The sulfide binding function of annelid hemoglobins: relic of an old biosystem?

Invertebrates living in sulfide-rich environments have developed different strategies of coping with sulfide toxicity. Some bivalves and annelids have hemoglobins that are capable of binding sulfide for detoxification and/or transporting it to internal bacterial symbionts. Annelids living in the sulfide-rich environments have giant (approximately 3.6 MDa) hemoglobin, consisting of 144 globin chains arranged in a hexagonal bilayer structure held together by 36 nonglobin linker chains. Some globin chains contain either a free cysteine residue at positions Cys+1 or at position Cys+11 relative to the E7 distal residue in the E helix and EF interhelical region, respectively, which bind sulfide. The hexagonal bilayer hemoglobins of annelids living in environments lacking sulfide, do not have the corresponding free cysteine residues and cannot bind sulphide. Given that the early stages of life occurred under anoxic conditions in the presence of sulfide, it is possible that the sulfide binding function from modern annelid globins inhabiting sulphide rich habitats is an evolutionary relic. This proposal seems supported by the recent finding of "protoglobins" which also have a corresponding cysteine residue in Archea known to exist in hyperthermophilic and sulfide-rich environments.     

Purification, characterization and sequence analyses of the extracellular giant hemoglobin from Oligobrachia mashikoi

We purified an extracellular hemoglobin with the molecular mass of ca. 440 kDa from the whole homogenates of Oligobrachia mashikoi (phylum Pogonophora) by a one-step gel-filtration. The preparation was pure to be crystallized. The P50 values of the hemoglobin and the fresh blood prepared from O. mashikoi were about 0.82 Torr and 0.9 Torr, respectively, which were much lower than the P50 value of human hemoglobin. However, the n values of the hemoglobin and the blood were about 1.2 and 1.1, respectively.Using the improved tricine SDS-PAGE, we could separate O. mashikoi hemoglobin into four kinds of the globin chains, A1, A2, B1 and B2, and succeeded for the first time in cloning and sequencing of the complete cDNA encoding B1 globin gene, in addition to A1, A2 and B2 globin genes in full length. We found that all globin genes have the extracellular signal sequences in each molecule and the distal His of the B1 globin chain is replaced to Gln. Finally, we constructed phylogenetic trees of the hemoglobins from Pogonophora, Vestimentifera and Annelida.     

Mass spectral analyses of hemoglobin adducts formed after in vitro exposure of erythrocytes to hydroxymethylvinyl ketone

Hydroxymethylvinyl ketone (HMVK) is a reactive oxidation product of 3-butene-1,2-diol, a metabolite of 1,3-butadiene. The potential for HMVK (0.1 and 1mM) to form hemoglobin (Hb) adducts in erythrocytes from Sprague-Dawley rats was investigated at physiological conditions (pH 7.4, 37 degrees C) using electrospray ionization mass spectrometry (ESI/MS). With the 0.1mM HMVK globin samples, the results indicate HMVK adduction on the alpha2, beta2 and beta3 chains. With the 1.0mM HMVK globin samples, adducts were detected on the beta2 and beta3 chains. However, no correlation was observed between incubation time and the extent of adduct formation, and additional adducts were detected when globin samples were fractionated by HPLC before the ESI/MS analyses. For specific localizations of adducts on the globin chains, trypsin digested peptides from the 1mM HMVK globin samples were subjected to liquid chromatography/mass spectrometry analyses. The results, which are consistent with formation of HMVK adducts on several specific peptides within the alpha- and beta-chains, suggest selectivity in the interaction of HMVK with the different cysteine residues in Hb. Because adducts were also detected in peptides containing no cysteine residues and multiple HMVK moieties were detected on some of the cysteine-containing peptides, the results suggest other amino acids may be also reactive with HMVK. Adduct profiles and their relative intensities were consistent between the 1 and 2h samples providing evidence for the HMVK reactions being fast and selective. The finding that fewer peptides were adducted in the 0.1mM HMVK globin samples provides further evidence for selectivity of the HMVK reaction. Collectively, the results show HMVK readily and selectively forms adducts on Hb. Characterization of these adducts will facilitate development of useful biomarkers of exposure to HMVK and its precursor 1,3-butadiene.     

The amino acid sequences of chains a, b, and c that form the trimer subunit of the extracellular hemoglobin from Lumbricus terrestris

The extracellular hemoglobin of Lumbricus terrestris comprises four major heme-containing chains, a, b, c, and d in equal proportions. We have determined the amino acid sequences of chains a, b, and c which form a disulfide-linked trimer. Chains a, b, and c have 151, 145, and 153 residues and calculated molecular weights of 17,525, 16,254, and 17,289, respectively. The sequence of chain b, reported previously (Garlick, R. L., and Riggs, A. F. (1982) J. Biol. Chem. 287, 9005-9015) has been completely redetermined and found to contain 12 fewer residues than originally reported. Chains a and c both contain unusual, highly polar NH2-terminal extensions of 7 residues before the A helix. These segments must be close together because they are joined by a disulfide bond. We suggest that this structure, with seven negatively charged groups, may be part of a functionally important Ca2+-binding site in the trimer. Comparison of the sequences of chains a, b, and c with those of chain d (Shishikura, F., Snow, J. W., Gotoh, T., Vinogradov, S. N., and Walz, D. A. (1987) J. Biol. Chem. 262, 3123-3131) and the four chains of the hemoglobin of Tylorrhynchus heterochaetus (Suzuki, T., and Gotoh, T. (1986) J. Biol. Chem. 261, 9257-9267) shows that the number and positions of the cysteinyl residues are all conserved. This suggests that the extracellular hemoglobins from both the Oligochaeta and Polychaeta have the same number and configuration of disulfide bonds within the molecule. Phylogenetic analysis suggests that gene duplication first generated an intracellular hemoglobin branch and an extracellular hemoglobin branch. DNA coding for a signal peptide would have been acquired by the extracellular globin gene after this event. At least two further gene duplications are required to account for the present four polypeptide chains.     

Evolution of the sulfide-binding function within the globin multigenic family of the deep-sea hydrothermal vent tubeworm Riftia pachyptila

The giant extracellular hexagonal bilayer hemoglobin (HBL-Hb) of the deep-sea hydrothermal vent tube worm Riftia pachyptila is able to transport simultaneously O(2) and H(2)S in the blood from the gills to a specific organ: the trophosome that harbors sulfide-oxidizing endosymbionts. This vascular HBL-Hb is made of 144 globins from which four globin types (A1, A2, B1, and B2) coevolve. The H(2)S is bound at a specific location (not on the heme site) onto two of these globin types. In order to understand how such a function emerged and evolved in vestimentiferans and other related annelids, six partial cDNAs corresponding to the six globins known to compose the multigenic family of R. pachyptila have been identified and sequenced. These partial sequences (ca. 120 amino acids, i.e., 80% of the entire protein) were used to reconstruct molecular phylogenies in order to trace duplication events that have led to the family organization of these globins and to locate the position of the free cysteine residues known to bind H(2)S. From these sequences, only two free cysteine residues have been found to occur, at positions Cys + 1 (i.e., 1 a.a. from the well-conserved distal histidine) and Cys + 11 (i.e., 11 a.a. from the same histidine) in globins B2 and A2, respectively. These two positions are well conserved in annelids, vestimentiferans, and pogonophorans, which live in sulfidic environments. The structural comparison of the hydrophobic environment that surrounds these cysteine residues (the sulfide-binding domain) using hydrophobic cluster analysis plots, together with the cysteine positions in paralogous strains, suggests that the sulfide-binding function might have emerged before the annelid radiation in order to detoxify this toxic compound. Moreover, globin evolutionary rates are highly different between paralogous strains. This suggests that either the two globin subfamilies involved in the sulfide-binding function (A2 and B2) have evolved under strong directional selective constraints (negative selection) and that the two other globins (A1 and B1) have accumulated more substitutions through positive selection or have evolved neutrally after a relaxation of selection pressures. A likely scenario on the evolution of this multigenic family is proposed and discussed from this data set.     

Observation of large, non-covalent globin subassemblies in the approximately 3600 kDa hexagonal bilayer hemoglobins by electrospray ionization time-of-flight mass spectrometry

A non-covalent globin subassembly comprising 12 globin chains (204 to 214 kDa) was observed directly by electrospray ionization time-of-flight mass spectrometry in the native hexagonal bilayer hemoglobins from the oligochaetes Lumbricus terrestris and Tubifex tubifex, the polychaetes Tylorrhynchus heterochaetus, Arenicola marina, Amphitrite ornata and Alvinella pompejana, the leeches Macrobdella decora, Haemopis grandis and Nephelopsis oscura and the chlorocruorin from the polychaete Myxicola infundibulum, over the pH range 3.5-7.0. The Hb from the deep-sea polychaete Alvinella exhibited in addition, peaks at approximately 107 kDa and at approximately 285 kDa, which were assigned to subassemblies of six globin chains and of 12 globin chains with three non-globin linker chains, respectively. The experimental masses decreased slightly with increased de-clustering potential (60 to 160 V) and were generally 0.1 to 0.2 % higher than the calculated masses, due probably to complexation with cations and water molecules.     

Amino acid sequence of the monomer subunit of the giant extracellular hemoglobin of the aquatic oligochaete, Tubifex tubifex

The extracellular hemoglobin of the aquatic oligochaete Tubifex tubifex consists of four subunits: a monomer of 16.5 kDa, a disulfide-bonded trimer of about 50 kDa and at least two subunits of about 30 kDa. The complete amino acid sequence of the monomeric subunit was determined: it consists of 141 amino acid residues and has a molecular mass of 16,286 Da including a heme group. 39 residues (28%) were found to be identical with those in the corresponding positions in the monomeric globin chains from Lumbricus terrestris, Pheretima sieboldi, and Tylorrhynchus heterochaetus. Tubifex and Lumbricus are most similar, with 75 amino acid identities (53%). There are eight invariant residues amongst these monomeric globins and the intracellular monomeric globin of Glycera and the human beta-globin. The monomeric globin from Tubifex aligns best with those of group A, globins which have a Cys in their second position and an invariant Lys-Val-Lys at positions 9-11 [Gotoh et al. (1987) Biochem. J. 241, 441-445]. The two cysteine residues, at positions 2 and 131, appear to be disulfide-bonded.     

The primary structure of three hemoglobin chains from the indigo snake (Drymarchon corais erebennus,Serpentes): first evidence for alphaD chains and two beta chain types in snakes

The hemoglobin of the indigo snake (Drymarchon corais erebennus, Colubrinae) consists of two components, HbA and HbD, in the ratio of 1:1. They differ in both their alpha and beta chains. The amino acid sequences of both a chains (alphaA and alphaD) and one beta chain (betaI) were determined. The presence of an alphaD chain in a snake hemoglobin is described for the first time. A comparison of all snake beta chain sequences revealed the existence of two paralogous beta chain types in snakes as well, which are designated as betaI and betaII type. For the discussion of the physiological properties of Drymarchon hemoglobin, the sequences were compared with those of the human alpha and beta chains and those of the closely related water snake Liophis milians where functional data are available. Among the heme contacts, the substitution alphaD58(E7)His-->Gln is unusual but most likely without any effect. The residues responsible for the main part of the Bohr effect are the same as in mammalian hemoglobins. In each of the three globin chains only two residues at positions involved in the alpha1/beta2 interface contacts, most important for the stability and the properties of the hemoglobin molecule, are substituted with regard to human hemoglobin. On the contrary, nine, eleven, and six alpha1/beta1 contact residues are replaced in the alphaA, alphaD, betaI chains, respectively.     

Identification of the globin chain and the amino acid residue responsible for polymerization of bullfrog (Rana catesbeiana) hemoglobin with iodo[14C]acetamide.

The bullfrog (Rana catesbeiana) major hemoglobin dissociates into its constituent globin chains (alpha and beta) which are separated by Sulfopropyl-Sephadex C-25 column chromatography after alkylation with iodo[14C]acetamide. Each globin chain has two cysteine residues and those of the beta-globin chain in the tetramer are preferentially alkylated with iodoacetamide.     

Axolotl hemoglobin: cDNA-derived amino acid sequences of two alpha globins and a beta globin from an adult Ambystoma mexicanum

Erythrocytes of the adult axolotl, Ambystoma mexicanum, have multiple hemoglobins. We separated and purified two kinds of hemoglobin, termed major hemoglobin (Hb M) and minor hemoglobin (Hb m), from a five-year-old male by hydrophobic interaction column chromatography on Alkyl Superose. The hemoglobins have two distinct alpha type globin polypeptides (alphaM and alpham) and a common beta globin polypeptide, all of which were purified in FPLC on a reversed-phase column after S-pyridylethylation. The complete amino acid sequences of the three globin chains were determined separately using nucleotide sequencing with the assistance of protein sequencing. The mature globin molecules were composed of 141 amino acid residues for alphaM globin, 143 for alpham globin and 146 for beta globin. Comparing primary structures of the five kinds of axolotl globins, including two previously established alpha type globins from the same species, with other known globins of amphibians and representatives of other vertebrates, we constructed phylogenetic trees for amphibian hemoglobins and tetrapod hemoglobins. The molecular trees indicated that alphaM, alpham, beta and the previously known alpha major globin were adult types of globins and the other known alpha globin was a larval type. The existence of two to four more globins in the axolotl erythrocyte is predicted.     

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.     

Production, Purification, and Characterization of Recombinant Human Hemoglobin Rainier Expressed inSaccharomyces cerevisiae

Hemoglobin Rainier is a naturally occurring hemoglobin variant in which the β145 tyrosine is substituted with cysteine. The α and β^Rainierglobin cDNAs were cloned in a high copy number vector and expressed inSaccharomyces cerevisiaeunder the control of galactose-regulated hybrid promoters. Using this system, we have expressed individual α and β^Rainierglobin chains. Coexpression of both α and β^RainiercDNAs resulted in the production of a functional hemoglobin molecule. Purification of the recombinant protein was accomplished by ion exchange chromatography. The N-termini of the α and β chains were correctly processed, and the molecular mass, as determined by mass spectrometry, indicated amino acid composition identical to that of natural hemoglobin Rainier. The chromatographic properties of the recombinant hemoglobin Rainier were similar to human-derived hemoglobin A₀. The purified recombinant hemoglobin molecule was shown to have an elevated oxygen affinity and a reduced cooperativity as previously reported for natural hemoglobin Rainier. Production of recombinant hemoglobin and especially hemoglobin variants like hemoglobin Rainier has the potential to facilitate use of hemoglobin as a blood substitute as well as in specific applications, such as for use as a therapeutic agent in the treatment of hypotension associated with septic shock.     

Primary structure, biochemical and physiological aspects of hemoglobin from South American lungfish (Lepidosiren paradoxus, Dipnoi)

The South American Lungfish has only one hemoglobin component. The complete amino-acid sequence of this hemoglobin is presented. A large quantity of carbonate dehydratase from the lungfish erythrocytes was also isolated. The carboxymethylated chains, obtained by separation of globin on DEAE-Sephacel, were submitted to tryptic digestion and chemical cleavage. The isolation of tryptic peptides was achieved either by Dowex-50 chromatography or by high performance liquid chromatography. The alignment of peptides was performed by homology with the previously established sequences of the carp and goldfish hemoglobins. The overlapping peptides confirmed this sequence. The alpha chains have 143 residues, the beta chains 147. The relation between the primary structure and the physiological properties of lungfish hemoglobin are discussed.     

Protein and gene structure of a chlorocruorin chain of Eudistylia vancouverii

The polychaete annelid, Eudistylia vancouverii, contains as oxygen carrier a hexagonal bilayer (HBL) chlorocruorin. One of the globin chains, chain a1, has 142 amino acids (Mr 16,054.99) and its sequence deviates strongly from other nonvertebrate globin sequences. Unprecedented, it displays a Phe at the distal position E7 as well as at position B10, creating a very hydrophobic heme pocket probably responsible for the low oxygen affinity of the native molecule. Phylogenetic analysis of annelid globin chains clearly proves that globin chain a1 belongs to type I of globin chains having a pattern of 3 cysteine residues essential for the aggregation into a HBL structure. The gene coding for globin chain a1 is interrupted by 2 introns at the conserved positions B12.2 and G7.0. Based on protein and gene structure it can therefore be concluded that the globin chains of chlorocruorins are not fundamentally different from other annelid globin chains.     

Monitoring exposure to acrylamide by the determination of S-(2-carboxyethyl)cysteine in hydrolyzed hemoglobin by gas chromatography-mass spectrometry

Acrylamide is a potent cumulative neurotoxin in animals and man. In vivo exposure to this electrophile results in the formation of a covalently bound reaction product with cysteine residues in hemoglobin. This adduct yields on acid hydrolysis S-(2-carboxyethyl)cysteine which has been analyzed by capillary gas chromatography with mass spectrometry. Globin isolated from the blood of rats exposed to acrylamide was spiked with an internal standard (globin treated in vitro with d3-acrylamide) and was then hydrolyzed with 6 N HCl. The protein hydrolysate was fractionated on a Dowex 50W H+ ion exchange column and the amino acids in the partially purified extract were determined as N-heptafluorobutyryl methyl esters using an OV-1701 fused silica capillary column. Quantitation was made by chemical ionization (isobutane) selective ion monitoring in which the ions m/z 386 (M-OCH3)+ derived from derivatized S-(2-carboxyethyl)cysteine in the sample and the corresponding ion m/z 389 from the added deuterium-labeled internal standard were monitored. The dose-response relationship between production of hemoglobin adduct and dose of acrylamide (0.1 mg/kg-5 mg/kg) is curved, showing an increasing slope with increasing doses of acrylamide.     

Amino Acid Sequence of Kalinowaski's Tinamou (Nothoprocta Kalinowskii) Hemoglobin and the Rate of Evolution of Bird αD-Globin

Two hemoglobin components are recognized in erythrocytes of the adult Tinamou. We determined the amino acid sequences of Tinamou α^D-, α^A-, and β-globins from intact globin chains and several chemically cleaved fragments. A remarkable feature of Tinamou hemoglobin was a deletion in the α^D-globin chain. This has not been reported in the literature, except in pigeon embryonic α^D-globin. The amino acid sequences of Tinamou globin were highly similar to those of Ostrich and Rhea hemoglobin. Comparison between Tinamou, Ostrich, and Rhea that suggested the evolution speed of globin, α^D = α^A > β, was related with the early appearance birds. The important residues in Tinamou hemoglobin as the heme contact and oxygen binding regions were highly conserved in other species.     

Two-dimensional electrophoresis of Arenicola marina extracellular hemoglobin: separation of chains with identical molecular mass but different isoelectric point.

1. On the basis of their molecular masses, four types of polypeptides (A, B, C, D) were obtained by SDS-PAGE of the extracellular hemoglobin of the polychaete annelid Arenicola marina. 2. On 2-dimensional polyacrylamide gel electrophoresis, the erythrocruorin dissociated into six different types of polypeptide chains; A1, A2, B1, B2, C and D. 3. A1 and B1 migrate in 2-dimensional electrophoresis at the same position as alpha and beta chains of human hemoglobin.     

Reassembly of Lumbricus terrestris hemoglobin: a study by matrix-assisted laser desorption/ionization mass spectrometry and 3D reconstruction from frozen-hydrated specimens

Dodecamers and four types of linker chains (L1-L4) were purified from dissociated hemoglobin of the earthworm Lumbricus terrestris. Various preparations comprising dodecamer of globin chains and linker chains were allowed to reassemble at neutral pH. They produced various oligomers that were purified by gel filtration, analyzed in matrix-assisted laser desorption/ionization mass spectrometry and submitted to 3D reconstruction from isolated particles observed in cryoelectron microscopy. Despite the impossibility to completely free the L2, L3, and L4 preparations from L1, the following conclusions were obtained. First, hemoglobin molecules indistinguishable from native hemoglobin at 25 A resolution were obtained in the absence of linker chains L2, L3, or L4. Second, the 3D reconstruction volumes of reassembled hemoglobins containing dodecamers and L1+L3 or dodecamers and L1+L4 demonstrate that reassembly of native-like structures can be obtained from at most two linker chains and dodecamers. Third, the 3D reconstruction volumes of native and reassembled hemoglobins containing dodecamers and (1) L1, L2, and L4, (2) L1, L3, and L4, (3) L1 and L4, and (4) L1 and L3 were highly similar. Since these structures comprise two types of substructures (one involved in the c3a, c3b, and c4 linking units of the hollow globular substructure and the other in the c5 connection and the toroid), it seems highly probable that the minimal number of linker chains required to reassemble native-like hemoglobin is at most two.     

The primary structure of the pallid bat (Antrozous pallidus, Chiroptera) hemoglobin

The complete primary structure of the hemoglobin from the Pallid Bat (Antrozous pallidus, Microchiroptera) is presented. This hemoglobin consists of two components with identical amino-acid sequences, differing, however, in the N-terminus which is formylated in 12.5% of the beta-chains. The alpha- and beta-chains were separated by reversed phase high performance liquid chromatography. The sequences of both chains were established by automatic Edman degradation with the film technique or gas phase method using the native chains and the tryptic peptides. The formylation of a part of the N-terminal peptide of the beta-chains was determined by mass spectrometric examination. Compared to the corresponding human chains we found 14 substitutions in the alpha-chains and 21 in the beta-chains. One substitution in the alpha-chains and three in the beta-chains are involved in alpha 1/beta 1-contacts. Among these the exchange beta 123(H1)Thr----Cys is unusual because cysteine was so far not found in this position of mammalian beta-chains. Compared to the hemoglobin of Myotis velifer, another representative of the family Vespertilionidae, 5 residues are replaced in the alpha-chains and 18 in the beta-chains.     

Hemoglobinase Activity of the Lysine Gingipain Protease (Kgp) of Porphyromonas gingivalis W83

Porphyromonas gingivalis, an important periodontal disease pathogen, forms black-pigmented colonies on blood agar. Pigmentation is believed to result from accumulation of iron protoporphyrin IX (FePPIX) derived from erythrocytic hemoglobin. The Lys-X (Lys-gingipain) and Arg-X (Arg-gingipain) cysteine proteases of P. gingivalis bind and degrade erythrocytes. We have observed that mutations abolishing activity of the Lys-X-specific cysteine protease, Kgp, resulted in loss of black pigmentation of P. gingivalis W83. Because the hemagglutinating and hemolytic potentials of mutant strains were reduced but not eliminated, we hypothesized that this protease played a role in acquisition of FePPIX from hemoglobin. In contrast to Arg-gingipain, Lys-gingipain was not inhibited by hemin, suggesting that this protease played a role near the cell surface where high concentrations of hemin confer the black pigmentation. Human hemoglobin contains 11 Lys residues in the alpha chain and 10 Lys residues in the beta chain. In contrast, there are only three Arg residues in each of the alpha and beta chains. These observations are consistent with human hemoglobin being a preferred substrate for Lys-gingipain but not Arg-gingipain. The ability of the Lys-gingipain to cleave human hemoglobin at Lys residues was confirmed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry of hemoglobin fragments resulting from digestion with the purified protease. We were able to detect several of the predicted hemoglobin fragments rendered by digestion with purified Lys-gingipain. Thus, we postulate that the Lys-gingipain of P. gingivalis is a hemoglobinase which plays a role in heme and iron uptake by effecting the accumulation of FePPIX on the bacterial cell surface.