The primary structure of hemoglobins of the rock hyrax (Procavia habessinica, Hyracoidea): insertion of glutamine in the alpha chains

The chromatography of the hemoglobin of the rock hyrax (Procavia habessinica) gives two components (73% HbI and 27% HbII). The amino-acid analysis and the sequences of the globin chains elucidated with the phenylthiohydantoin method, did not show any differences between the alpha I and alpha II or beta I and beta II chains, respectively. The different chromatographical behaviour cannot be explained. After chain separation by chromatography on CM-52 cellulose, all four primary structures were elucidated automatically in a sequenator on the chains and the tryptic peptides. In 20% of the beta I chains the N-terminal valine was blocked by acetyl. The alignment was performed by homology with the chains of human adult hemoglobin. The alpha chain of the rock hyrax has 142 amino-acid residues, i.e. one residue more than normal mammalian alpha chains, caused by an insertion of glutamine in the GH region supposed between positions 115 and 116. A comparison of human and hyrax hemoglobins shows an exchange of 21 amino-acid residues in the alpha chains and of 24 in the beta chains. Some substitutions in alpha 1 beta 1 contacts and in the surrounding of the heme are not supposed to effect the function of the hemoglobin. The phylogenetic relationship between the rock hyrax and the Indian elephant (Elephas maximus) on the one hand and with some Perissodactyla on the other, is discussed. Up to now the exchanges of alpha 110(G17)Ala leads to Ser and beta 56(D7)Gly leads to His have only been found in hyrax and elephant. This indicates a certain relationship between Hyracoidea and Proboscidea.     

The amino acid sequences of the alpha and beta chains of hemoglobin from the snake, Liophis miliaris

The hemoglobin of Liophis miliaris has unusual properties. The hemoglobin is dimeric in the oxy form, and the cooperativity of O2 binding is very low, but both the Bohr effect and cooperativity are greatly enhanced in the presence of ATP (Matsuura, M. S. A., Ogo, S. H., and Focesi, A., Jr. (1987) Comp. Biochem. Physiol. 86A, 683-687). Four unique chains (2 alpha, 2 beta) can be isolated from the hemolysate. The amino acid sequences of one alpha and one beta chain have been determined in an effort to understand the functional properties. Comparison of the sequences with those of the alpha and beta chains of human Hb shows the following. (i) All 7 of the residues in the beta chain normally conserved in globins are identical to those of the human chain: Gly(B6), Phe(CD1), His(E7), Leu(F4), His(F8), Lys(H10), and Tyr(HC2), except that the distal His(E7) has been replaced by Gln in the alpha chain. (ii) All heme contact residues in the beta chain are identical with those in the human chain, but two differences are present in the alpha chain: the distal His(E7) is replaced by Gln and Met(B13) by Leu. (iii) All residues that form the binding site for organic phosphates are identical to those in human Hb. (iv) The major residues that contribute to the normal Bohr effect in human Hb, Asp-beta 94, His-beta 146, and Val-alpha 1 are conserved. (v) All beta chain residues at the alpha 1 beta 2 interface are identical with those in the human chain except two: Glu(G3)----Val and Glu(CD2)----Thr; these differences in charged residues may explain the dissociation to dimers. (vi) The 23 residues of the alpha chain in the alpha 1 beta 2 contact region are identical with those of the human chain except three: Phe(B14)----Leu, Thr(C3)----Gln and Pro(CD2)----Ser. (vii) A total of 17 differences occur at the alpha 1 beta 1 interface, 11 in the alpha chain and 6 in the beta chain.     

Structure-function relationships of a membrane pore forming toxin revealed by reversion mutagenesis

Cyt2Aa1 is a haemolytic membrane pore forming toxin produced by Bacillus thuringiensis subsp. kyushuensis. To investigate membrane pore formation by this toxin, second-site revertants of an inactive mutant toxin Cyt2Aa1-I150A were generated by random mutagenesis using error-prone PCR. The decrease in side chain length caused by the replacement of isoleucine by alanine at position 150 in the alphaD-beta4 loop results in the loss of important van der Waals contacts that exist in the native protein between I150 and K199 and L203 on alphaE. 28 independent revertants of I150A were obtained and their relative toxicity can be explained by the position of the residue in the structure and the effect of the mutation on side-chain interactions. Analysis of these revertants revealed that residues on alphaA, alphaB, alphaC, alphaD and the loops between alphaA and alphaB, alphaD and beta5, beta6 and beta7 are important in pore formation. These residues are on the surface of the molecule suggesting that they may participate in membrane binding and toxin oligomerization. Changing the properties of the amino acid side-chains of these residues could affect the conformational changes required to transform the water-soluble toxin into the membrane insertion competent state.     

Perissodactyla: the primary structure of hemoglobins from the lowland tapir (Tapirus terrestris): glutamic acid in position 2 of the beta chains

The hemoglobins from a lowland tapir (Tapirus terrestris) were analysed and the complete primary structure is described. The globin chains were separated on CM cellulose column in 8M urea and the amino-acid sequences were determined in the liquid phase sequenator. The results show that globin consists of two alpha chains (alpha I and alpha II) and beta major and beta minor components. The alpha chains differ only at one position: alpha I contains aspartic acid and alpha II glycine. The beta chains are heterogeneous: aspartic and glutamic acid were found at position beta 21 and beta 73 of the beta major components and asparagine and serine at position beta 139. In the beta minor components four positions were found with more than one amino acid, namely beta 2, beta 4, beta 6 and beta 56. The sequences are compared with those of man, horse and rhinoceros. Four residues of horse methemoglobin, which are involved in the alpha 1 beta 1 contacts are substituted in tapir hemoglobins. In the alpha chains: alpha 107(G14)Ser----Val, alpha 111-(G18) Val----Leu, alpha 115(GH3) Asn----Asp or Gly; in the beta chains: beta 116(G18) Arg----Gln. The amino acid at beta 2 of the major components is glutamic acid while glutamine and histidine are found in the minor components. Although glutamic acid, a binding site for ATP, does not interact with 2,3-bisphosphoglycerate, glutamine and histidine in the minor components are responsible for the slight effect of 2,3-bisphosphoglycerate on tapir hemoglobin.     

Amino acid sequences of the alpha and beta chains of adult hemoglobin of the brown lemur, Lemur fulvus fulvus.

Globin prepared from hemoglobin of the brown lemur (Lemur fulvus fulvus) was separated into alpha and beta chains by chromatography on a CM 52 column. The S-aminoethylated alpha and beta chains were each digested with trypsin and resulting peptides were isolated. The amino acid sequences of the tryptic peptides were established. The ordering of these peptides in the alpha and beta chains was deduced from the homology of their amino acid sequences with that of human adult hemoglobin. The primary structure of brown lemur hemoglobin thus obtained differs from that of human hemoglobin in 15 amino acids in the alpha chain and 26 in the beta chain.     

Amino acid sequences of the aplpha and beta chains of adult hemoglobin of the tupai, Tupaia glis.

Globin prepared from hemoglobin of adult tupai (Tupaia glis) was separated into alpha and beta polypeptide chains by CM-cellulose column chromatography. The S-aminoethylated alpha polypeptide chain and S-carboxymethylated beta polypeptide chain were each digested with trypsin, and the sequences of all the peptides thus obtained were established. The ordering of these tryptic peptides in the alpha and beta polypeptide chains was deduced from the homology of their primary structures with that of human adult hemoglobin. In this way the primary structures of the alpha and beta polypeptide chains of tupai hemoglobin were established; 27 amino acids in the alpha polypeptide chain and 26 in the beta chain differ from those in human adult hemoglobin.     

Sea snake (Microcephalophis gracilis) hemoglobin: Primary structure and relationships to other forms

The hemoglobin of the sea snakeMicrocephalophis gracilis was purified and the primary structure of the α and β chains determined. This is the first sea snake hemoglobin structure characterized, and apparently also the first complete structure of any snake hemoglobin (an α chain of a viper was known), allowing judgments of reptilian variants. Variations between the sea snake form and other reptilian forms are large (52–65 differences for the α chains), of similar order as those between the sea snake and avian (56–65 differences) or human (58 differences) forms. Functionally, 19 residues at α/β contact areas and 7 at heme contacts are exchanged in relation to the human α and β chains. Four positions of the sea snake hemoglobin contain residues thus far unique to this form. However, all replacements appear compatible with conserved overall functional properties.     

Amino-acid sequence of the alpha and beta chains of adult hemoglobin of the harbor seal, Phoca vitulina

The complete amino-acid sequences of the alpha and beta chains of adult hemoglobin of harbor seal, Phoca vitulina that belong to carnivora were determined as follows. The alpha and beta chains isolated by chromatography on a CM-cellulose column were digested with trypsin after S-carboxymethylation. Amino-acid sequences of the tryptic peptides derived from both chains were analysed. Comparing the primary structures of the alpha and beta chains of the seal hemoglobin with those of human, dog, bear, badger and cat, 19, 12, 12, 11, and 16 substitutions, respectively, were recognized in the alpha chain, and 12, 10, 4, 6, and 19 (22) in the beta chain.     

Deoxygenation-linked association of a tetrameric component of chicken hemoglobin.

Deoxygenation-dependent association of hemoglobin tetramers appears to be widespread among amphibians, reptiles, and possibly all or most birds. The evidence for this conclusion depends largely on oxygen equilibria of whole blood which have Hill coefficients that reach values as high as 5-7 at 80-90% oxygenation. Computer simulation of the sedimentation velocity behavior of the major components A and D of chicken hemoglobin shows that component D but not A self-associates to form dimers of tetramers. The gradient profiles at pH 7.5 were satisfactorily fitted with an association constant of 1.26 x 10(4) M-1 and sedimentation coefficients of 4.63 and 7.35 S for tetramer and (tetramer)2, respectively. Since components A and D share common beta chains we conclude that tetramer-tetramer contacts must depend on surface residues of the alpha chains. Comparison of the amino acid sequences of the alpha D and alpha A chains of the hemoglobins from 12 avian species ranging from sparrow to ostrich shows that 20 residues are conserved in the alpha D chains but not in the alpha A chains. Nine of these (45%) are clustered between positions E20 and FG2. Four of the latter, Lys71 (E20), Asn75 (EF4), Gln78 (EF7), and Glu82 (F3) are conserved in all alpha D chains even though they do not appear to participate in intratetramer contacts. Molecular modeling indicates that residues Lys71, Gln78, and Glu82 of the alpha chain are strong candidates for the primary tetramer-tetramer contacts.     

Amino-acid sequences of the alpha and beta chains of adult hemoglobins of the Grand Galago, Galago crassicaudatus

The adult Grand Galago (Galago crassicaudatus) was found to have two hemoglobin components (Hb I and Hb II) which were separated by carboxymethyl cellulose column chromatography. The alpha and beta chains of each component were isolated. The tryptic peptides of the alpha and beta chains were each isolated and sequenced by the conventional method. The alignment of these peptides in each chain was deduced from the homology of their sequences with that of human adult hemoglobin. The alpha chains from Hb I and Hb II were considered to be identical. On the other hand, there was only one amino-acid difference between the two beta chains at the 125th residue from the N-terminus.     

Amino acid sequences of the alpha and beta chains of adult hemoglobin of the slender loris, Loris tardigradus.

alpha and beta chains from adult hemoglobin of the slender loris (Loris tardigradus) were isolated by Amberlite CG-50 column chromatography. After S-aminoethylation, both chains were digested with trypsin and the amino acid sequences of the tryptic peptides obtained were analyzed. Further, the order of these tryptic peptides in each chain was deduced from their homology with the primary structures of alpha and beta chains of human adult hemoglobin. Comparing the primary structures of the alpha and beta chains of adult hemoglobin of the slender loris thus obtained with those of adult hemoglobin of the slow loris, 4 amino acid substitutions in the alpha chains and 2 in the beta chains were recognized.     

Primary structure of alpha and beta chains from the major hemoglobin component of the magpie goose (Anseranas semipalmata, Anatidae)

The amino acid sequence of the alpha and beta chains from the major hemoglobin component (HbA) of Australian Magpie Goose (Anseranas semipalmata) is given. The minor component with the alpha D chains was detected, but only found in low concentrations. By homologous comparison, Greylag Goose hemoglobin (Anser anser) and Australian Magpie Goose alpha chains differ by 13 amino acids or 17 nucleotide (4 two point mutations) exchanges, beta chains by 6 exchanges. Seven alpha 1 beta 1 contacts are modified by substitutions in positions alpha 30-(B11)Glu leads to Gln, alpha 34(B15)Thr leads to Gln, alpha 35(B16)-Ala leads to Thr, alpha 36(B17)Tyr leads to Phe, beta 55(D6)Leu leads to Ile, beta 119(GH2)Ala leads to Ser and beta 125(H3)Glu leads to Asp. Further, one alpha 1 beta 2 contact point was changed in beta 39(C5)Gln leads to Glu. Mutation in this position, except in two abnormal human hemoglobins, was not found in any species. Amino acid exchanges between hemoglobin of Australian Magpie Goose and other birds are discussed.     

The amino acid sequences of two alpha chains of hemoglobins from Komodo dragon Varanus komodoensis and phylogenetic relationships of amniotes

To elucidate phylogenetic relationships among amniotes and the evolution of alpha globins, hemoglobins were analyzed from the Komodo dragon (Komodo monitor lizard) Varanus komodoensis, the world's largest extant lizard, inhabiting Komodo Islands, Indonesia. Four unique globin chains (alpha A, alpha D, beta B, and beta C) were isolated in an equal molar ratio by high performance liquid chromatography from the hemolysate. The amino acid sequences of two alpha chains were determined. The alpha D chain has a glutamine at E7 as does an alpha chain of a snake, Liophis miliaris, but the alpha A chain has a histidine at E7 like the majority of hemoglobins. Phylogenetic analyses of 19 globins including two alpha chains of Komodo dragon and ones from representative amniotes showed the following results: (1) The a chains of squamates (snakes and lizards), which have a glutamine at E7, are clustered with the embryonic alpha globin family, which typically includes the alpha D chain from birds; (2) birds form a sister group with other reptiles but not with mammals; (3) the genes for embryonic and adult types of alpha globins were possibly produced by duplication of the ancestral alpha gene before ancestral amniotes diverged, indicating that each of the present amniotes might carry descendants of the two types of alpha globin genes; (4) squamates first split off from the ancestor of other reptiles and birds.      

Carnivora: primary structure of the hemoglobins from ratel (Mellivora capensis)

The erythrocytes of adult ratel contain two hemoglobin components, with two alpha- and one beta-chains. In this paper, their complete amino acid sequences are presented. The two alpha-chains differ in one residue at position 34 (Ala----Val) only. The primary structure of the chains was determined by sequencing the N-terminal regions (45 steps) and the tryptic peptides after their isolation from the digests by reversed-phase high-performance liquid chromatography. The alignment of these peptides was deduced from homology with other carnivora globins. The alpha-chains show 21 and the beta-chains 11 exchanges compared with human globin chains. In the alpha-chains, one heme- and two alpha 1/beta 1 contacts are exchanged. In the beta-chains there are three exchanges which involve one alpha 1/beta 1-, one alpha 1/beta 2- and one heme-contact. Between the ratel hemoglobin and those of carnivora a high degree of homology was found.     

Structural adaptation of bird hemoglobins to high-altitude respiration and the primary sequences of black-headed gull (Larus ridibundus, Charadriiformes) alpha A- and beta/beta'-chains

Two hemoglobin components HbA (alpha A2 beta 2) and (alpha D2 beta 2) have been detected by analytical electrophoresis in the lysed erythrocytes of the adult Black-Headed Gull (Larus ridibundus). We report the complete primary structure of the alpha A- and beta-chains of the major hemoglobin component HbA. Following the chain separation and isolation of the tryptic peptides by RP-HPLC, the amino-acid sequence was established by automatic Edman degradation in spinning cup and gas-phase sequencers. The primary structures of alpha A- and beta-chains from the Black-Headed Gull HbA differ by 11 and by 6 amino-acid residues from the corresponding chains of Greylag Goose. These changes are randomly distributed over both alpha-helical and interhelical regions. The presence of beta/beta'-chains is indicated by the observation of Ile/Leu at position beta 78. An exchange at position beta 55 (D6)Leu-Asn which is known to be involved in the alpha 1 beta 1-interface with alpha 119(H2)Pro has been found. It is suggested that packing contacts in the alpha 1 beta 1-interface are important for high altitude respiration in birds.     

A new gene structure of the disintegrin family: a subunit of dimeric disintegrin has a short coding region

Disintegrin is a potent platelet aggregation inhibitor isolated from various snake venoms. The cDNA of the snake venom disintegrin family precursor is well-known to encode pre-peptide, metalloprotease, spacer, and disintegrin domains. Recently, new types of disintegrins, dimeric disintegrins, have been isolated, and their amino acid sequences were determined to be approximately 65 amino acid residues in each subunit. We isolated a novel heterodimeric disintegrin, acostatin, from the venom of Agkistrodon contortrix contortrix, which consisted of 63 and 64 amino acid residues in the alpha chain and beta chain, and both chains had the Arg-Gly-Asp (RGD) sequence for binding platelet GPIIb/IIIa. The cDNA lengths of the alpha chain and the beta chain of acostatin were 902 bp and 2031 bp, respectively. The acostatin alpha chain precursor, surprisingly, has the only disintegrin domain alone and lacked almost all of the pre-peptide and metalloprotease domains. The precursor of the acostatin beta chain belongs to a well-known motif of disintegrin precursors. Furthermore, both precursors of alpha and beta chains of another heterodimeric disintegrin, piscivostatin, also have the same domain structures as those of acostatin subunits. These results indicate that the cDNAs of heterodimeric disintegrin subunits have quite a different length of coding region and their precursors have a novel domain structure of disintegrin-family proteins.     

Studies on cobalt myoglobins and hemoglobins. Preparation of isolated chains containing cobaltous protoporphyrin IX and characterization of their equilibrium and kinetic properties of oxygenation and EPR spectra.

Human hemoglobin containing cobalt protoporphyrin IX or cobalt hemoglobin has been separated into two functionally active alpha and beta subunits using a new method of subunit separation, in which the -SH groups of the isolated subunits were successfully regenerated by treatment with dithiothreitol in the presence of catalase. Oxygen equilibria of the isolated subunit chains were examined over a wide range of temperature using Imai's polarographic method (Imai, K., Morimoto, H., Kotani, M., Watari, H., and Kuroda, M. (1970) Biochim. Biophys. Acta 200, 189-196). Kinetic properties of their reversible oxygenation were investigated by the temperature jump relaxation method at 16 degrees. Electron paramagnetic resonance characteristics of the molecules in both deoxy and oxy states were studies at 77K. The oxygen affinity of the individual regenerated chains was higher than that of the tetrameric cobalt hemoglobin and was independent of pH. The enthalpy changes of the oxygenation have been determined as -13.8 kcal/mol and -16.8 kcal/mol for the alpha and beta chains, respectively. The rates of oxygenation were similar to those reported for iron hemoglobin chains, whereas those of deoxygenation were about 10(2) times larger. The effects of metal substitution on oxygenation properties of the isolated chains were correlated with the results obtained previously on cobalt hemoglobin and cobalt myoglobin. The EPR spectrum of the oxy alpha chain showed a distinctly narrowed hyperfine structure in comparison with that of the oxy beta chain, indicating that the environment around the paramagnetic center (the bound oxygen) is different between these chains. In the deoxy form, EPR spectra of alpha and beta chains were indistinguishable. These observations suggest that one of the inequivalences between alpha and beta chains might exist near the distal histidine group.     

A "living fossil" sequence: primary structure of the coelacanth (Latimeria chalumnae) hemoglobin--evolutionary and functional aspects

The coelacanth (Latimeria chalumnae, Actinistia) has a single hemoglobin component. The primary structures of the alpha- and beta-chains are presented. They could be separated by reversed-phase HPLC. Peptides obtained by tryptic digestion of the native and oxidized chains were isolated by reversed-phase HPLC and sequenced in liquid and gas-phase sequenators. The alignment was achieved by employing the N-terminal sequences of the native chains and those of a beta-chain cyanogen bromide peptide as well as fragments obtained by acid hydrolysis. The Latimeria alpha-chains consist of 142 amino-acid residues, due to a fish-specific insertion between positions 46 and 47, whereas the beta-chains are of normal length (146 residues). Latimeria alpha- and beta-chains share 72 (51.1%) and 70 (47.9%) identical residues with human hemoglobin, respectively. Numerous heme contacts and positions involved in subunit interface contacts are replaced. The most interesting of them were studied by molecular modeling. The loss of an alpha 1/beta 2-contact by the exchanges alpha 92(FG4)Arg----Leu and beta 43(CD2)Glu----Lys might be responsible for the easy dissociation of the tetrameric hemoglobin molecule. A comparison of the residues replaced in contact positions with fishes and amphibians revealed the highest number of matches between Latimeria and tadpoles. The same result was obtained by the evaluation of other regions relevant for structure and function of the molecule, like exon-intron boundary regions, phosphate binding sites and salt bridges responsible for the Bohr effect.     

Relationship of the terminal sequences to the length of poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Immobilized tomato lectin interacts with high affinity with glycopeptides containing long poly-N-acetyllactosamine chains

To investigate the factors regulating the biosynthesis of poly-N-acetyllactosamine chains containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] in animal cell glycoproteins, we have examined the structures and terminal sequences of these chains in the complex-type asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Cells were grown in medium containing [6-3H]galactose, and radiolabeled glycopeptides were prepared and fractionated by serial lectin affinity chromatography. The glycopeptides containing the poly-N-acetyllactosamine chains in these cells were complex-type tri- and tetraantennary asparagine-linked oligosaccharides. The poly-N-acetyllactosamine chains in these glycopeptides had four different terminal sequences with the structures: I, Gal beta 1,4GlcNAc beta 1,3Gal-R; II, Gal alpha 1,3Gal beta 1,4GlcNac beta 1,3Gal-R; III, Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,3Gal-R; and IV, Sia alpha 2,6Gal beta 1,4GlcNAc beta 1,3Gal-R. We have found that immobilized tomato lectin interacts with high affinity with glycopeptides containing three or more linear units of the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] and thereby allows for a separation of glycopeptides on the basis of the length of the chain. A high percentage of the long poly-N-acetyllactosamine chains bound by immobilized tomato lectin were not sialylated and contained the simple terminal sequence of Structure I. In addition, a high percentage of the sialic acid residues that were present in the long chains were linked alpha 2,3 to penultimate galactose residues (Structure III). In contrast, a high percentage of the shorter poly-N-acetyllactosamine chains not bound by the immobilized lectin were sialylated, and most of the sialic acid residues in these chains were linked alpha 2,6 to galactose (Structure IV). These results indicate that there is a relationship in these cells between poly-N-acetyllactosamine chain length and the degree and type of sialylation of these chains.     

Molecular cloning and sequence analysis of aggretin, a collagen-like platelet aggregation inducer.

A cDNA library derived from the Malayan-pit-viper (Calloselasma rhodostoma) venom gland was constructed in the phagemid vector. Using the information of the N-terminal amino acid sequences of two subunits of aggretin, synthetic mixed-base oligonucleotides were employed as a screening probe for colony hybridization. Separate cDNA clones encoding for the alpha and beta chains of aggretin were isolated and sequenced. The results revealed that mature alpha and beta chains contain 136 and 123 amino acid residues, respectively. Aggretin subunits show high degrees of identity with respective subunits (50-60% for alpha, 49-58% for beta) of C-type lectin-like snake venoms. The identity to rattlesnake lectin is relatively lower (i.e., 39 and 30%). All cysteine residues in each chain of aggretin are well conserved and located at the positions corresponding to those of C-type lectins. Thus, three intracatenary disulfide bridges and an interchain disulfide bond between Cys83(alpha) and Cys75(beta) may be allocated. This is the first report regarding the entire sequence of venom GPIa/IIa agonist. According to the alignment of amino acid sequences, hypervariable regions among these C-type lectin-like proteins were revealed. These hypervariable regions are proposed to be the counterparts directly interacting with different receptors or different domains of a receptor on the surface of platelet.