Temperature-stress tolerance of the fungal strain Aspergillus niger 26: physiological and ultrastructural changes.

The study focuses on the morphological and physiological cell responses to oxidative stress induced by high temperature treatment in the industrially relevant fungus Aspergillus niger 26. Temperatures above 30 °C lead to growth suppression and changes in morphological characteristics: decrease in the size of hyphal elements and increase in "active length" by switching from slightly branched long filaments to a multitude of branched forms containing active cytoplasm. Transmission electron microscopy of fungal cultures heated at 40 °C demonstrated abnormal wavy septation with reduced amount of chitin (as shown by WGA-gold labelling), intrahyphal hyphae development, disintegration of mitochondria and extensive autolysis. Temperature-dependent decrease in the total intracellular protein content and a sharp increase (six to tenfold) in oxidatively damaged proteins were also demonstrated. Elevated temperatures caused a two and threefold increase in catalase and superoxide dismutase activities, respectively.

The cDNA sequence of three hemocyanin subunits from the garden snail Helix lucorum.

Hemocyanins are blue copper containing respiratory proteins residing in the hemolymph of many molluscs and arthropods. They can have different molecular masses and quaternary structures. Moreover, several molluscan hemocyanins are isolated with one, two or three isoforms occurring as decameric, didecameric, multidecameric or tubule aggregates. We could recently isolate three different hemocyanin isopolypeptides from the hemolymph of the garden snail Helix lucorum (HlH). These three structural subunits were named α(D)-HlH, α(N)-HlH and ß-HlH. We have cloned and sequenced their cDNA which is the first result ever reported for three isoforms of a molluscan hemocyanin. Whereas the complete gene sequence of α(D)-HlH and ß-HlH was obtained, including the 5' and 3' UTR, 180bp of the 5' end and around 900bp at the 3' end are missing for the third subunit. The subunits α(D)-HlH and ß-HlH comprise a signal sequence of 19 amino acids plus a polypeptide of 3409 and 3414 amino acids, respectively. We could determine 3031 residues of the α(N)-HLH subunit. Sequence comparison with other molluscan hemocyanins shows that α(D)-HlH is more related to Aplysia californicum hemocyanin than to each of its own isopolypeptides. The structural subunits comprise 8 different functional units (FUs: a, b, c, d, e, f, g, h) and each functional unit possesses a highly conserved copper-A and copper-B site for reversible oxygen binding. Potential N-glycosylation sites are present in all three structural subunits. We confirmed that all three different isoforms are effectively produced and secreted in the hemolymph of H. lucorum by analyzing a tryptic digest of the purified native hemocyanin by MALDI-TOF and LC-FTICR mass spectrometry.

Synthesis and inhibition properties of a series of pyranose derivatives towards a Zn-metalloproteinase from Saccharomonospora canescens.

The Zn-proteinase, isolated from Saccharomonospora canescens (NPS), shares many common features with thermolysin, but considerable differences are also evident, as far as the substrate recognition site is concerned. In substrates of general structure AcylAlaAlaPhe 4NA, this neutral proteinase cleaves only the arylamide bond (non-typical activity of Zn-proteinases), while thermolysin attacks the peptide bond Ala-Phe. Phosphoramidon is a powerful tight binding inhibitor for thermolysin and significantly less specific towards NPS. The K(i)-values (65 µM for NPS vs 0.034 µM for thermolysin) differ nearly 2000-folds. This implies significant differences in the specificity of the corresponding subsites. The carbohydrate moiety is supposed to accommodate in the S1-subsite and the series of arabinopyranosides and glucopyranosides (12 compounds), which are assayed as inhibitors in a model system: NPS with SucAlaAlaPhe4NA as a substrate could be considered as mapping the S1-subsite of NPS. Members of the series with an additional ring (3,4-epithio, 3,4-anhydro-derivatives) turned out to be reasonably good competitive inhibitors (K(i)≈0.1-0.2 mM are of the same order as the K(i) value for phosphoramidon). The structure of these compounds (8, 9, 11 and 12) seems to fit the size of the S1-subsite and due to an appropriately oriented OH-group in addition, to protect the active site Zn(2+).

Structure of hemocyanin from garden snail Helix lucorum.

Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs and arthropods with different quaternary structure. They are represented in the hemolymph of molluscs with one, two or three isoforms, as decameric, didecameric, multidecameric and tubules aggregates. We describe here the structure of the hemocyanin Helix lucorum (HlH), species in the series of molluscan hemocyanins. In contrast with other molluscan hemocyanins, three different hemocyanin isopolypeptides were isolated from the hemolymph of the garden snail H. lucorum, named as beta-HlH, alpha(D)-HlH and alpha(N)-HlH. Their molecular masses were determined by size exclusion chromatography to be 1068 kDa (beta-HlH) and 1079 kDa (alpha(D)-HlH, and alpha(N)-HlH). Native HlH exhibits a predominant didecameric structure as revealed by electron microscopy and additionally few tridecamers are shown in the electron micrographs of HlH resulting from the association of a further decamer with one didecamer. The three isoforms are represented mainly as homogeneous didecamers, but they have different behaviour after dissociation and reassociation in the pH-stabilizing buffer, containing 20 mM CaCl(2). All isoforms were reassociated into didecamers and tubules with different length, but in contrast to alpha(D)-HlH isoform, longer tubules were observed in beta-HlH. Moreover the structure of beta-HlH was analysed after limited proteolysis with trypsin followed by FPLC and HPLC separation of the cleavage products. Eight different functional units were identified by their N-terminal sequences and molecular masses. The protein characteristics, including UV absorption at 340 nm, fluorescence and CD spectra of the native molecule and its units confirmed the structure of multimer protein complexes.

Purification and partial characterization of Cu/Zn superoxide dismutase from Kluyveromyces marxianus yeast.

A new thermostable Cu/Zn SOD from a thermotolerant yeast strain Kluyveromyces marxianus NBIMCC 1984 has been purified and characterized. The purification procedure comprises thermal treatment and dialysis, ion-exchange chromatography and chromatofocusing. The methodology is a rapid, efficient and highly specific, generating pure preparation (specific activity 996 U mg of protein(-1)) with a yield of 53%. The purified enzyme is a homodimer with Mw of 34,034 Da and has high N-terminal homology with other yeasts' Cu/Zn SOD enzymes. The protein is characterized with some unique features such as-thermostability (t(1/2) at 70 degrees C=30 min), pH stability in the alkaline range (7.5-8.5) and resistance to inhibitors and variety of chemicals. These characteristics reveal possibilities for wide practical application of K. marxianus Cu/Zn SOD enzyme.

Identification of glycosylated sites in Rapana hemocyanin by mass spectrometry and gene sequence, and their antiviral effect.

Molluscan hemocyanins (Hcs) have recently received particular interest due to their significant immunostimulatory properties. This is mainly related to their high carbohydrate content and specific monosaccharide composition. We have now analyzed the oligosaccharides and the carbohydrate linkage sites of the Rapana venosa hemocyanin (RvH) using different approaches. We analyzed a number of glycopeptides by LC/ESI-MS/MS and identified the sugar chains and peptide sequences of 12 glycopeptides. Additionally, the potential carbohydrate linkage sites of 2 functional units, RvH-b and RvH-c, were determined by gene sequence analysis. Only RvH-c shows a potential N-glycosylation site. During this study, we discovered a highly conserved linker-intron, separating the coding exons of RVH-b and RvH-c. Following reports on antiviral properties from arthropod hemocyanin, we conducted a preliminary study of the antiviral activity of RvH and the functional units RvH-b and RvH-c. We show that the glycosylated FU RvH-c has antiviral properties against the respiratory syncytial virus (RSV), whereas native RvH and the nonglycosylated FU RvH-b have not. This is the first report of the fact that also molluscan hemocyanin functional units possess antiviral activity.

Simultaneous engagement of FcgammaIIb and CD22 inhibitory receptors silences targeted B cells and suppresses autoimmune disease activity.

All B cell targeting therapeutic approaches used at present are unspecific and there is an urgent need for agents that silence selectively pathological autoreactive B lymphocytes only. We hypothesized that this aim could be achieved by chimeric antibodies that cross-link B cell immunoglobulin receptors with inhibitory receptors on the surface of the same targeted disease-associated cell. A hybrid molecule was constructed by coupling copies of the DNA-mimicking DWEYSVWLSN peptide and of the CD22-binding STN epitope with a free terminal sialic acid to a mouse monoclonal IgG antibody backbone. The DNA mimotope peptide binds to the immunoglobulin B cell receptor of pathological DNA-specific B cells of lupus mice, the STN epitope - to CD22 and the IgG by its Fc fragment - to FcgammaIIb on the surface of the same cell. Mass-spectra analysis showed that 4 STN epitopes plus 5 DNA mimotope peptides were coupled to a single light immunoglobulin chain and 4 STN - and 2 DNA mimotopes - to a heavy chain. Both FcgammaIIb and CD22 receptors on spleen cells from lupus MRL/lpr mice were phosphorylated after exposure to the chimeric antibody, indicating the involvement of both inhibitory pathways. The constructed chimera suppressed specifically in vitro as well as in vivo anti-DNA IgM and IgG antibody production and delayed the development of glomerulonephritis in the lupus-prone animals. The use of chimeric antibodies targeting two independent inhibitory B lymphocyte receptors represents a novel approach for the selective suppression of pathological autoreactive B cells in autoimmune diseases.

Heat-shock-induced oxidative stress and antioxidant response in Aspergillus niger 26.

To extend the knowledge about the relationship between heat shock and oxidative stress in lower eukaryotes, the filamentous fungus Aspergillus niger 26 was chosen as a model system. Here, the response of A. niger cells to heat shock is reported. The temperature treatment significantly increased the levels of reactive oxygen species, superoxide anions (O2), and hydrogen peroxide and the rate of cyanide-resistant respiration as a marker of oxidative stress. Enhanced reactive oxygen species generation coincided with an increase in the content of oxidative damaged protein and in the accumulation of the storage carbohydrates trehalose and glycogen. Thermal survival of the A. niger cells corresponded to a significant increase in the levels of the antioxidant enzymes superoxide dismutase and catalase for all variants. These observations suggest that heat and oxidative stress have a common cellular effect.

Immunological potential of Helix vulgaris and Rapana venosa hemocyanins.

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.

Reversibility and "pH-T phase diagrams" of Rapana venosa hemocyanin and its structural subunits.

We have studied the stability and reassociation behaviour of native molecules of Rapana venosa hemocyanin and its two subunits, termed RvH1 and RvH2. In the presence of different concentrations of Ca(2+) and Mg(2+) ions and pH values, the subunits differ not only in their reassociation behaviour, but also in their formation of helical tubules and multidecamers. RvH1 revealed a greater stability at higher pH values compared to RvH2. Overall, the stability of reassociated RvH and its structural subunits was found to be pH-dependent. The increasing stability of native Hc and its subunits, shown by pH-induced CD transitions (acid and alkaline denaturation), can be explained with the formation of quaternary structure. The absence of a Cotton effect at temperatures 20-40 degrees C in the pH-transition curves of RvH2 indicates that this subunit is stabilized by additional "factors", e.g.: non-ionic/hydrophobic stabilization and interactions of carbohydrate moieties. A similar behaviour was observed for the T-transition curves in a wide pH interval for RvH and its structural subunits. At higher temperatures, many of the secondary structural elements are preserved especially at neutral pH, even at extreme high temperatures above 90 degrees C the protein structures resemble a "globule state".

Biochemical properties of Cu/Zn-superoxide dismutase from fungal strain Aspergillus niger 26.

The fungal strain Aspergillus niger produces two superoxide dismutases, Cu/Zn-SOD and Mn-SOD. The primary structure of the Cu/Zn-SOD has been determined by Edman degradation of peptide fragments derived from proteolytic digests. A single chain of the protein, consisting of 153 amino acid residues, reveals a very high degree of structural homology with the amino acid sequences of other Aspergillus Cu/Zn-SODs. The molecular mass of ANSOD, measured by MALDI-MS and ESI-MS, and calculated by its amino acid sequence, was determined to be 15821 Da. Only one Trp residue, at position 32, and one disulfide bridge were identified. However, neither a Tyr residue nor a carbohydrate chain occupying an N-linkage site (-Asn-Ile-Thr-) were found. Studies on the temperature and pH dependence of fluorescence, and on the temperature dependence of CD spectroscopic properties, confirmed that the enzyme is very stable, which can be explained by the stabilising effect of the disulfide bridge. The enzyme retains about 53% of its activity after incubation for a period of 30 min at 60 degrees C, and 15% at 85 degrees C.

o-Diphenol oxidase activity of molluscan hemocyanins.

Diphenoloxidase activities of two molluscan hemocyanins, isolated from the marine snails Rapana venosa and garden snails Helix vulgaris were studied using o-diphenol and L-Dopa as substrates. The dimers of H. vulgaris Hc show both, diphenol (K(m)=2.86 mM and K(cat)=4.48) and L-Dopa activity due to a more open active sites of the enzyme and better access of the substrates. The K(m) value of molluscan H. vulgaris Hc is very close to those of Helix pomatia and Sepia officinalis Hcs, but several times higher compared to those of Rapana and Octopus Hcs. Also HvH has a very high enzyme activity compared with other molluscan Hcs. Kinetic measurements with native RvH and both structural subunits, RvH1 and RvH2, show that RvH and only one structural subunit, RvH2, exhibited only o-diphenol activity, but no L-Dopa oxidizing activity.

Unusual location and characterization of Cu/Zn-containing superoxide dismutase from filamentous fungus Humicola lutea.

The present study aims to provide new information about the unusual location of Cu/Zn-superoxide dismutase (Cu/Zn-SOD) in lower eukaryotes such as filamentous fungi. Humicola lutea, a high producer of SOD was used as a model system. Subcellular fractions [cytosol, mitochondrial matrix, and intermembrane space (IMS)] were isolated and tested for purity using activity measurements of typical marker enzymes. Evidence, based on electrophoretic mobility, sensitivity to KCN and H(2)O(2) and immunoblot analysis supports the existence of Cu/Zn-SOD in mitochondrial IMS, and the Mn-SOD in the matrix. Enzyme activity is almost equally partitioned between both the compartments, thus suggesting that the intermembrane space could be one of the major sites of exposure to superoxide anion radicals. The mitochondrial Cu/Zn-SOD was purified and compared with the previously published cytosolic enzyme. They have identical molecular mass, cyanide- and H(2)O(2)-sensitivity, N-terminal amino acid sequence, glycosylation sites and carbohydrate composition. The H. lutea mitochondrial Cu/Zn-SOD is the first identified naturally glycosylated enzyme, isolated from IMS. These findings suggest that the same Cu/Zn-SOD exists in both the mitochondrial IMS and cytosol.

Oligosaccharide structure of a functional unit RvH1-b of Rapana venosa hemocyanin using HPLC/electrospray ionization mass spectrometry.

In the present study the structures of two glycopeptides (G1 and G1'), isolated from FU RvH(1)-b and two glycopeptides (G2 and G3), isolated from the structural subunit RvH(1) of Rapana venosa hemocyanin, were determined. To structurally characterize the site-specific carbohydrate heterogeneity and binding site of the N-linked glycopeptide(s), a combination of capillary reversed-phase chromatography and ion trap mass spectrometry was used. The amino acid sequences of glycopeptides G1 and G1' determined by Edman degradation and MS/MS sequencing demonstrated that the oligosaccharides are linked to N-glycosylation sites. Two peptides (a glycosylated (G1) and non-glycosylated one) were identified in this fraction and no linkage sites were observed in the latter one. Based on the sequencing of the glycosylated fractions G1, G1', G2 and G3, the carbohydrate structure Man(alpha1-6)Man(alpha1-3)Man(beta1-4)GlcNAc(beta1-4)[Fuc(alpha1-6)]GlcNAc-R could be identified for glycopeptides G1 and G3, and only the typical core structure Man(alpha1-6)Man(alpha1-3)Man(beta1-4)GlcNAc(beta1-4)GlcNAc-R was found for G1' and G2. The Fuc residue found in glycopeptides G1 and G3 is attached to N-acetyl-glucosamine of the carbohydrate core, as often found in other glycoproteins.

A challenging insight on the structural unit 1 of molluscan Rapana venosa hemocyanin.

Hemocyanins of mollusks are high molecular mass glycoproteins with a complex quaternary structure which still remains to be defined in detail for most of its species as far as number, spatial distribution and interactions of their structural units is concerned. In the present study, we isolated the functional units of the structural subunit RvH1 of Rapana venosa hemocyanin, combining enzymatic and non-enzymatic methods. Our results suggest that Hc's carbohydrate moieties play a basic role in the organization of the structural units, resulting from post-translational polymerization of the 50 kDa functional units and involving sugar moieties that link between them.

New insights in Rapana venosa hemocyanin N-glycosylation resulting from on-line mass spectrometric analyses.

The N-glycosylation of structural unit 1 of Rapana venosa hemocyanin was studied. Enzymatically liberated N-glycans were analyzed by matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis (CE)-MS following 8-aminopyrene-1,3,6-trisulfonate labeling and labeling with 3-aminopyrazole, a new dedicated sugar reagent. Structural information was obtained by exoglycosidase sequencing, on-line MS/MS, permethylation, and amidation. A mixture of high-mannose and complex glycans with so far unknown and unusual acidic terminal structures was revealed. As the hemocyanin protein sequence is currently unknown, de novo sequencing of the glycopeptides had to be carried out. The N-glycans were therefore enzymatically removed with simultaneous partial (50%) (18)O-labeling of glycosylated asparagine residues prior to proteolysis. Following nano-liquid chromatography-MALDI-TOF-MS, the originally glycosylated peptides could be revealed and their sequences determined by MS/MS. The site occupancies were subsequently elucidated by precursor ion scanning of the intact glycopeptides using a Q-Trap mass spectrometer.

Structure of hemocyanin subunit CaeSS2 of the crustacean Mediterranean crab Carcinus aestuarii.

Arthropodan hemocyanins are giant respiratory proteins responsible for oxygen transport. They exhibit unusual assemblies of up to 48 structural subunits. Hemocyanin from Carcinus aestuarii contains three major and two minor structural subunits. Here, we reveal the primary structure of the gamma-type 75 kDa subunit of Carcinus aestuarii hemocyanin, CaeSS2, and combine structure-based sequence alignments, tryptophan fluorescence, and glycosylation analyses to provide insights into the structural and functional organisation of CaeSS2. We identify three functional domains and three conserved histidine residues that most likely participate in the formation of the copper active site in domain 2. Oxygen-binding ability of Carcinus aestuarii Hc and its structural subunit 2 was studied using CD and fluorescence spectroscopy. Removing the copper dioxygen system from the active site led to a decrease of the melting temperature, which can be explained by a stabilizing effect of the binding metal ion. To study the quenching effect of the active site copper ions in hemocyanins, the copper complex Cu(II)(PuPhPy)2+ was used, which appears as a very strong quencher of the tryptophan emission. Furthermore, the structural localization was clarified and found to explain the observed fluorescence behavior of the protein. Sugar analysis reveals that CaeSS2 is glycosylated, and oligosaccharide chains connected to three O-glycosylated and one N-glycosylated sites were found.

Purification and characterization of a novel sialidase from a strain of Arthrobacter nicotianae.

The nonpathogenic strain Arthrobacter nicotianae produces two sialidase isoenzymes, NA1 and NA2, with molecular masses of 65 kDa and 54 kDa, respectively, as determined by 10% SDS-polyacrylamide gel electrophoresis. NA1 and NA2 exhibit maximum activities at pH 4 and 5, and both show clear thermal optima at 40 degrees C. They are stable at temperatures up to 50 degrees C. The critical temperatures (T (c) = 50 degrees C and 51 degrees C) for the two isoenzymes were determined by fluorescence spectroscopy and correlate well with the temperatures of melting (T (m) = 49 degrees C and 48 degrees C), determined by CD spectroscopy. The isoenzymes are less stable against denaturation with Gdn.HCl, and the free energy of stabilization in water was calculated to be 7.6 and 8.0 kJ mol(-1), respectively. The specific activity (K (m) value) toward glucomacropeptide as a substrate was calculated to be 0.126 mM for NA1 and 0.083 mM for NA2.

Structure and stability of arthropodan hemocyanin Limulus polyphemus.

In the hemolymph of many arthropodan species, respiratory copper proteins of high molecular weight, termed hemocyanins (Hcs) are dissolved. In this communication, we report on the protein stability of different hemocyanin species (Crustacea and Chelicerata) using fluorescence spectroscopy. Five to seven major electrophoretically separable protein chains (structural subunits) were purified by fast protein liquid chromatography (FPLC) ion exchange chromatography from different hemocyanins with very high sequence homology of the active site regions binding copper ions (CuA and CuB), and especially the relative sequence positions of histidine (His) and tryptophan (Trp) residues of these protein segments are in all cases identical. The conformational stabilities of the native dodecameric aggregates and their isolated structural subunits towards various denaturants (pH and guanidine hydrochloride (Gdn.HCl)) indicate that the quaternary structure is stabilized by hydrophilic and polar forces, whereby both, the oxy- and apo-forms of the protein are considered. These two classes of Crustacea and Chelicerata Hcs have the similar Trp-fluorescence quantum yields, but different values of lambda(max) emission (about 325 and 337 nm, respectively). Differences in the quantum yields are observed of the oxy- and apo-forms, which must be attributed to the fluorescence quenching effect of the two copper ions (CuA and CuB) in the active site. The position of emission maximum indicates tryptophan side chains are situated in a non-polar environment. Denaturation studies of Hcs by Gdn.HCl indicate that the denaturation process consists of two steps: dissociation of the native molecule into its structural subunits and denaturation of the subunits at concentrations >1.5M Gdn.HCl. Two steps of denaturation are also observed after keeping the protein in buffer solutions at different pH values with different pH-stability for holo-oxy and apo-Hc forms.

Structural and functional analysis of glycosylated Cu/Zn-superoxide dismutase from the fungal strain Humicola lutea 103.

The fungal strain Humicola lutea 103 produces a naturally glycosylated Cu/Zn-superoxide dismutase (Cu/ZnSOD) (HLSOD). To improve its yield, the effect of increased concentration of Cu2+ (from 1 to 750 microg/ml) on growth and enzyme biosynthesis was studied. The primary structure of this fungal enzyme has been determined by Edman degradation of peptide fragments derived from proteolytic digest. 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/ZnSODs. The difference of the molecular masses of H. lutea Cu/ZnSOD, 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. HLSOD protected mice from mortality after experimental influenza A/Aichi/2/68 (H3N2) virus infection. Using the glycosylated HLSOD, the survival rate is increased by 66% (protective index=86.1%) and the survival time prolonged by 5.2 days, similar to the application of ribavarin, while non-glycosylated bovine SOD conferred lower protection.