Biophysical insight into anti-amyloidogenic nature of novel ionic Co(II)(phen)(H2O)4]+[glycinate]- chemotherapeutic drug candidate against human lysozyme aggregation.

In the recent past, there has been an ever-increasing interest in the search for metal-based therapeutic drug candidates for protein misfolding disorders (PMDs) particularly neurodegenerative disorders such as Alzheimer's, Parkinson's, Prion's diseases, and amyotrophic lateral sclerosis. Also, different amyloidogenic variants of human lysozyme (HL) are involved in hereditary systemic amyloidosis. Metallo-therapeutic agents are extensively studied as antitumor agents, however, they are relatively unexplored for the treatment of non-neuropathic amyloidoses. In this work, inhibition potential of a novel ionic cobalt(II) therapeutic agent (CoTA) of the formulation [Co(phen)(H2O)4]+[glycinate]- is evaluated against HL fibrillation. Various biophysical techniques viz., dye-binding assays, dynamic light scattering (DLS), differential scanning calorimetry (DSC), electron microscopy, and molecular docking experiments validate the proposed mechanism of inhibition of HL fibrillation by CoTA. The experimental corroborative results of these studies reveal that CoTA can suppress and slow down HL fibrillation at physiological temperature and pH. DLS and 1-anilino-8-naphthalenesulfonate (ANS) assay show that reduced fibrillation in the presence of CoTA is marked by a significant decrease in the size and hydrophobicity of the aggregates. Fluorescence quenching and molecular docking results demonstrate that CoTA binds moderately to the aggregation-prone region of HL (Kb = 6.6 × 104 M-1), thereby, inhibiting HL fibrillation. In addition, far-UV CD and DSC show that binding of CoTA to HL does not cause any change in the stability of HL. More importantly, CoTA attenuates membrane damaging effects of HL aggregates against RBCs. This study identifies inorganic metal complexes as a therapeutic intervention for systemic amyloidosis.

Nonbonded Force Field Parameters from MBIS Partitioning of the Molecular Electron Density Improve Binding Affinity Predictions of the T4-Lysozyme Double Mutant.

The use of computer simulation for binding affinity prediction is growing in drug discovery. However, its wider use is constrained by the accuracy of the free energy calculations. The key sources of error are the force fields used to depict molecular interactions and insufficient sampling of the configurational space. To improve the quality of the force field, we developed a Python-based computational workflow. The workflow described here uses the minimal basis iterative stockholder (MBIS) method to determine atomic charges and Lennard-Jones parameters from the polarized molecular density. This is done by performing electronic structure calculations on various configurations of the ligand when it is both bound and unbound. In addition, we validated a simulation procedure that accounts for the protein and ligand degrees of freedom to precisely calculate binding free energies. This was achieved by comparing the self-adjusted mixture sampling and nonequilibrium thermodynamic integration methods using various protein and ligand conformations. The accuracy of predicting binding affinity is improved by using MBIS-derived force field parameters and a validated simulation procedure. This improvement surpasses the chemical precision for the eight aromatic ligands, reaching a root-mean-square error of 0.7 kcal/mol.

Probing the toxic effect of chlorpyrifos as an environmental pollutant on the structure and biological activity of lysozyme under physiological conditions.

The pervasive use of pesticides like chlorpyrifos (CPY) has been associated with deleterious effects on biomolecules, posing significant risks to environmental integrity, public health, and overall ecosystem equilibrium. Accordingly, in this study, we investigated the potential binding interaction between the well-conserved enzyme, lysozyme (LSZ), and CPY through various spectroscopic techniques and molecular modeling. The UV-vis absorption and fluorescence experiments confirmed the complex formation and static quenching of the intrinsic fluorescence intensity. LSZ revealed a singular binding site for CPY, with binding constants around 105 M-1 across different temperature ranges. Analysis of thermodynamic parameters showed the spontaneous nature of the complexation process, while also revealing the pivotal role of hydrophobic interactions in stabilizing the LSZ-CPY system. According to circular dichroism and Fourier transform infrared studies, CPY binding changed the secondary structure of LSZ by boosting α-helix presence and reducing the levels of ß-sheet and ß-turn content. Further, CPY decreased the stability and activity of LSZ. Computational docking delineated the specific and highly preferred binding site of CPY within the structure of LSZ. Molecular dynamic simulation indicated the enduring stability of the LSZ/CPY complex and revealed structural modifications in the LSZ after binding with CPY. This research provides a detailed understanding of the intermolecular dynamics between CPY and LSZ, concurrently elucidating the molecular-level implications for the potential hazards of pesticides in the natural environment.

Development of a drying method for proteins based on protein-hyaluronic acid precipitation.

This study aims to develop a new method to dry proteins based on protein-hyaluronic acid (HA) precipitation and apply the precipitation-redissolution technique to develop highly concentrated protein formulations. Lysozyme was used as a model protein and HA with various molecular weights (MW) were investigated. Under low ionic strength, low-MW HA (e.g., MW: around 5 K) did not induce lysozyme precipitation. Conversely, high-MW HA (e.g., MW: approximately from 40 K to 1.5 M) precipitated more than 90 % of lysozyme. The dried lysozyme-HA precipitates had moisture levels between 4 % and 5 %. The lysozyme-HA precipitates could be redissolved using PBS (pH 7.4, ionic strength: ∼ 163 mM). The viscosity of the reconstituted solution was dependent on HA MW, e.g., 4 cP for HA40K, and 155 cP for HA1.5 M, suggesting low-MW HA might be a proper excipient for highly concentrated solution formulations for subcutaneous/intraocular injection and high-MW HA may fit for other applications. The tertiary structure of lysozyme after the precipitation-redissolution steps had no significant difference from that of the original lysozyme as confirmed by fluorescence spectroscopy. The denaturation temperature of lysozyme after the precipitation-redissolution steps and that of the original lysozyme were close, indicating they possessed similar thermal stability. The accelerated stability study revealed that lysozyme stored in the dry precipitates was more physically stable than that in the buffer solution. Overall, this new drying technique is suitable for drying proteins and exhibits several benefits such as minimum energy consumption, cost effectiveness, high production yield, and mild processing conditions. In addition, the precipitation-redissolution technique proposed in this study can potentially be used to develop highly concentrated formulations, especially for proteins experiencing poor stability in the liquid state.

Investigating the interaction mechanism between gliadin and lysozyme through multispectroscopic analysis and molecular dynamic simulations.

The development of stable nanocomplexes based on gliadin and other biopolymers shows potential applications as delivery vehicles in the food industry. However, there is limited study specifically targeting the gliadin-lysozyme system, and their underlying interaction mechanism remains poorly understood. Therefore, the objective of this study was to investigate the binding mechanism between gliadin and lysozyme using a combination of multispectroscopic methods and molecular dynamic simulations. Stable gliadin-lysozyme complex nanoparticles were prepared using an anti-solvent precipitation method with a gliadin-to-lysozyme mass ratio of 2:1 and pH 4.0. The characteristic changes in the UV-visible spectrum of gliadin induced by lysozyme confirmed the complex formation. The analyses of fluorescence, FT-IR spectra, and dissociation tests demonstrated the indispensability of hydrophobic, electrostatic, and hydrogen bonding interactions in the preparation of the composites. Scanning electron microscopy revealed that the surface morphology of the nanoparticles changed from smooth and spherical to rough and irregular with the addition of lysozyme. Furthermore, molecular dynamic simulations suggested that lysozyme bound to the hydrophobic region of gliadin and hydrogen bonding was crucial for the stability of the complex. These findings contribute to the advancement of gliadin-lysozyme complex nanoparticles as an efficient delivery system for encapsulating bioactive compounds in food industry.

Lysozyme: A Natural Product with Multiple and Useful Antiviral Properties.

Lysozyme, especially the one obtained from hen's egg white, continues to show new pharmacological properties. The fact that only a few of these properties can be translated into therapeutic applications is due to the lack of suitable clinical studies. However, this lack cannot hide the evidence that is emerging from scientific research. This review for the first time examines, from a pharmacological point of view, all the relevant studies on the antiviral properties of lysozyme, analyzing its possible mechanism of action and its ability to block viral infections and, in some cases, inhibit viral replication. Lysozyme can interact with nucleic acids and alter their function, but this effect is uncoupled from the catalytic activity that determines its antibacterial activity; it is present in intact lysozyme but is equally potent in a heat-degraded lysozyme or in a nonapeptide isolated by proteolytic digestion. An analysis of the literature shows that lysozyme can be used both as a disinfectant for raw and processed foods and as a drug to combat viral infections in animals and humans. To summarize, it can be said that lysozyme has important antiviral properties, as already suspected in the initial studies conducted over 50 years ago, and it should be explored in suitable clinical studies on humans.

Functional convergence in gastric lysozymes of foregut-fermenting rodents, ruminants, and primates is not attributed to convergent molecular evolution.

Convergent evolution is a widespread phenomenon. While there are many examples of convergent evolution at the phenotypic scale, convergence at the molecular level has been more difficult to identify. A classic example of convergent evolution across scales is that of the digestive lysozyme found in ruminants and Colobine monkeys. These herbivorous species rely on foregut fermentation, which has evolved to function more optimally under acidic conditions. Here, we explored if rodents with similar dietary strategies and digestive morphologies have convergently evolved a lysozyme with digestive functions. At the phenotypic level, we find that rodents with bilocular stomach morphologies exhibited a lysozyme that maintained higher relative activities at low pH values, similar to the lysozymes of ruminants and Colobine monkeys. Additionally, the lysozyme of Peromyscus leucopus shared a similar predicted protonation state as that observed in previously identified digestive lysozymes. However, we found limited evidence of positive selection acting on the lysozyme gene in foregut-fermenting species and did not identify patterns of convergent molecular evolution in this gene. This study emphasizes that phenotypic convergence need not be the result of convergent genetic modifications, and we encourage further exploration into the mechanisms regulating convergence across biological scales.

Synthetic macromolecular peptide-mimetics with amino acid substructure residues as protein stabilising excipients.

The clinical use of protein and peptide biotherapeutics requires fabrication of stable products. This particularly concerns stability towards aggregation of proteins or peptides. Here, we tested a hypothesis that interactions between a synthetic peptide, which is an aggregation-prone region analogue, and its homologous sequence on a protein of interest, could be exploited to design excipients which stabilise the protein against aggregation. A peptide containing the analogue of lysozyme aggregation-prone region (GILQINSRW) was conjugated to a RAFT agent and used to initiate the polymerisation of N-hydroxyethyl acrylamide, generating a GILQINSRW-HEA90 polymer, which profoundly reduced lysozyme aggregation. Substitution of tryptophan in GILQINSRW with glycine, to form GILQINSRG, revealed that tryptophan is a critical amino acid in the protein stabilisation by GILQINSRW-HEA90. Accordingly, polymeric peptide-mimetics of tryptophan, phenylalanine and isoleucine, which are often present in aggregation-prone regions, were synthesized. These were based on synthetic oligomers of acrylamide derivatives of indole-3 acetic acid (IND), phenylacetic acid (PHEN), or 2-methyl butyric acid (MBA), respectively, conjugated with hydrophilic poly(N-hydroxyethyl acrylamide) blocks to form amphiphilic copolymers denoted as INDm-, PHENm- and MTBm-b-HEAn. These materials were tested as protein stabilisers and it was shown that solution properties and the abilities of these materials to stabilise insulin and the peptide IDR 1018 towards aggregation are dependent on the chemical nature of their side groups. These data suggest a structure-activity relationship, whereby the indole-based INDm-b-HEAn peptide-mimetic displays properties of a potential stabilising excipient for protein formulations.

Inhibitory Impacts of Fulvic Acid-Coated Iron Oxide Nanoparticles on the Amyloid Fibril Aggregations.

Alzheimer's disease is considered as multi-factor diseases, the main hallmarks of which are extracellular amyloid-beta and intracellular tau protein aggregations, leading to neural death. With this in mind, most of the studies have been focused on eliminating these aggregations. Fulvic acid is one of the polyphenolic compounds which exhibits strong anti-inflammation and anti-amyloidogenic effects. On the other hand, iron oxide nanoparticles are able to reduce/eliminate the amyloid aggregations. Here in, the effect of fulvic acid-coated iron-oxide nanoparticles on the commonly used in-vitro model for amyloid aggregation studies, i. e., lysozyme from chicken egg white was investigated. The chicken egg white lysozyme forms the amyloid aggregation under acidic pH and high heat. The average size of nanoparticles was 10.7±2.7 nm. FESEM, XRD, and FTIR confirmed that fulvic acid was coated onto the surface of the nanoparticles. The inhibitory effects of the nanoparticles were verified by Thioflavin T assay, CD, and FESEM analysis. Furthermore, the toxicity of the nanoparticles on the neuroblastoma SH-SY5Y was assessed through MTT assay. Our results indicate that these nanoparticles efficiently inhibit amyloid aggregation formation, while exhibiting no in-vitro toxicity. This data shed light on the anti-amyloid activity of the nanodrug; paving the way for future drug development for treating Alzheimer's disease.

Secondary structure and toxicity of lysozyme fibrils are determined by the length and unsaturation of phosphatidic acid.

A progressive aggregation of misfolded proteins is a hallmark of numerous pathologies including diabetes Type 2, Alzheimer's disease, and Parkinson's disease. As a result, highly toxic protein aggregates, which are known as amyloid fibrils, are formed. A growing body of evidence suggests that phospholipids can uniquely alter the secondary structure and toxicity of amyloid aggregates. However, the role of phosphatidic acid (PA), a unique lipid that is responsible for cell signaling and activation of lipid-gated ion channels, in the aggregation of amyloidogenic proteins remains unclear. In this study, we investigate the role of the length and degree of unsaturation of fatty acids (FAs) in PA in the structure and toxicity of lysozyme fibrils formed in the presence of this lipid. We found that both the length and saturation of FAs in PA uniquely altered the secondary structure of lysozyme fibrils. However, these structural differences in PA caused very little if any changes in the morphology of lysozyme fibrils. We also utilized cell toxicity assays to determine the extent to which the length and degree of unsaturation of FAs in PA altered the toxicity of lysozyme fibrils. We found that amyloid fibrils formed in the presence of PA with C18:0 FAs exerted significantly higher cell toxicity compared to the aggregates formed in the presence of PA with C16:0 and C18:1 FAs. These results demonstrated that PA can be an important player in the onset and spread of amyloidogenic diseases.

Interaction mechanism and binding mode of phycocyanin to lysozyme: Molecular docking and molecular dynamics simulation.

In this study, multispectral analysis and molecular simulations were performed to investigate the interaction mechanism between phycocyanin (PC) and lysozyme (Lys). The interaction was examined using surface plasmon resonance (SPR), and the structural changes were analyzed using Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), and transmission electron microscopy (TEM). The results suggest that the interaction between PC and Lys was primarily driven by electrostatic, hydrophobic, and hydrogen bonding forces. Molecular dynamics (MD) simulation revealed that Lys preferentially binds between the two subunits, alpha (α) and beta (ß), of PC, with residues ASP-13, GLU-106, and GLU-115 on PC and ARG-119, ARG-107, and ARG-98 on Lys being the main contributors to the binding interaction. Additionally, the formation of the PC-Lys complex resulted in increased kinetic and improved thermal stability of PC, which have important implications for PC applications.

Frequency dependence of ultrasonic effects on the kinetics of hen egg white lysozyme fibrillation.

Our study aimed to investigate the effects of ultrasound on the fibrillation kinetics of HEWL (hen egg white lysozyme) and its physicochemical properties. Ultrasound, a mechanical wave, can induce conformational changes in proteins. To achieve this, we developed an ultrasound exposure system and used various biophysical techniques, including ThT fluorescence spectroscopy, ATR-FTIR, Far-UV CD spectrophotometry, Fluorescence microscopy, UV-spectroscopy, and seeding experiments. Our results revealed that higher frequencies significantly accelerated the fibrillation of lysozyme by unfolding the native protein and promoting the fibrillation process, thereby reducing the lag time. We observed a change in the secondary structure of the sonicated protein change to the ß-structure, but there was no difference in the Tm of native and sonicated proteins. Furthermore, we found that higher ultrasound frequencies had a greater seeding effect. We propose that the effect of frequency can be explained by the impact of the Reynolds number, and for the Megahertz frequency range, we are almost at the transition regime of turbulence. Our results suggest that laminar flows may not induce any significant change in the fibrillation kinetics, while turbulent flows may affect the process.

Influence of water and trehalose on α- and ß-relaxation of freeze-dried lysozyme formulations.

Molecular mobility in form of alpha and beta relaxations is considered crucial for characterization of amorphous lyophilizates and reflected in the transition temperatures Tgα and Tgß. Based on an overview of applied methods to study beta relaxations, Dynamic Mechanical analysis was used to measure Tgα and Tgß in amorphous freeze-dried samples. Lysozyme and trehalose as well as their mixtures in varying ratios were investigated. Three different residual moisture levels, ranging from roughly 0.5-7 % (w/w), were prepared via equilibration of the freeze-dried samples. Known plasticising effects of water on Tgα were confirmed, also via differential scanning calorimetry. In addition and contrary to expectations, an influence of water on the Tgß also was observed. On the other hand, an increasing amount of trehalose lowered Tgα but increased Tgß showing that Tgα and Tgß are not paired. The findings were interpreted with regard to their underlying molecular mechanisms and a correlation with the known influences of water and trehalose on stability. The results provide encouraging hints for future stability studies of freeze-dried protein formulations, which are urgently needed, not least for reasons of sustainability.

Structural alterations and inhibition of lysozyme activity upon binding interaction with rotenone: Insights from spectroscopic investigations and molecular dynamics simulation.

The pervasive employment of pesticides such as rotenone on a global scale represents a substantial hazard to human health through direct exposure. Therefore, exploring the interactions between such compounds and body macromolecules such as proteins is crucial in comprehending the underlying mechanisms of their detrimental effects. The present study aims to delve into the molecular interaction between rotenone and lysozyme by employing spectroscopic techniques along with Molecular dynamics (MD) simulation in mimicked physiological conditions. The binding interaction resulted in a fluorescence quenching characterized by both dynamic and static mechanisms, with static quenching playing a prominent role in governing this phenomenon. The analysis of thermodynamic parameters indicated that hydrophobic interactions primarily governed the spontaneous bonding process. FT-IR and circular dichroism findings revealed structural alternations of lysozyme upon complexation with rotenone. Also, complexation with rotenone declined the biological activity of lysozyme, thus rotenone could be considered an enzyme inhibitor. Further, the binding interaction substantially decreased the thermal stability of lysozyme. Molecular docking studies showed the binding location and the key residues interacting with rotenone. The findings of the spectroscopic investigations were confirmed and accurately supported by MD simulation studies.

Lysozyme binding with amikacin and levofloxacin studied by tritium probe, fluorescence spectroscopy and molecular docking.

Lysozyme complexes with amikacin and levofloxacin were studied by spectroscopy approaches as well as using a tritium probe. Tritium was used as a labeling agent to trace labeled compound concentration in a system of two immiscible liquids and in the atomic form to determine the possible position of the binding site. Co-adsorption of protein and drug at the liquid-liquid interface was analyzed by scintillation phase method that allowed us to directly determine the amount of protein and drug in the mixed adsorption layer. Also, tensiometric measuring of the interfacial tension was used for calculation of binding parameters accordingly to Fainerman model. The treatment of complexes with atomic tritium followed by trypsinolysis and analysis of tritium distribution in the lysozyme peptides reveals the binding sites, binding energies in which were analyzed using molecular docking. Formation of complexes with amikacin and levofloxacin preserves secondar structure of protein. However, the formation of complex with amikacin leads to the almost total loss of the enzymatic activity of lysozyme and the redshift of the maximum on the lysozyme fluorescence band. A slight decrease in the distribution coefficient of lysozyme in the presence of amikacin assumes that the complex has higher hydrophilicity in comparison to lysozyme without additives. The most favorable for binding were the positions of the active centers that included amino acids Asp52 and Glu35, as well as in the vicinity of peptide His15-Arg21, with the participation of amino acids Tyr20, Arg14. In the case of levofloxacin, the formation of lysozyme-ligand complex in aqueous solution is possible without changing the microenvironment of the active center of the protein. Binding of levofloxacin to the active center of the enzyme was the most favorable, but Asp52 and Glu35 that are responsible for the enzymatic activity of lysozyme, were not affected.

Piezoelectric Amyloid Fibril for Energy Harvesting, Reactive Oxygen Species Generation, and Wireless Cell Therapy.

Biomolecular piezoelectric materials are envisioned for advanced biomedical applications for their robust piezoelectricity, biocompatibility, and flexibility. Here, we report the piezoelectric property of amyloid fibrils derived from three distinct proteins: lysozyme, insulin, and amyloid-ß. We found that piezoelectric properties are dependent on the extent of the ß-sheet structure and the extent of fibril anisotropy. We have observed the piezoelectric constant value in the range of 24-42 pm/V for fibrils made of lysozyme/insulin/amyloid-ß, and for the sheet/bundle-like structure of lysozyme aggregates, the value becomes 62 pm/V. These piezoelectric constant values are 4-10 times higher than the native lysozyme/insulin/amyloid proteins. Computational studies show that extension of the ß-sheet structure produces an asymmetric arrangement of charges (in creating dipole moment) and mechanical stress induces an aligned orientation of these dipoles that results in a piezoelectric effect. It is shown that these piezoelectric fibrils can harvest mechanical as well as ultrasound-based energy to produce a voltage of up to 1 V and a current of up to 13 nA. These fibrils are employed for reactive oxygen species (ROS) generation under ultrasound exposure and utilized for ultrasonic degradation of organic pollutants or killing of cancer cells via intracellular ROS generation under ultrasound exposure. Our findings demonstrate that the piezoelectric property of protein fibrils has potential for wireless therapeutic applications and may have physiological roles that are yet to be explored.

Investigating lysozyme amyloid fibril formation and structural variability dependence on its initial folding state under different pH conditions.

Protein fibril formation and accumulation are associated with dozens of amyloidoses, including the widespread and yet-incurable Alzheimer's and Parkinson's diseases. Currently, there are still several aspects of amyloid aggregation that are not fully understood, which negatively contributes to the development of disease-altering drugs and treatments. One factor which requires a more in-depth analysis is the effect of the environment on both the initial state of amyloidogenic proteins and their aggregation process and resulting fibril characteristics. In this work, we examine how lysozyme's folding state influences its amyloid formation kinetics and resulting aggregate structural characteristics under several different pH conditions, ranging from acidic to neutral. We demonstrate that both the initial state of the protein and the solution's pH value have a significant combined effect on the variability of the resulting aggregate secondary structures, as well as their stabilities, interactions with amyloid-specific dye molecules, and self-replication properties.

Plasmonic aptasensor with antifouling dual-functional surface layer for lysozyme detection in food.

Antifouling coatings are critically necessary for optical biosensors for various analytical application sectors, from medical diagnostics to foodborne pathogen detection. They help avoid non-specific protein/cell attachment on the active biosensor surface and catch the analytes directly in the complex media. Advances in antifouling plasmonic surfaces have been mainly focused on detecting clinical biomarkers in real biofluids, whereas developing antifouling coatings for direct analysis of analytes in complex media has been scarcely investigated for food quality control and safety. Herein, we propose a new low-fouling poly-l-lysine (PLL)-based surface layer for directly detecting an allergen protein, lysozyme, in the food matrix using surface plasmon resonance. The PLL-based polymer contains densely immobilized anionic oligopeptide side chains to create an electric charge-balanced layer able to repel the non-specific adsorption of undesired molecules on the biosensor surface. It also includes sparsely attached aptamer probes for capturing lysozyme directly in food sources with no pre-analytical sample treatment. We optimized the surface layer fabrication condition and tested the dual-functional surface to evaluate its ability to detect the target protein selectively. The developed analytical approach allowed for achieving a limit of detection of 0.04 µg mL-1 (2.95 nM) and a limit of quantification of 0.13 µg mL-1 (8.95 nM). Lysozyme was successfully quantified in milk samples using the plasmonic dual-functional aptasensor without sample pre-treatment or target isolation, illustrating the device's utility.

First Report of Lysozyme Amyloidosis with p.F21L/T88N Amino Acid Substitutions in a Russian Family.

Lysozyme amyloidosis is caused by an amino acid substitution in the sequence of this protein. In our study, we described a clinical case of lysozyme amyloidosis in a Russian family. In our work, we described in detail the histological changes in tissues that appeared as a result of massive deposition of amyloid aggregates that affected almost all organ systems, with the exception of the central nervous system. We determined the type of amyloidosis and mutations using mass spectrometry. Using mass spectrometry, the protein composition of tissue samples of patient 1 (autopsy material) and patient 2 (biopsy material) with histologically confirmed amyloid deposits were analyzed. Amino acid substitutions p.F21L/T88N in the lysozyme sequence were identified in both sets of samples and confirmed by sequencing of the lysozyme gene of members of this family. We have shown the inheritance of these mutations in the lysozyme gene in members of the described family. For the first time, we discovered a mutation in the first exon p.F21L of the lysozyme gene, which, together with p.T88N amino acid substitution, led to amyloidosis in members of the studied family.

Lysozyme Inhibitors as Tools for Lysozyme Profiling: Identification and Antibacterial Function of Lysozymes in the Hemolymph of the Blue Mussel.

Lysozymes are universal components of the innate immune system of animals that kill bacteria by hydrolyzing their main cell wall polymer, peptidoglycan. Three main families of lysozyme have been identified, designated as chicken (c)-, goose (g)- and invertebrate (i)-type. In response, bacteria have evolved specific protein inhibitors against each of the three lysozyme families. In this study, we developed a serial array of three affinity matrices functionalized with a c-, g-, and i-type inhibitors for lysozyme typing, i.e., to detect and differentiate lysozymes in fluids or extracts from animals. The tool was validated on the blue mussel (Mytilus edulis), whose genome carries multiple putative i-, g-, and c-type lysozyme genes. Hemolymph plasma of the animals was found to contain both i- and g-type, but not c-type lysozyme. Furthermore, hemolymph survival of Aeromonas hydrophila and E. coli strains lacking or overproducing the i- type or g-type lysozyme inhibitor, respectively, was analyzed to study the role of the two lysozymes in innate immunity. The results demonstrated an active role for the g-type lysozyme in the innate immunity of the blue mussel, but failed to show a contribution by the i-type lysozyme. Lysozyme profiling using inhibitor-based affinity chromatography will be a useful novel tool for studying animal innate immunity.