Fumio Shishikura

Purification, Characterization, and Amino Acid Sequence of an Embryonic Lectin in Perivitelline Fluid of the Horseshoe Crab

Hemagglutinating activity in perivitelline fluid of the horseshoe crab embryo dramatically increases during the third and fourth molt of the embryo. A 27-kDa lectin, which we named tachylectin-P (TL-P), was newly identified in perivitelline fluid of the horseshoe crab Tachypleus tridentatus. TL-P preferentially agglutinated human A-type erythrocytes, and the activity was inhibited by N-acetyl group-containing monosaccharides. The amino acid sequence analysis indicated that TL-P is almost structurally the same as a hemocyte-derived lectin with no hemagglutinating activity, tachylectin-1 (TL-1), and that 218 out of 221 amino acid residues in total were conserved between the two lectins. Despite the high sequence similarity, biological and biochemical characteristics of TL-P differed from those of TL-1: (i) unlike TL-1, TL-P agglutinates several animal-derived erythrocytes; (ii) unlike TL-1, TL-P has no significant affinity for bacterial lipopolysaccharides or antibacterial activity; (iii) Based on apparent molecular masses determined by gel filtration, TL-P forms a dimer in solution, while TL-1 is present as a monomer; (iv) and TL-P interacts with endogenous proteins of 13 and 14 kDa present in the perivitelline fluid; however, neither has any affinity for TL-1. We propose that TL-P may have an important role in completing embryonic development by interacting with endogenous glycoproteins orN-acetylhexosamines.

The Amino Acid Sequences of the α- and β-Globin Chains of Hemoglobin from the Aldabra Giant Tortoises, Geochelone gigantea

Abstract Tetrameric hemoglobins (Hbs) A and D were isolated from red blood cells of the Aldabra giant tortoises, Geochelone gigantea, by a hydrophobic interaction chromatography. After reduction and S-pyridylethylation, two sets of two types of α-chains (α-1 and α-2) and one β-chain were purified from the major Hb A and minor Hb D in molar ratios of about 1:1:2, respectively, by a reversed-phase column chromatography. The complete amino acid sequences of the three globin-chains from Hb A were determined: 141 amino acid residues for the two α-chains and 146 amino acid residues for the β-chain. Using computer analysis (amino acid maximum homology), the two α-chains shared a 96.5% sequence identity and had low sequence identities (37.8% for α-1 and 35.8% for α-2) with the β-chain of the same species, G. gigantea. We constructed a phylogenetic tree of 28 primary globin structures from Reptilia (7 species of squamates, 4 species of turtles, 3 species of crocodiles and 1 species of sphenodontids), including the three globins of G. gigantea Hb A. The following results were obtained: (1) The two terrestrial species of Geochelone (G. gigantea and G. carbonaria) were closely related: 139 amino acid residues (95.2%) of the two β-globin chains were conserved; (2) Based on the divergence patterns of globin-chains, the sea turtle Caretta caretta was shown to be unusual relatedness form the groups of terrestrial and freshwater species in turtles. The molecular relationships appearing on the phylogenetic tree also support the traditional classification of reptiles and partly confirm previous molecular studies of reptilian hemoglobin evolution.

Leech Extracellular Hemoglobin: Two Globin Strains That are Akin to Vertebrate Hemoglobin α and β Chains

Abstract Leech (Whitmania edentula, Haemadipsa zeylanica var. japonica and Erpobdella lineata) extracellular hemoglobins are basically composed of three constituent subunits, a dimer (D1 and D2 chains) and two monomers (M1 and M2 chains). We isolated these four chains from respective species by a combination of reversed-phase chromatography on a Resource RPC column and gel-filtration on a Superdex 75 column. The apparent molecular masses of the four globin chains were estimated by SDS-PAGE analysis to be 13 kDa (M1), 16 kDa (M2; 19 kDa in its reduced form) and about 27 kDa for the dimer subunit (13 kDa for D1;15 kDa for D2), regardless of the source. The amino (N)-terminal segments (21–30 residues) from twelve globin chains of the above three species were determined and aligned. It was found that the twelve sequences could be separated into two distinct globin groups A and B. This finding supports the original idea of “two globin strains in annelid hemoglobin”, which was proposed without any evidence for leech hemoglobins. Comparing the sequences in the three classes of Annelida, Hirudinea, Oligochaeta and Polychaeta, we found two invariant amino acids, Cys and Trp, which are interposed by eleven amino acid residues. Furthermore, the globin chains belonging to strain A were readily discernible as they had three more invariants, Ser-13, Asp-16 and Trp-28, while the globin chains of strain B had two more invariants, Lys-12 and Arg-27. Consequently, we propose that each of the three classes of Annelida have two distinct groups of globin chains that are akin to vertebrate hemoglobin α and β chains.

A comparative study on earthworm hemoglobins: an amino acid sequence comparison of monomer globin chains of two species, Pontodrilus matsushimensis and Pheretima communissima that belong to the family Megascolecidae.

Abstract The monomer subunits of giant extracellular hemoglobins from earthworms Pontodrilus matsushimensis and Pheretima communissima that belong to the family Megascolecidae, Oligochaeta, were purified by a reversed-phase column, Resource RPC, and sequenced. The complete amino acid sequences of the two monomeric globin chains were determined: 141 amino acid residues with a molecular weight of 16,366 Da for Pontodrilus matsushimensis and 140 amino acid residues with a molecular weight of 16,000 for Pheretima communissima, respectively. The Pontodrilus matsushimensis monomer globin has three cysteine residues, and the two located at positions 2 and 131 are conserved as those observed in all annelids and contribute to form a disulfide-bonded interchain. The third cysteine residue at position 73 is the first evidence for the annelid monomer globin subunits. The physiological functions of the third cysteine residue, however, are still unknown. The monomer sequences of the two species were aligned with those of five known sequences from annelids, including a polychaete, Tylorrhynchus heterochaetus, and four oligochaetes, Pheretima hilgendorfi, Pheretima sieboldi, Lumbricus terrestris and Tubifex tubifex. Using computer analysis, a 87.9% identity of the amino acid sequences between two monomeric subunits of Pheretima communissima and Pheretima hilgendorfi hemoglobins showed the highest degree of sequence similarity. A molecular phylogenetic tree of seven species of annelids has constructed, suggesting that the divergence times among the three species of Pheretima and between Pheretima and Pontodrilus were 50 to 100 and about 209 million years ago, respectively.

Involvement of the distal Arg residue in Cl⁻ binding of midge larval haemoglobin.

Monomeric haemoglobin component V (Hb V) from the larva of the midge Propsilocerus akamusi shows high Cl⁻ affinity under high salt concentrations at acidic pH. In order to understand the structural changes that depend on Cl⁻ binding, crystal structures of Hb V were determined under acidic high-salt conditions and the structural changes arising from different haem-bound ligands were simulated. Crystal structures of Hb V under acidic high-salt conditions indicated that the side chain of ArgE10 on the distal face of the haem contributes to stabilizing haem-bound Cl⁻. The conformation of the Arg side chain in the Cl⁻-bound form was almost identical to that in ligated Hb V at neutral pH but not to that in met Hb V under acidic salt-free conditions. Furthermore, preliminary molecular-dynamics simulations also indicated that the swinging of the Arg side chain into the haem pocket depends on Cl⁻ ligation. This result suggests that, like pH change, Cl⁻ binding affects the location of the distal Arg residue. Owing to the increased positive electrostatic potential observed in the haem pocket at acidic pH, it was concluded that electrostatic changes caused by pH change and anionic ligand binding may affect the behaviour of the polar Arg residue.