Exon-intron organization in genes of earthworm and vertebrate globins.

The structure of an invertebrate, intron-containing globin gene has been determined as part of a study of the evolution of hemoglobin. The gene encoding chain c of Lumbricus terrestris hemoglobin has the two-intron, three-exon structure characteristic of vertebrate globin genes, and the exact positions of the splice junctions are conserved. The two introns interrupting the coding sequence are longer than those of known hemoglobins but shorter than myoglobin introns. The gene encodes a secretory preglobin containing a 16-residue signal peptide, as expected for an extracellular hemoglobin. However, no intron separates the DNA encoding the signal sequence from that of the globin sequence. The 3' untranslated region of the Lumbricus gene is much longer than those of the genes for other hemoglobins and is similar to those found for myoglobins.

Lobster hemocyanin: properties of the minimum functional subunit and of aggregates.

Lobster hemocyanin dissociates into a functional subunit of 68,000 to 70,000 molecular weight when Ca(2+) ions are removed from an alkaline solution of low ionic strength. Succinylation results in a further dissociation into two nonfunctional subunits of approximately 34,000 to 35,000 molecular weight. Amino acid analysis and tryptic peptide patterns indicate that the functional subunit is composed of at least two polypeptide chains which are similar.

The structure of arthropod hemocyanins.

Hemocyanins are large multi-subunit copper proteins that transport oxygen in many arthropods and molluscs. Comparison of the amino acid sequence data for seven different subunits of arthropod hemocyanins from crustaceans and chelicerates shows many highly conserved residues and extensive regions of near identity. This correspondence can be matched closely with the three domain structure established by x-ray crystallography for spiny lobster hemocyanin. The degree of identity is particularly striking in the second domain of the subunit that contains the six histidines which ligate the two oxygen-binding copper atoms. The polypeptide architecture of spiny lobster hemocyanin appears to be the same in all arthropods. This structure must therefore be at least as old as the estimated time of divergence of crustaceans and chelicerates, about 540 to 600 million years ago.

Functional properties of hemoglobins from deep-sea fish: correlations with depth distribution and presence of a swimbladder.

The ligand binding properties of the hemoglobins of several deep-sea, bottom-living fish have been examined. These include five species of rattails (Macrouridae) and Antimora rostrata, all of which possess swimbladders, and two unrelated species without swimbladders, Bathysaurus mollis and Alepocephalus sp. All of the hemolysates of these fish exhibited the Root effect with a minimum ligand affinity at about pH 6 in the presence of organic phosphate. Under these conditions the hemolysates from fish which possess swimbladders exhibit two roughly equal populations of heme groups with markedly different ligand affinities. For the deeper-dwelling species the affinities for carbon monoxide differ by some 500-fold, the low-affinity population having a p50(CO) of 100 mmHg at 15 degrees C. This very low affinity is associated with a second-order rate constant for CO combination of the order of 10(3) M-1 X s-1. Those species without swimbladders have hemoglobins which do not have such heterogeneous binding sites, suggesting a relationship between these very-low-affinity heme groups and the pumping of oxygen into a swimbladder at high hydrostatic pressures.

Structure of chain d of the gigantic hemoglobin of the earthworm.

The extracellular hemoglobin of the earthworm has four major O2-binding chains, a, b, c and d, together with additional non-heme structural chains that are required for assembly. Although the abc trimer self-associates extensively at least to (abc)10, addition of chain d results in the formation of a discrete 280 kDa complex corresponding to (abcd)4. Thus a primary function of chain d is to cap the abc association and convert an abc trimer that binds O2 with weak cooperativity to a highly cooperative (abcd)4 complex. Amino-acid sequences of the major globin chains a, b, c have been determined previously by peptide and cDNA analysis. However, the peptide sequence reported for the major chain d (Shishikura, F., Snow, J.W., Gotoh, T., Vinogradov, S.N. and Walz, D.A. (1987) J. Biol. Chem., 262. 3123-3131), has a calculated molecular mass 134-167 Da higher than masses for components of chain d determined by mass spectrometry (Owrby, D.W., Zhu, H., Schneider, K., Beavis, R.C., Chait, B.T. and Riggs, A.F. (1993) J. Biol. Chem. 268, 13539-13547). Reverse-phase HPLC confirms the presence of two distinct polypeptides, d1 and d2, together with d'1, a variant of d1, cDNA derived amino acid sequences have been determined for chains d'1 and d2 by application of the polymerase chain reaction with primers based on the NH2-terminal sequences and oligo-dT. Each of the two cDNA-derived sequences has 140 residues and they differ by 28 substitutions. The data show that the sequence originally reported had been derived from peptides generated from both polypeptides.

Structural analysis of Urechis caupo hemoglobin.

The structure of Urechis caupo hemoglobin in the cyanomet state has been refined to R = 0.148 at 2.5 A resolution. Although the tertiary structure is similar to that of other vertebrate and invertebrate hemoglobins the quaternary structures of this tetramer is unique as suggested by the earlier determination of the 5.0 A resolution structure. The G and H helices of the hemoglobin are on the outside of the tetramer facing the solvent in contrast to human hemoglobin where the G and H helices form inter-subunit contacts. A substantial number of tightly bound water molecules help mediate interactions between subunits. The unusual arrangement of subunits is consistent with the general lack of co-operativity of oxygen uptake for Urechis caupo hemoglobin.

Self-association, cooperativity and supercooperativity of oxygen binding by hemoglobins.

Cooperative ligand binding by tetrameric vertebrate hemoglobins (Hbs) makes possible the delivery of oxygen at higher pressures than would otherwise occur. This cooperativity depends on changes in dimer-dimer interactions within the tetramer and is reflected in a 50 000-fold increase in the tetramer-dimer dissociation constant in human Hb upon oxygenation at pH 7.4, from approximately 2x10(-11)mol l-1 to approximately 10(-6)mol l-1. Hbs that undergo such ligand-dependent changes in association are widespread in non-vertebrates, where the mechanisms are very different from those in vertebrates. Oligomeric Hbs have been identified in organisms in five phyla (molluscs, echinoderms, annelids, phoronids and chordates) that dissociate to subunits upon oxidation of the heme iron and reassociate with the binding of ferric iron ligands such as CN-, N3- or NO2-. Thus, the valence and ligand state of the heme iron control the stability of a critical subunit interface. The broad distribution of this phenomenon suggests a common mechanism of communication between heme and interface that may be almost universal among non-vertebrate Hbs. This interaction may be similar to that known for the homodimeric Hb of the mollusc Scapharca inaequivalvis. Although muscle tissue Hbs or myoglobins (Mbs) are usually monomeric, with non-cooperative O2 binding, the radular muscles of gastropod molluscs and chitons have homodimeric Mbs that bind O2 cooperatively. Cooperative non-muscle tissue Hbs have also been identified. These include the neural Hb of the nemertean worm Cerebratulus lacteus and the Hb of the diving beetle Anisops assimilis, which exhibit deoxygenation-dependent self-association of monomers that is associated with high Hill coefficients. Calculations suggest that the 2-3 mmol l-1 concentration of Hb on a heme basis in the brain of Cerebratulus should substantially extend the time as an active predator in an anaerobic or hypoxic environment. Oxygen from the Hb of Anisops is delivered to a gas bubble and thereby controls the buoyant density. Many Hbs of amphibians, reptiles, birds and some embryonic mammals exhibit a further 'supercooperativity' of O2 binding which depends on reversible deoxygenation-dependent tetramer-tetramer association to form an assemblage with a very low affinity for O2. This phenomenon results in steeper O2-binding curves than exhibited by tetramers alone. The increased cooperativity should result in an increase in the amount of O2 delivered to the tissues and should be especially valuable for avian flight muscles.

Linker chain L1 of earthworm hemoglobin. Structure of gene and protein: homology with low density lipoprotein receptor.

The extracellular hemoglobins (Hbs) of annelids and tube worms are giant multisubunit proteins of up to approximately 200 polypeptides and molecular masses to at least 3,900 kDa. They differ from all other Hbs in having both O2-binding chains and "linker" chains. The latter are required for assembly and structural integrity of the protein and are deficient in or lack heme. We have determined the nucleotide sequences of the cDNA and gene for linker chain L1 of the hemoglobin of Lumbricus terrestris. The cDNA-derived amino acid sequence has 225 residues and a calculated molecular mass of 25,847 Da. The chain is 21-28% identical to linker chains of the related annelid Tylorrhynchus heterochaetus and the deep-sea tube worm Lamellibrachia sp. A remarkable feature of the linker chains is a conserved 38-39-residue segment that contains a repeating pattern of cysteinyl residues: (Cys-X6)3-Cys-X5-Cys-X10-Cys. This pattern, not present in any globin sequence, corresponds exactly to the cysteine-rich repeats of the ligand binding domains of the low density lipoprotein (LDL) receptors of man and Xenopus laevis. Furthermore, the cysteine-rich segment of linker chain L1 has the sequence Asp-Gly-Ser-Asp-Glu which is characteristic of LDL receptor repeats. Similar cysteine-rich sequences also occur in two other mammalian proteins, complement C9 and renal glycoprotein GP330. The results support the conclusion that the cysteine-rich motif of the LDL receptor and annelid Hbs is a multipurpose protein-binding unit of ancient origin which has been incorporated into diverse unrelated proteins, presumably by the process of exon shuffling.

The extracellular hemoglobin of the earthworm, Lumbricus terrestris. Oxygenation properties of isolated chains, trimer, and a reassociated product.

The extracellular hemoglobin of the earthworm Lumbricus terrestris has a two-tiered hexagonal structure that can be dissociated into 1/12 subunits. The Hb contains four major kinds of oxygen-binding chains, a, b, c, and d, of which a-c form a disulfide-linked trimer. Additional non-heme chains are necessary for the assembly of the intact 3800-kDa molecule of approximately 200 subunits. Oxygen equilibria have been measured for chains c and d, the abc trimer, the partially reassembled product of addition of chain d to the trimer, and the intact molecule. The results show that oxygenation of the trimer but not the isolated c or d subunits is modulated by both pH and Ca2+ ions. Cooperativity of oxygen binding by the trimer is low (Hill coefficient approximately 1.3). However, addition of chain d results in a substantial decrease in oxygen affinity and a large increase in cooperativity so that the oxygen equilibrium becomes indistinguishable from that of the intact native molecule at pH 6.8. Light-scattering data show that the smallest observed trimeric abc unit is the dimer (abc)2 at pH 6.8. Analysis of the major sedimentation velocity boundary of the product of the abc unit and chain d in the CO form in the absence of calcium surprisingly can be accounted for entirely in terms of a nondissociating dimer, (abc)2, and chain d. The data for the CO form in the presence of calcium are best fitted in terms of (abc)2.d. Although both subunits c and d also form dimers, oxygen binding by subunit c, but not d, is highly cooperative. These observations, taken together, suggest that the two dimers (abc)2 and d2 are likely to be the major participants in forming the primary functional unit, (abcd)2, which at pH 7.4 is partially dissociated when in the CO form. Subunit d is clearly necessary for the formation of a cooperative unit. The hypothesis that (abcd)2 is a primary functional unit is consistent with a stoichiometry of 2 (abcd)2 units per 1/12 subunit or 24 such units in each molecule of Hb which would contain, in all, 192 heme-containing chains.

Non-heme protein in the giant extracellular hemoglobin of the earthworm Lumbricus terrestris.

The protein/heme mass ratio for the extracellular hemoglobin of the earthworm Lumbricus terrestris has been redetermined. We find a value of 19,000 g of protein per mol of heme. Four major, heme-containing chains (a, b, c, and d), present in equal proportions, have a total molecular mass, with four hemes, of 69,664 Da based on their sequences. The intact hemoglobin comprises 12 subunits that form a two-layered hexagonal structure of about 3.8 MDa. This value, together with our determination of the protein/heme ratio, requires that 4 abcd units are present in each 1/12th subunit and that 192 heme-containing chains are present in the hemoglobin molecule. Our data indicate that approximately 2200 g of non-heme protein is present for each mole of heme-containing chain, or about 35,200 g per 1/12th subunit. This conclusion is consistent with the observation that chains of 31-37 kDa are present. On this basis the intact molecule would have 12 non-heme chains and 204 chains in all to give a total molecular mass of 3.77 MDa, close to that observed.

The hemoglobin of Urechis caupo. The cDNA-derived amino acid sequence.

The nucleotide sequence of a cDNA transcript containing part of the 5' noncoding region, the entire coding region, and the entire 3' noncoding region has been determined. The protein sequence predicted from the coding region matches almost exactly the aminoterminal sequence and the sequence of several peptides from Urechis caupo F-I globin. Only 11-20% of the amino acid positions are identical with those of other known globins.

Yeast flavohemoglobin is an ancient protein related to globins and a reductase family.

The hemoglobin of yeast is a two-domain protein with both heme and flavin prosthetic groups. The nucleotide sequences of the cDNA and genomic DNA encoding the protein from Saccharomyces cerevisiae show that introns are absent and that both domains are homologous with a flavoheme protein from Escherichia coli. The heme domains are also homologous with those of O2-binding heme proteins from several other distantly related bacteria, plants, and animals; all appear to be members of the same globin superfamily. Although the homologous hemoglobin of the bacterium Vitreoscilla sp. is a single-domain protein, several bacteria have related O2-binding heme proteins whose second domains have different structures and enzymatic activities: dihydropteridine reductase (E. coli), cytochrome c reductase (Alcaligenes eutrophus), and kinase in the O2 sensor of Rhizobium meliloti. This indicates that one evolutionary pathway of hemoglobin is that of a multipurpose domain attached to a variety of unrelated proteins to form molecules with different functions. The flavin domain of yeast hemoglobin is homologous with members of a flavoprotein family that includes ferredoxin reductase, nitric oxide synthase, and cytochrome P-450 reductase. The correspondence of yeast and E. coli flavohemoglobins indicates that the two-domain protein has been conserved intact for as long as 1.8 billion years, the estimated time of divergence of prokaryotes and eukaryotes provided that cross-species gene transfer has not occurred.

The structure of the hemocyanin from the horseshoe crab, Limulus polyphemus. The amino acid sequence of the largest cyanogen bromide fragment.

The amino acid sequence of the largest CNBr fragment, CNBrIa, from hemocyanin II of Limulus polyphemus has been determined. This fragment contains 203 residues, has Mr = 23,600, and is the NH2-terminal segment of the molecule. Comparison of this sequence with the first 87 residues of the NH2-terminal sequence of the alpha chain of hemocyanin from the Japanese horseshoe crab, Tachypleus tridentatus, of the Western Pacific (Nemoto, T., and Takagi, T. (1981) Biochim. Biophys. Acta 670, 79-83) shows that 46% of the residues are identical. Residues 172-177, -His-His-Trp-His-Trp-His-, appear to constitute at least part of the copper-binding site. This conclusion is supported by our finding that 40% of the residues in the sequence 172-186 are identical to residues 188-202 in tyrosinase from Neurospora; residues 188 and 193 in tyrosinase are known to be copper-binding histidines (Pfiffner, E., and Lerch, K. (1981) Biochemistry 20, 6029-6035). Model building suggests that histidines 172 and 175 might bind one copper atom and histidines 173 and 177 might bind the second copper. These observations suggest that tyrosinase and hemocyanin may have a common evolutionary origin, but we have not so far seen other correspondences.

The amino acid sequence of a major polypeptide chain of earthworm hemoglobin.

The amino acid sequence has been determined for polypeptide chain AIII of the major component of the hemoglobin (erythrocruorin) from the earthworm, Lumbricus terrestris. The chain has 157 residues and a molecular weight of 17,496. Although the extent of sequence identity with other globin chains is only 17-18%, analysis of the sequence leads to the conclusion that the secondary structure of the chain is very similar to those of vertebrate globins and that about 60-70% of the amino acid residues are in alpha helices. The D helix appears to be missing, as it is from the alpha chain of human hemoglobin and from the monomeric hemoglobin of Glycera dibranchiata, another annelid worm. This is the first sequence obtained from one of the "giant" extracellular hemoglobins of invertebrate animals.

The structure of the gene encoding chain c of the hemoglobin of the earthworm, Lumbricus terrestris.

The complete nucleotide sequence of the gene for chain c of hemoglobin of the earthworm Lumbricus terrestris has been determined. The sequence of 4037 base pairs (bp) includes about 310 bp of 5'-flanking sequence and 110 bp 3' to the poly(A) site. Comparison of cDNA and genomic sequences shows four silent differences in codons that suggest the presence of at least two genes. The coding sequence is split by two introns of 1344 and 1169 bp at highly conserved positions (Jhiang, S. M., Garey, J. R., and Riggs, A. F. (1988) Science 240, 334-336). The first intron possesses the unusual 5' splice junction sequence GC instead of GT. Many tandem triplet repeats based on (GAT) and (CCT) are present in the first intron. The second intron has nine tandem repeats based on the consensus sequence AAGGAAGGAGGTC. Each intron has several exact inverted repeats of 9-10 bp that might result in loops of 78-140 nucleotides in the RNA prior to splicing. The sequences in the second intron, at positions 2423-2644 are about 65% identical with parts of several genes found in yeast mitochondria and in DNA from several other organisms.

Structure and function of hemoglobin from Urechis caupo.

The hemoglobin of the erythrocytes of Urechis caupo is tetrameric. Three chromatographic fractions have been isolated. Fractions F-I (the major fraction) and F-II are each composed of at least five electrophoretic components. Minor fractions F-III has an amino acid composition which is very different from those of fractions F-I and F-II. Amino acid analysis and peptide mapping suggests that fractions F-I and F-II are very similar, and may differ only in a few post-translational modifications. The major fraction, F-I, appears to consist of tetramers in which all subunits are almost identical. Oxygen binding is noncooperative, with a pressure of half-saturation (p50) of 12 mm Hg at 20 degrees C and pH 7.5 in Tris buffers. The oxygen equilibria of undialyzed lysate and of hemoglobin purified by chromatography on Sephadex G-100 are identical. This observation indicates that no allosteric modulator is present. The oxygen equilibrium is not significantly affected by ATP, Cl-, Ca2+, Mg2+, or CO2. The pH dependence of oxygen binding is extremely small: delta Log p50/delta pH is only +0.06 between pH 6 and 7.5. The p50 value changes by no more than 15% between 6.8 microM and 4.3 mM (heme). The apparent enthalpy of oxygenation, delta H, is -11 kcal/mol. Deoxygenation, oxidation, dilution, or pH changes have no significant effect on the state of aggregation. These oxygen-binding properties are consistent with the suggestion that the primary function of the hemoglobin is to store oxygen during the hypoxia which occurs in the burrows of the animal at low tide.

Oxygen binding and aggregation of bullfrog hemoglobin.

The hemoglobin of the bullfrog, Rana catesbeiana, forms aggregates larger than tetramers in two ways. The first, which results from intermolecular disulfide bonds, can be prevented by treatment with iodoacetamide. The second way results from the association of the deoxygenated forms of the two major components, B and C, to form reversibly an aggregate which is believed to be a trimer, BC2. The sedimentation velocity data show that the stoichiometry of the aggregate cannot be 1:1. The electrophoretic pattern of the deoxygenated B/C mixture suggests that the association is not indefinite. No significant aggregation of the separate deoxygenated tetramers of the components nor of the oxygenated components or mixture occurs. Gel chromatography of the oxygenated forms of components B and C and of mixtures indicates that the B and C tetramers both form dimers upon dilution with a dissociation constant of 2-3 micron. The oxygen-binding data indicate that the B/C aggregate has a much lower oxygen affinity than its constituent tetramers. Dissociation of the low affinity B/C aggregate to higher affinity B and C tetramers with increasing oxygenation gives rise to enhanced cooperativity as measured by the Hill coefficient which is maximal near 75-80% oxygenation and is as high as 4.1 at a heme concentration of 15 mM.

The hemoglobins of the bullfrog Rana catesbeiana. The structure of the beta chain of component C and the role of the alpha chain in the formation of intermolecular disulfide bonds.

The adult bullfrog Rana catesbeiana has two major hemoglobin components, B and C. Component C polymerizes by disulfide bond formation between tetramers but component B does not. The amino acid sequence of the first 112 residues of the beta chain of component C has been reported (Baldwin, T. O., and Riggs, A. (1974) J. Biol. Chem. 249, 6110-6118). We have completed the sequence of the beta chain of component C by determining the last 28 residues. This segment contains the 2 cysteinyl residues of the chain. Examination of models indicates that neither of these is in a readily accessible position for the formation of intertetramer disulfide bonds. Reactive sulfhydryl groups of the alpha chains are shown to be responsible for the initial formation of disulfide bonds between tetramers. The beta chains within the tetramers form disulfide bonds only when the hemoglobin molecules are subjected to prolonged incubation at 37 degrees C under oxygen. The beta chains of components B and C appear to be identical; the alpha chains are clearly quite different. This suggests that the alpha B and alpha C subunits interact in the association of the deoxygenated tetramers B and C to form what appears to be a BC2 molecule.