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María Inés Becker - One of the best experts on this subject based on the ideXlab platform.
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N-Glycosylation of mollusk Hemocyanins contributes to their structural stability and immunomodulatory properties in mammals
The Journal of biological chemistry, 2019Co-Authors: Michelle L. Salazar, Javiera Villar, Augusto Manubens, Jose Jimenez, Maira Rivera, Mauricio Baez, María Inés BeckerAbstract:Hemocyanins are widely used as carriers, adjuvants, and nonspecific immunostimulants in cancer because they promote Th1 immunity in mammals. Hemocyanins also interact with glycan-recognizing innate immune receptors on antigen-presenting cells, such as the C-type lectin immune receptors mannose receptor (MR), macrophage galactose lectin (MGL), and the Toll-like receptors (TLRs), stimulating proinflammatory cytokine secretion. However, the role of N-linked oligosaccharides on the structural and immunological properties of Hemocyanin is unclear. Mollusk Hemocyanins, such as Concholepas concholepas (CCH), Fissurella latimarginata (FLH), and Megathura crenulata (KLH), are oligomeric glycoproteins with complex dodecameric quaternary structures and heterogeneous glycosylation patterns, primarily consisting of mannose-rich N-glycans. Here, we report that enzyme-catalyzed N-deglycosylation of CCH, FLH, and KLH disrupts their quaternary structure and impairs their immunogenic effects. Biochemical analyses revealed that the deglycosylation does not change Hemocyanin secondary structure but alters their refolding mechanism and dodecameric structure. Immunochemical analyses indicated decreased binding of N-deglycosylated Hemocyanins to the MR and MGL receptors and TLR4 and reduced endocytosis concomitant with an impaired production of tumor necrosis factor α, and interleukins 6 and 12 (IL-6 and IL-12p40, respectively) in macrophages. Evaluating the function of N-deglycosylated Hemocyanins in the humoral immune response and their nonspecific antitumor effects in the B16F10 melanoma model, we found that compared with native Hemocyanins N-deglycosylated Hemocyanins elicited reduced antibody titers, as well as partially diminished antitumor effects and altered carrier activities. In conclusion, the glycan content of Hemocyanins is, among other structural characteristics, critically required for their immunological activities and should be considered in biomedical applications.
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molluskan Hemocyanins activate the classical pathway of the human complement system through natural antibodies
Frontiers in Immunology, 2017Co-Authors: Javier Pizarrobauerle, Mercedes N Lopez, Flavio Salazaronfray, Ismael Maldonado, Eduardo Sosoniukroche, Gerardo Vallejos, Lorena Aguilarguzman, Carolina Valck, Arturo Ferreira, María Inés BeckerAbstract:Molluskan Hemocyanins are enormous oxygen-carrier glycoproteins that show remarkable immunostimulatory properties when inoculated in mammals, such as the generation of high levels of antibodies, a strong cellular reaction and generation of non-specific anti-tumor immune responses in some types of cancer, particularly for superficial bladder cancer. These proteins have the ability to bias the immune response towards a Th1 phenotype. However, despite all their current uses with beneficial clinical outcomes, a clear mechanism explaining these properties is not available. Taking into account reports of natural antibodies against the Hemocyanin of the gastropod Megathura crenulata (KLH) in humans as well as other vertebrate species, we report here for the first time, the presence, in sera from unimmunized healthy donors, of antibodies recognizing, in addition to KLH, two other Hemocyanins with documented immunomodulatory capacities: CCH and FLH (the Hemocyanins from the gastropods Concholepas concholepas and Fissurella latimarginata, respectively). Through an ELISA screening, we found IgM and IgG antibodies reactive with these Hemocyanins. When the capacity of these antibodies to bind deglycosylated Hemocyanins was studied, no decreased interaction was detected. Moreover, in the case of FLH, deglycosylation increased antibody binding. We evaluated through an in vitro complement deposition assay whether these antibodies activated the classical pathway of the human complement system. The results showed that all three Hemocyanins and their deglycosylated counterparts elicited this activation, mediated by C1 binding to immunoglobulins. Thus, this work contributes to the understanding on how the complement system could participate in the immunostimulatory properties of Hemocyanins, through natural, complement-activating antibodies reacting with these proteins. Although a role for carbohydrates cannot be completely ruled out, in our experimental setting, glycosylation status had a limited effect. Finally, our data open possibilities for further studies leading to the design of improved Hemocyanin-based research tools for diagnosis and immunotherapy.
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Hemocyanins Stimulate Innate Immunity by Inducing Different Temporal Patterns of Proinflammatory Cytokine Expression in Macrophages
Journal of immunology (Baltimore Md. : 1950), 2016Co-Authors: Ta-ying Zhong, Javiera Villar, Miguel Del Campo, Sergio Arancibia, Augusto Manubens, Raimundo Born, Ricardo Tampe, María Inés BeckerAbstract:Hemocyanins induce a potent Th1-dominant immune response with beneficial clinical outcomes when used as a carrier/adjuvant in vaccines and nonspecific immunostimulant in cancer. However, the mechanisms by which Hemocyanins trigger innate immune responses, leading to beneficial adaptive immune responses, are unknown. This response is triggered by a proinflammatory signal from various components, of which macrophages are an essential part. To understand how these proteins influence macrophage response, we investigated the effects of mollusks Hemocyanins with varying structural and immunological properties, including Hemocyanins from Concholepas concholepas, Fissurella latimarginata, and Megathura crenulata (keyhole limpet Hemocyanin), on cultures of peritoneal macrophages. Hemocyanins were phagocytosed and slowly processed. Analysis of this process showed differential gene expression along with protein levels of proinflammatory markers, including IL-1β, IL-6, IL-12p40, and TNF-α. An extended expression analysis of 84 cytokines during a 24-h period showed a robust proinflammatory response for F. latimarginata Hemocyanin in comparison with keyhole limpet Hemocyanin and C. concholepas Hemocyanin, which was characterized by an increase in the transcript levels of M1 cytokines involved in leukocyte recruitment. These cytokine genes included chemokines (Cxcl1, Cxcl3, Cxcl5, Ccl2, and Ccl3), ILs (Il1b and Ifng), growth factors (Csf2 and Csf3), and TNF family members (Cd40lg). The protein levels of certain cytokines were increased. However, every Hemocyanin maintains downregulated key M2 cytokine genes, including Il4 and Il5. Collectively, our data demonstrate that Hemocyanins are able to trigger the release of proinflammatory factors with different patterns of cytokine expression, suggesting differential signaling pathways and transcriptional network mechanisms that lead to the activation of M1-polarized macrophages.
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Hemocianinas, una herramienta inmunológica de la biomedicina actual
Revista medica de Chile, 2011Co-Authors: Miguel Del Campo, Jorge Ferreira, Pablo De ,ioannes, Sergio Arancibia, Esteban Nova, Fabián Salazar, Andrea González, Bruno Moltedo, Augusto Manubens, María Inés BeckerAbstract:Hemocyanins, the giant oxygen transporter glycoproteins of diverse mollusks, are xenogenic to the mammalian immune system and they display a remarkable immuno-genicity. Therefore they are ideal non-specific immunostimulants to treat some types of cancer. They are used as an alternative therapy for superficial urinary bladder cancer (SBC), that has been traditionally treated with Bacillus Calmette-Guerin (BCG). In contrast to BCG, Hemocyanins do not cause side-effects, making them ideal for long-term repetitive treatments. Hemocyanins have also been exploited as carriers to develop antibodies against hapten molecules and peptides, as carrier-adjuvants for cutting-edge vaccines against cancer, drug addiction, and infectious diseases and in the diagnosis of parasitic diseases, such as Schistosomiasis. The Hemocyanin from Megathura crenulata, also known as keyhole limpet Hemocyanin (KLH), has been used for over thirty years for the purposes described above. More recently, hemoc yanin from the Chilean mollusk Concholepas concholepas (CCH) has proved to be a reliable alternative to KLH, either as carrier protein, and as a likely alternative for the immunotherapy of SBC. Despite KLH and CCH differ significantly in their origin and structure, we have demonstrated that both Hemocyanins stimulate the immune system of mammals in a similar way by inducing a potent Thl-polarized cellular and humoral response.
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Immunotherapeutic effect of Concholepas Hemocyanin in the murine bladder cancer model: evidence for conserved antitumor properties among Hemocyanins.
The Journal of urology, 2006Co-Authors: Bruno Moltedo, Pablo De ,ioannes, Fernando Faunes, Denise Haussmann, Alfredo E De Ioannes, Javier Puente, María Inés BeckerAbstract:We determined the antitumor properties of a newly available Hemocyanin obtained from the Chilean gastropod Concholepas concholepas (Biosonda Corp., Santiago, Chile) in a syngeneic heterotopic mouse bladder carcinoma model. Since keyhole limpet Hemocyanin (Pierce, Rockford, Illinois) is used increasingly in biomedicine as a carrier for vaccines and an immunotherapeutic agent for bladder transitional cell carcinoma, there is a growing interest in finding new substances that share its potent immunomodulatory properties. Considering that keyhole limpet Hemocyanin and Concholepas concholepas Hemocyanin differ significantly, it was not possible to predict a priori the antitumor properties of Concholepas concholepas Hemocyanin. C3H/He mice were primed with Concholepas concholepas Hemocyanin before subcutaneous implantation of mouse bladder tumor-2 cells. Treatment consisted of a subcutaneous dose of Concholepas concholepas Hemocyanin (1 mg or 100 mug) at different intervals after implantation. Keyhole limpet Hemocyanin and phosphate buffered saline served as positive and negative controls, respectively. In addition, experiments were designed to determine which elements of the immune response were involved in its adjuvant immunostimulatory effect. Mice treated with Concholepas concholepas Hemocyanin showed a significant antitumor effect, as demonstrated by decreased tumor growth and incidence, prolonged survival and lack of toxic effects. These effects were similar to those achieved with keyhole limpet Hemocyanin. We found that each Hemocyanin increased natural killer cell activity but the effect of Concholepas concholepas Hemocyanin was stronger. Analysis of serum from treated mice showed an increased interferon-gamma and low interleukin-4, which correlated with antibody isotypes, confirming that Hemocyanins induce a T helper type 1 cytokine profile. To our knowledge our results are the first demonstration of the antitumor effect of a Hemocyanin other than keyhole limpet Hemocyanin. They suggest that this is an ancient conserved immunogenic mechanism shared by those Hemocyanins that is able to enhance T helper type 1 immunity and lead to antitumor activity. Therefore, Concholepas concholepas Hemocyanin may be an alternative candidate for providing safe and effective immunotherapy for human superficial bladder cancer.
Heinz Decker - One of the best experts on this subject based on the ideXlab platform.
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The Cupredoxin-like Domains in Hemocyanins
Biochemical Journal, 2010Co-Authors: Elmar Jaenicke, Jurgen Markl, Heinz Decker, Kay Büchler, Thomas Rm BarendsAbstract:Hemocyanins are multimeric oxygen transport proteins which bind oxygen to type 3 copper sites. Arthropod Hemocyanins are composed of 75 kDa subunits, while molluscan Hemocyanins contain 350-400 kDa subunits with 7-8 different 50 kDa “functional units” (FU‑a to FU‑h), each with an active site. FU‑h possesses a tail of 100 amino acids not present in the other FUs. Here we show by X-ray crystallography of FU-h of keyhole limpet Hemocyanin isoform 1 (KLH1) that the structure of the tail domain is cupredoxin-like but contains no copper. The copper-free domain #3 in arthropod Hemocyanin subunits has recently also been reinterpreted as being cupredoxin-like. We propose that the cupredoxin-like domain in both Hemocyanin types once served to upload copper to the active site of the oxygen-binding domain.
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Is activated Hemocyanin instead of phenoloxidase involved in immune response in woodlice
Developmental and comparative immunology, 2009Co-Authors: Elmar Jaenicke, Heinz Decker, Sebastian Fraune, Sandra May, Pinar Irmak, René Augustin, Christian Meesters, Martin ZimmerAbstract:In the Common woodlouse Porcellio scaber (Crustacea: Isopoda: Oniscidea), experimental immune challenge did not induce the expression of pro-phenoloxidase that, in most other invertebrates studied thus far, can be activated into phenoloxidase via an activation cascade upon immune challenge. Instead, Porcellio Hemocyanin proved to exhibit catecholoxidase activity upon activation. However, none of the activating factors known from other invertebrates other than SDS-treatment resulted in activation of Hemocyanin into a functional phenoloxidase in vitro. The distinct characteristics of isopod Hemocyanin are reflected by the quaternary structure of the Hemocyanin dodecamers that differs from that of other crustacean Hemocyanins in that the two hexamers share a common 3-fold rotation axis and have an angular offset of 60° against each other. Accordingly, the sequence of Porcellio Hemocyanin can be distinguished clearly from other crustacean Hemocyanins and in a phylogenetic analysis forms a cluster with other isopod and amphipod Hemocyanins. We propose a peracarid-type Hemocyanin that may have evolved in response to its required multiple functions in respiration and immune response, while phenoloxidase sensu strictu is lacking.
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Kinetic properties of catecholoxidase activity of tarantula Hemocyanin
The FEBS journal, 2008Co-Authors: Elmar Jaenicke, Heinz DeckerAbstract:Phenoloxidases occur in almost all organisms, being essentially involved in various processes such as the immune response, wound healing, pigmentation and sclerotization in arthropods. Many Hemocyanins are also capable of phenoloxidase activity after activation. Notably, in chelicerates, a phenoloxidase has not been identified in the hemolymph, and thus Hemocyanin is assumed to be the physiological phenoloxidase in these animals. Although phenoloxidase activity has been shown for Hemocyanin from several chelicerate species, a characterization of the enzymatic properties is still lacking. In this article, the enzymatic properties of activated Hemocyanin from the tarantula Eurypelma californicum are reported, which was activated by SDS at concentrations above the critical micellar concentration. The activated state of Eurypelma Hemocyanin is stable for several hours. Dopamine is a preferred substrate of activated Hemocyanin. For dopamine, a KM value of 1.45 ± 0.16 mm and strong substrate inhibition at high substrate concentrations were observed. Typical inhibitors of catecholoxidase, such as l-mimosine, kojic acid, tyramine, phenylthiourea and azide, also inhibit the phenoloxidase activity of activated Hemocyanin. This indicates that the activated Hemocyanin behaves as a normal phenoloxidase.
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Minireview: Recent progress in Hemocyanin research.
Integrative and comparative biology, 2007Co-Authors: Heinz Decker, Bernhard Lieb, Elmar Jaenicke, Nadja Hellmann, Ulrich Meissner, Jurgen MarklAbstract: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 latter takes into account the structural hierarchies in the oligomeric architecture. Recently, we applied these models to interpret the influence of allosteric effectors in detailed terms. Effectors shift the allosteric equilibria but have no influence on the oxygen affinities characterizing the various conformational states. We have shown that Hemocyanins from species living at different environmental temperatures have a cooperativity optimum at the typical temperature of their natural habitat. Besides being oxygen carriers, some Hemocyanins function as a phenoloxidase (tyrosinase/catecholoxidase) which, however, requires activation. Chelicerates such as spiders and scorpions lack a specific phenoloxidase, and in these animals activated Hemocyanin might catalyse melanin synthesis in vivo. We propose a similar activation mechanism for arthropod Hemocyanins, molluscan Hemocyanins and tyrosinases: amino acid(s) that sterically block the access of phenolic compounds to the active site have to be removed. The catalysis mechanism itself can now be explained on the basis of the recently published crystal structure of a tyrosinase. In a series of recent publications, we presented the complete gene and primary structure of various Hemocyanins from different molluscan classes. From these data, we deduced that the molluscan Hemocyanin molecule evolved ca. 740 million years ago, prior to the separation of the extant molluscan classes. Our recent advances in the 3D cryo-electron microscopy of Hemocyanins also allow considerable insight into the oligomeric architecture of these proteins of high molecular mass. In the case of molluscan Hemocyanin, the structure of the wall and collar of the basic decamers is now rapidly becoming known in greater detail. In the case of arthropod Hemocyanin, a 10-A structure and molecular model of the Limulus 8 × 6mer shows the amino acids at the various interfaces between the eight hexamers, and reveals histidine-rich residue clusters that might be involved in transferring the conformational signals establishing cooperative oxygen binding.
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Cooperative Transition in the Conformation of 24-Mer Tarantula Hemocyanin upon Oxygen Binding
The Journal of biological chemistry, 2005Co-Authors: Wolfgang Erker, Ute Beister, Heinz DeckerAbstract:Abstract Hemocyanins are large respiratory proteins of arthropods and mollusks, which bind oxygen with very high cooperativity. Here, we investigated the relationship between oxygen binding and structural changes of the 24-mer tarantula Hemocyanin. Oxygen binding of the Hemocyanin was detected following the fluorescence intensity of the intrinsic tryptophans. Under the same conditions, structural changes were monitored by the non-covalently bound fluorescence probe Prodan (6-propionyl-2-(dimethylamino)-naphthalene), which is very sensitive to its surroundings. Upon oxygen binding of the Hemocyanin a red shift of 5 nm in the emission maximum of the label was observed. A comparison of oxygen binding curves recorded with tryptophan and Prodan emission revealed that structural changes in tarantula Hemocyanin lag behind oxygen binding at the beginning of oxygenation. Analyses based on the nested two-state model, which describes cooperative oxygen binding of Hemocyanins, indicated that the transition monitored by Prodan emission is closely related to one of the four conformations (rR) predicted for the allosteric unit. Earlier, the allosteric unit of tarantula Hemocyanin was found to be the 12-mer half-molecule. Here, fluorescence titration revealed that the number of Prodan binding sites/24-mer tarantula Hemocyanin is ∼2, matching the number of allosteric units/Hemocyanin. Based on the agreement between oxygen binding curves and fluorescence titration we concluded that Prodan monitors a conformational transition of the allosteric unit.
Thorsten Burmester - One of the best experts on this subject based on the ideXlab platform.
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Identification and characterisation of Hemocyanin of the fish louse Argulus (Crustacea: Branchiura)
Journal of Comparative Physiology B, 2016Co-Authors: Pauline Pinnow, Christian Pick, Andrej Fabrizius, Thorsten BurmesterAbstract:Hemocyanin transports oxygen in the hemolymph of many arthropod species. Within the crustaceans, this copper-containing protein was thought to be restricted to Malacostraca, while other crustacean classes were assumed to employ hemoglobin or lack any respiratory protein. Only recently it has become evident that Hemocyanins also occur in Remipedia and Ostracoda. Here we report for the first time the identification and characterisation of Hemocyanin in the fish louse Argulus , which belongs to the class of Branchiura. This finding indicates that Hemocyanin was the principal oxygen carrier in the stem lineage of the pancrustaceans, but has been lost independently multiple times in crustacean taxa. We obtained the full-length cDNA sequences of two Hemocyanin subunits of Argulus foliaceus by a combination of RT-PCR, RACE and Illumina sequencing of the transcriptome. In addition, one full-length and one partial cDNA sequence were derived from the transcriptome data of Argulus siamensis. Western blot analysis confirmed the presence of at least two Hemocyanin subunits in A. foliaceus , which are expressed at the mRNA level at a 1:3.5 ratio. The addition to the branchiuran Hemocyanin subunits to a multiple sequence alignment of arthropod, Hemocyanins improved the phylogenetic resolution within the pancrustacean Hemocyanins. Malacostracan, ostracod and branchiuran Hemocyanins are distinct from the hexapod and remipede Hemocyanins, reinforcing the hypothesis of a close relationship of Remipedia and Hexapoda. Notably, the ostracod Hemocyanins are paraphyletic with respect to the branchiuran Hemocyanins, indicating ancient divergence and differential loss of distinct subunit types.
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Occurrence of Hemocyanin in Ostracod Crustaceans
Journal of Molecular Evolution, 2014Co-Authors: Julia C. Marxen, Todd H Oakley, Christian Pick, Thorsten BurmesterAbstract:Hemocyanin is a copper-containing protein that transports O_2 in the hemolymph of many arthropod species. Within the crustaceans, Hemocyanin appeared to be restricted to Malacostraca but has recently been identified in Remipedia. Here, we report the occurrence of Hemocyanin in ostracods, indicating that this respiratory protein is more widespread within crustaceans than previously thought. By analyses of expressed sequence tags and by RT-PCR, we obtained four full length and nine partial Hemocyanin sequences from six of ten investigated ostracod species. Hemocyanin was identified in Myodocopida ( Actinoseta jonesi , Cypridininae sp., Euphilomedes morini , Skogsbergia lerneri , Vargula tsujii ) and Platycopida ( Cytherelloidea californica ) but not in Podocopida. We found no evidence for the presence of hemoglobin in any of these ostracod species. Like in other arthropods, we identified multiple Hemocyanin subunits (up to six) to occur in a single ostracod species. Bayesian phylogenetic analyses showed that ostracod Hemocyanin subunit diversity evolved independently from that of other crustaceans. Ostracod Hemocyanin subunits were found paraphyletic, with myodocopid and platycopid subunits forming distinct clades within those of the crustaceans. This pattern suggests that ostracod Hemocyanins originated from distinct subunits in the pancrustacean stemline.
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Structure, diversity and evolution of myriapod Hemocyanins
The FEBS journal, 2014Co-Authors: Christian Pick, Jurgen Markl, Samantha Scherbaum, Elöd Hegedüs, Andreas Bernhard Meyer, Michael Saur, Ruben Neumann, Thorsten BurmesterAbstract:Oxygen transport in the hemolymph of many arthropods is mediated by Hemocyanins, large copper-containing proteins that are well-studied in Chelicerata and Crustacea, but had long been considered unnecessary in the subphylum of Myriapoda. Only recently has it become evident that Hemocyanins are present in Scutigeromorpha (Chilopoda) and Spirostreptida (Diplopoda). Here we present evidence for a more widespread occurrence of Hemocyanin in the myriapods. By means of RT-PCR, western blotting and database searches, Hemocyanins were identified in the symphylans Hanseniella audax and Symphylella vulgaris, the chilopod Scolopendra subspinipes dehaani and the diplopod Polydesmus angustus. No Hemocyanins were found in the diplopods Polyxenus lagurus, Cylindroiulus punctatus, Glomeris marginata, Glomeris pustulata and Arthrosphaera brandtii, or the chilopods Lithobius forficatus, Geophilus flavus and Strigamia maritima. This suggests multiple independent losses in myriapod taxa. Two independent Hemocyanin subunits were found that were already present in the myriapod stem line. We specifically investigated the structure of the Hemocyanin of P. angustus, which consists of three distinct subunits that occur in an approximately equimolar ratio. As deduced by 3D electron microscopy, the quaternary structure is a 3 × 6-mer that resembles the half structure of the 6 × 6-mer Hemocyanin from Scutigera coleoptrata. It was analyzed more closely by homology modeling of 1 × 6-mers and their rigid-body fitting to the electron density map of the 3 × 6-mer. In addition, we obtained the cDNA sequence of a putative myriapod phenoloxidase. Phenoloxidases are related to the arthropod Hemocyanins, but diverged before radiation of the arthropod subphyla.
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Evolution and Adaptation of Hemocyanin Within Spiders
Spider Ecophysiology, 2012Co-Authors: Thorsten BurmesterAbstract:Spiders and most other chelicerates use Hemocyanin for the transport of oxygen in the hemolymph or may lack any respiratory protein. Arthropod Hemocyanins are large, oligo-hexameric copper proteins (n × 6). The evolution of Hemocyanin subunit composition and assembly is essentially taxon specific. The early chelicerate Hemocyanin was probably a simple homo-hexamer, as found today in sea spiders (Pycnogonida). The first Hemocyanin oligo-hexamer, consisting of probably four distinct subunit types, emerged already before the separation of Arachnida and Xiphosura. Further gene duplications led to the formation of a 4 × 6-mer Hemocyanin in the arachnids. The xiphosuran (horseshoe crab) 8 × 6-mer Hemocyanin evolved independently. A 4 × 6-mer Hemocyanin, composed of seven distinct subunit types named a through g, was the original respiratory protein of the first spiders. This Hemocyanin type is still present in the Mygalomorphae (Orthognatha) and is conserved in many Araneomorphae (Labidognatha). However, in the entelegyne spiders of the so-called RTA clade, gene losses and independent gene duplications gave rise to a novel 2 × 6-mer Hemocyanin, which consists of six distinct g-type subunits that evolved 230–120 million years ago. However, in contrast to their crustacean paralogs, chelicerate Hemocyanins show only little variations in subunit compositions throughout the evolution and the life cycle. The high number of distinct subunits in the spider Hemocyanin permits cooperative binding and thus an efficient transport of oxygen. Spider Hemocyanins are multifunctional proteins: In addition to the oxygen transport, Hemocyanin may also be involved in immune response and transport of the molting hormone ecdysone.
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The diversity and evolution of chelicerate Hemocyanins
BMC Evolutionary Biology, 2012Co-Authors: Peter Rehm, Jurgen Markl, Christian Pick, Janus Borner, Thorsten BurmesterAbstract:Background Oxygen transport in the hemolymph of many arthropod species is facilitated by large copper-proteins referred to as Hemocyanins. Arthropod Hemocyanins are hexamers or oligomers of hexamers, which are characterized by a high O_2 transport capacity and a high cooperativity, thereby enhancing O_2 supply. Hemocyanin subunit sequences had been available from horseshoe crabs (Xiphosura) and various spiders (Araneae), but not from any other chelicerate taxon. To trace the evolution of Hemocyanins and the emergence of the large Hemocyanin oligomers, Hemocyanin cDNA sequences were obtained from representatives of selected chelicerate classes. Results Hemocyanin subunits from a sea spider, a scorpion, a whip scorpion and a whip spider were sequenced. Hemocyanin has been lost in Opiliones, Pseudoscorpiones, Solifugae and Acari, which may be explained by the evolution of trachea (i.e., taxon Apulmonata). Bayesian phylogenetic analysis was used to reconstruct the evolution of Hemocyanin subunits and a relaxed molecular clock approach was applied to date the major events. While the sea spider has a simple hexameric Hemocyanin, four distinct subunit types evolved before Xiphosura and Arachnida diverged around 470 Ma ago, suggesting the existence of a 4 × 6mer at that time. Subsequently, independent gene duplication events gave rise to the other distinct subunits in each of the 8 × 6mer Hemocyanin of Xiphosura and the 4 × 6mer of Arachnida. The Hemocyanin sequences were used to infer the evolutionary history of chelicerates. The phylogenetic trees support a basal position of Pycnogonida, a sister group relationship of Xiphosura and Arachnida, and a sister group relationship of the whip scorpions and the whip spiders. Conclusion Formation of a complex Hemocyanin oligomer commenced early in the evolution of euchelicerates. A 4 × 6mer Hemocyanin consisting of seven subunit types is conserved in most arachnids since more than 400 Ma, although some entelegyne spiders display selective subunit loss and independent oligomerization. Hemocyanins also turned out to be a good marker to trace chelicerate evolution, which is, however, limited by the loss of Hemocyanin in some taxa. The molecular clock calculations were in excellent agreement with the fossil record, also demonstrating the applicability of Hemocyanins for such approach.
Jurgen Markl - One of the best experts on this subject based on the ideXlab platform.
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structure of mega Hemocyanin reveals protein origami in snails
Structure, 2015Co-Authors: Christos Gatsogiannis, Jurgen Markl, Oliver Hofnagel, Stefan RaunserAbstract:Mega-Hemocyanin is a 13.5 MDa oxygen transporter found in the hemolymph of some snails. Similar to typical gastropod Hemocyanins, it is composed of 400 kDa building blocks but has additional 550 kDa subunits. Together, they form a large, completely filled cylinder. The structural basis for this highly complex protein packing is not known so far. Here, we report the electron cryomicroscopy (cryo-EM) structure of mega-Hemocyanin complexes from two different snail species. The structures reveal that mega-Hemocyanin is composed of flexible building blocks that differ in their conformation, but not in their primary structure. Like a protein origami, these flexible blocks are optimally packed, implementing different local symmetries and pseudosymmetries. A comparison between the two structures suggests a surprisingly simple evolutionary mechanism leading to these large oxygen transporters.
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Structure, diversity and evolution of myriapod Hemocyanins
The FEBS journal, 2014Co-Authors: Christian Pick, Jurgen Markl, Samantha Scherbaum, Elöd Hegedüs, Andreas Bernhard Meyer, Michael Saur, Ruben Neumann, Thorsten BurmesterAbstract:Oxygen transport in the hemolymph of many arthropods is mediated by Hemocyanins, large copper-containing proteins that are well-studied in Chelicerata and Crustacea, but had long been considered unnecessary in the subphylum of Myriapoda. Only recently has it become evident that Hemocyanins are present in Scutigeromorpha (Chilopoda) and Spirostreptida (Diplopoda). Here we present evidence for a more widespread occurrence of Hemocyanin in the myriapods. By means of RT-PCR, western blotting and database searches, Hemocyanins were identified in the symphylans Hanseniella audax and Symphylella vulgaris, the chilopod Scolopendra subspinipes dehaani and the diplopod Polydesmus angustus. No Hemocyanins were found in the diplopods Polyxenus lagurus, Cylindroiulus punctatus, Glomeris marginata, Glomeris pustulata and Arthrosphaera brandtii, or the chilopods Lithobius forficatus, Geophilus flavus and Strigamia maritima. This suggests multiple independent losses in myriapod taxa. Two independent Hemocyanin subunits were found that were already present in the myriapod stem line. We specifically investigated the structure of the Hemocyanin of P. angustus, which consists of three distinct subunits that occur in an approximately equimolar ratio. As deduced by 3D electron microscopy, the quaternary structure is a 3 × 6-mer that resembles the half structure of the 6 × 6-mer Hemocyanin from Scutigera coleoptrata. It was analyzed more closely by homology modeling of 1 × 6-mers and their rigid-body fitting to the electron density map of the 3 × 6-mer. In addition, we obtained the cDNA sequence of a putative myriapod phenoloxidase. Phenoloxidases are related to the arthropod Hemocyanins, but diverged before radiation of the arthropod subphyla.
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The diversity and evolution of chelicerate Hemocyanins
BMC Evolutionary Biology, 2012Co-Authors: Peter Rehm, Jurgen Markl, Christian Pick, Janus Borner, Thorsten BurmesterAbstract:Background Oxygen transport in the hemolymph of many arthropod species is facilitated by large copper-proteins referred to as Hemocyanins. Arthropod Hemocyanins are hexamers or oligomers of hexamers, which are characterized by a high O_2 transport capacity and a high cooperativity, thereby enhancing O_2 supply. Hemocyanin subunit sequences had been available from horseshoe crabs (Xiphosura) and various spiders (Araneae), but not from any other chelicerate taxon. To trace the evolution of Hemocyanins and the emergence of the large Hemocyanin oligomers, Hemocyanin cDNA sequences were obtained from representatives of selected chelicerate classes. Results Hemocyanin subunits from a sea spider, a scorpion, a whip scorpion and a whip spider were sequenced. Hemocyanin has been lost in Opiliones, Pseudoscorpiones, Solifugae and Acari, which may be explained by the evolution of trachea (i.e., taxon Apulmonata). Bayesian phylogenetic analysis was used to reconstruct the evolution of Hemocyanin subunits and a relaxed molecular clock approach was applied to date the major events. While the sea spider has a simple hexameric Hemocyanin, four distinct subunit types evolved before Xiphosura and Arachnida diverged around 470 Ma ago, suggesting the existence of a 4 × 6mer at that time. Subsequently, independent gene duplication events gave rise to the other distinct subunits in each of the 8 × 6mer Hemocyanin of Xiphosura and the 4 × 6mer of Arachnida. The Hemocyanin sequences were used to infer the evolutionary history of chelicerates. The phylogenetic trees support a basal position of Pycnogonida, a sister group relationship of Xiphosura and Arachnida, and a sister group relationship of the whip scorpions and the whip spiders. Conclusion Formation of a complex Hemocyanin oligomer commenced early in the evolution of euchelicerates. A 4 × 6mer Hemocyanin consisting of seven subunit types is conserved in most arachnids since more than 400 Ma, although some entelegyne spiders display selective subunit loss and independent oligomerization. Hemocyanins also turned out to be a good marker to trace chelicerate evolution, which is, however, limited by the loss of Hemocyanin in some taxa. The molecular clock calculations were in excellent agreement with the fossil record, also demonstrating the applicability of Hemocyanins for such approach.
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The diversity and evolution of chelicerate Hemocyanins
BMC evolutionary biology, 2012Co-Authors: Peter Rehm, Jurgen Markl, Christian Pick, Janus Borner, Thorsten BurmesterAbstract:Oxygen transport in the hemolymph of many arthropod species is facilitated by large copper-proteins referred to as Hemocyanins. Arthropod Hemocyanins are hexamers or oligomers of hexamers, which are characterized by a high O2 transport capacity and a high cooperativity, thereby enhancing O2 supply. Hemocyanin subunit sequences had been available from horseshoe crabs (Xiphosura) and various spiders (Araneae), but not from any other chelicerate taxon. To trace the evolution of Hemocyanins and the emergence of the large Hemocyanin oligomers, Hemocyanin cDNA sequences were obtained from representatives of selected chelicerate classes. Hemocyanin subunits from a sea spider, a scorpion, a whip scorpion and a whip spider were sequenced. Hemocyanin has been lost in Opiliones, Pseudoscorpiones, Solifugae and Acari, which may be explained by the evolution of trachea (i.e., taxon Apulmonata). Bayesian phylogenetic analysis was used to reconstruct the evolution of Hemocyanin subunits and a relaxed molecular clock approach was applied to date the major events. While the sea spider has a simple hexameric Hemocyanin, four distinct subunit types evolved before Xiphosura and Arachnida diverged around 470 Ma ago, suggesting the existence of a 4 × 6mer at that time. Subsequently, independent gene duplication events gave rise to the other distinct subunits in each of the 8 × 6mer Hemocyanin of Xiphosura and the 4 × 6mer of Arachnida. The Hemocyanin sequences were used to infer the evolutionary history of chelicerates. The phylogenetic trees support a basal position of Pycnogonida, a sister group relationship of Xiphosura and Arachnida, and a sister group relationship of the whip scorpions and the whip spiders. Formation of a complex Hemocyanin oligomer commenced early in the evolution of euchelicerates. A 4 × 6mer Hemocyanin consisting of seven subunit types is conserved in most arachnids since more than 400 Ma, although some entelegyne spiders display selective subunit loss and independent oligomerization. Hemocyanins also turned out to be a good marker to trace chelicerate evolution, which is, however, limited by the loss of Hemocyanin in some taxa. The molecular clock calculations were in excellent agreement with the fossil record, also demonstrating the applicability of Hemocyanins for such approach.
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The Cupredoxin-like Domains in Hemocyanins
Biochemical Journal, 2010Co-Authors: Elmar Jaenicke, Jurgen Markl, Heinz Decker, Kay Büchler, Thomas Rm BarendsAbstract:Hemocyanins are multimeric oxygen transport proteins which bind oxygen to type 3 copper sites. Arthropod Hemocyanins are composed of 75 kDa subunits, while molluscan Hemocyanins contain 350-400 kDa subunits with 7-8 different 50 kDa “functional units” (FU‑a to FU‑h), each with an active site. FU‑h possesses a tail of 100 amino acids not present in the other FUs. Here we show by X-ray crystallography of FU-h of keyhole limpet Hemocyanin isoform 1 (KLH1) that the structure of the tail domain is cupredoxin-like but contains no copper. The copper-free domain #3 in arthropod Hemocyanin subunits has recently also been reinterpreted as being cupredoxin-like. We propose that the cupredoxin-like domain in both Hemocyanin types once served to upload copper to the active site of the oxygen-binding domain.
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a novel immunomodulatory Hemocyanin from the limpet fissurella latimarginata promotes potent anti tumor activity in melanoma
PLOS ONE, 2014Co-Authors: Sergio Arancibia, Jorge Ferreira, Pablo De ,ioannes, Miguel Del Campo, Fabián Salazar, Bruno Moltedo, Ta-ying Zhong, Ricardo Tampe, Cecilia Espinoza, Ed C LavelleAbstract:Hemocyanins, the huge oxygen-transporting glycoproteins of some mollusks, are used as immunomodulatory proteins with proven anti-cancer properties. The biodiversity of Hemocyanins has promoted interest in identifying new anti-cancer candidates with improved immunological properties. Hemocyanins promote Th1 responses without known side effects, which make them ideal for long-term sustained treatment of cancer. In this study, we evaluated a novel Hemocyanin from the limpet/gastropod Fissurella latimarginata (FLH). This protein has the typical hollow, cylindrical structure of other known Hemocyanins, such as the keyhole limpet Hemocyanin (KLH) and the Concholepas Hemocyanin (CCH). FLH, like the KLH isoforms, is composed of a single type of polypeptide with exposed N- and O-linked oligosaccharides. However, its immunogenicity was significantly greater than that of KLH and CCH, as FLH induced a stronger humoral immune response and had more potent anti-tumor activity, delaying tumor growth and increasing the survival of mice challenged with B16F10 melanoma cells, in prophylactic and therapeutic settings. Additionally, FLH-treated mice demonstrated increased IFN-γ production and higher numbers of tumor-infiltrating CD4+ lymphocytes. Furthermore, in vitro assays demonstrated that FLH, but not CCH or KLH, stimulated the rapid production of pro-inflammatory cytokines (IL-6, IL-12, IL-23 and TNF-α) by dendritic cells, triggering a pro-inflammatory milieu that may explain its enhanced immunological activity. Moreover, this effect was abolished when deglycosylated FLH was used, suggesting that carbohydrates play a crucial role in the innate immune recognition of this protein. Altogether, our data demonstrate that FLH possesses increased anti-tumor activity in part because it activates a more potent innate immune response in comparison to other known Hemocyanins. In conclusion, FLH is a potential new marine adjuvant for immunization and possible cancer immunotherapy.
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Hemocianinas, una herramienta inmunológica de la biomedicina actual
Revista medica de Chile, 2011Co-Authors: Miguel Del Campo, Jorge Ferreira, Pablo De ,ioannes, Sergio Arancibia, Esteban Nova, Fabián Salazar, Andrea González, Bruno Moltedo, Augusto Manubens, María Inés BeckerAbstract:Hemocyanins, the giant oxygen transporter glycoproteins of diverse mollusks, are xenogenic to the mammalian immune system and they display a remarkable immuno-genicity. Therefore they are ideal non-specific immunostimulants to treat some types of cancer. They are used as an alternative therapy for superficial urinary bladder cancer (SBC), that has been traditionally treated with Bacillus Calmette-Guerin (BCG). In contrast to BCG, Hemocyanins do not cause side-effects, making them ideal for long-term repetitive treatments. Hemocyanins have also been exploited as carriers to develop antibodies against hapten molecules and peptides, as carrier-adjuvants for cutting-edge vaccines against cancer, drug addiction, and infectious diseases and in the diagnosis of parasitic diseases, such as Schistosomiasis. The Hemocyanin from Megathura crenulata, also known as keyhole limpet Hemocyanin (KLH), has been used for over thirty years for the purposes described above. More recently, hemoc yanin from the Chilean mollusk Concholepas concholepas (CCH) has proved to be a reliable alternative to KLH, either as carrier protein, and as a likely alternative for the immunotherapy of SBC. Despite KLH and CCH differ significantly in their origin and structure, we have demonstrated that both Hemocyanins stimulate the immune system of mammals in a similar way by inducing a potent Thl-polarized cellular and humoral response.
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Hemocyanins as immunostimulants Hemocianinas, una herramienta inmunológica de la biomedicina actual
2011Co-Authors: Del Campo Miguel, Arancibia Sergio, Nova Esteban, Salazar Fabián, González Andrea, Moltedo Bruno, Pablo De ,ioannes, Ferreira Jorge, Manubens Augusto, Becker, María InésAbstract:Hemocyanins, the giant oxygen transporter glycoproteins of diverse mollusks, are xenogenic to the mammalian immune system and they display a remarkable immunogenicity. Therefore they are ideal non-specific immunostimulants to treat some types of cancer. They are used as an alternative therapy for superficial urinary bladder cancer (SBC), that has been traditionally treated with Bacillus Calmette-Guèrin (BCG). In contrast to BCG, Hemocyanins do not cause side-effects, making them ideal for long-term repetitive treatments. Hemocyanins have also been exploited as carriers to develop antibodies against hapten molecules and peptides, as carrier-adjuvants for cutting-edge vaccines against cancer, drug addiction, and infectious diseases and in the diagnosis of parasitic diseases, such as Schistosomiasis. The Hemocyanin from Megathura crenulata, also known as keyhole limpet Hemocyanin (KLH), has been used for over thirty years for the purposes described above. More recently, Hemocyanin from t
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Immunotherapeutic effect of Concholepas Hemocyanin in the murine bladder cancer model: evidence for conserved antitumor properties among Hemocyanins.
The Journal of urology, 2006Co-Authors: Bruno Moltedo, Pablo De ,ioannes, Fernando Faunes, Denise Haussmann, Alfredo E De Ioannes, Javier Puente, María Inés BeckerAbstract:We determined the antitumor properties of a newly available Hemocyanin obtained from the Chilean gastropod Concholepas concholepas (Biosonda Corp., Santiago, Chile) in a syngeneic heterotopic mouse bladder carcinoma model. Since keyhole limpet Hemocyanin (Pierce, Rockford, Illinois) is used increasingly in biomedicine as a carrier for vaccines and an immunotherapeutic agent for bladder transitional cell carcinoma, there is a growing interest in finding new substances that share its potent immunomodulatory properties. Considering that keyhole limpet Hemocyanin and Concholepas concholepas Hemocyanin differ significantly, it was not possible to predict a priori the antitumor properties of Concholepas concholepas Hemocyanin. C3H/He mice were primed with Concholepas concholepas Hemocyanin before subcutaneous implantation of mouse bladder tumor-2 cells. Treatment consisted of a subcutaneous dose of Concholepas concholepas Hemocyanin (1 mg or 100 mug) at different intervals after implantation. Keyhole limpet Hemocyanin and phosphate buffered saline served as positive and negative controls, respectively. In addition, experiments were designed to determine which elements of the immune response were involved in its adjuvant immunostimulatory effect. Mice treated with Concholepas concholepas Hemocyanin showed a significant antitumor effect, as demonstrated by decreased tumor growth and incidence, prolonged survival and lack of toxic effects. These effects were similar to those achieved with keyhole limpet Hemocyanin. We found that each Hemocyanin increased natural killer cell activity but the effect of Concholepas concholepas Hemocyanin was stronger. Analysis of serum from treated mice showed an increased interferon-gamma and low interleukin-4, which correlated with antibody isotypes, confirming that Hemocyanins induce a T helper type 1 cytokine profile. To our knowledge our results are the first demonstration of the antitumor effect of a Hemocyanin other than keyhole limpet Hemocyanin. They suggest that this is an ancient conserved immunogenic mechanism shared by those Hemocyanins that is able to enhance T helper type 1 immunity and lead to antitumor activity. Therefore, Concholepas concholepas Hemocyanin may be an alternative candidate for providing safe and effective immunotherapy for human superficial bladder cancer.