Taro Nakagawa

Purification, Characterization and Sequence Analyses of the Extracellular Giant Hemoglobin from Oligobrachia mashikoi

Abstract 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.

External morphology of the posterior end, the "opisthosoma", of the beard worm Oligobrachia mashikoi (Pogonophora).

Abstract The entire length of the beard worm, Oligobrachia mashikoi (Pogonophora), including the posterior end, the “opisthosoma” was collected successfully. This species is exclusive to Tsukumo Bay in Ishikawa Prefecture, Japan. Although the portion preceding the opisthosoma was similar to a fine filament, it abruptly assumed a shape similar to a shovel and appeared to be composed of many segmental structures. The number of segments exceeded 50. The dorsal side of the opisthosoma differed from that of the ventral side in morphology. The opisthosoma was equipped with 4 lines of setae arranged longitudinally and a sucker on the tip. When considering the fact that the Family Oligobrachiidae is the most primitive group of pogonophores, the external morphology of the opisthosoma is interesting as it may be reminiscent of the ancestral condition. This is the first report of the opisthosoma in Oligobrachiidae.

Structural Basis for the Heterotropic and Homotropic Interactions of Invertebrate Giant Hemoglobin

The oxygen binding properties of extracellular giant hemoglobins (Hbs) in some annelids exhibit features significantly different from those of vertebrate tetrameric Hbs. Annelid giant Hbs show cooperative oxygen binding properties in the presence of inorganic cations, while the cooperativities of vertebrate Hbs are enhanced by small organic anions or chloride ions. To elucidate the structural basis for the cation-mediated cooperative mechanisms of these giant Hbs, we determined the crystal structures of Ca2+- and Mg2+-bound Hbs from Oligobrachia mashikoi at 1.6 and 1.7 A resolution, respectively. Both of the metal-bound structures were determined in the oxygenated state. Four Ca2+-binding sites and one Mg2+-binding site were identified in each tetramer subassembly. These cations are considered to stabilize the oxygenated form and increase affinity and cooperativity for oxygen binding, as almost all of the Ca2+ and Mg2+ cations were bound at the interface regions, forming either direct or hydrogen bond-mediated interactions with the neighboring subunits. A comparison of the structures of the oxygenated form and the partially unliganded form provides structural insight into proton-coupled cooperativity (Bohr effect) and ligand-induced transitions. Two histidine residues are assumed to be primarily associated with the Bohr effect. With regard to the ligand-induced cooperativity, a novel quaternary rotation mechanism is proposed to exist at the interface region of the dimer subassembly. Interactions among conserved residues Arg E10, His F3, Gln F7, and Val E11, together with the bending motion of the heme molecules, appear to be essential for quaternary rearrangement.

Structure of the partially unliganded met state of 400 kDa hemoglobin: insights into ligand-induced structural changes of giant hemoglobins.

Recent crystallographic studies have revealed the structures of some invertebrate extracellular giant hemoglobins of 3,600 kDa or 400 kDa and their common quaternary structure of dodecameric subassembly composed of four kinds of globin subunits (A1, A2, B1, and B2). These results have provided insight into the mechanisms of their unique functional properties of oxygen binding and sulfide binding. All of these structures were solved with oxygenated or CO‐liganded forms at low or moderate resolutions. We have determined the crystal structure of 400 kDa Hb from a polychaete Oligobrachia mashikoi at 1.95 Å resolution. The electron densities at higher resolution confirm the existence of an isoform of the B1 subunit because of the inconsistency with the model that was built from the formerly known amino acid sequence. The brownish color of the crystals used in this study and the absorption spectrum from the dissolved crystals strongly indicated that the obtained structure was a ferric met state, whereas completele absence of electron density around the distal heme pockets were observed at the A2, B1, and B2 subunits. We concluded that the obtained structure was in unliganded met forms at three of four globin subunits in the 24mer assembly and in oxygenated forms at the remaining A1 subunits. The partially unliganded structure showed remarkable structural changes at the AB loop regions causing quaternary rearrangements of the EF‐dimer structure. In contrast, few changes occurred at the interface regions composed of the E and F helices. These results suggest that the ligand‐induced structural changes of Oligobrachia Hb are quite different from those of the well‐studied mollusk Hb having the same EF‐dimer structure. The structural rearrangements make the dodecameric subassembliy form a tighter conformation than those of fully oxygenated or CO‐liganded dodecamer structure. Proteins 2008.

The structure of a deoxygenated 400 kDa haemoglobin reveals ternary- and quaternary-structural changes of giant haemoglobins

The quaternary structures of invertebrate haemoglobins (Hbs) are quite different from those of vertebrate Hbs. The extracellular giant Hbs of molecular masses of about 400 and 3600 kDa are composed of a dome-shaped dodecameric subassembly which consists of four individual globin subunits. Several crystal structures of 400 kDa Hbs from annelids have been reported, including structures in oxygenated and partially unliganded states, but the structure of the fully deoxygenated state has not been reported. In the present study, crystal structures of V2Hb from the tube worm Lamellibrachia satsuma have been determined in both the fully oxygenated and deoxygenated states. A glycosylation site and novel metal-binding sites for divalent cations were clearly observed with no intersubunit interactions in V2Hb. A comparison of the oxygenated and the deoxygenated forms of V2Hb reveals that the ternary- and quaternary-structural changes occur in a manner that maintains the molecular D3 symmetry. These structures suggest that the mechanisms of quaternary-structural changes between the oxy and deoxy states for the giant Hbs are identical across species.

Purification and Characterization of Coacervate-Forming Cuticular Proteins from Papilio xuthus Pupae

The Papilio xuthus (Lepidoptera: Papilionidae) pupa expresses novel soluble proteins that undergo reversible temperature-dependent coacervate-formation. We purified two coacervate-forming proteins, PX-1 and PX-4, from the wings of pharate adults. PX-1 and PX-4 form coacervates upon warming. Transmission electron microscopy analysis revealed that these proteins assemble ordered bead-like ultrastructures. We cloned and sequenced PX-1 and PX-4 cDNAs. The PX-1 and PX-4 amino acid sequences contain many hydrophobic residues and show homologies to insect cuticular proteins. Moreover, when recombinant PX-1 and PX-4 were overexpressed in Escherichia coli, both recombinant proteins exhibited temperature-dependent coacervation. Furthermore, analyses of truncated mutants of PX-1 suggest that both the Val/Pro-rich region and Gly/lle-rich regions of PX-1 are involved in such coacervation.

Direct Evidence That Extracellular Giant Hemoglobin is Produced in Chloragogen Tissues in a Beard Worm, Oligobrachia mashikoi (Frenulata, Siboglinidae, Annelida)

Abstract In Oligobrachia mashikoi, a mouthless and gutless polychaete known as a beard worm, sites of production of extra-cellular giant hemoglobin were examined with whole-mount in-situ hybridization and semi-quantitative RT-PCR. An RNA probe was prepared from mRNA of the A2-globin subunit. Clear signals were obtained from a peritoneal membrane covering the trophosome in the posterior body in all seven individuals examined in this study. In addition, weak signals were observed in the peritoneal membrane covering tissues in the middle part of the body in some individuals. Furthermore, in one individual, signals were obtained in complicated bodies invaginated into the dorsal vessel from a peritoneal membrane that also released signals. The results of RT-PCR regarding the expression levels of four kinds of globin-subunit genes suggest that the main site of hemoglobin production is the peritoneal membrane in the posterior body.

Ligand-induced structural changes of giant hemoglobin

with the DNA template in the active site available for pairing with the substrate. DNA strand separation occurs one position downstream of the active site, implying that only one substrate at a time can bind to the EC. At the upstream edge of the RNA/DNA hybrid, the first displaced RNA base is trapped within a protein pocket, suggesting a mechanism for RNA displacement. The displaced RNA resides in the RNA exit channel and adopts a conformation mimicking that of a double helix, providing insight into a mechanism for hairpin-dependent pausing and termination. The mechanism of substrate loading in multi-subunit RNA polymerases is crucial for understanding the general principles of transcription. We have determined the EC structures (3Å) with a non-hydrolysable substrate analogue (AMPcPP), and with AMPcPP plus the antibiotic streptolydigin (Stl). In the EC/AMPcPP structure, the substrate binds to the active (‘insertion’) site closed through refolding of the trigger loop (TL). In contrast, the EC/AMPcPP/Stl structure reveals an inactive (‘preinsertion’) open substrate intermediate stabilized by Stl-induced displacement of the TL. Our data suggest three main implications. First, the two-step preinsertion/insertion mechanism of substrate loading may be universal for all RNA polymerases. Second, freezing of the preinsertion state is an attractive target for the drug design. Last, the TL emerges as a regulatory target whose refolding can be modulated by transcription factors. 1. Vassylyev et al. (2007a) Nature, 448, 157-162. 2. Vassylyev et al. (2007b) Nature, 448, 163-168.