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Nature Communications Jul 2018The 6,6-quinolone scaffolds on which viridicatin-type fungal alkaloids are built are frequently found in metabolites that display useful biological activities. Here we...
The 6,6-quinolone scaffolds on which viridicatin-type fungal alkaloids are built are frequently found in metabolites that display useful biological activities. Here we report in vitro and computational analyses leading to the discovery of a hemocyanin-like protein AsqI from the Aspergillus nidulans aspoquinolone biosynthetic pathway that forms viridicatins via a conversion of the cyclopenin-type 6,7-bicyclic system into the viridicatin-type 6,6-bicyclic core through elimination of carbon dioxide and methylamine through methyl isocyanate.
Topics: Alkaloids; Aspergillus nidulans; Binding Sites; Biosynthetic Pathways; Carbon Dioxide; Cloning, Molecular; Crystallography, X-Ray; Cyclization; Escherichia coli; Fungal Proteins; Gene Expression; Genetic Vectors; Hemocyanins; Hydroxyquinolines; Isocyanates; Kinetics; Methylamines; Models, Molecular; Protein Binding; Protein Conformation, alpha-Helical; Protein Interaction Domains and Motifs; Quinolones; Recombinant Proteins; Substrate Specificity; Zinc
PubMed: 30026518
DOI: 10.1038/s41467-018-05221-5 -
The FEBS Journal Apr 2014Oxygen transport in the hemolymph of many arthropods is mediated by hemocyanins, large copper-containing proteins that are well-studied in Chelicerata and Crustacea, but...
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.
Topics: Amino Acid Sequence; Animals; Arthropods; Evolution, Molecular; Hemocyanins; Models, Molecular; Molecular Sequence Data
PubMed: 24520955
DOI: 10.1111/febs.12742 -
The Journal of Experimental Medicine Sep 1966Groups of rabbits were injected with either bovine serum albumin, sheep red cell stroma, or keyhole limpet hemocyanin to which 2,4-dinitrophenyl and/or p-azophenyl...
Groups of rabbits were injected with either bovine serum albumin, sheep red cell stroma, or keyhole limpet hemocyanin to which 2,4-dinitrophenyl and/or p-azophenyl arsonate groups had been coupled. Groups of animals received either doubly coupled antigen or an equivalent mixture of singly coupled antigens. Materials were injected intravenously as a solution or subcutaneously and intramuscularly in complete Freund's adjuvant. The presence of dinitrophenyl groups on the immunizing antigen could suppress, partially or completely, the antibody response to p-azophenyl arsonate when this hapten was located on the same molecule. Suppression was dependent on the ratio of haptenic groups on the molecule, appeared to be greatly affected by the method of immunization, and could be demonstrated in all three antigen systems. Partial suppression was manifested in decreased frequency and delayed appearance of the response as well as decreased maximal antibody titers. These findings appear irreconcilable with the possibility of direct clonal selection of antibody-producing cells by unprocessed antigen.
Topics: Animals; Antibody Formation; Arsenicals; Azo Compounds; Dinitrophenols; Erythrocytes; Haptens; Hemocyanins; Immunodiffusion; Mollusca; Rabbits; Serum Albumin, Bovine
PubMed: 4958801
DOI: 10.1084/jem.124.3.293 -
STAR Protocols Mar 2022Keyhole limpet hemocyanin (KLH) is a glycosylated multi-subunit metalloprotein that elicits a strong nonspecific immune activation, thus inducing both cellular and...
Keyhole limpet hemocyanin (KLH) is a glycosylated multi-subunit metalloprotein that elicits a strong nonspecific immune activation, thus inducing both cellular and humoral immune responses. The exceptional immunogenicity of this protein can be leveraged to vaccinate mice against self-antigens that otherwise would not induce an autoimmune response. This protocol describes the covalent conjugation of KLH with acyl-coenzyme A-binding protein (ACBP), the autovaccination of mice with ACBP-KLH conjugate together with a potent adjuvant, and the detection of the produced anti-ACBP autoantibodies. For complete details on the use and execution of this profile, please refer to Bravo-San Pedro et al. (2019c).
Topics: Animals; Carrier Proteins; Hemocyanins; Immunization; Mice; Peptides; Vaccination
PubMed: 35059656
DOI: 10.1016/j.xpro.2021.101095 -
Insect Molecular Biology Oct 2019Haemocyanins constitute a group of copper-containing respiratory proteins, and hexamerins were derived from hexapod haemocyanin but lost the ability to transport oxygen...
Haemocyanins constitute a group of copper-containing respiratory proteins, and hexamerins were derived from hexapod haemocyanin but lost the ability to transport oxygen and serve as storage proteins. Although hexamerins have been reported in most insect species, none of them has been identified in Collembola, one of the most primitive hexapod lineages, thereby preventing us from exploring relevant evolutionary scenarios regarding the origin and evolution of hexamerins in hexapods. Here we report on collembolan hexamerins for the first time, and investigated the temporal expression profiles of hexamerin and haemocyanin in the collembolan Folsomia candida. Haemocyanin was expressed over the entire life cycle, with higher expression at the embryonic stage than at other stages, whereas hexamerin expression was restricted to embryos, unlike insect hexamerins, which are generally expressed from larval to adult stages. A phylogenetic analysis and molecular clock estimation suggested that all investigated hexapod hexamerins have a single and ancient origin (~423 Ma), coincident with the rise of atmospheric oxygen levels in the Silurian-Devonian period, indicating a physiological link between molecular evolution and Palaeozoic oxygen changes.
Topics: Amino Acid Sequence; Animals; Arthropod Proteins; Arthropods; Evolution, Molecular; Hemocyanins; Insect Proteins; Larva; Phylogeny; Transcriptome
PubMed: 30953580
DOI: 10.1111/imb.12585 -
Zeitschrift Fur Naturforschung. C,... 2001Penaeus monodon (class Crustacea, order Decapoda) is one of the largest shrimps of the Penaeidea family from the Indo-West Pacific region. The dioxygen-transporting... (Comparative Study)
Comparative Study
Penaeus monodon (class Crustacea, order Decapoda) is one of the largest shrimps of the Penaeidea family from the Indo-West Pacific region. The dioxygen-transporting protein hemocyanin, isolated from the hemolymph of this invertebrate, is composed of three 75-76 kDa structural/functional subunits designated as Pm1, Pm2 and Pm3. The N-terminal sequences of the chains were determined and compared with those of other decapodan hemocyanin subunits. Pm2 and Pm3 are highly homologous and electrophoretically undistinguishable polypeptides. In comparison to Pml, they have an extension of six residues. Pm1 is closely related to the subunit Pv2 of the Penaeus vannamei hemocyanin. Probably, subunits like Pm1 and Pv2 are family-specific for the Penaeidea hemocyanins and the other subunits are species-specific. Comparison of N-terminal sequences of respiratory proteins from the sub-orders Natantia and Reptantia demonstrated family- and sub-order-specific sequences. A melting point of 69 degrees C, lower than those for the di-hexameric decapodan hemocyanins, was determined from the temperature dependence of ellipticity of the mono-hexameric Penaeus monodon hemocyanin. Thermostability of decapodan hemocyanins depends on their aggregation state.
Topics: Amino Acid Sequence; Animals; Crustacea; Hemocyanins; Molecular Sequence Data; Penaeidae; Protein Denaturation; Protein Subunits; Sequence Alignment; Sequence Homology, Amino Acid; Thermodynamics
PubMed: 11421459
DOI: 10.1515/znc-2001-5-616 -
PloS One 2012Hemocyanins are giant oxygen transport proteins found in the hemolymph of several invertebrate phyla. They constitute giant multimeric molecules whose size range up to...
Hemocyanins are giant oxygen transport proteins found in the hemolymph of several invertebrate phyla. They constitute giant multimeric molecules whose size range up to that of cell organelles such as ribosomes or even small viruses. Oxygen is reversibly bound by hemocyanins at binuclear copper centers. Subunit interactions within the multisubunit hemocyanin complex lead to diverse allosteric effects such as the highest cooperativity for oxygen binding found in nature. Crystal structures of a native hemocyanin oligomer larger than a hexameric substructure have not been published until now. We report for the first time growth and preliminary analysis of crystals of the 24-meric hemocyanin (M(W) = 1.8 MDa) of emperor scorpion (Pandinus imperator), which diffract to a resolution of 6.5 Å. The crystals are monoclinc with space group C 1 2 1 and cell dimensions a = 311.61 Å, b = 246.58 Å and c = 251.10 Å (α = 90.00°, β = 90.02°, γ = 90.00°). The asymmetric unit contains one molecule of the 24-meric hemocyanin and the solvent content of the crystals is 56%. A preliminary analysis of the hemocyanin structure reveals that emperor scorpion hemocyanin crystallizes in the same oxygenated conformation, which is also present in solution as previously shown by cryo-EM reconstruction and small angle x-ray scattering experiments.
Topics: Animals; Arthropod Proteins; Crystallization; Crystallography, X-Ray; Electrons; Hemocyanins; Models, Molecular; Protein Multimerization; Protein Structure, Quaternary; Scorpions
PubMed: 22403673
DOI: 10.1371/journal.pone.0032548 -
PloS One 2020Structural knowledge of gastropod hemocyanins is scarce. To better understand their evolution and diversity we studied the hemocyanin of a caenogastropod, Pomacea...
Structural knowledge of gastropod hemocyanins is scarce. To better understand their evolution and diversity we studied the hemocyanin of a caenogastropod, Pomacea canaliculata (PcH). Through a proteomic and genomic approach, we identified 4 PcH subunit isoforms, in contrast with other gastropods that usually have 2 or 3. Each isoform has the typical Keyhole limpet-type hemocyanin architecture, comprising a string of eight globular functional units (FUs). Correspondingly, genes are organized in eight FUs coding regions. All FUs in the 4 genes are encoded by more than one exon, a feature not found in non- caenogastropods. Transmission electron microscopy images of PcH showed a cylindrical structure organized in di, tri and tetra-decamers with an internal collar structure, being the di and tri-decameric cylinders the most abundant ones. PcH is N-glycosylated with high mannose and hybrid-type structures, and complex-type N-linked glycans, with absence of sialic acid. Terminal β-N-GlcNAc residues and nonreducing terminal α-GalNAc are also present. The molecule lacks O-linked glycosylation but presents the T-antigen (Gal-β1,3-GalNAc). Using an anti-PcH polyclonal antibody, no cross-immunoreactivity was observed against other gastropod hemocyanins, highlighting the presence of clade-specific structural differences among gastropod hemocyanins. This is, to the best of our knowledge, the first gene structure study of a Caenogastropoda hemocyanin.
Topics: Animals; Evolution, Molecular; Gastropoda; Gene Expression Profiling; Genomics; Hemocyanins; Mass Spectrometry; Microscopy, Electron, Transmission; Models, Molecular; Protein Conformation; Protein Domains; Protein Isoforms; Proteomics
PubMed: 31999773
DOI: 10.1371/journal.pone.0228325 -
The Journal of Biological Chemistry Dec 2019Hemocyanins are widely used as carriers, adjuvants, and nonspecific immunostimulants in cancer because they promote Th1 immunity in mammals. Hemocyanins also interact...
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 -linked oligosaccharides on the structural and immunological properties of hemocyanin is unclear. Mollusk hemocyanins, such as (CCH), (FLH), and (KLH), are oligomeric glycoproteins with complex dodecameric quaternary structures and heterogeneous glycosylation patterns, primarily consisting of mannose-rich -glycans. Here, we report that enzyme-catalyzed -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 -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 -deglycosylated hemocyanins in the humoral immune response and their nonspecific antitumor effects in the B16F10 melanoma model, we found that compared with native hemocyanins -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.
Topics: Adjuvants, Immunologic; Animals; Cell Line; Cytokines; Galactose; Glycosylation; Hemocyanins; Immunity, Humoral; Lectins; Lectins, C-Type; Macrophages; Mannose Receptor; Mannose-Binding Lectins; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mollusca; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Polysaccharides; Protein Folding; Protein Structure, Quaternary; Protein Structure, Secondary; Receptors, Cell Surface
PubMed: 31719148
DOI: 10.1074/jbc.RA119.009525 -
Expert Opinion on Biological Therapy Aug 2009Conventional therapies for glioblastoma multiforme (GBM) fail to target tumor cells exclusively, resulting in non-specific toxicity. Immune targeting of tumor-specific... (Review)
Review
Conventional therapies for glioblastoma multiforme (GBM) fail to target tumor cells exclusively, resulting in non-specific toxicity. Immune targeting of tumor-specific mutations may allow for more precise eradication of neoplastic cells. EGFR variant III (EGFRvIII) is a tumor-specific mutation that is widely expressed in GBM and other neoplasms and its expression enhances tumorigenicity. This in-frame deletion mutation splits a codon, resulting in a novel glycine at the fusion junction producing a tumor-specific epitope target for cellular or humoral immunotherapy. We have previously shown that vaccination with a peptide that spans the EGFRvIII fusion junction (PEPvIII-KLH/CDX-110) is an efficacious immunotherapy in syngeneic murine models. In this review, we summarize our results in GBM patients targeting this mutation in multiple, multi-institutional Phase II immunotherapy trials. These trials demonstrated that a selected population of GBM patients who received vaccines targeting EGFRvIII had an unexpectedly long survival time. Further therapeutic strategies and potential pitfalls of using this approach are discussed.
Topics: Animals; Brain Neoplasms; Cancer Vaccines; Central Nervous System; Clinical Trials as Topic; ErbB Receptors; Glioblastoma; Hemocyanins; Humans; Immunotherapy; Mice; Mutation; Peptides; Prognosis
PubMed: 19591631
DOI: 10.1517/14712590903124346