Amino acid sequence of myoglobin from the chitonLiolophura japonica and a phylogenetic tree for molluscan globins

Myoglobin was isolated from the radular muscle of the chitonLiolophura japonica, a primitive archigastropodic mollusc.Liolophura contains three monomeric myoglobins (I, II, and III), and the complete amino acid sequence of myoglobin I has been determined. It is composed of 145 amino acid residues, and the molecular mass was calculated to be 16,070 D. The E7 distal histidine, which is replaced by valine or glutamine in several molluscan globins, is conserved inLiolophura myoglobin. The autoxidation rate at physiological conditions indicated thatLiolophura oxymyoglobin is fairly stable when compared with other molluscan myoglobins. The amino acid sequence ofLiolophura myoglobin shows low homology (11–21%) with molluscan dimeric myoglobins and hemoglobins, but shows higher homology (26–29%) with monomeric myoglobins from the gastropodic molluscsAplysia, Dolabella, andBursatella. A phylogenetic tree was constructed from 19 molluscan globin sequences. The tree separated them into two distinct clusters, a cluster for muscle myoglobins and a cluster for erythrocyte or gill hemoglobins. The myoglobin cluster is divided further into two subclusters, corresponding to monomeric and dimeric myoglobins, respectively.Liolophura myoglobin was placed on the branch of monomeric myoglobin lineage, showing that it diverged earlier from other monomeric myoglobins. The hemoglobin cluster is also divided into two subclusters. One cluster contains homodimeric, heterodimeric, tetrameric, and didomain chains of erythrocyte hemoglobins of the blood clamsAnadara, Scapharca, andBarbatia. Of special interest is the other subcluster. It consists of three hemoglobin chains derived from the bacterial symbiont-harboring clamsCalyptogena andLucina, in which hemoglobins are supposed to play an important role in maintaining the symbiosis with sulfide bacteria.     

Amino acid sequence of myoglobin from the chitonLiolophura japonica and a phylogenetic tree for molluscan globins

Myoglobin was isolated from the radular muscle of the chitonLiolophura japonica, a primitive archigastropodic mollusc.Liolophura contains three monomeric myoglobins (I, II, and III), and the complete amino acid sequence of myoglobin I has been determined. It is composed of 145 amino acid residues, and the molecular mass was calculated to be 16,070 D. The E7 distal histidine, which is replaced by valine or glutamine in several molluscan globins, is conserved inLiolophura myoglobin. The autoxidation rate at physiological conditions indicated thatLiolophura oxymyoglobin is fairly stable when compared with other molluscan myoglobins. The amino acid sequence ofLiolophura myoglobin shows low homology (11–21%) with molluscan dimeric myoglobins and hemoglobins, but shows higher homology (26–29%) with monomeric myoglobins from the gastropodic molluscsAplysia, Dolabella, andBursatella. A phylogenetic tree was constructed from 19 molluscan globin sequences. The tree separated them into two distinct clusters, a cluster for muscle myoglobins and a cluster for erythrocyte or gill hemoglobins. The myoglobin cluster is divided further into two subclusters, corresponding to monomeric and dimeric myoglobins, respectively.Liolophura myoglobin was placed on the branch of monomeric myoglobin lineage, showing that it diverged earlier from other monomeric myoglobins. The hemoglobin cluster is also divided into two subclusters. One cluster contains homodimeric, heterodimeric, tetrameric, and didomain chains of erythrocyte hemoglobins of the blood clamsAnadara, Scapharca, andBarbatia. Of special interest is the other subcluster. It consists of three hemoglobin chains derived from the bacterial symbiont-harboring clamsCalyptogena andLucina, in which hemoglobins are supposed to play an important role in maintaining the symbiosis with sulfide bacteria.     

Evidence of met-form myoglobin from Theliostyla albicilla radular muscle

Gastropod mollusc myoglobins provide interesting clues to the evolution of this family of proteins. In addition to conventional monomeric myoglobins, this group also has dimeric and unusual indoleamine dioxygenase-like myoglobins. We isolated myoglobin from the radular muscle of living gastropod mollusc Theliostyla albicilla. The myoglobin appeared to be present in an oxidized met-form, a physiologically inactive form that is not capable of binding oxygen. Under the same extraction conditions, myoglobins mainly of the physiologically active oxy-form have been isolated from other molluscs. The complete amino acid sequence of 157 residues of Theliostyla myoglobin shows that it has a long N-terminal extension of seven residues and contains three functional key residues: CD1-Phe, E7-His, and F8-His. The metmyoglobin can easily be reduced to a ferrous state with Na(2)S(2)O(4). The autoxidation rate of the oxy-form was comparable to other molluscan myoglobins over a wide pH range, and Theliostyla myoglobin was shown to be stable as an oxygen-binding protein. Thus, the predominantly met-form of myoglobin in Theliostyla can be attributed to the incomplete functioning of the myoglobin reduction system in the radular muscle. Although the function of Theliostyla myoglobin is unclear, it may be a scavenger of H(2)O(2).     

Molecular mechanism for ligand stabilization in the mollusc myoglobin possessing the distal Val residue.

Myoglobin extracted from the triturative stomach of Dolabella auricularia, a common mollusc found on the Japanese coast, possesses naturally occurring substitution at the distal E7 position (Val-E7) and its oxygen affinity is only slightly lower than those of the common mammalian myoglobins possessing the usual His-E7. 1H nuclear magnetic resonance studies of Dolabella met-cyano myoglobin have revealed that a guanidino NH proton of Arg-E10 is hydrogen-bonded to the Fe-bound CN-. The role of Arg-E10 as a hydrogen-bond donor for Fe-bound ligand in the present myoglobin appears to be responsible for its relatively high ligand affinity.     

Metastable CO binding sites in the photoproduct of a novel cooperative dimeric hemoglobin.

The infrared absorption spectrum of the CO-photoproduct from Scapharca inaequivalvis hemoglobin (Hbl) at 10 K yields only a single line in the "B" state region at 2132 cm-1. This is the same frequency as the B1 line observed in photodissociated vertebrate HbCO and MbCO. No evidence was found for the B2 line detected in vertebrate hemoglobins and myoglobin in the 2118-2120 cm-1 region. These data demonstrate that the protein does not have the same conformationally accessible ligand-binding sites as do vertebrate hemoglobins and myoglobins. The absence of the B2 line indicates that only a single weak site is accessible to the photolyzed CO molecule. These results are in accord with geminate rebinding experiments and ligand escape pathway calculations which have shown that the distal properties of Hbl are distinct from those of tetrameric hemoglobins and vertebrate myoglobins.     

The Soret magnetic circular dichroism of ferric high-spin myoglobins. A probe for the distal histidine residue.

To find a simple criterion for the presence of the distal (E7) histidine residue in myoglobins and hemoglobins, the Soret magnetic-circular-dichroic spectra were examined for ferric metmyoglobins from various species. A distinct and symmetric dispersion-type curve was obtained for myoglobins containing the distal histidine, whereas a relatively weak and unsymmetric pattern was observed for myoglobins lacking this residue, such as those from three kinds of gastropodic sea molluscs, a shark and the African elephant. The magnetic-circular-dichroic spectra obtained would thus be a direct reflection of the presence or absence of a water molecule at the sixth coordinate position of the heme iron(III), this axial water ligand being stabilized by hydrogen-bond formation to the distal histidine residue. On the basis of these Soret magnetic-circular-dichroic signals, we also examined the structure of a protozoan myoglobin (or a monomeric hemoglobin) from Paramecium caudatum of particular interest for the evolution of these proteins from protozoa to higher animals.     

Nearest-neighbour analysis of myoglobin antigenic sites. Nearest-neighbour residues whose replacement can alter the environment of binding-site residue(s) and thus change their characteristics and binding capability.

By using the known antigenic structure of sperm-whale myoglobin previously determined in this laboratory and the X-ray co-ordinates for the myoglobin molecule, we have calculated the nearest-atom distances between each of the residues of the antigenic sites and all the other amino acids of the myoglobin molecule. These calculations have enabled us to identify the nearest-neighbour residues to each of the residues in the five antigenic sites, and which thus describe the immediate molecular environment of the sites. The influences of chemical changes or replacements in these environmental residues on the binding capacity of an antigenic site, when considered together with replacements directly in the antigenic sites, are expected to account for the major effects and will be extremely useful in explaining the cross-reactions of myoglobins from various species. However, it is stressed that the analysis has limitations due to the qualitative estimates of the effects, the influences of substitutions of once-removed or even at more distant locations (especially when they are cumulative) and finally the influences of any conformational re-adjustments when these occur as a result of the replacement(s).     

Properties of monoclonal antibodies specific for determinants of a protein antigen, myoglobin

Monoclonal hybridoma antibodies specific for the protein antigen sperm whale myoglobin were produced using hyperimmune spleen cells from mice with the genetic trait of high responsiveness to myoglobin. Antibodies from the several clones tested were found to produce linear Scatchard plots, as predicted for homogeneous antibodies, and to possess high affinities for the immunogen (KA congruent to 10(9) M-1). None of the monoclonal antibodies tested reacted with either fragment (1-55) or fragment (132-153) of sperm whale myoglobin. Competitive binding assays using human and horse myoglobins suggested that several of these monoclonal antibodies, which can readily distinguish these myoglobins, recognize different antigenic determinants on the myoglobin molecule. Studies using additional myoglobin sequence variants as competitors should be able to more closely define these antigenic determinants.     

Amino acid sequence of myoglobin from the mollusc Dolabella auricularia

The complete amino acid sequence of the myoglobin from Dolabella auricularia, a common gastropodic mollusc on the Japanese coast, has been determined. The myoglobin is composed of 146 amino acid residues, is acetylated at the NH2 terminus, and contains a single histidine residue at position 95 which most likely corresponds to the heme-binding proximal histidine. The sequence of Dolabella myoglobin shows strong homology (72-77%) with those of Aplysia myoglobins. The autoxidation rate of Dolabella oxymyoglobin (MbO2) was examined in 0.1 M buffer at 25 degrees C over pH range 4.8-12. Dolabella MbO2 was extremely unstable between pH 7 and 11, and the pH dependence of the stability was quite different from that of sperm whale MbO2. This property may be partly due to the absence of a distal (E7) histidine in Dolabella myoglobin.     

Crystal structures of modified myoglobins. II. Relation between oxygen affinity properties and structural changes around heme in myoglobins reconstituted with 2,4-diisopropyldeuteroheme, 2-isopropyl-4-vinyldeuteroheme, and 2-vinyl-4-isopropyldeuteroheme

The crystal structures of sperm whale metmyoglobins reconstituted with three kinds of modified hemes, 2,4-diisopropyldeuteroheme, 2-isopropyl-4-vinyldeuteroheme, and 2-vinyl-4-isopropyldeuteroheme, have been determined and refined at 2.2 A resolution to R = 0.216, 0.219, and 0.195, respectively. All the crystals of these myoglobins are isomorphous with that of native metmyoglobin. The 2-vinyl-4-isopropyldeuteroheme was found to be in a reverse orientation, in which the heme plane is rotated by 180 degrees about an axis through the alpha-gamma-meso carbons, whereas the orientations of the other two hemes were the same as that of protoheme in native myoglobin. In the myoglobins with 2,4-diisopropyldeuteroheme and 2-vinyl-4-isopropyldeuteroheme, both of which have lower oxygen affinities than native myoglobin, the bulky isopropyl side chain pushes Phe 43 0.7 A toward His 64 (the distal histidine) in the former, and the whole E helix at most 1.5 A, including a 0.7 A shift of the His 64 imidazole ring, in the latter. The changes of the structures prevent His 64 from forming a hydrogen bond with the liganded oxygen molecule, so that these two modified myoglobins show low oxygen affinities. On the other hand, there is no such drastic displacement in myoglobin with 2-isopropyl-4-vinyldeuteroheme, which has a slightly higher oxygen affinity than native myoglobin.     

The myoglobin of rodents:Lagostomus maximus (viscacha) andSpalax ehrenbergi (mole rat)

The primary sequences of the myoglobins of two rodents (the South American viscacha and the Mediterranean mole rat) have been determined. Both myoglobins exhibit one polymorphism. The two rodent sequences have been compared with each other and with other known myoglobins. The myoglobin of the viscacha is similar to those of the diving mammals and penguin in having a high arginine content. Among mammalian sequences, the arginines at positions 77 (in one of the viscacha myoglobins) and 79 have been found only in the myoglobin from viscacha. Mole rat myoglobin has a lysine at position 31, where arginine or serine is found in all other known vertebrate myoglobins.     

The myoglobin of rodents:Lagostomus maximus (viscacha) andSpalax ehrenbergi (mole rat)

The primary sequences of the myoglobins of two rodents (the South American viscacha and the Mediterranean mole rat) have been determined. Both myoglobins exhibit one polymorphism. The two rodent sequences have been compared with each other and with other known myoglobins. The myoglobin of the viscacha is similar to those of the diving mammals and penguin in having a high arginine content. Among mammalian sequences, the arginines at positions 77 (in one of the viscacha myoglobins) and 79 have been found only in the myoglobin from viscacha. Mole rat myoglobin has a lysine at position 31, where arginine or serine is found in all other known vertebrate myoglobins.     

The antibody response to myoglobin is independent of the immunized species. Analysis in terms of replacements in the antigenic sites and in environmental residues of the cross-reactions of fifteen myoglobins with sperm-whale myoglobin antisera raised in different species.

The recent determination of the entire antigenic structure of sperm-whale myoglobin with rabbit and goat antisera has permitted the examination of whether the antigenic structure recognized by antibodies depends on the species in which the antisera are raised. Also, by knowledge of the antigenic structure, the molecular factors that determine and influence antigenicity can be better understood in terms of the effects of amino acid substitutions occurring in the antigenic sites and in the environmental residues of the sites. In the present work, the myoglobins from finback whale, killer whale, horse, chimpanzee, sheep, goat, bovine, echidna, viscacha, rabbit, dog, cape fox, mouse and chicken were examined for their ability to cross-react with antisera to sperm-whale myoglobin. By immunoadsorbent titration studies with radioiodinated antibodies, each of these myoglobins was able to bind antibodies to sperm-whale myoglobin raised in goat, rabbit, chicken, cat, pig and outbred mouse. It was found that the extent of cross-reaction of a given myoglobin was not dependent on the species in which the antisera were raised. This indicated that the antibody response to sperm-whale myoglobin (i.e. its antigenic structure) is independent of the species in which the antisera are raised and is not directed to regions of sequence differences between the injected myoglobin and the myoglobin of the immunized host. Indeed, in each antiserum from a given species examined, that antiserum reacted with the myoglobin of that species. The extent of this auto-reactivity for a given myoglobin was comparable with the general extent of cross-reactivity shown by that myoglobin with antisera raised in other species. The cross-reactivities and auto-reactivities (both of which are of similar extents for a given myoglobin) can be reasonably rationalized in terms of the effects of amino acid substitutions within the antigenic sites and within the residues close to these sites. These findings confirm that the antigenicity of the sites is inherent in their three-dimensional locations.     

Kinetic Characterization of Myoglobins from Vertebrates with Vastly Different Body Temperatures

Fish myoglobins are structurally distinct from the previously characterized mammalian myoglobins. Teleost fishes express generally lower levels of myoglobin than those found in mammals. Although the oxygen binding affinity is essentially the same as mammalian myoglobins, oxygen dissociation rates and carbon monoxide combination rates of the teleost myoglobins studied are significantly faster. Thus, the kinetic parameters of myoglobin from two Antarctic teleost species, measured close to their body temperature of −1°C, are comparable to those of mammalian myoglobins with higher body temperatures. These data suggest myoglobins from Antarctic teleosts may function at extreme environmental temperatures.     

Hydrophilicity and antigenicity of proteins — A case study of myoglobin and hemoglobin

Hydrophilicity index is used to locate antigenic determinants on two related groups of proteins-myoglobin and hemoglobin. The data on 41 species (including 34 mammals) of myoglobin show that average hydrophilicity for the complete myoglobin molecules as well as the average hydrophilicity for all hydrophilic regions put together seem to remain constant; the variation in the size and location of the antigenic determinants in these species is very small indicating that the antigenic sites are not shifted during evolution. In the case of both the proteins there is a good agreement between the antigenic sites picked up by using hydrophilicity index and the experimentally determined antigenic sites. The data on 56 species of hemoglobin α-chains and 44 species of hemoglobinβ-chains showed that although there are few sites on hemoglobin which have remained invariant during evolution, there is a significant variation in other sites in terms of either a splitting of a site, or a drastic change in the hydrophilicity values and/or a length of the site. Comparison of the hydrophilicity data on these two groups of proteins suggests that hemoglobins which perform a variety of functions as compared to myoglobins are evolving faster than myoglobins supporting the contention of earlier workers.     

Aplysia myoglobins with an unusual amino acid sequence

The complete amino acid sequence of the myoglobin from Aplysia juliana, a species distributed world-wide, has been determined and compared with the sequence of the myoglobin of Aplysia limacina, a Mediterranean species, and of Aplysia kurodai, a Japanese and Asian species. Unlike mammalian myoglobins, Aplysia myoglobins contain only a single histidine residue, lacking the distal one, the homology being 76% between A. juliana and A. limacina, 74% between A. juliana and A. kurodai, and 83% between A. limacina and A. kurodai. The hydropathy profiles of the Aplysia myoglobins are very similar, but completely different from that of sperm whale myoglobin, taken as the reference.     

Electrostatic effects in myoglobin. Application of the modified Tanford-Kirkwood theory to myoglobins from horse, California grey whale, harbor seal, and California sea lion.

The modified Tanford-Kirkwood electrostatic theory (Shire et al., 1974a) was applied to ferrimyoglobins from the following animal species: sperm whale (Physeter catodon), horse, California grey whale (Eschrichtius gibbosus), harbor seal (Phoca vitulina), and California sea lion (Zalophus californianus). Computations were made of the overall hydrogen ion titration curves of the proteins, and of pH and ionic strength variations of ionization equilibria for individual groups in the protein, with particular reference to the hemic acid ionization of the iron bound water molecule. Coordinates and static solvent accessibility were estimated in terms of the sperm whale myoglobin structure. Where possible, theoretical results and experimental data are compared. Some comparative features of charge and ionization properties among the various myoglobins are presented.     

Antigenic specificity of monoclonal antibodies to human myoglobin

Two monoclonal antibodies directed against different sites of the human myoglobin molecule have been tested for their cross-reactivities against several myoglobins including seven from mammalian species. The relation between their cross-reactivities and their amino acid sequences had led to a possible localization of two antigenic domains in human myoglobin. Each domain includes residues previously considered not to be directly involved in the antigenic structure of myoglobin. Unlike polyclonal serum antibodies, monoclonal hybridoma antibodies directed to a native protein often fail to bind to supposedly antigenic protein fragments. This is explicable in terms of the concept of antigenic domains. Such domains are numerous and overlapping, each comprising a number of contributory amino acid side chains which need not necessarily include continuous sequences of amino acids and which need not exhibit measurable antigenicity in isolation from the rest of the domain.     

Structure-dynamics-function relationships in Asian elephant (Elephas maximus) myoglobin. An optical spectroscopy and flash photolysis study on functionally important motions.

In this work we report the thermal behavior (10-300 K) of the Soret band lineshape of deoxy and carbonmonoxy derivatives of Asian elephant (Elephas maximus) and horse myoglobins together with their carbon monoxide recombination kinetics after flash photolysis; the results are compared to analogous data relative to sperm whale myoglobin. The Soret band profile is modeled as a Voigt function that accounts for the coupling with high and low frequency vibrational modes, while inhomogeneous broadening is taken into account with suitable distributions of purely electronic transition frequencies. This analysis makes it possible to isolate the various contributions to the overall lineshape that; in turn, give information on structural and dynamic properties of the systems studied. The optical spectroscopy data point out sizable differences between elephant myoglobin on one hand and horse and sperm whale myoglobins on the other. These differences, more pronounced in deoxy derivatives, involve both the structure and dynamics of the heme pocket; in particular, elephant myoglobin appears to be characterized by larger anharmonic contributions to soft modes than the other two proteins. Flash photolysis data are analyzed as sums of kinetic processes with temperature-dependent fractional amplitudes, characterized by discrete pre-exponentials and either discrete or distributed activation enthalpies. In the whole temperature range investigated the behavior of elephant myoglobin appears to be more complex than that of horse and sperm whale myoglobins, which is in agreement with the increased anharmonic contributions to soft modes found in the former protein. Thus, to satisfactorily fit the time courses for CO recombination to elephant myoglobin five distinct processes are needed, only one of which is populated over the whole temperature range investigated. The remarkable convergence and complementarity between optical spectroscopy and flash photolysis data confirms the utility of combining these two experimental techniques in order to gain new and deeper insights into the functional relevance of protein fluctuations.     

Preparation of artificial 2-, 3-, 4- and 8-domain myoglobins and comparison of their autoxidation rates

Although most hemoglobins and myoglobins consist of 15-kDa single-domain subunits, structurally unusual hemoglobins, such as Artemia 9-domain and Barbatia 2-domain hemoglobins, occur naturally in several invertebrates. These hemoglobins appear to be the result of gene duplication and fusion. Using cDNA coding for the open reading frame of Aplysia kurodai myoglobin, artificial cDNA inserts corresponding to contiguous dimer, trimer, tetramer and octamer myoglobins (2-, 3-, 4- and 8-domain myoglobins) were prepared and cloned into pMAL or pQE plasmids. These artificial myoglobins and wild-type single-domain myoglobins were successfully expressed in Escherichia coli in the heme-attached, oxygenated form. Myoglobin was purified partially by ammonium sulfate fractionation and gel filtration, and autoxidation rates were examined. The autoxidation rates of recombinant wild-type myoglobins with MBP or hexameric His tag were comparable to those of native myoglobin, suggesting that the recombinant proteins appear to be properly folded and that the N-terminal MBP or His tag does not have an affect on the rate. On the other hand, the rates were significantly decreased in the 2- and 3-domain myoglobins (50% and 30% of the single-domain myoglobins, respectively). The rates for 4- and 8-domain myoglobins were similar to those for 3-domain myoglobin. These results indicate that the artificial poly-domain structure of myoglobin is more stable than the usual single-domain myoglobin from the viewpoint of storage of bound dioxygen.