Structures of two molluscan hemocyanin genes: significance for gene evolution.

We present here the description of genes coding for molluscan hemocyanins. Two distantly related mollusks, Haliotis tuberculata and Octopus dofleini , were studied. The typical architecture of a molluscan hemocyanin subunit, which is a string of seven or eight globular functional units (FUs, designated a to h, about 50 kDa each), is reflected by the gene organization: a series of eight structurally related coding regions in Haliotis , corresponding to FU-a to FU-h, with seven highly variable linker introns of 174 to 3,198 bp length (all in phase 1). In Octopus seven coding regions (FU-a to FU-g) are found, separated by phase 1 introns varying in length from 100 bp to 910 bp. Both genes exh…

Respiratory proteins in Sipunculus nudus--implications for phylogeny and evolution of the hemerythrin family.

Three major classes of respiratory proteins are known, hemoglobin, molluscan and arthropod hemocyanin, and hemerythrin (Hr). Similar to hemoglobin, respiratory Hr is packed into erythrocytes floating in the coelomic fluid and is only known from sipunculids, brachiopods, and priapulids. Owing to this scattered distribution, the presence of Hr is generally assumed to be the plesiomorphic condition without phylogenetic importance. By sequencing 2000 Expressed Sequence Tags (ESTs) from Sipunculus nudus, we found 75 Hr-coding ESTs assembled to 20 cDNA contigs classified as four distinct Hr isoforms: three polymeric Hrs (subunit A, A', and B) and the monomeric myo-hemerythrin (myoHr). Phylogeneti…

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 si…

Minireview: Recent progress in hemocyanin research

This review summarizes recent highlights of our joint work on the structure, evolution, and function of a family of highly complex proteins, the hemocyanins. They are blue-pigmented oxygen carriers, occurring freely dissolved in the hemolymph of many arthropods and molluscs. They are copper type-3 proteins and bind one dioxygen molecule between two copper atoms in a side-on coordination. They possess between 6 and 160 oxygen-binding sites, and some of them display the highest molecular cooperativity observed in nature. The functional properties of hemocyanins can be convincingly described by either the Monod-Wyman-Changeux (MWC) model or its hierarchical extension, the Nested MWC model; the…

Quantification of DNA and RNA: A Spectrophotometric Method

Spectophotometric quantification is a very exact method of quantifying the content of nucleic acids in any sample. The purity of the sample can be simultaneously checked very easily. Keywords: DNA; RNA; spectrophotometric quantification

One precursor, three apolipoproteins: The relationship between two crustacean lipoproteins, the large discoidal lipoprotein and the high density lipoprotein/β-glucan binding protein

The novel discoidal lipoprotein (dLp) recently detected in the crayfish, differs from other crustacean lipoproteins in its large size, apoprotein composition and high lipid binding capacity, We identified the dLp sequence by transcriptome analyses of the hepatopancreas and mass spectrometry. Further de novo assembly of the NGS data followed by BLAST searches using the sequence of the high density lipoprotein/1-glucan binding protein (HDL-BGBP) of Astacus leptodactylus as query revealed a putative precursor molecule with an open reading frame of 14.7 kb and a deduced primary structure of 4889 amino acids. The presence of an N-terminal lipid bind- ing domain and a DUF 1943 domain suggests the…

Recombinant expression of gastropod (Biomphalaria glabrata) haemoglobin

A new haemocyanin in cuttlefish (Sepia officinalis) eggs: sequence analysis and relevance during ontogeny

Abstract Background Haemocyanin is the respiratory protein of most of the Mollusca. In cephalopods and gastropods at least two distinct isoforms are differentially expressed. However, their physiological purpose is unknown. For the common cuttlefish Sepia officinalis, three isoforms are known so far, whereas for only two of them the complete mRNA sequences are available. In this study, we sequenced the complete mRNA of the third haemocyanin isoform and measured the relative expression of all three isoforms during embryogenesis to reveal a potential ontogenetic relevance. Results The cDNA of isoform 3 clearly correlates to the known Sepia officinalis haemocyanin subunits consisting of eight …

Responsiveness of metallothionein and hemocyanin genes to cadmium and copper exposure in the garden snail Cornu aspersum.

Abstract Terrestrial gastropods express metal‐selective metallothioneins (MTs) by which they handle metal ions such as Zn2+, Cd2+, and Cu+/Cu2+ through separate metabolic pathways. At the same time, they depend on the availability of sufficient amounts of Cu as an essential constituent of their respiratory protein, hemocyanin (Hc). It was, therefore, suggested that in snails Cu‐dependent MT and Hc pathways might be metabolically connected. In fact, the Cu‐specific snail MT (CuMT) is exclusively expressed in rhogocytes, a particular molluscan cell type present in the hemocoel and connective tissues. Snail rhogocytes are also the sites of Hc synthesis. In the present study, possible interacti…

Quantification of DNA and RNA: a Quantitative Visual Comparison

The analytical procedure for the quantification of DNA and RNA described is the quantitative visual comparison of an unknown sample with known standards. This is quick and easy to do, suitable for amounts of sample as small as 10 ng. Keywords: DNA; RNA; rapid quantification

Influence of Temperature, Hypercapnia, and Development on the Relative Expression of Different Hemocyanin Isoforms in the Common CuttlefishSepia officinalis

The cuttlefish Sepia officinalis expresses several hemocyanin isoforms with potentially different pH optima, indicating their reliance on efficient pH regulation in the blood. Ongoing ocean warming and acidification could influence the oxygen-binding properties of respiratory pigments in ectothermic marine invertebrates. This study examined whether S. officinalis differentially expresses individual hemocyanin isoforms to maintain optimal oxygen transport during development and acclimation to elevated seawater pCO2 and temperature. Using quantitative PCR, we measured relative mRNA expression levels of three different hemocyanin isoforms in several ontogenetic stages (embryos, hatchlings, juv…

Selecting ribosomal protein genes for invertebrate phylogenetic inferences: how many genes to resolve the Mollusca?

Summary 1. Multi-gene analyses are currently the gold standard in phylogenetics, despite limited taxon sampling. To facilitate broad taxon representation on an economically tolerable level, we optimize the gene selection for future PCR-based sampling strategies. 2. Highly expressed ribosomal proteins (RP) were sampled chiefly for molluscs, the second largest metazoan phylum with largely unknown internal relationships. Thirty-two new sequences for Lepidochitona cinerea (Polyplacophora) were integrated into a data-matrix of 79 RP genes, comprising 16 mollusc species (five classes). The resulting maximum likelihood tree was used to evaluate each single-gene tree according to its topological fi…

Tracing keratin evolution: catalog, expression patterns and primary structure of shark (Scyliorhinus stellaris) keratins.

We have studied individual keratins of an elasmobranch, the shark Scyliorhinus stellaris (the lesser-spotted dogfish). From various shark tissues, notably skin and stomach, cytoskeletal proteins were isolated and then separated by two-dimensional polyacrylamide gel electrophoresis. Using complementary keratin blot-binding assays and immunoblotting, among these proteins we identified a variety of type I and type II keratins. According to their tissue-specific expression, we distinguished Is and IIs keratins from IE and IIE keratins ("S" and "E" from "simple epithelial" and "epidermal", respectively). Guinea pig antibodies which in immunoblots specifically labeled the entire range of identifi…

Phylogenomic analyses unravel annelid evolution

Annelida, the ringed worms, is a highly diverse animal phylum that includes more than 15,000 described species and constitutes the dominant benthic macrofauna from the intertidal zone down to the deep sea. A robust annelid phylogeny would shape our understanding of animal body-plan evolution and shed light on the bilaterian ground pattern. Traditionally, Annelida has been split into two major groups: Clitellata (earthworms and leeches) and polychaetes (bristle worms), but recent evidence suggests that other taxa that were once considered to be separate phyla (Sipuncula, Echiura and Siboglinidae (also known as Pogonophora)) should be included in Annelida(1-4). However, the deep-level evoluti…

A new metallothionein gene from the giant keyhole limpet Megathura crenulata.

Abstract Metallothioneins (MTs) are small soluble proteins ubiquitously expressed in animals and plants. Different isoforms are present in deuterostomes and protostomes. They do not differ greatly in primary structure, but are clearly distinguishable. Here, I present the gene and the complete cDNA of a novel MT from the mollusk Megathura crenulata . This protein is closely related to the Cu-inducible MTs of the vineyard snail Helix pomatia , but has also some minor sequence features typical of Cd-inducible isoforms of H . pomatia and other molluscs. Overall, the deduced primary structure is similar to the known molluscan MTs, but in addition possesses an insertion of 5 amino acids not found…

Phylogenomics reveals deep molluscan relationships.

Evolutionary relationships among the eight major lineages of Mollusca have remained unresolved despite their diversity and importance. Previous investigations of molluscan phylogeny, based primarily on nuclear ribosomal gene sequences1–3 or morphological data4, have been unsuccessful at elucidating these relationships. Recently, phylogenomic studies using dozens to hundreds of genes have greatly improved our understanding of deep animal relationships5. However, limited genomic resources spanning molluscan diversity has prevented use of a phylogenomic approach. Here we use transcriptome and genome data from all major lineages (except Monoplacophora) and recover a well-supported topology for …

Spiralian phylogenomics supports the resurrection of Bryozoa comprising Ectoprocta and Entoprocta.

Phylogenetic analyses based on 79 ribosomal proteins of 38 metazoans, partly derived from 6 new expressed sequence tag projects for Ectoprocta, Entoprocta, Sipuncula, Annelida, and Acanthocephala, indicate the monophyly of Bryozoa comprising Ectoprocta and Entoprocta, 2 taxa that have been separated for more than a century based on seemingly profound morphological differences. Our results also show that bryozoans are more closely related to Neotrochozoa, including molluscs and annelids, than to Syndermata, the latter comprising Rotifera and Acanthocephala. Furthermore, we find evidence for the position of Sipuncula within Annelida. These findings suggest that classical developmental and mor…

cDNA sequences of two arylphorin subunits of an insect biliprotein: phylogenetic differences and gene duplications during evolution of hexamerins-implications for hexamer formation

Arylphorins represent a conserved class of hexameric ∼500 kDa insect hemolymph glycoproteins, rich in aromatic amino acids, which are produced in large quantities at the larval stage as reserves for metamorphosis and egg development. The recently isolated arylphorin from the moth Cerura vinula is unique in being complexed to a novel farnesylated bilin. Protein sequencing suggested the presence of two different ∼85 kDa subunits. Here, we report the complete coding sequences of two cDNAs encoding two arylphorins subunits with 67% identity and calculated physicochemical characteristics in agreement with the isolated holoprotein. Our phylogenetic analyses of the hexamerins revealed monophyletic…

Recombinant functional multidomain hemoglobin from the gastropod Biomphalaria glabrata

The extracellular hemoglobin multimer of the planorbid snail Biomphalaria glabrata, intermediate host of the human parasite Schistosoma mansoni, is presumed to be a 1.44 MDa complex of six 240 kDa polypeptide subunits, arranged as three disulfide-bridged dimers. The complete amino acid sequence of two subunit types (BgHb1 and BgHb2), and the partial sequence of a third type (BgHb3) are known. Each subunit encompasses 13 paralogus heme domains, and N-terminally a smaller plug domain responsible for subunit dimerization. We report here the recombinant expression of different functional fragments of BgHb2 in Escherichia coli, and of the complete functional subunits BgHb1 and BgHb2 in insect ce…

Abalone (Haliotis tuberculata) hemocyanin type 1 (HtH1) . Organization of the = 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 unit…

Red blood with blue-blood ancestry: Intriguing structure of a snail hemoglobin

The phylogenetic enigma of snail hemoglobin, its isolated occurrence in a single gastropod family, the Planorbidae, and the lack of sequence data, stimulated the present study. We present here the complete cDNA and predicted amino acid sequence of two hemoglobin polypeptides from the planorbid Biomphalaria glabrata (intermediate host snail for the human parasite Schistosoma mansoni ). Both isoforms contain 13 different, cysteine-free globin domains, plus a small N-terminal nonglobin “plug” domain with three cysteines for subunit dimerization (total M r ≈ 238 kDa). We also identified the native hemoglobin molecule and present here a preliminary 3D reconstruction from electron microscopical …

Hemocyanin in mollusks--a molecular survey and new data on hemocyanin genes in Solenogastres and Caudofoveata.

The most common respiratory protein of mollusks is the blue, copper-containing hemocyanin (van Holde and Miller, 1995). It is not bound to hemocytes but suspended in the hemolymph. Its molecular mass ranges from 3500 10 to 8000 10 Da (dalton) or even more (Herskovits, 1988). These differences in molecular weight are due to the fact that the basic decamers that constitute the barrel-shaped protein may aggregate to didecamers or multidecameric elongated particles (Herskovits, 1988). In cephalopods and chitons (Polyplacophora), there are exclusively decamers, whereas in protobranch bivalves and gastropods the predominantly observed aggregation state is didecamers (Herskovits, 1988; van Holde a…

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 Octo…

Randomised double-blind placebo-controlled intervention study on the nutritional efficacy of a food for special medical purposes (FSMP) and a dietary supplement in reducing the symptoms of veisalgia

ObjectiveTo assess whether the symptoms of veisalgia can be reduced by intense water supply and the intake of antioxidative supplements and plant extracts.MethodsWe performed the world’s largest randomised double-blind placebo-controlled intervention study (214 participants) on the efficacy of a food for special medical purposes (FSMP) against veisalgia symptoms. We analysed the effectiveness of: (1) an FSMP, including distinct plant extracts, vitamins and minerals, and additional (antioxidative) compounds; (2) a dietary supplement only comprising vitamins and minerals and additional (antioxidative) compounds; and (3) a placebo containing only glucose. The study followed the CONSORT (Consol…

The metal binding abilities of Megathura crenulata metallothionein (McMT) in the frame of gastropoda MTs.

Metallothioneins (MTs) are proteins that play a major role in metal homeostasis and/or detoxification in all kind of organisms. The MT gene/protein system of gastropod molluscs provides an invaluable model to study the diversification mechanisms that have enabled MTs to achieve metal-binding specificity through evolution. Most pulmonate gastropods, particularly terrestrial snails, harbor three paralogous isogenes encoding three MT isoforms with different metal binding preferences: the highly specific CdMT and CuMT isoforms, for cadmium and copper respectively, and the unspecific Cd/CuMT isoform. Megathura crenulata is a non-pulmonate gastropod in which only one MT isogene has so far been re…

Effects of temperature and CO2 on relative haemocyanin expression of the common cuttlefish Sepia officinalis

Marine tumor vaccine carriers: structure of the molluscan hemocyanins KLH and htH.

Keyhole limpet hemocyanin (KLH) is a well-established immune stimulant and hapten carrier, and Haliotis tuberculata hemocyanin (HtH) is a related product. Biologically, KLH and HtH are blue copper proteins which serve as oxygen carriers in the blood of the keyhole limpet Megathura crenulata and the abalone H. tuberculata, respectively, two marine gastropods. Both hemocyanins occur as two distinct isoforms, termed KLH1 KLH2, HtH1, and HtH2. Each of these molecules is based on a very large polypeptide chain, the subunit (molecular mass ca 400 kDa), which is folded into a series of eight globular functional units (molecular mass ca 50 kDa each). Twenty copies of this subunit form a cylindrical…

Mitochondrial genome sequence and gene order of Sipunculus nudus give additional support for an inclusion of Sipuncula into Annelida.

Abstract Background Mitochondrial genomes are a valuable source of data for analysing phylogenetic relationships. Besides sequence information, mitochondrial gene order may add phylogenetically useful information, too. Sipuncula are unsegmented marine worms, traditionally placed in their own phylum. Recent molecular and morphological findings suggest a close affinity to the segmented Annelida. Results The first complete mitochondrial genome of a member of Sipuncula, Sipunculus nudus, is presented. All 37 genes characteristic for metazoan mtDNA were detected and are encoded on the same strand. The mitochondrial gene order (protein-coding and ribosomal RNA genes) resembles that of annelids, b…

Hemocyanin genes as indicators of habitat shifts in Panpulmonata?

Hemocyanin is the primary respiratory protein for the majority of the Mollusca and therefore directly interfaces with the physiological requirements of each species and the environments to which they are adapted. Hemocyanin is therefore likely to have been evolutionarily imprinted by significant habitat shifts. In the gastropod clade Panpulmonata (>30,000 species) major realm transitions have occurred multiple times independently and may have contributed to the diversification of this group. Yet, little is known about the adaptive changes linked to these habitat shifts. In order to gain deeper insight into the evolution of panpulmonate hemocyanins and to infer possible impacts associated wi…

Developmental expression of two Haliotis asinina hemocyanin isoforms

Hemocyanins are large copper-containing respiratory proteins that play a role in oxygen transport in many molluscs. In some species only one hemocyanin isoform is present while in others two are expressed. The physiological relevance of these isoforms is unclear and the developmental and tissue-specific expression of hemocyanin genes is largely unknown. Here we show that two hemocyanin genes in the gastropod Haliotis asinina, which encode H. asinina hemocyanin (HaH1) and HaH2 isoforms, are developmentally expressed. These genes initially are expressed in a small number of mesenchyme cells at trochophore and pre-torsional veliger stages, with HaH1 expression slightly preceding HaH2. These ce…

3D-Ultrastructure, Functions and Stress Responses of Gastropod (Biomphalaria glabrata) Rhogocytes

Rhogocytes are pore cells scattered among the connective tissue of different body parts of gastropods and other molluscs, with great variation in their number, shape and size. They are enveloped by a lamina of extracellular matrix. Their most characteristic feature is the "slit apparatus", local invaginations of the plasma membrane bridged by cytoplasmic bars, forming slits of ca. 20 nm width. A slit diaphragm creates a molecular sieve with permeation holes of 20×20 nm. In blue-blooded gastropods, rhogocytes synthesize and secrete the respiratory protein hemocyanin, and it has been proposed-though not proven-that in the rare red-blooded snail species they might synthesize and secrete the he…

Blood cells and the biosynthesis of hemocyanin in Sepia embryos.

Molluscan hemocyanins: News and views

Evolution of molluscan hemocyanins as deduced from DNA sequencing

Gene structure and hemocyanin isoform HtH2 from the mollusc Haliotis tuberculata indicate early and late intron hot spots.

Abstract We have cloned and sequenced cDNAs coding for the complete primary structure of HtH2, the second hemocyanin isoform of the marine gastropod Haliotis tuberculata. The deduced protein sequence comprises 3399 amino acids, corresponding to a molecular mass of 392 kDa. It shares only 66% of structural identity with the previously analysed first isoform HtH1, and according to a molecular clock, the two isoforms of Haliotis hemocyanin separated ca. 320 million years ago. By genomic polymerase chain reaction and 5′ race, we have also sequenced the complete gene of HtH2 (18,598 bp), except of the 5′ region in front of the secreted protein. It encompasses 15 exons and 14 introns and shows se…

Phylogenomics of Lophotrochozoa with Consideration of Systematic Error.

Phylogenomic studies have improved understanding of deep metazoan phylogeny and show promise for resolving incongruences among analyses based on limited numbers of loci. One region of the animal tree that has been especially difficult to resolve, even with phylogenomic approaches, is relationships within Lophotrochozoa (the animal clade that includes molluscs, annelids, and flatworms among others). Lack of resolution in phylogenomic analyses could be due to insufficient phylogenetic signal, limitations in taxon and/or gene sampling, or systematic error. Here, we investigated why lophotrochozoan phylogeny has been such a difficult question to answer by identifying and reducing sources of sys…

Assessing divergence time of Spirulida and Sepiida (Cephalopoda) based on hemocyanin sequences

Abstract The phylogenetic position of the mesopelagic decabrachian cephalopod Spirula is still a matter of debate. Since hemocyanin has successfully been used to calibrate a molecular clock for many molluscan species, a molecular clock was calculated based on this gene with special attention to the cephalopod genera Spirula and Sepia. The obtained partial sequence comprising ca., one third (3567 bp) of the complete gene is similar to that of Sepia officinalis. The molecular clock was calibrated using the splits of Gastropoda–Cephalopoda (ca. 550 ± 50 mya) and Heterobranchia–Vetigastropoda (ca. 380 ± 10 mya). The resulting hemocyanin-based molecular clock is stable, and the estimated diverge…

Type II keratin cDNAs from the rainbow trout: implications for keratin evolution.

From a teleost fish, the rainbow trout Oncorhynchus mykiss, we have cloned and sequenced cDNAs encoding five different type II keratins. The corresponding protein spots, as separated by 2D-PAGE of trout cytoskeletal preparations, have been identified by peptide mass mapping using MALDI mass spectrometry. Three of the sequenced keratins are expressed in the epidermis (subtype IIe), and two in simple epithelia and mesenchymal cells (subtype IIs). The IIs keratins are both orthologs of human K8. This leaves unsequenced only the trace component S3 of the biochemically established trout keratin catalog. A phylogenetic tree has been constructed from a multiple alignment of the rod domains of the …

Data from: Phylogenomics of Lophotrochozoa with consideration of systematic error

Phylogenomic studies have improved understanding of deep metazoan phylogeny and show promise for resolving incongruences among analyses based on limited numbers of loci. One region of the animal tree that has been especially difficult to resolve, even with phylogenomic approaches, is relationships within Lophotrochozoa (the animal clade that includes molluscs, annelids, and flatworms among others). Lack of resolution in phylogenomic analyses could be due to insufficient phylogenetic signal, limitations in taxon and/or gene sampling, or systematic error. Here, we investigated why lophotrochozoan phylogeny has been such a difficult question to answer by identifying and reducing sources of sys…

Data from: Phylogenomics of Lophotrochozoa with consideration of systematic error

Phylogenomic studies have improved understanding of deep metazoan phylogeny and show promise for resolving incongruences among analyses based on limited numbers of loci. One region of the animal tree that has been especially difficult to resolve, even with phylogenomic approaches, is relationships within Lophotrochozoa (the animal clade that includes molluscs, annelids, and flatworms among others). Lack of resolution in phylogenomic analyses could be due to insufficient phylogenetic signal, limitations in taxon and/or gene sampling, or systematic error. Here, we investigated why lophotrochozoan phylogeny has been such a difficult question to answer by identifying and reducing sources of sys…