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.

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.

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 and 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. Some bioactive compounds in a mucus 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.

As observed in most molluscan hemocyanins, high-mannose type glycans were identified in hemocyanins from Rapana venosa (RvH), Helix lucorum (HlH) and keyhole limpet (Megatura crenulata). In addition, a glycan with a branching structure containing xylose, fucose and terminal methyl hexose was identified in β-HlH. We have examined the immuno-adjuvant properties of hemocyanins, their derivatives and conjugates associated with the cell mediated immunity in experimental tumor-bearing animals with ascites tumor of Guerin. After immunization of the animals with the experimental vaccine preparations, the highest values of splenic lymphocytes were observed in groups immunized with the conjugates RvH-TAg, β-HlH-TAg and KLH-TAg (42.3%; 40.8% and 40.58%, respectively) than with the native hemocyanins (36.5%; 35.1% and 32.4%, respectively). The immunization of rats with the hemocyanins β-HlH, RvH and KLH and their conjugates, prolonged the median survival time of tumor-bearing animals compared with non-immunized animals (39, 33, 31 and 7 days, respectively). Both hemocyanins β-HlH and RvH activate the immune system of the experimental animals and therefore could be a good alternative for KLH. For this reason they could be included into the composition of non-specific anti-tumor vaccines to enhance their effectiveness.

Hemocyanins are oxygen-transporting glycoproteins in the hemolymph of some invertebrate species that attracted scientific interest as potential anticancer agents. The present study aims to assess the in vitro and in vivo anticancer activity of hemocyanins isolated from Helix aspersa, Helix lucorum, and Rapana venosa in the Graffi myeloid tumor model. The in vitro antitumor activity of the hemocyanins was determined by a MTT test and cytomorphological analysis by fluorescent and transmission electron microscopy. The in vivo effects of the hemocyanins were examined in hamsters transplanted with Graffi tumor. The serum antibody titers against the tested hemocyanins and tumor antigen were determined by ELISA. Histopathological assessment of the morphological features related to antitumor effect, immune system response, and toxicity in some internal organs was performed. The results of in vitro studies indicated that the tested hemocyanins induced significant antiproliferative and apoptogenic effects. The in vivo investigations demonstrated a protective antitumor effect, expressed in reduced transplantability, suppression of tumor growth and metastasis, reduced mortality, prolonged survival time, and absence of toxic side effects. The present study indicated that the antitumor activity of the studied hemocyanins was due to both immune stimulation and direct effects on the tumor cells, and they displayed their potential as therapeutic agents against hematological malignances.