Multidomain Structure of the Rapana thomasiana (Gastropod) Hemocyanin Structural Subunit RHSS1

The Rapana thomasiana hemocyanin structural subunit RHSS1 is composed of eight functional dioxygen-binding domains. To determine the multidomain structure, the polypeptide chain of RHSS1 was subjected to limited proteolysis with TPCK-trypsin, elastase and other proteinases. Individual functional units and fragments, containing two or three domains, were isolated and characterized. All domains and fragments were N-terminally sequenced and the order of the dioxygen-binding units in the polypeptide chain of RHSS1 was established.     

Low-resolution molecular structures of isolated functional units from arthropodan and molluscan hemocyanin

Synchrotron x-ray scattering measurements were performed on dilute solutions of the purified hemocyanin subunit (Bsin1) from scorpion (Buthus sindicus) and the N-terminal functional unit (Rta) from a marine snail (Rapana thomasiana). The model-independent approach based on spherical harmonics was applied to calculate the molecular envelopes directly from the scattering profiles. Their molecular shapes in solution could be restored at 2-nm resolution. We show that these units represent stable, globular building blocks of the two hemocyanin families and emphasize their conformational differences on a subunit level. Because no crystallographic or electron microscopy data are available for isolated functional units, this study provides for the first time structural information for isolated, monomeric functional subunits from both hemocyanin families. This has been made possible through the use of low protein concentrations (< or = 1 mg/ml). The observed structural differences may offer advantages in building very different overall molecular architectures of hemocyanin by the two phyla.     

Carbohydrate content and monosaccharide composition of Rapana thomasiana grosse (Gastropoda) hemocyanin and its structural subunits. Comparison with gastropodan hemocyanins

The hemocyanin of Rapana thomasiana grosse (marine snail, gastropod) is a glycoprotein with a carbohydrate content of 8.9% (w/w) and monosaccharide constituents xylose, fucose, 3-OOmethylgalactose, mannose, galactose, N-acetylgalactosamine and N-acetylglucosamine residues. The two structural subunits of this oxygen carrier, RHSS1 and RHSS2, are unevenly glycosylated. On subtracting the carbohydrate contribution from the M_r values of 250 and 450 kDa attributed to the two subunits, values of 2.18 × 10⁵ daltons and 4.30 × 10⁵ daltons were calculated for the polypeptide part of the “light” and “heavy” subunits, respectively. Comparison of the monosaccharide compositions of gastropodan hemocyanins revealed qualitative similarities, as well as relationships between the quantities, of the individual monosaccharides: Man 3MeGal > GlcNAc GalNAc and Fuc Xyl     

The Hemocyanin from a Living Fossil, the Cephalopod Nautilus pompilius: Protein Structure, Gene Organization, and Evolution

By electron microscopic and immunobiochemical analyses we have confirmed earlier evidence that Nautilus pompilius hemocyanin (NpH) is a ring-like decamer (M_r = ∼3.5 million), assembled from 10 identical copies of an ∼350-kDa polypeptide. This subunit in turn is substructured into seven sequential covalently linked functional units of ∼50 kDa each (FUs a–g). We have cloned and sequenced the cDNA encoding the complete polypeptide; it comprises 9198 bp and is subdivided into a 5′ UTR of 58 bp, a 3′ UTR of 365 bp, and an open reading frame for a signal peptide of 21 amino acids plus a polypeptide of 2903 amino acids (M_r = 335,881). According to sequence alignments, the seven FUs of Nautilus hemocyanin directly correspond to the seven FU types of the previously sequenced hemocyanin “OdH” from the cephalopod Octopus dofleini. Thirteen potential N-glycosylation sites are distributed among the seven Nautilus hemocyanin FUs; the structural consequences of putatively attached glycans are discussed on the basis of the published X-ray structure for an Octopus dofleini and a Rapana thomasiana FU. Moreover, the complete gene structure of Nautilus hemocyanin was analyzed; it resembles that of Octopus hemocyanin with respect to linker introns but shows two internal introns that differ in position from the three internal introns of the Octopus hemocyanin gene. Multiple sequence alignments allowed calculation of a rather robust phylogenetic tree and a statistically firm molecular clock. This reveals that the last common ancestor of Nautilus and Octopus lived 415 ± 24 million years ago, in close agreement with fossil records from the early Devonian. [Reviewing Editor: Dr. Axel Meyer] The sequence reported in this paper has been deposited in the EMBL/GenBank database under accession number AJ619741.     

Hexamers of subunit II from Limulus hemocyanin (a 48-mer) have the same quaternary structure as whole Panulirus hemocyanin molecules

Hemocyanins are copper-containing proteins that transport oxygen in a variety of invertebrates. Considerable evidence has accumulated that arthropodan hemocyanins are multimers of a fundamental hexameric unit. X-Ray crystallographic structure determination has revealed that the hemocyanin molecule from the spiny lobster Panulirus interruptus is a single hexamer having 32 point group symmetry. Using crystals of subunit II, one of 8 polypeptide types comprising the octahexameric hemocyanin of the horseshoe crab Limulus polyphemus, and the molecular replacement method for crystallographic phase determination we show that subunit II forms assemblies with the same hexameric quaternary structure as the whole Panulirus hemocyanin molecule. Observation of the same hexameric motif in two widely separated species provides strong additional evidence that this quaternary structural unit is a universal building block of arthropodan hemocyanins.     

Purification and Characterization of a Variant of Rhodocetin from Calloselasma rhodostoma (Malayan Pit Viper) Venom

Rhodocetin is a novel C-type lectin-related protein (CLP) purified from the venom of Calloselasma rhodostoma. Thus far, it is the only reported CLP whose and subunits are not linked by an interdisulfide bond. We report here the isolation of a variant of rhodocetin from a different source of venom. This variant of rhodocetin exhibited a different elution profile in reverse-phase HPLC as compared to the rhodocetin reported in our original publication [Wang et al., (1999), Biochemistry 38, 7584–7593]. Specifically, the subunit of the variant was eluted at a considerably lower percentage of acetonitrile, which suggested a less hydrophobic polypeptide chain as compared to the original rhodocetin. Using a combination of microcharacterization techniques such as peptide mapping, mass spectrometry, and amino acid sequencing, we identified an amino acid substitution, I63K, in the polypeptide chain that could account for the difference in elution behavior of the subunit. In addition, we also found a conserved E88D substitution in the chain which was not apparent during reverse-phase HPLC. However, neither of these substitutions resulted in the alteration of the functional properties of the rhodocetin variant.     

Abalone (Haliotis tuberculata) hemocyanin type 1 (HtH1). Organization of the approximately 400 kDa subunit, and amino acid sequence of its functional units f, g and h.

We have identified two separate hemocyanin types (HtH1 and HtH2) in the European abalone Haliotis tuberculata. HtH1/HtH2 hybrid molecules were not found. By selective dissociation of HtH2 we isolated HtH1 which, as revealed by electron microscopy and SDS/PAGE, is present as didecamers of a approximately 400 kDa subunit. Immunologically, HtH1 and HtH2 correspond to keyhole limpet hemocyanin (KLH)1 and KLH2, respectively, the two well-studied hemocyanin types of the closely related marine gastropod Megathura crenulata. On the basis of limited proteolytic cleavage, two-dimensional immunoelectrophoresis, SDS/PAGE and N-terminal sequencing, we identified eight different 40-60 kDa functional units in HtH1, termed HtH1-a to HtH1-h, and determined their linear arrangement within the elongated subunit. From Haliotis mantle tissue, rich in hemocyanin-producing pore cells, we isolated mRNA and constructed a cDNA library. By expression screening with HtH-specific rabbit antibodies, a cDNA clone was isolated and sequenced which codes for the three C-terminal functional units f, g and h of HtH1. Their sequences were aligned to those available from other molluscs, notably to functional unit f and functional unit g from the cephalopod Octopus dofleini. HtH1-f, which is the first sequenced functional unit of type f from a gastropod hemocyanin, corresponds to functional unit f from Octopus. Also functional unit g from Haliotis and Octopus correspond to each other. HtH1-h is a gastropod hemocyanin functional unit type which is absent in cephalopods and has not been sequenced previously. It exhibits a unique tail extension of approximately 95 amino acids, which is lacking in functional units a to g and aligns with a published peptide sequence of 48 amino acids from functional unit h of Helix pomatia hemocyanin. The new Haliotis sequences are discussed with respect to their counterparts in Octopus, the 15 A three-dimensional reconstruction of the KLH1 didecamer from electron micrographs, and the recent 2.3 A X-ray structure of functional unit g from Octopus hemocyanin.     

The cDNA sequence of three hemocyanin subunits from the garden snail Helix lucorum

Hemocyanins are blue copper containing respiratory proteins residing in the hemolymph of many molluscs and arthropods. They can have different molecular masses and quaternary structures. Moreover, several molluscan hemocyanins are isolated with one, two or three isoforms occurring as decameric, didecameric, multidecameric or tubule aggregates. We could recently isolate three different hemocyanin isopolypeptides from the hemolymph of the garden snail Helix lucorum (HlH). These three structural subunits were named α_D-HlH, α_N-HlH and β-HlH. We have cloned and sequenced their cDNA which is the first result ever reported for three isoforms of a molluscan hemocyanin. Whereas the complete gene sequence of α_D-HlH and β-HlH was obtained, including the 5′ and 3′ UTR, 180 bp of the 5′ end and around 900 bp at the 3′ end are missing for the third subunit. The subunits α_D-HlH and β-HlH comprise a signal sequence of 19 amino acids plus a polypeptide of 3409 and 3414 amino acids, respectively. We could determine 3031 residues of the α_N-HLH subunit. Sequence comparison with other molluscan hemocyanins shows that α_D-HlH is more related to Aplysia californicum hemocyanin than to each of its own isopolypeptides. The structural subunits comprise 8 different functional units (FUs: a, b, c, d, e, f, g, h) and each functional unit possesses a highly conserved copper-A and copper-B site for reversible oxygen binding. Potential N-glycosylation sites are present in all three structural subunits. We confirmed that all three different isoforms are effectively produced and secreted in the hemolymph of H. lucorum by analyzing a tryptic digest of the purified native hemocyanin by MALDI-TOF and LC-FTICR mass spectrometry.     

Crystallization and Preliminary X-Ray Diffraction Analysis of the 190-Å-Long Coiled-Coil Dimerization Domain of the Actin-Bundling Protein Cortexillin I fromDictyostelium discoideum

We have crystallized the ≈190-Å-long parallel two-stranded coiled-coil oligomerization domain of the actin-bundling protein cortexillin I fromDictyostelium discoideum. The orthorhombic crystals belong to the space group C222₁with unit cell dimensions ofa= 71.3 Å,b= 127.8 Å, andc= 91.6 Å. As both native and selenomethionine-substituted protein crystals diffract to 3.0 and 2.85 Å resolution, respectively, using synchrotron radiation, they are suitable for the first high-resolution structural analysis of a two-stranded coiled coil comprising more than six heptad repeats. Moreover, because the polypeptide chain fragment contains a recently identified two-heptad-repeat long sequence that is indispensable for the assembly of the cortexillin I coiled-coil oligomerization domain, its high-resolution structure should enable us to extend our knowledge on the molecular mechanisms underlaying coiled-coil formation and to establish the precise manner in which the two “trigger” sequences interact with one another in the dimer.     

The sequence of a gastropod hemocyanin (HtH1 from Haliotis tuberculata)

The eight functional units (FUs), a-h, of the hemocyanin isoform HtH1 from Haliotis tuberculata (Prosobranchia, Archaeogastropoda) have been sequenced via cDNA, which provides the first complete primary structure of a gastropod hemocyanin subunit. With 3404 amino acids (392 kDa) it is the largest polypeptide sequence ever obtained for a respiratory protein. The cDNA comprises 10,758 base pairs and includes the coding regions for a short signal peptide, the eight different functional units, a 3'-untranslated region of 478 base pairs, and a poly(A) tail. The predicted protein contains 13 potential sites for N-linked carbohydrates (one for HtH1-a, none for HtH1-c, and two each for the other six functional units). Multiple sequence alignments show that the fragment HtH1-abcdefg is structurally equivalent to the seven-FU subunit from Octopus hemocyanin, which is fundamental to our understanding of the quaternary structures of both hemocyanins. Using the fossil record of the gastropod-cephalopod split to calibrate a molecular clock, the origin of the molluscan hemocyanin from a single-FU protein was calculated as 753 +/- 68 million years ago. This fits recent paleontological evidence for the existence of rather large mollusc-like species in the late Precambrian.     

Nautilus pompilius hemocyanin: 9 A cryo-EM structure and molecular model reveal the subunit pathway and the interfaces between the 70 functional units

Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs. Nautilus pompilius (Cephalopoda) hemocyanin is a cylindrical decamer of a 350 kDa polypeptide subunit that in turn is a “pearl-chain” of seven different functional units (FU-a to FU-g). Each globular FU has a binuclear copper centre that reversibly binds one O₂ molecule, and the 70-FU decamer is a highly allosteric protein. Its primary structure and an 11 Å cryo-electron microscopy (cryo-EM) structure have recently been determined, and the crystal structures of two related FU types are available in the databanks. However, in molluscan hemocyanin, the precise subunit pathway within the decamer, the inter-FU interfaces, and the allosteric unit are still obscure, but this knowledge is crucial to understand assembly and allosterism of these proteins. Here we present the cryo-EM structure of Nautilus hemocyanin at 9.1 Å resolution (FSC_1/2-bit criterion), and its molecular model obtained by rigid-body fitting of the individual FUs. In this model we identified the subunit dimer, the subunit pathway, and 15 types of inter-FU interface. Four interface types correspond to the association mode of the two protomers in the published Octopus FU-g crystal. Other interfaces explain previously described morphological structures such as the fenestrated wall (which shows D5 symmetry), the three horizontal wall tiers, the major and minor grooves, the anchor structure and the internal collar (which unexpectedly has C5 symmetry). Moreover, the potential calcium/magnesium and N-glycan binding sites have emerged. Many interfaces have amino acid constellations that might transfer allosteric interaction between FUs. From their topologies we propose that the prime allosteric unit is the oblique segment between major and minor groove, consisting of seven FUs from two different subunits. Thus, the 9 Å structure of Nautilus hemocyanin provides fundamentally new insight into the architecture and function of molluscan hemocyanins.     

Molecular characterization of the alpha subunit of complement component C8 (GcC8α) in the nurse shark (Ginglymostoma cirratum)

Target cell lysis by complement is achieved by the assembly and insertion of the membrane attack complex (MAC) composed of glycoproteins C5b through C9. The lytic activity of shark complement involves functional analogues of mammalian C8 and C9. Mammalian C8 is composed of α, β, and γ subunits. The subunit structure of shark C8 is not known. This report describes a 2341 nucleotide sequence that translates into a polypeptide of 589 amino acid residues, orthologue to mammalian C8α and has the same modular architecture with conserved cysteines forming the peptide bond backbone. The C8γ-binding cysteine is conserved in the perforin-like domain. Hydrophobicity profile indicates the presence of hydrophobic residues essential for membrane insertion. It shares 41.1% and 47.4% identity with human and Xenopus C8α respectively. Southern blot analysis showed GcC8α exists as a single copy gene expressed in most tissues except the spleen with the liver being the main site of synthesis. Phylogenetic analysis places it in a clade with C8α orthologs and as a sister taxa to the Xenopus.     

Characterization of hemocyanin from the peacock mantis shrimp Odontodactylus scyllarus (Malacostraca:Hoplocarida)

Hemocyanin is the blue respiratory protein of many arthropod species. While its structure, evolution, and physiological function have been studied in detail in Decapoda, there is little information on hemocyanins from other crustacean taxa. Here, we have investigated the hemocyanin of the peacock mantis shrimp Odontodactylus scyllarus, which belongs to the Stomatopoda (Hoplocarida). O. scyllarus hemocyanin forms a dodecamer (2 × 6-mer), which is composed of at least four distinct subunit types. We obtained the full-length cDNA sequences of three hemocyanin subunits, while a fourth cDNA was incomplete at its 5′ end. The complete full-length cDNAs of O. scyllarus hemocyanin translate into polypeptides of 650–662 amino acids, which include signal peptides of 16 or 17 amino acids. The predicted molecular masses of 73.1–75.1 kDa correspond well with the main hemolymph proteins detected by SDS-PAGE and Western blotting using various anti-hemocyanin antibodies. Phylogenetic analyses show that O. scyllarus hemocyanins belong to the β-type of malacostracan hemocyanin subunits, which diverged from the other subunits before the radiation of the malacostracan subclasses around 520 million years ago. Molecular clock analysis revealed an ancient and complex pattern of hemocyanin subunit evolution in Malacostraca and also allowed dating divergence times of malacostracan taxa.     

C-terminal functional unit of Rapana thomasiana (marine snail, gastropod) hemocyanin isoform RtH1: isolation and characterization

Rapana thomasiana hemocyanin (RtH) is a mixture of two hemocyanin (Hc) isoforms termed RtH1 and RtH2. Both subunit types are built up of eight functional units (FUs). The C-terminal functional unit (RtH1-h) of the Rapana Hc subunit 1 has been isolated by limited trypsinolysis of the subunit polypeptide chain. The oxy- and apo-forms of the unit are characterized by fluorescence spectroscopy. Upon excitation of RtH1-h at 295 or 280 nm, tryptophyl residues buried in the hydrophobic interior of the protein globule determine the fluorescence emission. This is confirmed by quenching experiments with acrylamide, cesium chloride and potassium iodide. The copper-dioxygen system at the binuclear active site quenches the indole emission of the oxy-RtH1-h. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophyl residues in the apo-RtH1-h. The thermal stability of the apo-RtH1-h is characterized fluorimetrically by the "melting" temperature T(m) (65 degrees C) and by the transition temperature T(m) (83 degrees C) obtained by differential scanning calorimetry for oxy-RtH1-h. The results confirm the role of the copper-dioxygen complex for the stabilization of the Hc structure in solution.     

Structure of arthropod hemocyanin

Hemocyanins are large multi-subunit copper proteins that transport oxygen in many arthropods and molluscs. The amino acid sequence of subunit a of Panulirus interruptus hemocyanin (657 residues) has been completed and fitted to the electron-density map (3.2 Å resolution). Comparison of amino acid sequence data for several different hemocyanin subunits of arthropod species indicated that the general features of the polypeptide architecture as found in spiny lobster hemocyanin occur in all arthropods. This structure must therefore be at least as old as the estimated time of divergence of crustaceans and chelicerates, 540–600 million years ago.     

Isolation and partial characterization of the N-terminal functional unit of subunit RtH1 from Rapana thomasiana grosse hemocyanin

Two different structural subunits were identified in Rapana thomasiana hemocyanin: RtH1 and RtH2. RtH1-a is the N-terminal functional unit in the subunit RtH1 and its stability toward temperature and chemical denaturation by guanidinium hydrochloride (Gdn.HCl) are studied and compared with the structural subunit RtH1 and the whole Rapana hemocyanin molecule. The conformational changes, induced by the various treatments, were monitored by CD and fluorescence spectroscopy. The critical temperatures (T(c)) for RtH1-a, the structural subunits and the native Hc, determined by fluorescence spectroscopy, coincide closely with the melting temperatures (T(m)), determined by CD spectroscopy. The free energy of stabilization in water, DeltaG(D)(H(2)O), determined from (Gdn. HCl) denaturation studies, is about two times higher for the structural subunit RtH1 and the whole hemocyanin molecule as compared to the functional unit RtH1-a. The oligomerization between the structural subunits or the eight functional units, assembled in subunit RtH1, has a stabilizing effect on the whole molecule as well as the structural subunits.     

The complete amino acid sequence of the antenna polypeptide B806-866-β from the cytoplasmic membrane of the green bacterium Chloroflexus auranliacus

The bacteriochlorophyll a-binding polypeptide B806–866-β was extracted from membranes of the green thermophilic bacterium Chloroflexus aurantiacus with chloroform/methanol/ammonium acetate. Purification of the antenna polypeptide (6.3 kDa) was achieved by chromatography on Sephadex LH-60, Whatman DE-32 and by FPLC. The complete amino acid sequence (53 amino acid residues) was determined. The B806–866-β polypeptide is sequence homologous to the antenna β-polypeptides of purple bacteria (27–40%) and exhibits the characteristic three domain structure of the B870, B800–850 and B800–820 antenna complexes. The two typical His residues, conserved in all antenna β-polypeptides of purple bacteria, were found: His-24 lies within the N-terminal hydrophilic domain and His-42 within the central hydrophobic domain. This polypeptide together with the previously described α-polypeptide form the basic structural unit of the B806–866 antenna complex from C. aurantiacus.     

cDNA Sequence, Protein Structure, and Evolution of the Single Hemocyanin from Aplysia californica, an Opisthobranch Gastropod

By protein immunobiochemistry and cDNA sequencing, we have found only a single hemocyanin polypeptide in an opisthobranch gastropod, the sea hare Aplysia californica, which contrasts with previously studied prosobranch gastropods, which express two distinct isoforms of this extracellular respiratory protein. We have cloned and sequenced the cDNA encoding the complete polypeptide of Aplysia californica hemocyanin (AcH). The cDNA comprises 11,433 bp, encompassing a 5UTR of 77 bp, a 3UTR of 1057 bp, and an open reading frame for a signal peptide of 20 amino acids plus a polypeptide of 3412 amino acids (M_r ca. 387 kDa). This polypeptide is the subunit of the cylindrical native hemocyanin (M_r ca. 8 MDa). It comprises eight different functional units (FUs: a, b, c, d, e, f, g, h) that have been identified immunobiochemically after limited proteolysis of AcH purified from the hemolymph. Each FU shows a highly conserved copper-A and copper-B site for reversible oxygen binding. FU AcH-h carries a specific C-terminal extension of ca. 100 amino acids that include two cysteines that may be utilized for disulfide bridge formation. Potential N-glycosylation sites are present in six FUs but lacking in AcH-b and AcH-c. On the basis of multiple sequence alignments, phylogenetic trees and a statistically firm molecular clock were calculated. The latter suggests that the last common ancestor of Haliotis and Aplysia lived 373±47 million years ago, in convincing agreement with fossil records from the early Devonian. However, the gene duplication yielding the two distinct hemocyanin isoforms found today in Haliotis tuberculata occurred 343±43 million years ago.[Reviewing Editor: Dr. Axel Meyer]The sequence reported in this paper has been deposited in the GenBank database under accession number AJ556169.     

The refined structure of functional unit h of keyhole limpet hemocyanin (KLH1-h) reveals disulfide bridges

Hemocyanins are multimeric oxygen-transport proteins in the hemolymph of many arthropods and mollusks. The overall molecular architecture of arthropod and molluscan hemocyanin is very different, although they possess a similar binuclear type 3 copper center to bind oxygen in a side-on conformation. Gastropod hemocyanin is a 35 nm cylindrical didecamer (2 × 10-mer) based on a 400 kDa subunit. The latter is subdivided into eight paralogous "functional units" (FU-a to FU-h), each with an active site. FU-a to FU-f contribute to the cylinder wall, whereas FU-g and FU-h form the internal collar complex. Atomic structures of FU-e and FU-g, and a 9 ? cryoEM structure of the 8 MDa didecamer are available. Recently, the structure of keyhole limpet hemocyanin FU-h (KLH1-h) was presented as a C(α) -trace at 4 ? resolution. Unlike the other seven FU types, FU-h contains an additional C-terminal domain with a cupredoxin-like fold. Because of the resolution limit of 4 ?, in some loops, the course of the protein backbone could not be established with high certainty yet. Here, we present a refined atomic structure of FU-h (KLH1-h) obtained from low-resolution refinement, which unambiguously establishes the course of the polypeptide backbone and reveals the disulfide bridges as well as the orientation of bulky amino acids.