Molecular Cloning, Structure and Phylogenetic Analysis of a Hemocyanin Subunit from the Black Sea Crustacean Eriphia verrucosa (Crustacea, Malacostraca).

Hemocyanins are copper-binding proteins that play a crucial role in the physiological processes in crustaceans. In this study, the cDNA encoding hemocyanin subunit 5 from the Black sea crab Eriphia verrucosa (EvHc5) was cloned using EST analysis, RT-PCR and rapid amplification of the cDNA ends (RACE) approach. The full-length cDNA of EvHc5 was 2254 bp, consisting of a 5' and 3' untranslated regions and an open reading frame of 2022 bp, encoding a protein consisting of 674 amino acid residues. The protein has an N-terminal signal peptide of 14 amino acids as is expected for proteins synthesized in hepatopancreas tubule cells and secreted into the hemolymph. The 3D model showed the presence of three functional domains and six conserved histidine residues that participate in the formation of the copper active site in Domain 2. The EvHc5 is O-glycosylated and the glycan is exposed on the surface of the subunit similar to Panulirus interruptus. The phylogenetic analysis has shown its close grouping with γ-type of hemocyanins of other crustacean species belonging to order Decapoda, infraorder Brachyura.

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".

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.

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.

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.

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.

Characterization of the carbohydrate moieties of the functional unit RvH1-a of Rapana venosa haemocyanin using HPLC/electrospray ionization MS and glycosidase digestion.

The primary structures of two biantennary N -glycans of the glycoprotein Rapana venosa (marine snail) haemocyanin were determined. Two different structural subunits have been found in R. venosa haemocyanin: RvH1 and RvH2. The carbohydrate content of the N-terminal functional unit RvH1-a of RvH1 was studied and compared with the N-terminal functional unit RvH2-a of RvH2. Oligosaccharide fragments were released from the glycoprotein by Smith degradation of a haemocyanin pronase digest and separated on a Superdex 300 column. The glycopeptide fragments, giving a positive reaction for the orcinol/H2SO4 method, were separated by HPLC. In order to determine the linked sugar chains to the hinge glycopeptides isolated from the functional unit RvH1-a, several techniques were applied, including capillary electrophoresis, matrix-assisted laser desorption ionization-MS and electrospray ionization-MS in combination with glycosidase digestion. On the basis of these results and amino acid sequence analysis, we concluded that the functional unit RvH1-a contains 7% oligosaccharides N-glycosidically attached to Asn262 and Asn401, and the following structures were suggested:[structure: see text]