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Alzheimer's disease (AD) is the most widespread neurodegenerative disorder. Recently, it was found that mucus extract from Cornu aspersum has beneficial effects on memory and cognitive processes in a rat scopolamine model of AD. The present study elucidated the mechanisms of action of standardized mucus snail extract (SE) enriched with a fraction above 20 kDa on Alzheimer-type dementia in rats. Using proteomic analysis on two-dimensional polyacrylamide gel electrophoresis (2D–PAGE) on rat cortex extracts, we compared protein expression in both groups: the first group was treated intraperitoneally with scopolamine (Sco, 2 mg/kg, 11 days) and the second (Sco + SE) group was treated intraperitoneally with Sco (Sco, 2 mg/kg) and protected by SE (0.5 mL/100 g bw) applied daily orally for 11 days. Brain cortex was separated and the expressions of various proteins related to memory and cognitive functions were identified. We found that the expression of Ubiquitin carboxyl-terminal hydrolase isozyme L1, Calbindin, Vacuolar ATP synthase catalytic subunit A, Tropomyosin beta chain, 14-3-3 zeta/delta, Kinesin-1 heavy chain, and Stathmin-4 significantly differs in SE-protected rats as compared to dement animals treated only by Sco, and these brain proteins might be potential therapeutic targets for Alzheimer's-type dementia treatment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The structural stability of the hеmocyanin purified from the hеmolymph of gаrden snаils Helix lucorum (HlH) was investigated by means of far-UV circular dichroism (CD), differential scanning calorimetry (DSC) and transmission electron microscopy (TEM). For the first time, TEM analyses showed the presence of tubular polymers in hemocyanins after three-day dialysis against a stabilizing buffer containing high concentrations of Ca2+ and Mg2+ ions (100 mmol L−1). The conformational stability study of native HlH by means of CD in a wide pH range (2.5-11.5) defined the pH stability region of HlH at pH 6.5 − 8.0. DSC analyses demonstrated the thermal stability of this hemocyanin. One transition, with an apparent transition tеmperature (Tm) at 82.3 °C, was detected in the heаt capacity curve of HlH in 50 mmol L−1 Tris-HCl buffer, pH 7.2, at a heating rate of 1.0 °C min−1. The calorimetrically observed thermal transition correlates well with the unfolding transition monitored by CD measurements. The two-state kinetic model was used to analyse the process of irreversible thermal denaturation of HlH; Еa of 451 ± 4 kJ mol−1 was calculated. The obtained results on the conformational stability of HlH will facilitate the further investigation of the properties and potential biomedical applications of this respiratory protein. [ABSTRACT FROM AUTHOR]

Various aspects of biomedical applications of molluscan hemocyanins, associated with their immunogenic properties and antitumor activity, promoted us to perform structural studies on these glycoproteins. The stability and reassociation behavior of native Cornu aspersum hemocyanin (CaH) are studied in the presence of different concentrations of Ca2+ and Mg2+ ions and pH values using electron microscopy. Higher concentrations of those ions led to a more rapid reassociation of CaH, resulting in stable multidecamers with different lengths. The conformational changes of native CaH are investigated within a wide pH-temperature range by UV circular dichroism. The relatively small changes of initial [θ]λ indicated that many secondary structural elements are preserved, even at high temperatures above 80°C, especially at neutral pH. The mechanism of thermal unfolding of CaH has a complicated character, and the process is irreversible. The conformational stability of the native didecameric aggregates of CaH toward various denaturants indicates that hydrophilic and polar forces stabilize the quaternary structure. For the first time, the unfolding of native CaH in water solutions in the presence of four different denaturants is investigated. The free energy of stabilization in water, ∆GDH2O, was calculated in the range of 15.48–16.95 kJ mol−1. The presented results will facilitate the further investigation of the properties and potential applications of CaH. [ABSTRACT FROM AUTHOR]

Molluscan hemocyanins (Hcs) have recently particular interest due to their significant immunostimulatory properties. This is mainly related to their high carbohydrate content and specific monosaccharide composition. The oligosaccharide structures of the structural subunits RvH2 from Rapana venosa hemocyanin (RvH) and βc-Helix lucorum hemocyanin (βc-HlH) were comparative investigated by mass spectrometry. Our study revealed a highly heterogeneous mixture of N-glycans with compositions Hex3-7HexNAc2-5MeHex0-4Pent0-1Fuc0-1 and Hex0-9HexNAc2-4MeHex0-1HexA0-1Pent0-1Fuc0-3, isolation from βc-HlH and RvH2, respectively. N-glycans from βc-HlH contain mainly a terminal methyl-hexose (MeHex) residue, in some cases even more than one. Several carbohydrate moieties from βc-HlH are core-fucosylated, and also possess on methylation. In contrast with RvH2, N-glycans with an internal fucose residue, substituted at two positions with N-acetylhexosamine and hexuronic acid, as and glycans containing internal fucose were not found in βc-HlH. The glycosylation sites occupancy was subsequently elucidated by precursor ion scanning of the intact glycopeptides from RvH and βc-HlH using a nano-ESI mass spectrometry and Q-trap-LC/MS system. The oligosaccharide moieties found in HlH and RvH reveal a complex N-glycan pattern combining typical structural features of different higher organisms. Moreover, they are a potential source of novel N-glycans that are important for the stimulation of the immune response and/or for the production of antibodies used in diagnosis and therapy. [ABSTRACT FROM AUTHOR]

The fungal strain, Penicillium griseofulvum P29, isolated from a soil sample taken from Terra Nova Bay, Antarctica, was found to be a good producer of sialidase (P29). The present study was focused on the purification and structural characterization of the enzyme. P29 enzyme was purified using a Q-Sepharose column and fast performance liquid chromatography separation on a Mono Q column. The determined molecular mass of the purified enzyme of 40 kDa by SDS-PAGE and 39924.40 Da by matrix desorption/ionization mass spectrometry (MALDI-TOF/MS) analysis correlated well with the calculated mass (39903.75 kDa) from the amino acid sequence of the enzyme. P29 sialidase shows a temperature optimum of 37 °C and low-temperature stability, confirming its cold-active nature. The enzyme is more active towards α(2 → 3) sialyl linkages than those containing α(2 → 6) linkages. Based on the determined amino acid sequence and 3D structural modeling, a 3D model of P29 sialidase was presented, and the properties of the enzyme were explained. The conformational stability of the enzyme was followed by fluorescence spectroscopy, and the new enzyme was found to be conformationally stable in the neutral pH range of pH 6 to pH 9. In addition, the enzyme was more stable in an alkaline environment than in an acidic environment. The purified cold-active enzyme is the only sialidase produced and characterized from Antarctic fungi to date., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Background: Some molluscan hemocyanins (Hcs) have significant immunological and antitumor potential, enabling their application in oncology. The antitumor activity of Hcs from marine snails Rapana venosa (RvH), giant keyhole limpet Megathura crenulata (KLH) and garden snails Helix lucorum (HlH), as well as their different derivatives, were studied in vitro on a permanent T24 cell line of bladder cancer and normal urothelial cell line HL 10/29 compared to doxorubicin., Methods: The antiproliferative activity of the tested Hcs was determined using the WST-1 assay and BrdU ELISA assay. Morphological changes in both urothelial cell lines were confirmed by fluorescence microscopy. The proteomic analysis of a bladder cancer cell line before and after treatment with functional unit (FU) βc-HlH-h using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry revealed differences in the expression of some proteins., Results: Studies prove that the T24 tumor cell line is dose- and time-dependent, sensitive to the action of the tested isoforms, and it glycosylated FU of these hemocyanins. Selective inhibition of T24 cell growth was observed after incubation with structural subunits (βc-HlH, RvHI and RvHII) and FUs (βc-HlH-h and RvHII-e). Additionally, fluorescent microphotographs did not show apoptotic or necrotic alterations in the normal urothelial cell line HL 10/29. The FU βc-HlH-h demonstrated the highest antiproliferative effect (similarly to doxorubicin), in which predominantly apoptotic and less late apoptotic or necrotic changes in the tumor cells were observed. Several downand up-regulated proteins identified by proteome analysis may be associated with the apoptosis pathway., Conclusion: The present study illustrated the selectivity of the cytotoxic effect of Hcs against the Т24 cancer cell line. This is the first report of protein expression in T24 human bladder cancer cells under the influence of FU βc-HlH-h. That is probably due to the specific oligosaccharide structures rich in methylated hexoses exposed on the surface of βc-HlH-h., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Metabolic profiling based on 1 H nuclear magnetic resonance (NMR) spectroscopy was applied with the aim to investigate the functional role of the metabolites in lyophilized mucus from the garden snail Helix aspersa . Twenty metabolites were unambiguously identified by 1 H, 1D TOCSY, 2D J-resolved, 2D COSY, and 2D HSQC NMR spectra with water suppression. The metabolic profiles of two fractions with low molecular weight (Mw < 1 kDa and Mw < 3 kDa) are very similar. Metabolites with known antioxidant, antibacterial, and antimicrobial activity were detected by NMR metabolic analysis of mucus samples from Helix aspersa . Some of them were confirmed by mass spectrometric analysis. The primary structure of several peptides was identified in low molecular weight fractions (Mw < 1 kDa) by tandem mass spectrometry.

The understanding of the function of macromolecular complexes is mainly related to a precise knowledge of their structure. Recently, the development of suitable mass spectrometric techniques (electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI)) and multi-angle laser light scattering has enabled mass determination of native complexes and of their subunits. By these techniques, the structure and association/dissociation behavior of huge molecules of molluscan Octopus vulgaris, Sepia officinalis and Rapana venosa have been characterized. Molecular masses of the native and dissociated molecule of cephalopodan Hcs O. vulgaris (3545 and 359.3 kDa, respectively) and S. officinalis (4134 and 443.8 kDa, respectively) revealed that only one type subunit organizes their molecules, while the presence of two isoforms with different masses (422.8 and 400.0 kDa) has been determined for gastropodan R. venosa Hc, aggregated into didecamers. The difference of their structural subunits was also established after limited proteolysis with TPCK-trypsin. Eight functional units (FUs) with masses of ~ 50 kDa were isolated from both subunits of RvH and isoform of Sepia officinalis, while seven FUs were purified from OvH. Further characterization of proteins by ESI-mass spectrometry (MS) and MALDI-MS, methods gave insights into post-translational modifications such as glycosylation. Glycosylation of O. vulgaris and S. officinalis Hcs was suggested based on the differences (11.6 and 40.0 kDa, respectively) between the masses measured by ESI-MS and those calculated by their gene sequences., (Copyright © 2012 John Wiley & Sons, Ltd.)