Struktur, Funktion und Eigenschaften von kupferhaltigen Proteinen: Hämocyanine und Superoxiddismutasen

The purpose of this thesis was to study the structure and properties of the metal - containing proteins, hemocyanins and superoxide dismutase. Both proteins contain copper ions in their active site and are useful in immunotherapy of various diseases. 1. The oligomeric stability of gastropodan hemocyanins Rapana venosa (RvH) and Helix vulgaris and their structural subunits RvH1 and RvH2 has been investigated. The reassociation of these two RvH isoforms and the native molecule was characterized in buffers with different pH values and concentrations of Ca2+ and Mg2+ chloride. Mixed RvH subunits reassociate into didecamers and multidecamers over a period of 2 days of dialysis using a stabilizing buffer (SB). RvH1 and RvH2 are biochemically and immunologically different and have also different dissociation properties. The reassociation, performed at pH 9.6 with 2 mM CaCl2 and MgCl2 over a period of several weeks, led to the formation of decameric oligomers, while didecamers formed predominately in the stabilizing buffer (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. 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. 2. Using Zn2+ ions as new method, several FUs have been isolated from molluscan Hc Rapana venosa without formation of non-functional proteolytic side products. N-terminal sequences of these fragments in comparison with FUs from other gastropodan Hcs show a very high degree of structural identity. Only four FUs, purified from enzyme-treated structural subunits RvH1 and RvH2 show identical N-terminal sequences compared to fragments isolated after treatment with Zn2+ ions. 3. Two isoforms alpha-hemocyanin and beta-hemocyanin of the garden snail Helix vulgaris have been isolated from the hemocyanin which do not reassociate completely in 50 mM CaCl2 and MgCl2 buffer, pH 7.0 4. It is generally accepted that the sugar constituents of the hemocyanin are likely to be implicated in their antigenicity. Carbohydrate moieties of molluscan Rapana venosa and arthropodan Carcinus aestuarii hemocyanins have been determined using several techniques: capillary electrophoresis, MALDI-MS, ESI-MS in combination with glycosidase digestions. Oligosaccharide composition of two FUs from the first structural subunit of Rapana venosa Hc, RvH1-a and RvH1-f, have been analysed. 5. The native subunit CaSS2 of crab Carcinus aestuarii is a polypeptide of 650 amino acid residues with a molecular weight determined to be 75036 Da which agrees with the SDS-PAGE results. The primary structure of CaeSS2 shows a high degree of sequence similarity with the other crustacean hemocyanins and seems to be more homologous with Cancer magister subunit 6 (73%). 10 Trp and 28 Tyr residues were identified in CaeSS2 and classified into three classes with fluorescence lifetimes around 0.11-0.15, 0.33 and 3.1-3.5 ns, respectively. 6. The conformation stability of arthropodan Hc Limulus polyphemus has been studied by CD and fluorescence spectroscopy. The conformational stabilities of the native dodecameric aggregates and their five isolated structural subunits towards various denaturants (pH and guanidine hydrochloride) indicate that the quaternary structure is stabilized by hydrophilic and polar forces, whereby both, the oxy- and apo-forms of the proteins are considered. Gnd.HCl-induced equilibrium denaturation of Hcs yielded a structural, partially unfolded state and a rapid conformational transition. 7. The primary structure of glycosylated Cu/Zn-superoxide dismutase from the fungal strain Humicola lutea was determined by Edman degradation. A single chain of the protein, consisting of 152 amino acid residues, reveals a very high degree (74–85%) of structural homology in comparison to the amino acid sequences of other fungal Cu/Zn-SODs. The difference of the molecular masses of H. lutea Cu/Zn-SOD, measured by MALDI-MS (15,935 Da) and calculated by its amino acid sequence (15,716 Da), is attributed to the carbohydrate chain of one mole of N-acetylglucosamine, attached to the N-glycosylation site Asn23-Glu-Ser. HL-SOD protected mice from mortality after experimental influenza A/Aichi/2/68 virus infection. Using the glycosylated HL-SOD, the survival rate is increased by 66% (protective index = 86.1%) and the survival time prolonged by 5 days, similar to the application of ribavarin, while non-glycosylated bovine SOD conferred lower protection. The results of this thesis are a further contribution to the structure, function and phylogenetic evolution of two clones of copper-containing proteins, the hemocyanins and superoxide dismutases.Das Ziel dieser Arbeit war es die Struktur und Eigenschaften von kupferhaltigen Proteinen, Hämocyanine und Superoxiddismutasen, zu untersuchen. Beide Proteine enthalten Kupferionen in ihren Aktivzentrums und sind hilfreich in der Immunotherapie verschiedener Krankheiten. 1. Die oligomere Stabilität der Gastropoden-Hämocyanine von Rapana venosa (RvH) und Helix vulgaris und deren strukturelle Untereinheiten wurden untersucht. Zwei strukturelle Untereinheiten RvH1 und RvH2 vom Rapana venosa-Hämocyanin wurden auf einer Ionenaustauschersäule mit Hilfe von FPLC getrennt. Die Reassoziation der beiden RvH-Isoformen und des nativen Hämocyanins wurden in Puffern bei verschiedenen pH-Werten und Kalzium- und Magnesiumchlorid-Konzentrationen untersucht. Gemische von RvH–Untereinheiten reassoziieren zu Didacdecameren und Multidecameren bei Dialyse innerhalb von zwei Tagen unter Verwendung eines Dialysestabilisierungspuffers von pH 7 in Gegenwart von 100 mM Kalziumchlorid und Magnesiumchlorid. RvH1 und RvH2 unterscheiden sich sowohl biochemisch wie immonologisch; sie haben außerdem verschiedene Dissoziationseigenschaften. Für die Reassoziation, die bei pH 6 mit 2 mM Calciumclorid und Magnesiumchlorid bei 4° C innerhalb einer bis mehreren Wochen durchgeführt wurden, bilden sich Dekamere; Didekamere traten hauptsächlich mit stabilisierendem Puffer bei pH 7 auf. Höhere Kalzium- und Magnesiumionen-Konzentrationen führten zu einer rascheren Reassization von RvH1, wobei stabile Multidekamere und helikale Assoziale sich bildeten, welche bei höheren pH-Werten zu kürzeren Multidekameren und Dekameren dissoziierten. Die Reassoziationen von strukturellen Untereinheiten von RvH2 erfolgten in denselben Puffern langsamer und führten zur Bildung von Didekameren, kürzeren Aggregaten und langen Multidekameren, die jedoch bei höheren pH-Werten weniger stabil sind. Es wurde weiterhin beobachtet, dass der Rückbildungsprozess von RvH1- und RvH2- Untereinheiten mehr dem von KLH ähnelt als dem von Haliotis tuberculata-Hämocyaninuntereinheiten. 2. Es wurde eine neue Methode etabliert, um vom nativen Rapana venosa Hämocyanin funktionelle Einheiten mit Hilfe von Zinkionen zu gewinnen. Die N-terminalen Sequenzen dieser funktionellen Einheiten zeigen eine hohe Identität mit denen anderer Gastropodenhämocyanine. Nur vier funktionelle Untereinheiten, welche nach Trypsinbehandlung von RvH1 und RvH2 gewonnen wurden, zeigten eine identische N-terminale Sequenz mit denen nach Zinkbehandlung gewonnenen. 3. Aus der Gartenschnecke Helix vulgaris wurden zwei Hämocyanin-Isoformen isoliert, welche jedoch in 50 mM Calciumchlorid- und Magnesiumchloridpuffer bei pH 7 nicht vollständig reassoziierten. 4. Es wird allgemein angenommen, dass die Kohlenhydratkomponenten von Hämocyaninen für die Stimulation des Immunsystems verantwortlich sind. Von Rapana venosa- und Carcinus aestuarii-Hemocyanin wurden die Strukturen von Oligosaccharidketten durch MALDI-MS, ESI-MS in Kombination mit verschiedenen Glykosidasen bestimmt. 5. Die Primärstruktur von der Untereinheit 2 von C. aestuarii zeigt eine hohe Sequenzidentität mit der Untereinheit 1 von P.vulgaris und Untereinheit c von P.interruptus. 10 Tryptophan- und 28 Tyrosinreste wurden in der Untereinheit 2 von C. aestuarii identifiziert und durch Fluoreszenzspektroskopie untersucht. 6. Vom Limulus polyphemus-Hämacyanin wurde die Konformationsstabilität mit Circulardichroismus und Fluoreszenzspektroskopie untersucht. Dabei können fünf Proteinketten elektrophoretisch getrennt werden. Es zeigte sich, dass die strukturellen Untereinheiten eine geringere Thermostabilität aufweisen, als das native Molekül. Auch Entfernung des Kupfers aus dem Aktivzentrum führt zu einer geringeren Thermostabiltät. 7. Aus dem Pilz H. lutea wurde eine glykolisierte Kupfer-Zink-Superoxiddismutase isoliert und deren Primärstruktur bestimmt. Das Protein besitzt 152 Aminosäurereste und ist zu 74 - 85 Prozent strukturhomolog mit anderen Kupfer-Zink-Superoxid-dismutasen aus Pilzen. Durch Edman-Abbau und MALDI-Massenspektrometrie konnte nachgewiesen werden, dass ein N-Acetylglucoseminrest mit der N-Glykosy-lierungsstelle Asn23-Glu-Ser verknüpft ist. Durch Tierversuche konnte gezeigt werden, dass diese glycosylierte Kupfer-Zink-Superoxiddismutase bei Mäusen die Überlebensrate nach Virusinfektionen erhöht. Die Ergebnisse dieser Dissertation liefern einen Beitrag zur Struktur, Funktion und Phylogenese von Hämocyaninen und Superoxiddismutasen

Isolation and characterization of novel tyrosinase from Laceyella sacchari

We here describe the isolation and characterization of a tyrosinase from a newly isolated soil bacterium. 16S rDNA sequence analysis revealed that the bacterium most probably belongs to the species Laceyella sacchari (Ls) (>99.9% identity). The tyrosinase extracellular enzymatic activity was induced in the presence of L-methionine and CuSO4. The crude enzyme was first purified by centrifugation followed by ammonium sulphate precipitation and ultrafiltration. After removal of a brown pigment, probably melanin, a purified enzyme was obtained by further separation of the crude protein mixture using size exclusion chromatography. Some 10.5 mg of pure tyrosinase (LsTyr) was isolated with a molecular mass of 30 910 Da, based on MALDI mass spectrometry. Together with the observed enzymatic activity, N-terminal chemical sequence analysis confirmed that the isolated enzyme is homologous to other tyrosinases. The kinetic parameters for the diphenol substrates L-DOPA and dopamine and for the monophenol substrate L-tyrosine were determined to be K-M = 4.5 mM, 1.5 mM and 0.055 mM, and k(cat)/K-M = 261.5 mM (1) s (1), 30.6 mM (1) s (1) and 56.3 mM(-1) s(-1), respectively. Maximal activities of the purified enzyme were found to occur at pH 6.8

Antibacterial Properties of Peptide and Protein Fractions from <i>Cornu aspersum</i> Mucus

The discovery and investigation of new natural compounds with antimicrobial activity are new potential strategies to reduce the spread of antimicrobial resistance. The presented study reveals, for the first time, the promising antibacterial potential of two fractions from Cornu aspersum mucus with an MW 20 kDa against five bacterial pathogens—Bacillus cereus 1085, Propionibacterium acnes 1897, Salmonella enterica 8691, Enterococcus faecalis 3915, and Enterococcus faecium 8754. Using de novo sequencing, 16 novel peptides with potential antibacterial activity were identified in a fraction with an MW 20 kDa were determined via a proteomic analysis on 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and bioinformatics. High homology with proteins and glycoproteins was found, with potential antibacterial activity in mucus proteins named aspernin, hemocyanins, H-lectins, and L-amino acid oxidase-like protein, as well as mucins (mucin-5AC, mucin-5B, mucin-2, and mucin-17). We hypothesize that the synergy between the bioactive components determined in the composition of the fraction > 20 kDa are responsible for the high antibacterial activity against the tested pathogens in concentrations between 32 and 128 µg/mL, which is comparable to vancomycin, but without cytotoxic effects on model eukaryotic cells of Saccharomyces cerevisiae. Additionally, a positive effect, by reducing the levels of intracellular oxidative damage and increasing antioxidant capacity, on S. cerevisiae cells was found for both mucus extract fractions of C. aspersum. These findings may serve as a basis for further studies to develop a new antibacterial agent preventing the development of antibiotic resistance

Antitumour activity of Helix hemocyanin against bladder carcinoma permanent cell lines

Hemocyanins are oxygen-transporting glycoproteins in molluscs and arthropods. In this study, we assayed the biomacromolecules from the molluscs Helix lucorum (HlH), Rapana venosa (RvH) and Megatura crenulata (KLH) including their functional units (FUs), for therapy of bladder cancer permanent cells. In vitro studies antitumour activities of these proteins were performed with bladder cancer permanent cell line CAL-29 and the normal urothelial cell line HL 10/29 in comparison to doxorubicin. The obtained results showed that the human tumour CAL-29 cell line is sensitive to the action of the tested hemocyanins and their isoforms. We observed a dose- and time-dependent inhibition of tumour cell growth after incubation with native HlH and two FUs (βc-HlH-a and FU βc-HlH-h); and of particular significance, FU βc-HlH-h showed a surprisingly stronger effect than that the doxorubicin-treated cells. Cells treated with βc-HlH-h, showed both, apoptotic and necrotic cells. In addition, two-dimensional polyacrylamide gel electrophoresis (PAGE) found for differential up-regulation of several proteins after hemocyanin treatment. Eight different down-regulated and two up-regulated proteins were identified, which may be associated with the apoptosis pathway. No inhibition of the normal urothelial cell line HL 10/29 was observed after treatment with HlH and its isoforms. The most effective inhibition of CAL-29 tumour cells was observed after treatment with βc-HlH-h, probably caused by a specific oligosaccharide structure of HlH with methylated hexoses. These results suggest that hemocyanin glycosylation plays an important role in its anticancer activity

Effect and Mechanisms of Antibacterial Peptide Fraction from Mucus of C. aspersum against Escherichia coli NBIMCC 8785

Peptides isolated from the mucus of Cornu aspersum could be prototypes for antibiotics against pathogenic bacteria. Information regarding the mechanisms, effective concentration, and methods of application is an important tool for therapeutic, financial, and ecological regulation and a holistic approach to medical treatment. A peptide fraction with MW &lt; 10 kDa was analyzed by MALDI-TOF-TOF using Autoflex&trade; III. The strain Escherichia coli NBIMCC 8785 (18 h and 48 h culture) was used. The changes in bacterial structure and metabolic activity were investigated by SEM, fluorescent, and digital image analysis. This peptide fraction had high inhibitory effects in surface and deep inoculations of E. coli of 1990.00 and 136.13 mm2/mgPr/&micro;Mol, respectively, in the samples. Thus, it would be effective in the treatment of infections involving bacterial biofilms and homogenous cells. Various deformations of the bacteria and inhibition of its metabolism were discovered and illustrated. The data on the mechanisms of impact of the peptides permitted the formulation of an algorithm for the treatment of infections depending on the phase of their development. The decrease in the therapeutic concentrations will be more sparing to the environment and will lead to a decrease in the cost of the treatment

Hemocyanins from Helix and Rapana Snails Exhibit in Vitro Antitumor Effects in Human Colorectal Adenocarcinoma

Hemocyanins are oxygen-transporting glycoproteins in the hemolymph of arthropods and mollusks that attract scientific interest with their diverse biological activities and potential applications in pharmacy and medicine. The aim of the present study was to assess the in vitro antitumor activity of hemocyanins isolated from marine snail Rapana venosa (RvH) and garden snails Helix lucorum (HlH) and Helix aspersa (HaH), as well the mucus of H. aspersa snails, in the HT-29 human colorectal carcinoma cell line. The effects of the hemocyanins on the cell viability and proliferation were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the alterations in the tumor cell morphology were examined by fluorescent and transmission electron microscopy. The results of the MTT assay showed that the mucus and &alpha;-subunit of hemocyanin from the snail H. aspersa had the most significant antiproliferative activity of the tested samples. Cytomorphological analysis revealed that the observed antitumor effects were associated with induction of apoptosis in the tumor cells. The presented data indicate that hemocyanins and mucus from H. aspersa have an antineoplastic activity and potential for development of novel therapeutics for treatment of colorectal carcinoma

Antitumor Activity of Bioactive Compounds from Rapana venosa against Human Breast Cell Lines

This study is the first report describing the promising antitumor activity of biologically active compounds isolated from the hemolymph of marine snail Rapana venosa&mdash;a fraction with Mw between 50 and 100 kDa and two structural subunits (RvH1 and RvH2), tested on a panel of human breast cell lines&mdash;six lines of different molecular subtypes of breast cancer MDA-MB-231, MDA-MB-468, BT-474, BT-549, SK-BR-3, and MCF-7 and the non-cancerous MCF-10A. The fraction with Mw 50&ndash;100 kDa (HRv 50&ndash;100) showed good antitumor activity manifested by a significant decrease in cell viability, altered morphology, autophagy, and p53 activation in treated cancer cells. An apparent synergistic effect was observed for the combination of HRv 50&ndash;100 with cis-platin for all tested cell lines. The combination of HRv 50&ndash;100 with cisplatin and/or tamoxifen is three times more effective compared to treatment with classical chemotherapeutics alone. The main proteins in the active fraction, with Mw at ~50 kDa, ~65 kDa, ~100 kDa, were identified by MALDI-MS, MS/MS analyses, and bioinformatics. Homology was established with known proteins with antitumor potential detected in different mollusc species: peroxidase-like protein, glycoproteins Aplysianin A, L-amino acid oxidase (LAAO), and the functional unit with Mw 50 kDa of RvH. Our study reveals new perspectives for application of HRv 50&ndash;100 as an antitumor agent used alone or as a booster in combination with different chemotherapies

Structural and functional characterization of cold-active sialidase isolated from Antarctic fungus Penicillium griseofulvum P29

The fungal strain, Penicillium griseofulvum P29, isolated from a soil sample taken from Terra Nova Bay, Antarctica, was found to be a good producer of sialidase (P29). The present study was focused on the purification and structural characterization of the enzyme. P29 enzyme was purified using a Q-Sepharose column and fast performance liquid chromatography separation on a Mono Q column. The determined molecular mass of the purified enzyme of 40 kDa by SDS-PAGE and 39924.40 Da by matrix desorption/ionization mass spectrometry (MALDI-TOF/MS) analysis correlated well with the calculated mass (39903.75 kDa) from the amino acid sequence of the enzyme. P29 sialidase shows a temperature optimum of 37 °C and low-temperature stability, confirming its cold-active nature. The enzyme is more active towards α(2 → 3) sialyl linkages than those containing α(2 → 6) linkages.Based on the determined amino acid sequence and 3D structural modeling, a 3D model of P29 sialidase was presented, and the properties of the enzyme were explained. The conformational stability of the enzyme was followed by fluorescence spectroscopy, and the new enzyme was found to be conformationally stable in the neutral pH range of pH 6 to pH 9. In addition, the enzyme was more stable in an alkaline environment than in an acidic environment. The purified cold-active enzyme is the only sialidase produced and characterized from Antarctic fungi to date