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.     

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.     

The First Complete cDNA Sequence of the Hemocyanin from a Bivalve,the Protobranch <Emphasis Type="Italic">Nucula nucleus</Emphasis>

By cDNA sequencing we have achieved the first, and complete, hemocyanin sequence of a bivalve (Nucula nucleus). This extracellular oxygen-binding protein consists of two immunologically distinguishable isoforms, here termed NnH1 and NnH2. They share a mean sequence identity of 61%, both contain a linear arrangement of eight paralogous, ca.50-kDa functional units (FUs a-h), and in both isoforms the C-terminal FU-h possesses an extension of ca. 100 amino acids. The cDNA of NnH1 comprises 11,090 bp, subdivided into a 5′utr of 75 bp, a 3′utr of 791 bp, and an open reading frame for a signal peptide of 19 amino acids plus a polypeptide of 3389 amino acids (M _r = 385 kDa). The cDNA of NnH2 comprises 10,849 bp, subdivided into a 5′utr of 47 bp, a 3′utr of 647 bp, and an open reading frame for a signal peptide of 16 amino acids plus a polypeptide of 3369 amino acids (M _r = 387 kDa). In contrast to other molluscan hemocyanins, which are highly glycosylated, the bivalve hemocyanin sequence exhibits only four potential N-glycosylation sites, and within both isoforms a peculiar indel is present, surrounding the highly conserved copper-binding site CuA. Phylogenetic analyses of NnH1 and NnH2, compared to the known hemocyanin sequences of gastropods and cephalopods, reveal a statistically sound closer relationship between gastropod and protobranch hemocyanin than to cephalopod hemocyanin. Assuming a molecular clock, the last common ancestor of protobranch and gastropods lived 494 million ± 50 million years ago, in conformity with fossil records from the late Cambrian. [Reviewing Editor: Dr. Rüdiger Cerff] The sequence reported in this paper has been deposited in the EMBL/GenBank database under accession number AJ786639 for NnH1 and AJ786640 for NnH2.     

Keyhole Limpet Hemocyanin Type 2 (KLH2): Detection and Immunolocalization of a Labile Functional Unit h

Keyhole limpet hemocyanin (KLH) is a mixture of two hemocyanin isoforms, termed KLH1 and KLH2. Within KLH1 eight oxygen-binding functional units (FUs), 1-a to 1-h, have been identified, in contrast to KLH2, which was previously thought to be organized in seven FUs (2-a to 2-g). By limited proteolysis of KLH2 subunits, isolation of the polypeptide fragments, and N-terminal sequencing, we have now identified an eighth FU of type h, with a molecular mass of 43 kDa. This is unusually small for a FU h from a gastropodan hemocyanin. It is also shown that KLH2 didecamers can be split into a stable and homogeneous population of decamers by dialysis against 50 mM Tris/HCl, pH 7.5, in the absence of divalent cations. Electron microscopic immunolocalization using a specific monoclonal antibody reveals that FU KLH2-h is located at the collar of the decamer.     

Subunit organization of the abalone Haliotis tuberculata hemocyanin type 2 (HtH2), and the cDNA sequence encoding its functional units d, e, f, g and h.

We have developed a HPLC procedure to isolate the two different hemocyanin types (HtH1 and HtH2) of the European abalone Haliotis tuberculata. On the basis of limited proteolytic cleavage, two-dimensional immunoelectrophoresis, PAGE, N-terminal protein sequencing and cDNA sequencing, we have identified eight different 40-60-kDa functional units (FUs) in HtH2, termed HtH2-a to HtH2-h, and determined their linear arrangement within the elongated 400-kDa subunit. From a Haliotis cDNA library, we have isolated and sequenced a cDNA clone which encodes the five C-terminal FUs d, e, f, g and h of HtH2. As shown by multiple sequence alignments, defg of HtH2 correspond structurally to defg from Octopus dofleini hemocyanin. HtH2-e is the first FU of a gastropod hemocyanin to be sequenced. The new Haliotis hemocyanin sequences are compared to their counterparts in Octopus, Helix pomatia and HtH1 (from the latter, the sequences of FU-f, FU-g and FU-h have recently been determined) and discussed in relation to the recent 2.3 A X-ray structure of FU-g from Octopus hemocyanin and the 15 A three-dimensional reconstruction of the Megathura crenulata hemocyanin didecamer from electron micrographs. This data allows, for the first time, an insight into the evolution of the two functionally different hemocyanin isoforms found in marine gastropods. It appears that they evolved several hundred million years ago within the Prosobranchia, after separation of the latter from the branch leading to the Pulmonata. Moreover, as a structural explanation for the inefficiency of the type 1 hemocyanin to form multidecamers in vivo, the additional N-glycosylation sites in HtH1 compared to HtH2 are discussed.     

Molecular cloning, expression and characterization of the zebrafish bram1 gene, a BMP receptor-associated molecule

Summary We have identified a cDNA clone encoding BMP receptor-associated molecule 1 (BRAM1) from the zebrafish expressed sequence tag (EST) database. The 2606 bp full-length bram1 cDNA was cloned, and further confirmed by nucleotide sequencing. The zebrafish sequence encodes a protein of 195 amino acids with an evolutionarily conserved MYND domain, which displays ∼ ∼98% homology with human and mouse BRAM1, and ∼ ∼64% homology with C. elegans BRA-1 and BRA-2. The bram1 gene, composed of five exons and four introns, spans ∼ ∼14 kb on linkage group 14 of the zebrafish genome. RT-PCR and whole mount in situ hybridization analyses disclosed that zebrafish BRAM1 is a maternal factor. The protein interacts directly with zebrafish BMP Receptor type IA, as observed from GST-pull down and co-immunoprecipitation assays. Furthermore, cotransfection of zebrafish BRAM1 with the corresponding BMP receptor resulted in down-regulation of BMP-mediated signaling. Our results collectively indicate that BRAM1 plays a biological role during zebrafish development.     

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.     

Cloning,characterization and TBT exposure response of CuZn superoxide dismutase from Haliotis diversicolor supertexta

The full-length cDNA and genomic DNA of a cytoplasmic copper, zinc superoxide dismutase (CuZn-sod) were cloned from the hepatopancreas of small abalone Haliotis diversicolor supertexta by RT-PCR, RACE and TAIL PCR. The full-length cytoplasmic CuZn-sod cDNA (designated sasod) comprises 984 bp. Its ORF encodes a polypeptide of 154 amino acids with a predicted molecular mass of 15.7 kDa and theoretical isoelectric point of 6.30. The deduced amino acid (designated saSOD) shares a common consensus pattern with the SODs of vertebrate and invertebrate animals. The full-length sasod genomic DNA comprises 5,574 bp, containing five exons and four introns. The splice donor and acceptor sequence of the four introns is 5′GT-AG3′. Real time quantitative PCR analysis revealed that sasod expression level in hepatopancreas of small abalone was no significant difference at 2, 6, 48 and 192 h post TBT exposure (P > 0.05). However, the sasod expression level at 12 and 24 h post TBT exposure was decreased significantly (P < 0.05).     

Molecular cloning and expression analysis of a heat shock protein (Hsp90) gene from black tiger shrimp (<Emphasis Type="Italic">Penaeus monodon</Emphasis>)

The techniques of homology cloning and anchored PCR were used to clone the Hsp90 gene from black tiger shrimp. The full length cDNA of black tiger shrimp Hsp90 (btsHsp90) contained a 5′ untranslated region (UTR) of 72 bp, an ORF (open reading frame) of 2160 bp encoding a polypeptide of 720 amino acids with an estimated molecular mass of 83-kDa and a 3′ UTR of 288 bp. The sequence of the coding region showed 90 and 84% homology with that of the Chiromantes haematocheir and Homo sapiens, respectively. Conserved signature sequences of Hsp90 gene family were found in the btsHsp90 deduced amino acid sequence. The temporal expressions of Hsp90 gene were constitutively in the black tiger shrimp tissues including liver, ovary, muscle, brain stomach, and heart, and their levels were markedly enhanced after 30-min heat treatment at 37°C. In ovarian maturation stages, the expression of btsHsp90 was strongest in the second stage, weaker in the fourth and first stage.     

The Hemocyanin of the Squid Sepioteuthis lessoniana: Structural Comparison with Other Cephalopod Hemocyanins

The subunit structure of the hemocyanin from the squid Sepioteuthis lessoniana has been characterized by analytical sedimentation. The protein exists free in the hemolymph as a 60S, multisubunit structure with a molecular mass of approximately 4.0 × 10⁶ gm/mol. At extremes of pH this can be dissociated into 10 monomers, of mass 3.8 × 10⁵ gm/mol each.At neutral pH, if 5 mM Mg²⁺ or less is present, the monomers associate to form a dimer of molecular mass 7.7 × 10⁵ gm/mol. In the pH range from 10.5 to 7.5, formation of the dimer is facilitated by the binding of one proton per monomer. If the magnesium concentration is raised to 25 mM or more, the dimers reversibly and completely associate into decamers. This process is shown to require the binding of 10 magnesium ions.In its size, subunit structure, and behavior with respect to association–dissociation processes, Sepioteuthis hemocyanin resembles the corresponding proteins of other squid much more than it does those of Octopus or Nautilus.     

Cloning and characterization of squalene synthase gene from <Emphasis Type="Italic">Fusarium fujikuroi</Emphasis> (Saw.) Wr.

The gene encoding squalene synthase (GfSQS) was cloned from Fusarium fujikuroi (Gibberella fujikuroi MP-C) and characterized. The cloned genomic DNA is 3,267 bp in length, including the 5′-untranslated region (UTR), 3′-UTR, four exons, and three introns. A noncanonical splice-site (CA-GG, or GC-AG) was found at the first intron. The open reading frame of the gene is 1,389 bp in length, corresponding to a predicted polypeptide of 462 amino acid residues with a MW 53.4 kDa. The predicted GfSQS shares at least four conserved regions involved in the enzymatic activity with the SQSs of varied species. The recombinant protein was expressed in E. coli and detected by SDS–PAGE and western blot. GC–MS analysis showed that the wild-type GfSQS could catalyze the reaction from farnesyl diphosphate (FPP) to squalene, while the mutant mGfSQS (D82G) lost total activity, supporting the prediction that the aspartate-rich motif (DTXED) in the region I of SQS is essential for binding of the diphosphate substrate.     

Keyhole limpet hemocyanin type 2 (KLH2): detection and immunolocalization of a labile functional unit h

Keyhole limpet hemocyanin (KLH) is a mixture of two hemocyanin isoforms, termed KLH1 and KLH2. Within KLH1 eight oxygen-binding functional units (FUs), 1-a to 1-h, have been identified, in contrast to KLH2, which was previously thought to be organized in seven FUs (2-a to 2-g). By limited proteolysis of KLH2 subunits, isolation of the polypeptide fragments, and N-terminal sequencing, we have now identified an eighth FU of type h, with a molecular mass of 43 kDa. This is unusually small for a FU h from a gastropodan hemocyanin. It is also shown that KLH2 didecamers can be split into a stable and homogeneous population of decamers by dialysis against 50 mM Tris/HCl, pH 7.5, in the absence of divalent cations. Electron microscopic immunolocalization using a specific monoclonal antibody reveals that FU KLH2-h is located at the collar of the decamer.     

Analysis of <Emphasis Type="Italic">Acropora muricata</Emphasis> Calmodulin (<Emphasis Type="Italic">CaM</Emphasis>) Indicates That Scleractinian Corals Possess the Ancestral Exon/Intron Organization of the Eumetazoan <Emphasis Type="Italic">CaM</Emphasis> Gene

Calmodulin (CaM), belonging to the tropinin C (TnC) superfamily, is one of the calcium-binding proteins that are highly conserved in their protein and gene structure. Based on the structure comparison among published vertebrate and invertebrate CaM, it is proposed that the ancestral form of eumetazoan CaM genes should have five exons and four introns (four-intron hypothesis). In this study, we determined the gene structure of CaM in the coral Acropora muricata, an anthozoan cnidarian representing the basal position in animal evolution. A CaM clone was isolated from a cDNA library constructed from the spawned eggs of A. muricata. This clone was composed of 908 nucleotides, including 162 base pairs (bp) of 5′-untranslated region (UTR), 296 bp of 3′-UTR, and an open reading frame 450 bp in length. The deduced amino acid indicated that the Acropora CaM protein is identical to that of the actiniarian, Metridinium senile, and has four putative calcium-binding domains highly similar to those of other vertebrate or invertebrate CaMs. Southern blot analysis revealed that Acropora CaM is a putative single-copy gene in the nuclear genome. Genomic sequencing showed that Acropora CaM was composed of five exons and four introns, with intron II not corresponding to any region in the actiniarian CaM gene, which possesses only four exons and three introns. Our results highlight that the coral CaM gene isolated from A. muricata has four introns at the predicted positions of the early metazoan CaM gene organization, providing the first evidence from the basal eumetazoan phylum to support the four-intron hypothesis.     

Genetic diversity of isolated populations of Nautilus pompilius (Mollusca, Cephalopoda) in the Great Barrier Reef and Coral Sea

Nautilus species are the only remaining cephalopods with an external shell. Targeted heavily by the shell trade across their distribution area, these species have a poorly known population structure and genetics. Molecular techniques have been used to assess levels of inter- and intra-population genetic diversity in isolated populations of Nautilus in the northern sections of the Great Barrier Reef (GBR), Australia and in the Coral Sea. Distinct populations, physically separated by depths in excess of 1,000 m were examined. RAPD analysis of genetic differences showed limited differentiation of the “Northern GBR” populations and the “Coral Sea” populations. Discrimination between the two geographic groups was observed from these data. In addition, partial sequencing of the CoxI gene region, yielded 575 bp of sequence, which was aligned for 43 samples and phylogenetic trees constructed to examine genetic relationships. Two distinct clades were resolved in the resulting trees, representing the “Northern GBR” and “Coral Sea” population groups. Inter- and intra-population relationships are presented and discussed. The differentiation of the Nautilus populations from the Northern section of the Great Barrier Reef and those from the Coral Sea were supported by two distinctly different methodologies and the significance of this separation and the potential evolutionary divergence of these two population groups is discussed.     

Patterns of nuclear and mitochondrial DNA variation in Iberian populations of <Emphasis Type="Italic">Emys orbicularis</Emphasis> (Emydidae): conservation implications

The European pond turtle (Emys orbicularis) is threatened and in decline in several regions of its natural range, due to habitat loss combined with population fragmentation. In this work, we have focused our efforts on studying the genetic diversity and structure of Iberian populations with a fine-scale sampling (254 turtles in 10 populations) and a representation from North Africa and Balearic island populations. Using both nuclear and mitochondrial markers (seven microsatellites, ∼1048 bp nDNA and ∼1500 bp mtDNA) we have carried out phylogenetic and demographic analyses. Our results show low values of genetic diversity at the mitochondrial level although our microsatellite dataset revealed relatively high levels of genetic variability with a latitudinal genetic trend decreasing from southern to northern populations. A moderate degree of genetic differentiation was estimated for Iberian populations (genetic distances, F _ST values and clusters in the Bayesian analysis). The results in this study combining mtDNA and nDNA, provide the most comprehensive population genetic data for E. orbicularis in the Iberian Peninsula. Our results suggest that Iberian populations within the Iberian–Moroccan lineage should be considered as a single subspecies with five management units, and emphasize the importance of habitat management rather than population reinforcement (i.e. captive breeding and reintroduction) in this long-lived species.     

Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis)

A tissue-specific cDNA library was constructed using polyA⁺ RNA from pituitary glands of the Indian catfishHeteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence ofH. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.     

Glycan structures of the structural subunit (HtH1) of <Emphasis Type="Italic">Haliotis tuberculata</Emphasis> hemocyanin

The oligosaccharide structures of the structural subunit HtH1 of Haliotis tuberculata hemocyanin (HtH) were studied by mass spectral sequence analysis of the glycans. The proposed structures are based on MALDI-TOF-MS data before and after treatment with the specific exoglycosidases β1-3,4,6-galactosidase and α1-6(>2,3,4) fucosidase followed by sequence analysis via electrospray ionization MS/MS-spectra. In total, 15 glycans were identified as a highly heterogeneous group of structures. As in most molluscan hemocyanins, the glycans of HtH1 contain a terminal MeHex, but more interestingly, a novel structural motif was observed: MeHex[Fuc(α1-3)-]GlcNAc, including thus MeHex and (α1-3)-Fuc residues being linked to an internal GlcNAc residue. While the functional unit (FU) c (HtH1-c) is completely lacking any potential glycosylation site, FU-h possesses a second exposed sugar attachment site between beta-strands 8 and 9 within the beta sandwich domain compared to the other FUs. The glycosylation pattern/sites show a high degree of conservation. In FU-h two prominent potential glycosylation sites can be detected. The finding that HtH1 is not able to form multidecameric structures in vivo could be explained by the presence of the exposed glycan on the surface of FU-h.     

Molecular characterization,chromosomal localization and association analysis with back-fat thickness of porcine <Emphasis Type="Italic">LPIN2</Emphasis> and <Emphasis Type="Italic">LPIN3</Emphasis>

The products of mammalian LPIN2 and LPIN3 are phosphatidate phosphatase type 1 enzymes, which play an important role in the de novo biosynthesis of triacylglycerol, phosphatidylcholine and phosphatidylethanolamine. In this study, we obtained a 2,985-bp cDNA sequence of porcine LPIN2, which contains a 2,676-bp open reading frame flanked by an 11-bp 5′UTR and a 298-bp 3′UTR, and a 2,843-bp cDNA sequence of porcine LPIN3, which contains a 111-bp 5′UTR, a 2,580-bp open reading frame and a 152-bp 3′UTR. RT-PCR analysis showed that both LPIN2 and LPIN3 mRNA were ubiquitously expressed with a very high level in liver. By using the somatic cell hybrid panel (SCHP) and the radiation hybrid (IMpRH) panel, porcine LPIN2 and LPIN3 were assigned to 6q24-(1/2)q31 and 17(1/2)q21-q23, respectively. One T2193C single nucleotide polymorphism in LPIN2 was identified and was detected by Hin6I PCR-RFLP. Association analysis showed that different genotypes of LPIN2 were associated with back-fat thickness between the 6th and 7th ribs (P < 0.01).