. We report the cloning and analysis of two rice (Oryza sativa L.) hb genes, hb1 and hb2, that code for plant Hbs. Rice hb1 and hb2 genes contain four exons and three introns, as with all of the known plant hb genes. At least three copies of the hb gene were detected in rice DNA, and analysis of gene expression shows that hb1 and hb2 are expressed in leaves but only hb1 is expressed in roots. A cDNA for rice Hb1 was expressed in Escherichia coli, and the recombinant Hb (rHb1) shows an unusually high affinity for O2 because of a very low dissociation constant. The absorbance spectra of the ferric and deoxyferrous rHb1 indicate that, in contrast to symbiotic Hbs, a distal ligand is coordinated to the ligand-binding site. Mutation of the distal His demonstrates that this residue coordinates the heme Fe of ferric and deoxyferrous rHb1 and stabilizes O2 in oxy-rHb1. The biochemical properties of rice rHb1 suggest that this protein probably does not function to facilitate the diffusion of O2.     

Cloning of a full-length symbiotic hemoglobin cDNA and in situ localization of the corresponding mRNA in Casuarina glauca root nodule

We have characterized a full-length cDNA ( hb -Cg1F) that represents symbiotic mRNA hemoglobin ( hb ) from Casuarina glauca root nodules. In situ hybridization was used to examine the correlation between hb -Cg1F mRNA and the state of the Frankia infection process. The efficiency of in situ hybridization using DIG-labeled vs [³⁵S]-labeled probes was compared. The expression of hb -Cg1F gene is induced in young infected host cells prior to the detection of Frankia nif H mRNA. Since Frankia does not form vesicles in C. glauca nodules, it is proposed that Hb is necessary to reduce the O₂ concentration in the cytoplasm of the host cells before the nif genes are expressed.     

The role of Bicoid cooperative binding in the patterning of sharp borders in Drosophila melanogaster

In Drosophila embryonic development, the Bicoid (Bcd) protein establishes positional information of downstream developmental genes like hunchback (hb), which has a strong anterior expression and a sharp on-off boundary in the mid-embryo. The role of Bcd cooperative binding in the positioning of the Hb pattern has been previously demonstrated. However, there are discrepancies in the reported results about the role of this mechanism in the sharp Hb border. Here, we determined the Hill coefficient (n(H)) required for Bcd to generate the sharp border of Hb in wild-type (WT) embryos. We found that an n(H) of approximately 6.3 (s.d. 1.4) and 10.8 (s.d. 4.0) is required to account for Hb sharpness at early and late cycle 14A, respectively. Additional mechanisms are possibly required because the high n(H) is likely unachievable for Bcd binding to the hb promoter. To test this idea, we determined the n(H) required to pattern the Hb profile of 15 embryos expressing an hb(14F) allele that is defective in self-activation and found n(H) to be 3.0 (s.d. 1.0). This result indicates that in WT embryos, the hb self-activation is important for Hb sharpness. Corroborating our results, we also found a progressive increase in the required value of n(H) spanning from 4.0 to 9.2 by determining this coefficient from averaged profiles of eight temporal classes at cycle 14A (T1 to T8). Our results indicate that there is a transition in the mechanisms responsible for the sharp Hb border during cycle 14A: in early stages of this cycle, Bcd cooperative binding is primarily responsible for Hb sharpness; in late cycle 14A, hb self-activation becomes the dominant mechanism.     

Modeling the tertiary structure of a maize (Zea mays ssp. mays) non-symbiotic hemoglobin.

The tertiary structure of a maize (Zea mays ssp. mays) non-symbiotic hemoglobin (Hbm) was modeled using computer tools and the known tertiary structure of rice Hb1 as a template. This method was tested by predicting the tertiary structure of soybean leghemoglobin a (Lba) using rice Hb1 as a template. The tertiary structures of the predicted and native Lba were similar, indicating that our computer methods could reliably predict the tertiary structures of plant Hbs. We next predicted the tertiary structure of Hbm. Hbm appears to have a long pre-helix A and a large CD-loop. The positions of the distal and proximal His are identical in Hbm and rice Hb1, which suggests that heme-Fe is hexacoordinate in Hbm and that the kinetic properties of Hbm and rice Hb1 are expected to be very similar, i.e. that Hbm has a high O2-affinity. Thermostability analysis showed that Hbm CD-loop is unstable and may provide mobility to amino acids located at the heme pocket for both ligand binding and stabilization and heme-Fe coordination. Analysis of the C-terminal half of Hbm showed the existence of a pocket-like region (the N/C cavity) where interactions with organic molecules or proteins could be possible. Lys K94 protrudes into the N/C cavity, suggesting that K94 may sense the binding of molecules to the N/C cavity. Thus, it is likely that the instability of the CD-loop and the possibility of binding molecules to the N/C cavity are essential for positioning amino acids in the heme pocket and in regulating Hbm activity and function.     

水蕨血红蛋白基因的分子克隆和序列分析

植物血红蛋白(Hemoglobin)是一类由珠蛋白(Globin)和血红素(Ferroheme)组成的结合蛋白,在植物中广泛分布,迄今已在苔藓植物、裸子植物和被子植物中克隆到血红蛋白基因序列,但在蕨类植物中相关研究还未见报道。该研究采用热不对称交错PCR(TAIL-PCR)方法克隆了水蕨血红蛋白基因的全长序列。该基因的序列总长为949 bp,包含4个外显子和3个内含子,编码189个氨基酸。预测的蛋白质(命名为CtHb)的分子量为21.14 kDa,等电点(pI)为7.81。三维结构模拟表明CtHb具有植物血红蛋白典型的三级结构:即含有A、B、C、E、F、G和H螺旋,形成了3-on-3的"三明治"结构。和水稻血红蛋白的三级结构相比,CtHb的大部分结构(包括具有远端和近端组氨酸定位的E螺旋和F螺旋的位置等)同水稻的结构极为相似。两者的不同之处主要表现在:(1)CtHb含有较长的N-端区域;(2)两者CD-loop的折叠方式不同;(3)两者螺旋B和螺旋C的连接方式不同,CtHb是通过卷曲连接的,而水稻中借助的是螺旋。结构进化分析揭示了植物血红蛋白从非共生到共生进化过程中的一些关键改变,这些改变可能有助于非共生血红蛋白向共生血红蛋白结构的转变,特别是有助于豆血红蛋白共生功能的实现。     

Model for the robust establishment of precise proportions in the early Drosophila embryo

During embryonic development, a spatial pattern is formed in which proportions are established precisely. As an early pattern formation step in Drosophila embryos, an anterior-posterior gradient of Bicoid (Bcd) induces hunchback (hb) expression (Nature 337 (1989) 138; Nature 332 (1988) 281). In contrast to the Bcd gradient, the Hb profile includes information about the scale of the embryo. Furthermore, the resulting hb expression pattern shows a much lower embryo-to-embryo variability than the Bcd gradient (Nature 415 (2002) 798). An additional graded posterior repressing activity could theoretically account for the observed scaling. However, we show that such a model cannot produce the observed precision in the Hb boundary, such that a fundamentally different mechanism must be at work. We describe and simulate a model that can account for the observed precise generation of the scaled Hb profile in a highly robust manner. The proposed mechanism includes Staufen (Stau), an RNA binding protein that appears essential to precision scaling (Nature 415 (2002) 798). In the model, Stau is released from both ends of the embryo and relocalizes hb RNA by increasing its mobility. This leads to an effective transport of hb away from the respective Stau sources. The balance between these opposing effects then gives rise to scaling and precision. Considering the biological importance of robust precision scaling and the simplicity of the model, the same principle may be employed more often during development.     

Adaptive haemoglobin gene control in Daphnia pulex at different oxygen and temperature conditions

Hypoxia-induced haemoglobin (Hb) expression is a central regulatory mechanism in Daphnia in response to environmental hypoxia or warm temperatures. Changes in Hb concentration as well as Hb subunit composition, which modulate Hb oxygen affinity, guarantee the oxygen supply of tissues under these environmental conditions. Based on the sequenced D. pulex genome, Hb genes were related to the properties of haemolymph Hb, which included its concentration and oxygen affinity (both measured by spectrophotometry) as well as the Hb subunit composition (determined by 2-D gel electrophoresis and ESI-MS analysis). Permanent cultures of D. pulex acclimated to different oxygen conditions (normoxia and hypoxia) and temperatures (10°C, 20°C, and 24°C), showed characteristic changes in Hb concentration, subunit composition and oxygen affinity. Several subunits (Hb4, Hb7, Hb8, and Hb10) were obviously responsible for changes in oxygen affinity including those, which carry a number of hypoxia-responsive elements (HREs) upstream of the respective gene (hb4 and hb10). Analysing the effects of different oxygen- or temperature-acclimations on Hb subunit expression in D. pulex and D. magna on a common basis (Hb concentration or oxygen affinity) revealed a general pattern of oxygen and temperature effects on Hb, which implies that Hb quantity and quality are mostly influenced by the degree of tissue hypoxia. Differences between both species in the onset of hypoxia-induced differential Hb expression and Hb oxygen affinity, which are probably related to different HRE patterns and functionally important differences in the amino acid sequence of only a few subunits, cause a reduced ability of D. pulex to adjust Hb function to temperature changes in comparison to D. magna.     

A Role of Polycomb Group Genes in the Regulation of Gap Gene Expression in Drosophila

Anteroposterior polarity of the Drosophila embryo is initiated by the localized activities of the maternal genes, bicoid and nanos, which establish a gradient of the hunchback (hb) morphogen. nanos determines the distribution of the maternal Hb protein by regulating its translation. To identify further components of this pathway we isolated suppressors of nanos. In the absence of nanos high levels of Hb protein repress the abdomen-specific genes knirps and giant. In suppressor-of-nanos mutants, knirps and giant are expressed in spite of high Hb levels. The suppressors are alleles of Enhancer of zeste (E(z)) a member of the Polycomb group (Pc-G) of genes. We show that E(z), and likely other Pc-G genes, are required for maintaining the expression domains of knirps and giant initiated by the maternal Hb protein gradient. We have identified a small region of the knirps promoter that mediates the regulation by E(z) and hb. Because Pc-G genes are thought to control gene expression by regulating chromatin, we propose that imprinting at the chromatin level underlies the determination of anteroposterior polarity in the early embryo.     

Molecular cloning and characterization of a moss (Ceratodon purpureus) nonsymbiotic hemoglobin provides insight into the early evolution of plant nonsymbiotic hemoglobins

Nonsymbiotic hemoglobins (nsHbs) are widespread in plants including bryophytes. Bryophytes (such as mosses) are among the oldest land plants, thus an analysis of a bryophyte nsHb is of interest from an evolutionary perspective. However, very little is known about bryophyte nsHbs. Here, we report the cloning and characterization of an nshb gene (cerhb) from the moss Ceratodon purpureus. Sequence analysis showed that cerhb is interrupted by 3 introns in identical position as all known plant nshb genes, which suggests that the ancestral nshb gene was interrupted by 3 introns. Expression analysis showed that cerhb expresses in protonemas and gametophytes growing in normal conditions and that it overexpresses in protonemas subjected to osmotic (sucrose), heat-shock, cold-, and nitrate-stress conditions. Also, modeling of the Ceratodon nsHb (CerHb) tertiary structure suggests that CerHb is hexacoordinate and that it binds O(2) with high affinity. Comparative analysis of the predicted CerHb with native rice Hb1 and soybean leghemoglobin a structures revealed that the major evolutionary changes that probably occurred during the evolution of plant Hbs were 1) a hexacoordinate to pentacoordinate transition at the heme prosthetic group, 2) a length decrease at the CD-loop and N- and C-termini regions, and 3) the compaction of the protein into a globular structure.     

T-quaternary structure of oxy human adult hemoglobin in the presence of two allosteric effectors, L35 and IHP

The cooperative O(2)-binding of hemoglobin (Hb) have been assumed to correlate to change in the quaternary structures of Hb: T(deoxy)- and R(oxy)-quaternary structures, having low and high O(2)-affinities, respectively. Heterotropic allosteric effectors have been shown to interact not only with deoxy- but also oxy-Hbs causing significant reduction in their O(2)-affinities and the modulation of cooperativity. In the presence of two potent effectors, L35 and inositol hexaphosphate (IHP) at pH 6.6, Hb exhibits extremely low O(2)-affinities (K(T)=0.0085mmHg(-1) and K(R)=0.011mmHg(-1)) and thus a very low cooperativity (K(R)/K(T)=1.3 and L(0)=2.4). (1)H-NMR spectra of human adult Hb with these two effectors were examined in order to determine the quaternary state of Hb in solution and to clarify the correlation between the O(2)-affinities and the structural change of Hb caused by the heterotropic effectors. At pH 6.9, (1)H-NMR spectrum of deoxy-Hb in the presence of L35 and IHP showed a marker of the T-quaternary structure (the T-marker) at 14ppm, originated from inter- dimeric α(1)β(2)- (or α(2)β(1)-) hydrogen-bonds, and hyperfine-shifted (hfs) signals around 15-25ppm, caused by high-spin heme-Fe(II)s. Upon addition of O(2), the hfs signals disappeared, reflecting that the heme-Fe(II)s are ligated with O(2), but the T-marker signals still remained, although slightly shifted and broadened, under the partial pressure of O(2) (P(O2)) of 760mmHg. These NMR results accompanying with visible absorption spectroscopy and visible resonance Raman spectroscopy reveal that oxy-Hb in the presence of L35 and IHP below pH 7 takes the ligated T-quaternary structure under the P(O2) of 760mmHg. The L35-concentration dependence of the T-marker in the presence of IHP indicates that there are more than one kind of L35-binding sites in the ligated T-quaternary structure. The stronger binding sites are probably intra-dimeric binding sites between α(1)G- and β(1)G-helices, and the other weaker binding site causes the R→T transition without release of O(2). The fluctuation of the tertiary structure of Hb seems to be caused by both the structural perturbation of α(1)β(1) (or α(2)β(2)) intra-dimeric interface, where the stronger L35-binding sites exist, and by the IHP-binding to the α(1)α(2)- (or β(1)β(2)-) cavity. The tertiary structural fluctuation induced by the allosteric effectors may contribute to the significant reduction of the O(2)-affinity of oxy-Hb, which little depends on the quaternary structures. Therefore, the widely held assumptions of the structure-function correlation of Hb - [the deoxy-state]=[the T-quaternary structure]=[the low O(2)-affinity state] and [the oxy-state]=[the R-quaternary structure]=[the high O(2)-affinity state] and the O(2)-affiny of Hb being regulated by the T/R-quaternary structural transition - are no longer sustainable. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

Increased hemoglobin O2 affinity protects during acute hypoxia

Acclimatization to hypoxia requires time to complete the adaptation mechanisms that influence oxygen (O(2)) transport and O(2) utilization. Although decreasing hemoglobin (Hb) O(2) affinity would favor the release of O(2) to the tissues, increasing Hb O(2) affinity would augment arterial O(2) saturation during hypoxia. This study was designed to test the hypothesis that pharmacologically increasing the Hb O(2) affinity will augment O(2) transport during severe hypoxia (10 and 5% inspired O(2)) compared with normal Hb O(2) affinity. RBC Hb O(2) affinity was increased by infusion of 20 mg/kg of 5-hydroxymethyl-2-furfural (5HMF). Control animals received only the vehicle. The effects of increasing Hb O(2) affinity were studied in the hamster window chamber model, in terms of systemic and microvascular hemodynamics and partial pressures of O(2) (Po(2)). Pimonidazole binding to hypoxic areas of mice heart and brain was also studied. 5HMF decreased the Po(2) at which the Hb is 50% saturated with O(2) by 12.6 mmHg. During 10 and 5% O(2) hypoxia, 5HMF increased arterial blood O(2) saturation by 35 and 48% from the vehicle group, respectively. During 5% O(2) hypoxia, blood pressure and heart rate were 58 and 30% higher for 5HMF compared with the vehicle. In addition, 5HMF preserved microvascular blood flow, whereas blood flow decreased to 40% of baseline in the vehicle group. Consequently, perivascular Po(2) was three times higher in the 5HMF group compared with the control group at 5% O(2) hypoxia. 5HMF also reduced heart and brain hypoxic areas in mice. Therefore, increased Hb O(2) affinity resulted in hemodynamics and oxygenation benefits during severe hypoxia. This acute acclimatization process may have implications in survival during severe environmental hypoxia when logistic constraints prevent chronic acclimatization.     

O2 release from Hb vesicles evaluated using an artificial,narrow O2-permeable tube: comparison with RBCs and acellular Hbs

A phospholipid vesicle that encapsulates a concentrated hemoglobin (Hb) solution and pyridoxal 5'-phosphate as an allosteric effector [Hb vesicle (HbV) diameter, 250 nm] has been developed to provide an O2 carrying ability to plasma expanders. The O2 release from flowing HbVs was examined using an O2-permeable, fluorinated ethylenepropylene copolymer tube (inner diameter, 28 microm) exposed to a deoxygenated environment. Measurement of O2 release was performed using an apparatus that consisted of an inverted microscope and a scanning-grating spectrophotometer with a photon-count detector, and the rate of O2 release was determined based on the visible absorption spectrum in the Q band of Hb. HbVs and fresh human red blood cells (RBCs) were mixed in various volume ratios at a Hb concentration of 10 g/dl in isotonic saline that contained 5 g/dl albumin, and the suspension was perfused at the centerline flow velocity of 1 mm/s through the narrow tube. The mixtures of acellular Hb solution and RBCs were also tested. Because HbVs were homogeneously dispersed in the albumin solution, increasing the volume of the HbV suspension resulted in a thicker marginal RBC-free layer. Irrespective of the mixing ratio, the rate of O2 release from the HbV/RBC mixtures was similar to that of RBCs alone. On the other hand, the addition of 50 vol% of acellular Hb solution to RBCs significantly enhanced the rate of deoxygenation. This outstanding difference in the rate of O2 release between the HbV suspension and the acellular Hb solution should mainly be due to the difference in the particle size (250 vs. 7 nm) that affects their diffusion for the facilitated O2 transport.