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.     

Nonsymbiotic haemoglobins in plants.

General aspects regarding the presence of nonsymbiotic haemoglobin in plants are presented with the emphasis on those related to its function. As it becomes apparent that the nonsymbiotic haemoglobins are widespread across the plant kingdom and that they represent a more primitive and evolutionary older form of the plant globin genes, the question of their function becomes more attractive. While the physiological functions of the symbiotic haemoglobins in plants are well understood, almost nothing is known about their nonsymbiotic predecessors. Therefore, the known and hypothetical functions of haemoglobins in various systems are described along with information concerning properties and the regulation of expression of the nonsymbiotic haemoglobins. Interestingly, a number of nonsymbiotic haemoglobins have been shown to be hypoxia-inducible. The spatial and temporal pattern of this induction in barley may suggest that it is an integral part of the plants response to limiting oxygen stress.     

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.     

Molecular evolution of plant haemoglobin: two haemoglobin genes in Nymphaeaceae Euryale ferox

We isolated and sequenced two haemoglobin genes from the early-branching angiosperm Euryale ferox (Nymphaeaceae). The two genes belong to the two known classes of plant haemoglobin. Their existence in Nymphaeaceae supports the theory that class 1 haemoglobin was ancestrally present in all angiosperms, and is evidence for class 2 haemoglobin being widely distributed. These sequences allowed us to unambiguously root the angiosperm haemoglobin phylogeny, and to corroborate the hypothesis that the class 1/class 2 duplication event occurred before the divergence between monocots and eudicots. We addressed the molecular evolution of plant haemoglobin by comparing the synonymous and nonsynonymous substitution rates in various groups of genes. Class 2 haemoglobin genes of legumes (functionally involved in a symbiosis with nitrogen-fixing bacteria) show a higher nonsynonymous substitution rate than class 1 (nonsymbiotic) haemoglobin genes. This suggests that a change in the selective forces applying to plant haemoglobins has occurred during the evolutionary history of this gene family, potentially in relation with the evolution of symbiosis.     

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.     

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.     

Characterization of haemoglobin from Actinorhizal plants – An in silico approach

Plant haemoglobins (Hbs), found in both symbiotic and non-symbiotic plants, are heme proteins and members of the globin superfamily. Hb genes of actinorhizal Fagales mostly belong to the non-symbiotic type of haemoglobin; however, along with the non-symbiotic Hb, Casuarina sp. posses a symbiotic one (symCgHb), which is expressed specifically in infected cells of nodules. A thorough sequence analysis of 26 plant Hb proteins, currently available in public domain, revealed a consensus motif of 29 amino acids. This motif is present in all the members of symbiotic class II Hbs including symCgHb and non-symbiotic Class II Hbs, but is totally absent in Class I symbiotic and non-symbiotic Hbs. Further, we constructed 3D structures of Hb proteins from Alnus and Casuarina through homology modelling and peeped into their structural properties. Structure-based studies revealed that the Casuarina symbiotic haemoglobin protein shows distinct stereochemical properties from that of the other Casuarina and Alnus Hb proteins. It also showed considerable structural similarities with leghemoglobin structure from yellow lupin (pdb id 1GDI). Therefore, sequence and structure analyses point to the fact that symCgHb protein shows significant resemblance to symbiotic haemoglobin found in legumes and may thus eventually play a similar role in shielding the nitrogenase from oxygen as seen in the case of leghemoglobin.     

Hemoglobin,from microorganisms to man: a single structural motif,multiple functions

Haemoglobins from unicellular organisms, plants or animals, share a common structure, which results from the folding, around the heme group, of a polypeptide chain made from 6-8 helices. Nowadays, deciphering the genome of several species allows one to draw the evolutionary tree of this protein going back to 1800 millions of years, at a time when oxygen began to accumulate in the atmosphere. This permits to follow the evolution of the ancestral gene and of its product. It is likely that, only in complex multicellular species, transport and storage of oxygen became the main physiological function of this molecule. In addition, in unicellular organisms and small invertebrates, it is likely that the main function of this protein was to protect the organism from the toxic effect of O2, CO and NO*. The very high oxygen affinity of these molecules, leading them to act rather as a scavenger as an oxygen carrier, supports this hypothesis. Haemoglobins from microorganisms, which may probably be the closest vestiges to the ancestral molecules, are divided into three families. The first one is made from flavohaemoglobins, a group of chimerical proteins carrying a globin domain and an oxido-reduction FAD-dependant domain. The second corresponds to truncated haemoglobins, which are hexacoordinated with very high oxygen-affinity molecules, 20-40 residues shorter than classical haemoglobins. The third group is made from bacterial haemoglobins such as that of Vitreoscilla. Some specific structural arrangements in the region surrounding the heme are cause of their high oxygen affinity. In plants, two types of haemoglobins are present (non-symbiotic and symbiotic), that arose from duplication of an ancestral vegetal gene. Non-symbiotic haemoglobins, which are probably the oldest, are scarcely distributed within tissues having high energetic consumption. Conversely, symbiotic haemoglobins (also named leghaemoglobins) are present at a high concentration (mM) mostly in the rhizomes of legumes, where they are involved in nitrogen metabolism. In some species, haemoglobin was proposed to be an oxygen sensor bringing to the organism information to adjust metabolism or biosynthesis to the oxygen requirement. Elsewhere haemoglobin may act as final electron acceptors in oxido-reduction pathways. Evolution of haemoglobin in invertebrates followed a large variety of scenarios. Some surprising functions as sulphide acquisition in invertebrates living near hydrothermal vents, or a role in the phototrophism of worm need to be mentioned. In invertebrates, the size of haemoglobin varies from monomers to giant molecules associating up to 144 subunits, while in vertebrates it is always a tetramer. In some species, several haemoglobins, with completely different structure and function, may coexist. This demonstrates how hazardous may be to extrapolate the function of a protein from only structural data.     

Molecular evolution of the equilibrative nucleoside transporter family: identification of novel family members in prokaryotes and eukaryotes

Equilibrative nucleoside transporters (ENTs) are integral membrane proteins which enable the movement of hydrophilic nucleosides and nucleoside analogs down their concentration gradients across cell membranes. ENTs were only recently characterized at the molecular level, and little is known about the tertiary structure or distribution of these proteins in nonmammalian organisms. To identify conserved regions, residues, and motifs of ENTs that may indicate functionally important parts of the protein and to better understand the evolutionary history of this protein family, we conducted an exhaustive analysis to characterize and compare ENTs in taxonomically diverse organisms. We have identified novel ENT family members in humans, mice, fish, tunicates, slime molds, and bacteria. This greatly extends our knowledge on the distribution of the ENTs in eukaryotes, and we have identified, for the first time, family members in bacteria. The prokaryote ENTs are attractive models for future studies on transporter tertiary structure and mechanism of substrate translocation. Using sequence similarities, we have identified regions, residues, and motifs that are conserved across all family members. These areas are presumably correlated with function and therefore are important targets for future analysis. Finally, we propose an evolutionary history for the ENT family which clarifies the origin(s) of multiple isoforms in different taxa.     

Evolutionary trace analysis of CYP51 family: implication for site-directed mutagenesis and novel antifungal drug design

Lanosterol 14α-demethylase (CYP51) is an essential enzyme in the fungal life cycle and also an important target for the antifungal drug development. Based on the multiple sequence alignments of CYP51 family, an evolutionary tree of the CYP51 family was constructed by the evolutionary trace (ET) method. The identified trace residues could provide a reliable and rational guide to the design of CYP51 mutations and give more information about the detailed mechanism of substrate (drug) recognition and binding. The reliability of ET analysis to identify residues of functional importance was validated by the reported site-directed mutagenesis studies of CYP51s. Several residues in the active site were also validated by our mutagenesis studies. Mapping the identified trace residues onto the active site of the modeled structure of Candida albicans CYP51 (CACYP51) may provide useful information for the design of novel antifungal agents.     

Integration of Evolutionary Features for the Identification of Functionally Important Residues in Major Facilitator Superfamily Transporters

The identification of functionally important residues is an important challenge for understanding the molecular mechanisms of proteins. Membrane protein transporters operate two-state allosteric conformational changes using functionally important cooperative residues that mediate long-range communication from the substrate binding site to the translocation pathway. In this study, we identified functionally important cooperative residues of membrane protein transporters by integrating sequence conservation and co-evolutionary information. A newly derived evolutionary feature, the co-evolutionary coupling number, was introduced to measure the connectivity of co-evolving residue pairs and was integrated with the sequence conservation score. We tested this method on three Major Facilitator Superfamily (MFS) transporters, LacY, GlpT, and EmrD. MFS transporters are an important family of membrane protein transporters, which utilize diverse substrates, catalyze different modes of transport using unique combinations of functional residues, and have enough characterized functional residues to validate the performance of our method. We found that the conserved cores of evolutionarily coupled residues are involved in specific substrate recognition and translocation of MFS transporters. Furthermore, a subset of the residues forms an interaction network connecting functional sites in the protein structure. We also confirmed that our method is effective on other membrane protein transporters. Our results provide insight into the location of functional residues important for the molecular mechanisms of membrane protein transporters.     

Symbiotic and nonsymbiotic hemoglobin genes of Casuarina glauca.

Casuarina glauca has a gene encoding hemoglobin (cashb-nonsym). This gene is expressed in a number of plant tissues. Casuarina also has a second family of hemoglobin genes (cashb-sym) expressed at a high level in the nodules that Casuarina forms in a nitrogen-fixing symbiosis with the actinomycete Frankia. Both the nonsymbiotic and symbiotic genes retained their specific patterns of expression when introduced into the legume Lotus corniculatus. We interpret this finding to mean that the controls of expression of the symbiotic gene in Casuarina must be similar to the controls of expression of the leghemoglobin genes that operate in nodules formed during the interaction between rhizobia and legumes. Deletion analyses of the promoters of the Casuarina symbiotic genes delineated a region that contains nodulin motifs identified in legumes; this region is critical for the controlled expression of the Casuarina gene. The finding that the nonsymbiotic Casuarina gene is also correctly expressed in L. corniculatus suggests to us that a comparable non-symbiotic hemoglobin gene will be found in legume species.     

Evolutionary trace residues in noroviruses: importance in receptor binding, antigenicity, virion assembly, and strain diversity

Noroviruses cause major epidemic gastroenteritis in humans. A large number of strains of these single-stranded RNA viruses have been reported. Due to the absence of infectious clones of noroviruses and the high sequence variability in their capsids, it has not been possible to identify functionally important residues in these capsids. Consequently, norovirus strain diversity is not understood on the basis of capsid functions, and the development of therapeutic compounds has been hampered. To determine functionally important residues in noroviruses, we have analyzed a number of norovirus capsid sequences in the context of the Norwalk virus capsid crystal structure by using the evolutionary trace method. This analysis has identified capsid protein residues that uniquely characterize different norovirus strains and provide new insights into capsid assembly and disassembly pathways and the strain diversity of these viruses. Such residues form specific three-dimensional clusters that may be of functional importance in noroviruses. One of these clusters includes residues known to participate in the proteolytic cleavage of these viruses at high pH. Other clusters are formed in capsid regions known to be important in the binding of antibodies to noroviruses, thereby indicating residues that may be important in the antigenicity of these viruses. The highly variable region of the capsid shows a distinct cluster whose residues may participate in norovirus-receptor interactions.     

Different functional modulation by heterotropic ligands (2,3-diphosphoglycerate and chlorides) of the two haemoglobins from fallow-deer (Dama dama).

Two haemoglobin components have been identified and purified from fallow-deer (Dama dama) erythrocytes. They are present in similar amounts and the two tetrameric molecules share the same alpha chain, while two different beta chains are detected in the two components. The beta chains differ by 14 residues, even though they both have 145 amino-acid residues, which account for a molecular mass of 16,023 and 16,064 Da, respectively, while alpha chain has 141 residues, yielding a molecular mass of 15,142 Da. Compared with human Hb, the N-terminal region of both beta chains shows deletion of Val beta 1 and the replacement of His beta 2 by a methionyl residue, a modification which is common to most ruminant haemoglobins. Although both isolated components show a low intrinsic affinity for oxygen, meaningful differences between the two haemoglobins have been found with respect to the effect of heterotropic effectors, such as 2,3-diphosphoglycerate and chloride ions. In view of the high sequence homology between the two components, the different effect of heterotropic ligands has been tentatively correlated to possible localized structural variations between beta chains of the two haemoglobin components.     

Molecular adaptations in haemoglobins of notothenioid fishes

Since haemoglobins of all animal species have the same haem group, differences in their properties, including oxygen affinity, electrophoretic mobility and pH sensitivity, must result from the interaction of the prosthetic group with specific amino‐acid residues in the primary structure. For this reason, fish globins have been the subject of extensive studies in recent years, not only for their structural characteristics, but also because they offer the possibility to investigate the evolutionary history of these ancient molecules in marine and freshwater species living in a great variety of environmental conditions. This review summarizes the current knowledge on the structure, function and phylogeny of haemoglobins of notothenioid fishes. On the basis of crystallographic analysis, the evolution of the Root effect is analysed. Adaptation of the oxygen transport system in notothenioids seems to be based on evolutionary changes, involving levels of biological organization higher than the structure of haemoglobin. These include changes in the rate of haemoglobin synthesis or in regulation by allosteric effectors, which affect the amount of oxygen transported in blood. These factors are thought to be more important for short‐term response to environmental challenges than previously believed.     

Multiple globins and haemoglobins in the bass, Dicentrarchus labrax (L.) (Serranidae: Teleostei)

The haemoglobins and globins of bass, Dicentrarchus labrax (L.) have been studied by starch and polyacrylamide gel electrophoresis. Five haemoglobin components were found. These haemoglobins appear to result from the combination of four different globin monomers. The molecular weight of the pooled haemoglobin is about 54 400, confirming its tetrameric form. The evolutionary significance of multiple haemoglobins is discussed.     

真核翻译延伸因子1A蛋白家族功能位点的进化踪迹分析

真核翻译延伸因子1A（eEF1A）是真核生物蛋白质翻译过程中能将氨酰tRNA运送到核糖体A位点参与多肽延伸反应的多功能蛋白质. 本文主要利用多种生物信息学分析工具进行地中海涡虫翻译延伸因子1A（SmEF1A）蛋白序列的查找与eEF1A直系同源蛋白的搜索, 并基于90条直系同源蛋白进行eEF1A蛋白家族的进化踪迹分析和SmEF1A蛋白功能位点的比较研究. 结果表明,在eEF1A蛋白家族中共识别到338个踪迹残基位点和20个踪迹残基富集区域,SmEF1A蛋白的功能位点与踪迹残基位点密切相关,与GTP/Mg2+结合相关的S21、T72、D91、G94等重要位点均为全家族保守的踪迹残基,N 糖基化、磷酸化等蛋白修饰位点中踪迹残基位点往往是被修饰的部位或修饰功能发挥的关键辅助位点,而位于分子表面的配基结合口袋则与20个踪迹残基富集区域在分子表面形成的踪迹残基簇关系密切. eEF1A蛋白家族的进化踪迹分析为eEF1A蛋白重要功能区域关键残基的确定和未知功能位点的预测提供了重要信息.     

Evolutionary trace analysis of scorpion toxins specific for K-channels

Scorpion alpha-K(+) channel toxins are a large family of polypeptides with a similar structure but diverse pharmacological activities. Despite many structural and functional data available at present, little progress has been made in understanding the toxin's molecular basis responsible for the functional diversification. In this paper, we report the first complete cDNA sequences of toxins belonging to subfamily 6 and identify five new members, called alpha-KTx 6.6-6.10. By analyzing the rates of mutations that occurred in the corresponding cDNAs, we suggest that accelerated evolution in toxin-coding regions may be associated with the functional diversification of this subfamily. To pinpoint sites probably involved in the functional diversity of alpha-KTx family, we analyzed this family of sequences using the evolutionary trace method. This analysis highlighted one channel-binding surface common for all the members. This surface is composed of one conserved lysine residue at position 29 assisted by other residues at positions 10, 26, 27, 32, 34, and 36. Of them, the positions 29, 32, and 34 have been reported to be the most major determinants of channel specificity. Interestingly, another contrary surface was also observed at a higher evolutionary time cut-off value, which may be involved in the binding of ERG (ether-a-go-go-related gene) channel-specific toxins. The good match between the trace residues and the functional epitopes of the toxins suggested that the evolutionary trace results reported here can be applied to predict channel-binding sites of the toxins. Because, the side-chain variation in the trace positions is strongly linked with the functional alteration and channel-binding surface transfer of alpha-KTx family, we conclude that our findings should also be important for the rational design of new toxins targeting a given potassium channel with high selectivity.