Lyudmila Velkova

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-γ 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 ProTα[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 (ProTα[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.

Glycan structures of the structural subunit (HtH1) of Haliotis tuberculata hemocyanin

The oligosaccharide structures of the structural subunit HtH1 of Haliotis tuberculata hemocyanin (HtH) were studied by mass spectral sequence analysis of the glycans. The proposed structures are based on MALDI-TOF-MS data before and after treatment with the specific exoglycosidases β1-3,4,6-galactosidase and α1-6(>2,3,4) fucosidase followed by sequence analysis via electrospray ionization MS/MS-spectra. In total, 15 glycans were identified as a highly heterogeneous group of structures. As in most molluscan hemocyanins, the glycans of HtH1 contain a terminal MeHex, but more interestingly, a novel structural motif was observed: MeHex[Fuc(α1-3)-]GlcNAc, including thus MeHex and (α1-3)-Fuc residues being linked to an internal GlcNAc residue. While the functional unit (FU) c (HtH1-c) is completely lacking any potential glycosylation site, FU-h possesses a second exposed sugar attachment site between beta-strands 8 and 9 within the beta sandwich domain compared to the other FUs. The glycosylation pattern/sites show a high degree of conservation. In FU-h two prominent potential glycosylation sites can be detected. The finding that HtH1 is not able to form multidecameric structures in vivo could be explained by the presence of the exposed glycan on the surface of FU-h.

Antimicrobial Activity of Molluscan Hemocyanins from Helix and Rapana Snails

For the first time the antimicrobial activities of hemocyanins from the molluscs Rapana venosa (RvH) and Helix aspersa (HaH) have been tested. From the hemolymph of the garden snail H. aspersa one structural subunit (βc-HaH ) and eight functional units (FUs, βc-HaH-a to βc-HaH-h) were isolated, and their N-terminal sequences and molecular weights, ranging between 45 and 65 kDa, determined. The antimicrobial test of the hemocyanins against different bacteria showed that only two FUs from Rapana, RvH1-b and RvH1-e, exhibit a low inhibition effect against Staphylococcus aureus. In contrast and surprisingly, the structural subunit βc-HaH of H. aspersa not only shows strong antimicrobial activities against S. aureus and the likewise Gram-positive Streptococcus epidermidis, but also against the Gram-negative bacterium Escherichia coli. We suggest that this subunit therefore has the potential to become a substitute for the commonly used antibiotics against which bacterial resistance has gradually been developed.

Rapana Venosa Hemocyanin with Antiviral Activity

ABSTRACT Molluscan hemocyanins (Hcs) have recently particular interest due to their significant immunostimulatory properties. This is mainly related to their high carbohydrate content and highly specific monosaccharide composition. Our study revealed a highly heterogeneous mixture of different glycans isolation from structural subunit RvH2 of Rapana venosa hemocyanin at least 28 different compositions of Hex0-9 HexNAc2-4 Hex0-3 Pent0-3 Fuc0-3 and deoxyhexose and pentose residues. A novel type of N-glycan, with an internal Fuc connecting one GalNAc(β1–2) and one hexuronic acid, was detected in RvH2 as was previously found in subunit RvH1. We compared investigation on antiviral effects of several molluscas hemocyanins (keyhole limpet hemocyanin, Rapana venosa hemocyanin and Helix vulgaris hemocyanin) and the arthropod Carcinus aestuarii hemocanin. For the first time, we demonstrate here the inhibitory effect of one glycosylated functional unit of molluscan hemocyanin against viruses. The FU RvH-1 of Rapana venosa hemocyanin is the most effective inhibitor on the replication of Herpes simplex virus type 1, strain Vic, (HSV-1).

Positions of the Glycans in Molluscan Hemocyanin, Determined by Fluorescence Spectroscopy

Molluscan hemocyanins are glycoproteins with different quaternary and carbohydrate structures. It was suggested that the carbohydrate chains of some Hcs are involved in their antiviral and antitumor effect, as well in the organization of the quaternary structure of the molecules. Using a well-known complex for saccharide sensing, positions and access to the carbohydrate chains in the native hemocyanins from Rapana venosa (RvH) and Helix lucorum (HlH) and also their structural subunits (RvH1, RvH2 and βcHlH) and functional units (FUs) were analysed by fluorescence spectroscopy and circular dichroism. Almost no effect was observed in the fluorescence emission after titration of the complex with native RvH and HlH due to lack of free hydroxyl groups which are buried in the didecameric form of the molecules. Titration with the structural subunits βcHlH and RvH2, increasing of the emission indicates the presence of free hydroxyl groups compared to the native molecules. Complex titration with the structural subunit βc-HlH of H. lucorum Hcs leads to a 2.5 fold increase in fluorescence intensity. However, the highest emission was measured after titration of the complex with FU βcHlH-g. The result was explained by the structural model of βcHlH-g showing the putative position of the glycans on the surface of the molecule. The results of the fluorescent measurements are in good correlation with those of the circular dichroism data, applied to analyse the effect of titration on the secondary structure of the native molecules and functional units. The results also support our previously made suggestion that the N-linked oligosaccharide trees are involved in the quaternary organization of molluscan Hcs.

Thermodynamic Analysis and Molecular Modeling of Rapana Venosa Hemocyanin—Functional Unit RVH2-E

ABSTRACT pH-T diagram is typical “phase portrait” for stability of functional unit RvH2-e. Using different techniques the T-transition curves at different pH for RvH2-e were analyzed and the parameters of the thermodynamic functions were obtained. Increasing temperature and within the T range 25–55°C the reversibility increases and “opens a reversibility window” within the range of pH 5.5–9.0, for which were calculate at standard temperature the thermodynamic functions ΔHo and ΔGoexp. Molecular modeling of correct 3D structure of functional unit RvH2-e was done which allows us to fix most probably position of missing 9 residues now presented in existed x-ray model at very poor resolution of 3.30Å.

N-glycan structures of β-HlH subunit of Helix lucorum hemocyanin

The carbohydrate structures of molluscan hemocyanins have recently received particular interest due to their specific monosaccharide composition, as well as their immunostimulatory properties and application in clinical studies. For the first time, we investigated N-glycans of the structural subunit β-HlH of hemocyanin isolated from Helix lucorum. In total, 32 different glycans were enzymatically liberated and characterized by tandem mass spectrometry using a Q-Trap mass spectrometer. Our study revealed a highly heterogeneous mixture of glycans with composition Hex3-7HexNAc2-5MeHex0-4Pent0-1Fuc0-1. The oligosaccharide chains are mostly modified at the inner core by β1-2-linked xylose to β-mannose, by α1-6-fucosylation of the innermost GlcNAc residue (the Asn-bound GlcNAc), and by methylation. The glycans of β-HlH mainly contain a terminal MeHex residue; in some cases even two, three or four of these residues occur. Several carbohydrate chains in β-HlH are core-fucosylated without Xyl and also possess a high degree of methylation. This study shows the presence of mono- and bi-antennary N-glycans as well as hybrid type structures with or without core-fucosylation.