Structural analysis and molecular modeling of the RvH2-e functional unit of Rapana venosa hemocyanin.

Rapana venosa hemocyanin (RvH), a circulating glycoprotein of the marine snail, has a complex structure. To provide details on the stability of the protein, one functional unit, RvH2-e, was compared with the native molecule and the structural subunits, RvH1 and RvH2, via pH-T diagrams, typical phase portraits for stability and denaturation reversibility. By analyzing the T transition curves of RvH2-e at different pH values, several parameters of the thermodynamic functions were obtained. Increasing the temperature from 25°C to 55°C, the reversibility of the molecule of protein also increases, opening a reversibility window within the range of pH 4.0-8.0. On analyzing the pH transition curves, the start of the acid denaturation (below pH 6) and alkaline denaturation (above pH 9) was determined to be between 20°C and 35°C. For this range, the thermodynamic functions ΔH° and ΔG° for a standard temperature of 25°C were calculated.

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.

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.

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.