The evolution of extracellular hemoglobins of annelids, vestimentiferans, and pogonophorans

The evolution of extracellular hemoglobins of annelids, vestimentiferans, and pogonophorans was investigated by applying cladistic and distance-based approaches to reconstruct the phylogenetic relationships of this group of respiratory pigments. We performed this study using the aligned sequences of globin and linker chains that are the constituents of these complex molecules. Three novel globin and two novel linker chains of Sabella spallanzanii described in an accompanying paper (Pallavicini, A., Negrisolo, E., Barbato, R., Dewilde, S., Ghiretti-Magaldi, A., Moens, L., and Lanfranchi, G. (2001) J. Biol. Chem. 276, 26384--26390) were also included. Our results allowed us to test previous hypotheses on the evolutionary pathways of these proteins and to formulate a new most parsimonious model of molecular evolution. According to this novel model, the genes coding for the polypeptides forming these composite molecules were already present in the common ancestor of annelids, vestimentiferans, and pogonophorans.     

Linker chains of the gigantic hemoglobin of the earthworm Lumbricus terrestris: primary structures of linkers L2, L3, and L4 and analysis of the connectivity of the disulfide bonds in linker L1

The extracellular hemoglobin (Hb) of the earthworm, Lumbricus terrestris, has four major kinds of globin chains: a, b, c, and d, present in equimolar proportions, and additional non-heme, non-globin scaffolding chains called linkers that are required for the calcium-dependent assembly of the full-sized molecule. The amino acid sequences of all four of the globin chains and one of the linkers (L1) have previously been determined. The amino acid sequences via cDNA of each of the three remaining linkers, L2, L3, and L4, have been determined so that the sequences of all constituent polypeptides of the hemoglobin are now known. Each linker has a highly conserved cysteine-rich segment of approximately 40 residues that is homologous with the seven ligand-binding repeats of the human low-density lipoprotein receptor (LDLR). Analysis of linker L1 shows that the connectivity of the three disulfide bonds is exactly the same as in the LDLR ligand-binding repeats. The presence of a calcium-binding site comprising one glutamyl and three aspartyl residues in both the LDLR repeats and in the linkers supports the suggestion that calcium is required for the folding and disulfide connectivity of the linkers as in the LDLR repeats. Linker L2 is markedly heterogeneous and contains unusual glycine-rich sequences near the NH2-terminus and a polar zipper-like sequence with imperfect repeats of Asp-Asp-His at the carboxyl terminus. Similar Asp-Asp-His repeats have been found in a protein homologous to superoxide dismutase in the hemolymph of certain mussels. These repeats may function as metal-binding sites.     

Direct microinjection of rabbit globin mRNA into mouse 3T3 cells. Analysis of the polypeptides synthesized in vivo

3T3 cells grown attached to 9 mm² coverslips have been microinjected in the cytoplasm with total rabbit globin mRNA and the polypeptides synthesized after injection have been labelled with [³⁵S]-methionine under conditions in which the product of as few as 100 cells could be analysed by high resolution two-dimensional gel electrophoresis followed by 10 days' fluorography. Microinjection of rabbit globin mRNA results in the synthesis of a basic polypeptide of mol. wt 15 K that is not present in control cells, and that co-migrates with purified [³H]leucine-labelled globin as determined by high resolution two-dimensional gel electrophoresis (NEPHGE). Visual inspection of the fluorograms revealed that the injection of globin mRNA (up to 14000 molecules/cell) does not alter significantly the relative intensity of the major acidic (IEF) and basic (NEPHGE) polypeptides synthesized by the cells.     

Gene Structure and Molecular Phylogeny of the Linker Chains from the Giant Annelid Hexagonal Bilayer Hemoglobins

Giant extracellular hexagonal bilayer hemoglobin (HBL-Hb), found only in annelids, is an ∼3500-kDa heteropolymeric structure involved in oxygen transport. The HBL-Hbs are comprised of globin and linker chains, the latter being required for the assembly of the quaternary structure. The linker chains, varying in size from 225 to 283 amino acids, have a conserved cysteine-rich domain within their N-terminal moiety that is homologous to the cysteine-rich modules constituting the ligand binding domain of the low-density lipoprotein receptor (LDLR) protein family found in many metazoans. We have investigated the gene structure of linkers from Arenicola marina, Alvinella pompejana, Nereis diversicolor, Lumbricus terrestris, and Riftia pachyptila. We found, contrary to the results obtained earlier with linker genes from N. diversicolor and L. terrestris, that in all of the foregoing cases, the linker LDL-A module is flanked by two phase 1 introns, as in the human LDLR gene, with two more introns in the 3′ side whose positions varied with the species. In addition, we obtained 13 linker cDNAs that have been determined experimentally or found in the EST database LumbriBASE. A molecular phylogenetic analysis of the linker primary sequences demonstrated that they cluster into two distinct families of linker proteins. We propose that the common gene ancestor to annelid linker genes exhibited a four-intron and five-exon structure and gave rise to the two families subsequent to a duplication event.     

Molecular cloning and characterization of the human beta-like globin gene cluster

The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.     

Characterization, structure and function of linker polypeptides in phycobilisomes of cyanobacteria and red algae: an overview

Cyanobacteria and red algae have intricate light-harvesting systems comprised of phycobilisomes that are attached to the outer side of the thylakoid membrane. The phycobilisomes absorb light in the wavelength range of 500-650 nm and transfer energy to the chlorophyll for photosynthesis. Phycobilisomes, which biochemically consist of phycobiliproteins and linker polypeptides, are particularly wonderful subjects for the detailed analysis of structure and function due to their spectral properties and their various components affected by growth conditions. The linker polypeptides are believed to mediate both the assembly of phycobiliproteins into the highly ordered arrays in the phycobilisomes and the interactions between the phycobilisomes and the thylakoid membrane. Functionally, they have been reported to improve energy migration by regulating the spectral characteristics of colored phycobiliproteins. In this review, the progress regarding linker polypeptides research, including separation approaches, structures and interactions with phycobiliproteins, as well as their functions in the phycobilisomes, is presented. In addition, some problems with previous work on linkers are also discussed.     

Characterization, structure and function of linker polypeptides in phycobilisomes of cyanobacteria and red algae: An overview

Cyanobacteria and red algae have intricate light-harvesting systems comprised of phycobilisomes that are attached to the outer side of the thylakoid membrane. The phycobilisomes absorb light in the wavelength range of 500-650 nm and transfer energy to the chlorophyll for photosynthesis. Phycobilisomes, which biochemically consist of phycobiliproteins and linker polypeptides, are particularly wonderful subjects for the detailed analysis of structure and function due to their spectral properties and their various components affected by growth conditions. The linker polypeptides are believed to mediate both the assembly of phycobiliproteins into the highly ordered arrays in the phycobilisomes and the interactions between the phycobilisomes and the thylakoid membrane. Functionally, they have been reported to improve energy migration by regulating the spectral characteristics of colored phycobiliproteins. In this review, the progress regarding linker polypeptides research, including separation approaches, structures and interactions with phycobiliproteins, as well as their functions in the phycobilisomes, is presented. In addition, some problems with previous work on linkers are also discussed.     

Perturbation of chromatin structure in the region of the adult beta-globin gene in chicken erythrocyte chromatin

An EcoRI chromatin fragment containing the adult beta-globin gene and flanking sequences, isolated from chicken erythrocyte nuclei, sediments at a reduced rate relative to bulk chromatin fragments of the same size. We show that the specific retardation cannot be reversed by adding extra linker histones to native chromatin. When the chromatin fragments are unfolded either by removing linker histones or lowering the ionic strength, the difference between globin and bulk chromatin fragments is no longer seen. The refolded chromatin obtained by restoring the linker histones to the depleted chromatin, however, exhibits the original sedimentation difference. This difference is therefore due to a special property of the histone octamers on the active gene that determines the extent of its folding into higher-order structure. That it is not due to the differential binding of linker histones in vitro is shown by measurements of the protein to DNA ratios using CsCl density-gradients. Both before and after selective removal of the linker histones, the globin gene fragment and bulk chromatin fragments exhibit only a marginal difference in buoyant density. In addition, we show that cleavage of the EcoRI fragment by digestion at the 5' and 3' nuclease hypersensitive sites flanking the globin gene liberates a fragment from between these sites that sediments normally. We conclude that the hypersensitive sites per se are responsible for the reduction in sedimentation rate. The non-nucleosomal DNA segments appear to be too long to be incorporated into the chromatin solenoid and thus create spacers between separate solenoidal elements in the chromatin, which can account for its hydrodynamic behaviour.     

The organization of the tadpole and adult alpha globin genes of Xenopus laevis

Adult erythrocytes of X. laevis contain six electrophoretically resolvable globin polypeptides while tadpole erythrocytes contain four polypeptides, none of which comigrates with an adult protein. We show that three of the adult proteins are alpha globin polypeptides (alpha 1, alpha 2, alpha 3) and three are beta globin polypeptides (beta 1, beta 2, beta 3). We find that a tadpole alpha globin gene (alpha T1) is linked to the major adult locus in the sequence 5'-alpha T1-alpha 1-beta 1-3' with 5.2 kb separating alpha T1 from alpha 1. Another tadpole alpha globin gene (alpha T2) is linked to the minor adult locus in the sequence 5'-alpha T2-alpha 2-beta 2-3' with 10.7 kb separating alpha T2 from alpha 2. These linkage relationships are consistent with the major and minor loci having arisen by tetraploidization but the different separation of larval and adult globin genes at the two loci indicates the occurrence of some additional chromosomal rearrangement. Two alternative models are presented.     

Complete amino acid sequences of the major early embryonic alpha-like globins of the chicken

Vertebrate embryos contain hemoglobins composed of globin polypeptides structurally distinct from those of adults. Together with fetal and adult globin chains, these early embryonic globins are encoded by two developmentally regulated multigene families. To facilitate analysis of the structure and evolution of early embryonic alpha-globin genes, we have determined the complete amino acid sequences of the pi and pi' alpha-like globins of the chick embryo. While differing from each other by an alanine/glutamic acid interchange at position 124, this pair of sequences differs from the major and minor adult alpha-globins by 43%. The early embryonic and adult alpha-like sequences appear to have diverged following an ancient gene duplication. We discuss specific amino acid substitutions in functional positions as possible mediators of the reduced Bohr effect and elevated oxygen affinity, which are characteristic of early embryonic hemoglobins.     

Three-dimensional reconstruction of Lumbricus terrestris hemoglobin at 22 A resolution: intramolecular localization of the globin and linker chains.

A 3D reconstruction of the hemoglobin (Hb) of the earthworm Lumbricus terrestris was carried out by the 3D projection alignment method from electron microscopy images of a frozen-hydrated specimen at 22 A resolution. The results were analyzed by a new approach taking into account the evolution of the 210 densities forming the 3D volume as a function of the threshold of surface representation. The whole oligomer with D6point-group symmetry is comprised of 12 hollow globular substructures (HGS) with local 3-fold symmetry tethered to a complex network of linking subunits (linker complex). The 12 globin subunits of each HGS are distributed around local 3-fold axis in four layers of three subunits. The first layer, the most external, contains monomeric globin chains 2A, 3A, and 5A. The three trimers corresponding to the nine remaining subunits have one subunit in each of the second (2B, 3B, 5B), third (1A, 4A, 6A), and fourth (1B, 4B, 6B) layer. The distances between the centers of the globin chains forming the trimers are in the ranges 20-32 A and 45-52 A. The linker complex is made up of two types of linking units. The first type forms three loops connecting globin chains of the second, third and fourth layers. The average molecular mass (Mm) of these subunits was 25 kDa. The second type forms the central structure, termed hexagonal toroid, and its 12 connections to the HGS. This structure corresponds to a hexamer of a single linking unit with a Mm (31.2 kDa), size and a shape different from those of the HGS loops. A careful study of 3D volume architecture shows that each toroid linking unit is bound to the three loops of a HGS pair located in the upper and lower hexagonal layers, respectively. As shown in a model of architecture, hexagonal bilayered (HBL) Hbs can be built very simply from 144 globin chains and 42 linker chains belonging to two different types. We also propose a simple assembly sequence for the construction of HBL Hbs based on the architecture model.     

Structure, composition, and assembly of paracrystalline phycobiliproteins in Synechocystis sp. strain BO 8402 and of phycobilisomes in the derivative strain BO 9201.

The phycobiliproteins of the unicellular cyanobacterium Synechocystis sp. strain BO 8402 and its derivative strain BO 9201 are compared. The biliproteins of strain BO 8402 are organized in paracrystalline inclusion bodies showing an intense autofluorescence in vivo. These protein-pigment aggregates have been isolated. The highly purified complexes contain phycocyanin with traces of phycoerythrin, corresponding linker polypeptides LR35PC and LR33PE (the latter in a small amount), and a unique colored polypeptide with an M(r) of 55,000, designated L55. Allophycocyanin and the core linker polypeptides are absent. The substructure of the aggregates has been studied by electron microscopy. Repetitive subcomplexes of hexameric stacks of biliproteins form extraordinary long rods associated side by side in a highly condensed arrangement. Evidence that the linker polypeptides LR35PC and LR33PE stabilize the biliprotein hexamers is presented, while the location and function of the colored linker L55 remain uncertain. The derivative strain BO 9201 contains established hemidiscoidal phycobilisomes comprising phycoerythrin, phycocyanin, and allophycocyanin as well as the corresponding linker polypeptides. The core-membrane linker protein (LCM), and two polypeptides with M(r)s of 40,000 and 45,000 which are present in small amounts, exhibit strong cross-reactivity in Western blot (immunoblot) analysis using an antibody directed against the colored LCM of a Nostoc sp. In contrast, strain BO 8402 exhibits no polypeptide with a significant immunological cross-reactivity in Western blot analysis. Physiological and genetic implications of the unusual pigment compositions of both strains are discussed.     

Pattern of chick gene activation in chick erythrocyte heterokaryons

The reactivation of chicken erythrocyte nuclei in chick-mammalian heterokaryons resulted in the activation of chick globin gene expression. However, the level of chick globin synthesis was dependent on the mammalian parental cell type. The level of globin synthesis was high in chick erythrocyte-rat L6 myoblast heterokaryons but was 10-fold lower in chick erythrocyte-mouse A9 cell heterokaryons. Heterokaryons between chick erythrocytes and a hybrid cell line between L6 and A9 expressed chick globin at a level similar to that of A9 heterokaryons. Erythrocyte nuclei reactivated in murine NA neuroblastoma, 3T3, BHK and NRK cells, or in chicken fibroblasts expressed less than 5% chick globin compared with the chick erythrocyte-L6 myoblast heterokaryons. The amount of globin expressed in heterokaryons correlated with globin mRNA levels. Hemin increased beta globin synthesis two- to threefold in chick erythrocyte-NA neuroblastoma heterokaryons; however, total globin synthesis was still less than 10% that of L6 heterokaryons. Distinct from the variability in globin expression, chick erythrocyte heterokaryons synthesized chick constitutive polypeptides in similar amounts independent of the mammalian parental cell type. Approximately 40 constitutive chick polypeptides were detected in heterokaryons after immunopurification and two-dimensional gel electrophoresis. The pattern of synthesis of these polypeptides was similar in heterokaryons formed by fusing chicken erythrocytes with rat L6 myoblasts, hamster BHK cells, or mouse neuroblastoma cells. Three polypeptides synthesized by non-erythroid chicken cells but less so by embryonic erythrocytes were conspicuous in heterokaryons. Two abundant erythrocyte polypeptides were insignificant in non-erythroid chicken cells and in heterokaryons.     

Towards a resolution of the long-standing controversy regarding the molecular mass of extracellular erythrocruorin of the earthworm Lumbricus terrestris

The published molecular mass of erythrocruorin of Lumbricus terrestris and related earthworm species covers a bewildering range of 3.23-4.5 MDa. A critical reexamination reveals that some mass determinations were underestimated and the results do cluster, not at one, but at two values of the molecular mass. One cluster corresponds to approximately 3.6 MDa, as predicted for a stoichiometry of 144 globin and 36 linker chains-the Vinogradov model for the hexagonal bilayer (HBL) assembly of Lumbricus erythrocruorin-and as estimated from the crystal structure of HBL at 5.5 A resolution [Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 7107]. The other cluster corresponds to approximately 4.4 MDa. In addition, a molecular mass of 4.1 MDa, determined by multiangle laser light scattering (MALLS), stands apart of the two clusters, separated from the masses obtained by other methods of molecular mass determination.We propose a stoichiometry of 192 globin and 36 linker chains for the 4.4-MDa molecule. The 36 linkers and 144 out of 192 globin chains are identified with the HBL and the remaining 48 globins are allotted equally to the two halves of the axial cavity above and below the central torus of the structure. The proposed model is supported by the occurrence in some annelid species of erythrocruorin with centrally placed subunits [Biochim. Biophys. Acta 359 (1974) 210], and by the oxidation-dependent shedding of subunits in Lumbricus erythrocruorin. We propose further that the 4.1 MDa determination represents the weight average molecular mass of a population of molecules resulting from a partial dissociation of 4.4-MDa erythrocruorin. This interpretation seems reasonable on the background of the very low protein concentrations ( approximately 100 microg/ml and lower) prevailing at the MALLS experiment.     

Maturation of rabbit reticulocytes: degradation of specific reticulocyte proteins.

As analyzed by polyacrylamide-gel electrophoresis, rabbit reticulocyte cytoplasm contains, in addition to globin, seven predominant polypeptides. The amounts of these range from 0.1 to 1.2% of globin. Rabbit erythrocytes contain only three of these nonglobin polypeptides. The loss of the four other polypeptides is correlated with maturation of the reticulocytes to erythrocytes. We also fractionated reticulocytes according to age by equilibrium centrifugation through a gradient of metrizamide and showed that younger reticulocytes contain much more of these four polypeptides than do more mature reticulocytes.The four polypeptides that are lost during differentiation are also very sensitive to in vitro destruction by chymotrypsin or trypsin under conditions where globin and the three reticulocyte nonglobin peptides that remain during reticulocyte maturation are completely resistant. Our results are consistent with the notion that the degradative rate of a reticulocyte cytoplasmic protein is determined by its susceptibility to general intracellular proteases.     

Interdomain linkage in the polymeric hemoglobin molecule of Artemia

Artemia has evolved the longest known concatenation of hemoglobin domains, the subunit containing nine domains and the subunit having a similar size. Translation of the cDNA sequence of the subunit reveals eight regions of inter-domain polypeptide linking together the nine heme-binding domains, together with partially analogous sequences preceding the first domain and following the last. Analysis of the structural possibilities of the linker sequences suggests how the domains may be organized in the subunit.The interdomain linker sequences were 14%–64% identical (62%–91% similar by Dayhoff substitution matrix) and approximately 14 residues in length including a consensus -Val-Asp-Pro-Val-Thr-Gly-Leu-. The linker composition resembled that of the 11 amino acid pre-A leader sequence of Petromyzon marinus (lamprey) hemoglobin V, the structure of which is known. Prediction of structure from the Artemia linker sequences indicated a nonhelical, turn-associated linker which could be modeled to the Petromyzon leader. Measurements confirmed that such a structure could support the packing of nine Artemia domains into a polymeric subunit of annular shape, two of which subunits (which can be similar or dissimilar) comprise the physiological molecule.The position of interdomain introns and the character of a variable residue early in the linker are compatible with the nine-domain polymer having evolved through gene duplication reflected in globin domain fusion incorporating an extension specifically of the N-terminus. The multiplication of an original single-domain globin gene to give the present nine is estimated from sequence differences, allowing for multiple mutations at individual sites, to have occurred in a period at least 500–700 million years ago.Correspondence to: C.N.A. Trotman 1444     

Structure of the genes encoding the rod-core linker polypeptides of Mastigocladus laminosus phycobilisomes and functional aspects of the phycobiliprotein/linker-polypeptide interactions.

The 3' portion of the cpc operon in Mastigocladus laminosus encloses the genes 5'-cpcF-cpcG1-cpcG2-cpcG3 3'. The three cpcG genes encode different phycocyanin-associated rod-core linker polypeptides of the phycobilisomes with predicted 279, 247 and 254 amino acids in length. The gene products CpcG show a high similarity at their N-terminal domains (190 amino acids) and an overall identity of 47-53% to one another. Each of the three CpcG polypeptides is highly related to one of the four CpcG gene products of Anabaena sp. PCC 7120 (66-81% identity). It is suggested that these pairs of rod-core linker polypeptides mediate the same specific type of phycocyanin----allophycocyanin interaction in the similar phycobilisomes of M. laminosus and Anabaena sp. PCC 7120. The similarity of the CpcG1, CpcG2 and CpcG3 polypeptides to the single CpcG rod-core linker polypeptide of Synechococcus sp. PCC 7002 (36-41% identity) is lower. The rod-core linker polypeptides are more distantly related to the rod linker polypeptides associated with phycocyanin or phycoerythrin. However, six conserved domains were identified within the N-terminal 190 amino acids of these linker proteins, which bear similar amino acid sequences, including highly conserved basic amino acids. A similar amino acid sequence but with conserved acidic amino acids can be found in the beta subunits of phycocyanin, phycoerythrin and phycoerythrocyanin, which is protruding into the central cavity of the phycobiliprotein hexamers. It is suggested that these domains are sites of phycobiliprotein-hexamer/rod and rod-core linker interactions.