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Jurgen Markl - One of the best experts on this subject based on the ideXlab platform.
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Hemocyanin from the keyhole limpet Megathura crenulata (KLH) carries a novel type of N-glycans with Gal(β1–6)Man-motifs
European journal of biochemistry, 2002Co-Authors: Tomofumi Kurokawa, Jurgen Markl, Manfred Wuhrer, Günter Lochnit, Hildegard Geyer, Rudolf GeyerAbstract:Keyhole limpet (Megathura crenulata) hemocyanin (KLH), an extracellular respiratory protein, is widely used as hapten carrier and immune stimulant. Although it is generally accepted that the sugar constituents of this glycoprotein are likely to be implicated in the antigenicity and biomedical properties of KLH, knowledge of its carbohydrate structure is still limited. Therefore, we have investigated the N-linked oligosaccharides of KLH. Glycan chains were enzymatically liberated from tryptic glycopeptides, pyridylaminated and separated by two-dimensional HPLC. Only neutral oligosaccharides were obtained and characterized by carbohydrate constituent and methylation analyses, MALDI-TOF-MS, ESI-ion trap-MS and sequential exoglycosidase digestion. The results revealed that KLH is carrying high mannose-type glycans and truncated sugar chains derived thereof. As a characteristic feature, a number of the studied N-glycans contained a Gal(β1–6)Man-unit which has not been found in glycoprotein-N-glycans so far. Hence, our studies demonstrate that this marine mollusk glycoprotein is characterized by a unique oligosaccharide pattern comprising, in part, novel structural elements.
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Topology of the 10 subunits within the decamer of KLH, the hemocyanin of the marine gastropod Megathura crenulata.
Journal of structural biology, 2002Co-Authors: Wolfgang Gebauer, J. Robin Harris, Jurgen MarklAbstract:Immunoelectron microscopy has been performed using negatively stained immune complexes of keyhole limpet hemocyanin isoform 1 (KLH1) decamers and a functional unit-specific monoclonal antibody anti-KLH1-c1. The antibody links hemocyanin molecules at both the collar and the collarless edge of the decamer, indicating a peripheral localization of functional units c. In isoform 2 (KLH2) the positions of functional units c have been identified with the peanut agglutinin (PNA), which has previously been shown to exclusively bind to KLH2-c. Ferritin linked to PNA was used to visualize labeled molecules electron microscopically. The pattern of labeling also indicates a peripheral localization of the c functional units. The data presented in this paper support only one of two possible models for the subunit orientation within the hemocyanin decamer.
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Marine tumor vaccine carriers: structure of the molluscan hemocyanins KLH and HtH
Journal of Cancer Research and Clinical Oncology, 2001Co-Authors: Jurgen Markl, Benjamin Altenhein, Wolfgang Gebauer, Bernhard Lieb, Ulrich Meissner, J. Robin HarrisAbstract: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, HIM, 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 quaternary structure (molecular mass ca 8 MDa). This article reviews the recent data on the biosynthesis, quaternary structure, subunit architecture, amino acid sequence, gene structure, and recombinant production of KLH and HtH.
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rhogocytes pore cells as the site of hemocyanin biosynthesis in the marine gastropod haliotis tuberculata
Cell and Tissue Research, 2001Co-Authors: Ute Albrecht, Henning Keller, Wolfgang Gebauer, Jurgen MarklAbstract:Rhogocytes (pore cells) are specific molluscan cell types that are scattered throughout the connective tissues of diverse body parts. We have identified rhogocytes in large numbers in tissue taken from mantle, foot and midgut gland of the abalone Haliotis tuberculata (Vetigastropoda). Within cisternae of the endoplasmic reticulum, particles are visible that resemble, in shape and size, hemocyanin molecules, the respiratory protein of many molluscs. Immunohistochemical experiments using hemocyanin-specific antibodies demonstrated that these cells contain hemocyanin. In situ hybridization with a cDNA probe specific for Haliotis hemocyanin showed that hemocyanin-specific mRNA is present in rhogocytes, which confirmed that they are the site of hemocyanin biosynthesis in this gastropod. A possible path of hemocyanin release into the hemolymph is discussed. Also in the vetigastropod Megathura crenulata, many rhogocytes could be detected. However, they lacked hemocyanin molecules which, together with published data, indicates a seasonal expression of hemocyanin in this animal.
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Hemocyanin Subunit Organization of the Gastropod Rapana thomasiana
Archives of biochemistry and biophysics, 1999Co-Authors: Wolfgang Gebauer, Stanka Stoeva, Wolfgang Voelter, Enrico Dainese, Benedetto Salvato, Mariano Beltramini, Jurgen MarklAbstract:Abstract RtH1 and RtH2, the two hemocyanin isoforms of the prosobranch gastropod Rapana thomasiana, have been purified by anion-exchange chromatography and studied by SDS–PAGE and immunoelectrophoresis. Both subunit types are built up of eight functional units (FUs). Under reducing conditions subunit RtH2 splits into two fragments, RtH2- a – f and RtH2- gh, suggesting the presence of a disulfide bridge between FU2- f and FU2- g. By proteolytic cleavage of the subunits into three-, two-, and single-FU fragments, purification of fragments by HPLC, N-terminal sequencing of the peptides, and crossed-line immunoelectrophoresis, FUs- a – h of RtH2 and FU- a, FU- d, FU- e, and FU- f of RtH1 were identified and correlated to the eight-FUs pattern of immunoelectrophoresis. FU- a, FU- e, and FU- f of RtH1 and RtH2 are very closely related immunologically. RtH1 and RtH2 both correspond immunologically to KLH2, one of the two hemocyanin isoforms of the prosobranch gastropod Megathura crenulata.
Wolfgang Voelter - One of the best experts on this subject based on the ideXlab platform.
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Immunological Potential of Helix vulgaris and Rapana venosa Hemocyanins
Immunological investigations, 2008Co-Authors: Pavlina Dolashka-angelova, Tsetanka Stefanova, Evangelia Livaniou, Lyudmila Velkova, Persefoni Klimentzou, Stefan Stevanovic, B. Salvato, Hristo Neychev, Wolfgang VoelterAbstract:A new hemocyanin was isolated from the hemolymph of garden snails Helix vulgaris, composed of two isoforms, HvH1 and HvH2 separated on an ion exchange column DEAE-Sepharose 6CL. Structural and immunological properties of Helix vulgaris hemocyanin were studied in comparison with molluscan Hcs Rapana venosa and Megathura crenulata. The possibility of using HvH and RvH as carriers of small molecules (haptens) in immunizing protocols was studied in comparison with KLH, which is a widely used, highly immunogenic carrier protein. By using HvH as a carrier of the well-known hapten TNBS (2,4,6-trinitrobenzene sulfonic acid), an increasing with time production of hapten-specific TFN-gamma was detected in splenocyte cultures of mice, which lasted longer than in case of KLH and RvH carriers. Also, use of HvH or RvH as a carrier of the hapten ProT alpha[101-109] (i.e., the synthetic C-terminal fragment of the poorly immunogenic protein prothymosin alpha) showed that antisera of higher titres than that of the control conjugate (ProT alpha[101-109]-KLH) were obtained immediately after the second bleeding. HvH and RvH may prove to be useful for the development of new antiviral, antibacterial and antitumor vaccines, since they seem to launch strong and specific immune response against the conjugated antigens.
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of Keyhole Limpet Hemocyanin
2005Co-Authors: Aleksander Dolashki, Wolfgang Voelter, Jürgen Schütz, Rumyana Hristova, Pavlina Dolashka, G. BonchevAbstract:2 Abstract: The functional unit KLH2-c, isolated from the structural subunit KLH2 of keyhole limpet Megathura crenulata hemocyanin, was studied to explain the effect of the oligosaccharide component for the conformational stability of the protein. The properties of this FU were characterized by fluorescence spectroscopy, combined with fluorescence quenching studies, using acrylamide, caesium chloride and potassium iodide as tryptophan quenchers. Using circular dichroism and fluorescence spectroscopy, the thermostability of the non-glycosylated FU KLH2-c was studied in comparison to the glycosylated native molecule, the structural subunits KLH1, KLH2 and the FU RvH1-a of Rapana venosa Hc. The melting T (45°C) m as well as the critical temperatures T (46) were found to be less for KLH2-c compared to the glycosylated forms c which could be explained to be caused by the presence of oligosaccharide side chains and aggregation effects.
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Glycosylation of Rapana thomasiana hemocyanin. Comparison with other prosobranch (gastropod) hemocyanins.
Comparative biochemistry and physiology. Part B Biochemistry & molecular biology, 2004Co-Authors: Krassimira Idakieva, Stanka Stoeva, Wolfgang Voelter, Constant GielensAbstract:Abstract The carbohydrate content and composition of hemocyanins (Hcs) of three prosobranchs (gastropods), Rapana thomasiana , Megathura crenulata and Haliotis tuberculata , were compared. The analyses were performed by gas–liquid chromatography after methanolysis, re- N -acetylation and trimethylsilylation. The two structural subunits of R. thomasiana Hc, Rt H1 and Rt H2, both showed 2.6% (w/w) carbohydrate content with very similar monosaccharide composition, indicative for N -glycosylation. The two isoforms of M. crenulata Hc (KLH), KLH1 and KLH2, on the other hand, definitely differed in glycosylation: KLH2 (3.4% carbohydrate, w/w) comprised relatively less mannose and more N -acetylgalactosamine than KLH1 (3.0% carbohydrate, w/w), in agreement with the fact that O -glycosylation has been observed in a functional unit (FU) of KLH2. For the Hc of the abalone H. tuberculata , with 4.5% (w/w) carbohydrate, appreciable amounts of 3- O -methyl- d -mannose and 3- O -methyl- d -galactose were detected, showing that the occurrence of methylated sugars is not restricted to the Hcs of pulmonates. From the structural subunit Rt H2 of Rapana Hc the FUs Rt H2- b and R tH2- d were isolated. On the basis of amino acid sequence analysis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of the respective native and PNGase-F-treated glycopeptides, one N -glycosylation site was found for each FU. This site was located at Asn-405 for Rt H2- b and at Asn-394 for Rt H2- d ; the carbohydrate moiety corresponded to GlcNAc 2 Man 6 and GlcNAc 2 Man 5 , respectively. A comparison was made with the N -glycosylation sites of other FUs of Rapana Hc.
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Oligomeric stability of Rapana venosa hemocyanin (RvH) and its structural subunits.
Biochimica et biophysica acta, 2003Co-Authors: Pavlina Dolashka-angelova, Stefan Stevanovic, Heinz Schwarz, Aleksandar Dolashki, Miriam Fecker, Muhammad Saeed, Wolfgang VoelterAbstract:The two structural subunits RvH1 and RvH2 were separated after overnight dialysis of Rapana venosa Hc against 130 mM Gly/NaOH buffer, pH 9.6, on an ion exchange column Hiload 26/10 Sepharose Q using a fast performance liquid chromatography (FPLC) system. The reassociation characteristics of these two RvH isoforms and the native molecule were studied in buffers with different pH values and concentrations of Ca(2+) and Mg(2+). Reassociation of mixed RvH subunits was performed over a period of several days using a stabilizing buffer (SB) of pH 7.0 containing different concentrations of Ca(2+) and Mg(2+) ions. After 2 days of dialysis, an RvH subunit mixture of didecamers and multidecamers was observed in the presence of 100 mM CaCl(2) and MgCl(2), though RvH1 and RvH2 are biochemically and immunologically different and have also different dissociation properties. The reassociation, performed at pH 9.6 with 2 mM CaCl(2) and MgCl(2) at 4 degrees C over a period of one to several weeks, led to the formation of decameric oligomers, while didecamers formed predominantly in the SB at pH 7.0. Higher concentrations of calcium and magnesium ions led to a more rapid reassociation of RvH1 resulting in long stable multidecamers and helical tubules, which were stable and slowly dissociated into shorter multidecamers and decamers at higher pH values. The reassociation of the RvH2 structural subunit in the same buffers processed slowly and yielded didecamers, shorter tubule polymers and long multidecamers which are less stable at higher pH values. The stability of RvH isoforms under varying ionic conditions is compared with the stability of keyhole limpet (KLH, Megathura crenulata) hemocyanin (KLH) and Haliotis tuberculata hemocyanin (HtH) isoforms. The process of dissociation and reassociation is connected with changes of the fluorescence intensity at 600 nm, which can be explained by differences in opalescence of the solutions of these two isoforms. The solutions of longer tubule polymers and multidecamers of RvH1 show a higher opalescence compared to the solutions of shorter helical tubules and multidecamers of RvH2.
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Isolation and spectroscopic characterization of the structural subunits of keyhole limpet hemocyanin.
Biochimica et biophysica acta, 2001Co-Authors: Jürgen Schütz, Pavlina Dolashka-angelova, Rosen Abrashev, Peter Nicolov, Wolfgang VoelterAbstract:Keyhole limpet hemocyanin is a respiratory glycoprotein of high molecular weight from the gastropod mollusc Megathura crenulata. Two subunits, KLH1 and KLH2, were isolated using ion exchange chromatography and their physical properties are compared with the parent molecule. The various proteins are characterized by fluorescence spectroscopy, combined with fluorescence quenching studies, using acrylamide, cesium chloride and potassium iodide as tryptophan quenchers. The conformational stability of the native aggregate and its isolated structural subunits are also studied by circular dichroism and fluorescence spectroscopy as a function of temperature, as well as in the presence of guanidinium hydrochloride and urea. The associated subunits in the hemocyanin aggregates increase considerably the melting temperature to 67°C and the free energy of stabilization in water, ΔGH2OD, towards guanidinium hydrochloride is higher for the decamer as compared to the isolated subunits; this difference can be accounted for by the stabilizing effects of intra-subunit interactions exerted within the oligomer. The copper-dioxygen complex at the active site additionally stabilizes the molecule, and removing of the copper ions increases the tryptophan emission and the quantum yield of the fluorescence.
Wolfgang Gebauer - One of the best experts on this subject based on the ideXlab platform.
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Topology of the 10 subunits within the decamer of KLH, the hemocyanin of the marine gastropod Megathura crenulata.
Journal of structural biology, 2002Co-Authors: Wolfgang Gebauer, J. Robin Harris, Jurgen MarklAbstract:Immunoelectron microscopy has been performed using negatively stained immune complexes of keyhole limpet hemocyanin isoform 1 (KLH1) decamers and a functional unit-specific monoclonal antibody anti-KLH1-c1. The antibody links hemocyanin molecules at both the collar and the collarless edge of the decamer, indicating a peripheral localization of functional units c. In isoform 2 (KLH2) the positions of functional units c have been identified with the peanut agglutinin (PNA), which has previously been shown to exclusively bind to KLH2-c. Ferritin linked to PNA was used to visualize labeled molecules electron microscopically. The pattern of labeling also indicates a peripheral localization of the c functional units. The data presented in this paper support only one of two possible models for the subunit orientation within the hemocyanin decamer.
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Marine tumor vaccine carriers: structure of the molluscan hemocyanins KLH and HtH
Journal of Cancer Research and Clinical Oncology, 2001Co-Authors: Jurgen Markl, Benjamin Altenhein, Wolfgang Gebauer, Bernhard Lieb, Ulrich Meissner, J. Robin HarrisAbstract: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, HIM, 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 quaternary structure (molecular mass ca 8 MDa). This article reviews the recent data on the biosynthesis, quaternary structure, subunit architecture, amino acid sequence, gene structure, and recombinant production of KLH and HtH.
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rhogocytes pore cells as the site of hemocyanin biosynthesis in the marine gastropod haliotis tuberculata
Cell and Tissue Research, 2001Co-Authors: Ute Albrecht, Henning Keller, Wolfgang Gebauer, Jurgen MarklAbstract:Rhogocytes (pore cells) are specific molluscan cell types that are scattered throughout the connective tissues of diverse body parts. We have identified rhogocytes in large numbers in tissue taken from mantle, foot and midgut gland of the abalone Haliotis tuberculata (Vetigastropoda). Within cisternae of the endoplasmic reticulum, particles are visible that resemble, in shape and size, hemocyanin molecules, the respiratory protein of many molluscs. Immunohistochemical experiments using hemocyanin-specific antibodies demonstrated that these cells contain hemocyanin. In situ hybridization with a cDNA probe specific for Haliotis hemocyanin showed that hemocyanin-specific mRNA is present in rhogocytes, which confirmed that they are the site of hemocyanin biosynthesis in this gastropod. A possible path of hemocyanin release into the hemolymph is discussed. Also in the vetigastropod Megathura crenulata, many rhogocytes could be detected. However, they lacked hemocyanin molecules which, together with published data, indicates a seasonal expression of hemocyanin in this animal.
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Hemocyanin Subunit Organization of the Gastropod Rapana thomasiana
Archives of biochemistry and biophysics, 1999Co-Authors: Wolfgang Gebauer, Stanka Stoeva, Wolfgang Voelter, Enrico Dainese, Benedetto Salvato, Mariano Beltramini, Jurgen MarklAbstract:Abstract RtH1 and RtH2, the two hemocyanin isoforms of the prosobranch gastropod Rapana thomasiana, have been purified by anion-exchange chromatography and studied by SDS–PAGE and immunoelectrophoresis. Both subunit types are built up of eight functional units (FUs). Under reducing conditions subunit RtH2 splits into two fragments, RtH2- a – f and RtH2- gh, suggesting the presence of a disulfide bridge between FU2- f and FU2- g. By proteolytic cleavage of the subunits into three-, two-, and single-FU fragments, purification of fragments by HPLC, N-terminal sequencing of the peptides, and crossed-line immunoelectrophoresis, FUs- a – h of RtH2 and FU- a, FU- d, FU- e, and FU- f of RtH1 were identified and correlated to the eight-FUs pattern of immunoelectrophoresis. FU- a, FU- e, and FU- f of RtH1 and RtH2 are very closely related immunologically. RtH1 and RtH2 both correspond immunologically to KLH2, one of the two hemocyanin isoforms of the prosobranch gastropod Megathura crenulata.
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Primary structure and unusual carbohydrate moiety of functional unit 2-c of keyhole limpet hemocyanin (KLH).
Biochimica et Biophysica Acta, 1999Co-Authors: Stanka Stoeva, Wolfgang Gebauer, Jurgen Markl, Jürgen Schütz, Tonia Hundsdörfer, Christine Manz, Wolfgang VoelterAbstract:Abstract The complete amino acid sequence of the Megathura crenulata hemocyanin functional unit KLH2-c was determined by direct sequencing and matrix-assisted laser desorption ionization mass spectrometry of the protein, and of peptides obtained by cleavage with EndoLysC proteinase, chymotrypsin and cyanogen bromide. This is the first complete primary structure of a functional unit c from a gastropod hemocyanin. KLH2-c consists of 420 amino acid residues. Circular dichroism spectra indicated approx. 31% β-sheet and 29% α-helix contents. A multiple sequence alignment with other molluscan hemocyanin functional units revealed average identities between 41 and 49%, but 55% in case of Octopus hemocyanin functional unit c which is the structural equivalent to KLH2-c. KLH2-c has a molecular mass of approx. 48 kDa as calculated from its sequence and a measured mass of approx. 56 kDa; the mass difference is attributed to the sugar side chains usually decorating molluscan hemocyanin. However, inspection of the sequence of KLH2-c revealed no potential N-linked carbohydrate attachment sites, and this was supported by its inability to bind concanavalin A. Also KLH1-c was unreactive, whereas most, if not all, other functional units of KLH1 and KLH2 reacted positively to this lectin. On the other hand, peanut agglutinin specifically binds KLH2-c, indicating the presence of O-glycosidically linked carbohydrates in this functional unit. This contrasts to all other KLH functional units (including KLH1-c), which lack O-linked glycosides. The present results are discussed in view of the recent X-ray structure of the functional unit g from Octopus hemocyanin, and a published record of the Thomsen Friedenreich tumor antigenic epitope in KLH.
J. Robin Harris - One of the best experts on this subject based on the ideXlab platform.
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Topology of the 10 subunits within the decamer of KLH, the hemocyanin of the marine gastropod Megathura crenulata.
Journal of structural biology, 2002Co-Authors: Wolfgang Gebauer, J. Robin Harris, Jurgen MarklAbstract:Immunoelectron microscopy has been performed using negatively stained immune complexes of keyhole limpet hemocyanin isoform 1 (KLH1) decamers and a functional unit-specific monoclonal antibody anti-KLH1-c1. The antibody links hemocyanin molecules at both the collar and the collarless edge of the decamer, indicating a peripheral localization of functional units c. In isoform 2 (KLH2) the positions of functional units c have been identified with the peanut agglutinin (PNA), which has previously been shown to exclusively bind to KLH2-c. Ferritin linked to PNA was used to visualize labeled molecules electron microscopically. The pattern of labeling also indicates a peripheral localization of the c functional units. The data presented in this paper support only one of two possible models for the subunit orientation within the hemocyanin decamer.
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Marine tumor vaccine carriers: structure of the molluscan hemocyanins KLH and HtH
Journal of Cancer Research and Clinical Oncology, 2001Co-Authors: Jurgen Markl, Benjamin Altenhein, Wolfgang Gebauer, Bernhard Lieb, Ulrich Meissner, J. Robin HarrisAbstract: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, HIM, 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 quaternary structure (molecular mass ca 8 MDa). This article reviews the recent data on the biosynthesis, quaternary structure, subunit architecture, amino acid sequence, gene structure, and recombinant production of KLH and HtH.
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Controlled cleavage of KLH1 and KLH2 by the V8 protease from Staphylococcus aureus reassociation, electrophoretic and transmission electron microscopy study of peptide fragments.
European journal of biochemistry, 1999Co-Authors: Wolfgang Gebauer, J. Robin HarrisAbstract:The reassociation behaviour of protease V8-cleaved peptides from KLH1 and KLH2, the two hemocyanin isoforms from the giant keyhole limpet Megathura crenulata, has been studied by transmission electron microscopy of negatively stained specimens and SDS/PAGE. Reassociation of the complete mixture of protease cleavage products and of combinations of peptide fragments purified by HPLC was performed in the presence of 100 mm CaCl2 and 100 mm MgCl2 at pH 7.4, over a period of 1 to 4 weeks. The V8 protease splits KLH1 into peptide fragments containing the functional units abc, def, defg, defgh, g and h. This mixture of peptide fragments reassociated to form helical tubular polymers, with a diameter of approximately 25 nm. The single functional units g and h were not incorporated into the polymer. An essentially identical polymer was formed from the re-mixed HPLC-purified fragments abc, def and defg alone. As with uncleaved subunit, the tubular polymer of V8-cleaved KLH1 forms bundles. The combination of peptides def and defg led to the formation of short arc-like filamentous structures, which aggregated but showed little tendency to associate into larger polymers. The KLH1 peptide fragments abc and def alone, did not reassociate and in combination their potential to form polymers was very low. With KLH2, the V8 protease generated peptide fragments containing the functional units abc, defg, defgh and h, which in combination slowly reassociated to form a tubular polymer significantly different to that obtained from the KLH1 V8 fragments. The three-functional unit fragment abc from KLH2 showed no tendency to polymerize and the combination of peptides defg + defgh generated only disordered aggregates, with some indication of malformed tubules. The combination of biochemical and electron microscopical methods enabled the characterization of these polymers with respect to peptide composition and higher order structure.
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Negative Stains Containing Trehalose: Application to Tubular and Filamentous Structures
Microscopy and Microanalysis, 1996Co-Authors: J. Robin Harris, Max Gerber, Wolfgang Gebauer, Wolfgang Wernicke, Jurgen MarklAbstract:Several examples are presented that show the successful application of uranyl acetate and ammonium molybdate negative staining in the presence of trehalose for TEM studies of filamentous and tubular structures. The principal benefit to be gained from the inclusion of trehalose stems from the considerably reduced flattening of the large tubular structures and the greater orientational freedom of single molecules due to an increased depth of the negative stain in the presence of trehalose. Trehalose is likely to provide considerable protection to protein molecules and their assemblies during the drying of negatively stained specimens. Some reduction in the excessive density imparted by uranyl acetate around large assemblies is also achieved. Nevertheless, in the presence of 1% (w/v) trehalose, it is desirable to increase the concentration of negative stain to 5% (w/v) for ammonium molybdate and to 4% for uranyl acetate to produce satisfactory image contrast. In general, the ammonium molybdate-trehalose negative stain is more satisfactory than the uranyl acetate-trehalose combination, because of the greater electron beam sensitivity of the uranyl negative stain. Reassembled taxol-stabilized pig brain microtubules, together with collagen fibrils, sperm tails, helical filaments, and reassociated hemocyanin (KLH2), all from the giant keyhole limpet Megathura crenulata, have been studied by negative staining in the presence of trehalose. In all cases satisfactory TEM imaging conditions were readily obtained on the specimens, as long as regions of excessively deep stain were avoided.
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Keyhole limpet haemocyanin: negative staining in the presence of trehalose
Micron, 1995Co-Authors: J. Robin Harris, Wolfgang Gebauer, Jurgen MarklAbstract:Abstract Samples of unpurified and purified haemocyanin from the giant keyhole limpet Megathura crenulata have been studied by transmission electron microscopy (TEM) using mixtures of trehalose with the negative stains, uranyl acetate and ammonium molybdate. Trehalose is a known protein preservative during air and freeze drying, UV irradiation and high temperatures, and therefore offers the possibility of protecting proteins during the drying of negatively-stained specimens and their subsequent electron microscopical study. Evidence is presented that trehalose possesses satisfactory stability within the electron beam during conventional room temperature, negative-staining studies. The combination of negative stain and trehalose generates a deeper layer of amorphous stain around and within the keyhole limpet haemocyanin (KLH) di- and multidecamers, thereby increasing protein mobility and the generation of a range of tilted images, alongside the more usually observed end-on and side-on image projections. The concentration of uranyl acetate and ammonium molybdate has been increased to 4 and 5%, respectively, in the presence of 1% trehalose, in order to generate satisfactory image contrast. This is because of the reduction by trehalose of the net mass thickness of the dried negative stain-carbohydrate mixture compared to for an equivalent thickness of stain alone. The study of these specimens at low temperatures (−175°C) offers the possibility of superior structural preservation.
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, Augusto Manubens, Javiera Villar, 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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antitumor activity and carrier properties of novel hemocyanins coupled to a mimotope of gd2 ganglioside
European Journal of Medicinal Chemistry, 2018Co-Authors: Miriam Palacios, Ta-ying Zhong, Ricardo Tampe, Miguel Del Campo, Mercedes N Lopez, Flavio Salazaronfray, María Inés BeckerAbstract:Abstract Conjugation to carrier proteins is a way to improve the immunogenicity of peptides. Such is the case for peptides mimicking carbohydrate tumor-associated antigens in cancer vaccine development. The most used protein for this purpose is the keyhole limpet hemocyanin (KLH) from Megathura crenulata. Its limited bioavailability has prompted interest in finding new candidates; nevertheless, it is not known whether other hemocyanins might be equally efficient as carrier of carbohydrate peptide mimotopes to promotes anti-tumor responses. Here, we evaluated the carrier and antitumor activity of novel hemocyanins with documented immunogenicity obtained from Concholepas concholepas (CCH) and Fissurella latimarginata (FLH), coupled through sulfo-SMCC to P10, a mimetic peptide of GD2, the major ganglioside constituent of neuroectodermal tumors, and incorporating AddaVax as an adjuvant. The humoral immune responses of mice showed that CCH-P10 and FLH-P10 conjugates elicited specific IgM and IgG antibodies against P10 mimotope, similar to those obtained with KLH-P10, which was used as a positive control. The CCH-P10 and FLH-P10 antisera, exhibited cross-reactivity with murine and human melanoma cells, like anti-CCH and anti-FLH sera suggesting a cross-reaction of CCH and FLH glycosylations with carbohydrate epitopes on the tumor cell surfaces, similar to the KLH antisera. When mice were primed with each hemocyanin-P10 and challenged with melanoma cells, better antitumor effects were observed for FLH-P10 than for CCH-P10 and, as for KLH-P10, irrespective of conjugation. These data demonstrate that CCH and FLH are useful carriers of carbohydrate mimotopes; however, the best antitumor activity of FLH preparations, indicate that is a suitable candidate for further cancer vaccines research.
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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, Miguel Del Campo, Sergio Arancibia, Augusto Manubens, Raimundo Born, Ricardo Tampe, Javiera Villar, 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, Pablo De ,ioannes, Sergio Arancibia, Esteban Nova, Fabián Salazar, Andrea González, Bruno Moltedo, Jorge Ferreira, 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.