Oxidation of the Tryptophan 32 Residue of Human Superoxide Dismutase 1 Caused by Its Bicarbonate-dependent Peroxidase Activity Triggers the Non-amyloid Aggregation of the Enzyme

The role of oxidative post-translational modifications of human superoxide dismutase 1 (hSOD1) in the amyotrophic lateral sclerosis (ALS) pathology is an attractive hypothesis to explore based on several lines of evidence. Among them, the remarkable stability of hSOD1^WT and several of its ALS-associated mutants suggests that hSOD1 oxidation may precede its conversion to the unfolded and aggregated forms found in ALS patients. The bicarbonate-dependent peroxidase activity of hSOD1 causes oxidation of its own solvent-exposed Trp³² residue. The resulting products are apparently different from those produced in the absence of bicarbonate and are most likely specific for simian SOD1s, which contain the Trp³² residue. The aims of this work were to examine whether the bicarbonate-dependent peroxidase activity of hSOD1 (hSOD1^WT and hSOD1^G93A mutant) triggers aggregation of the enzyme and to comprehend the role of the Trp³² residue in the process. The results showed that Trp³² residues of both enzymes are oxidized to a similar extent to hSOD1-derived tryptophanyl radicals. These radicals decayed to hSOD1-N-formylkynurenine and hSOD1-kynurenine or to a hSOD1 covalent dimer cross-linked by a ditryptophan bond, causing hSOD1 unfolding, oligomerization, and non-amyloid aggregation. The latter process was inhibited by tempol, which recombines with the hSOD1-derived tryptophanyl radical, and did not occur in the absence of bicarbonate or with enzymes that lack the Trp³² residue (bovine SOD1 and hSOD1^W32F mutant). The results support a role for the oxidation products of the hSOD1-Trp³² residue, particularly the covalent dimer, in triggering the non-amyloid aggregation of hSOD1.     

Transition temperatures in the sensitivity of escherichia coli B/r to lysozyme

Lysozyme attacked Escherichia coli B/r in the absence of EDTA or imposed osmotic shocks when the cells were rapidly cooled below specific temperatures. Cells subjected to lysozyme while being cooled to below 20°C began to lose ability to subsequently form colonies. This sensitivity increased with decreasing temperatures and almost all cells cooled to 0°C were affected. Slightly hypertonic solutions did not improve survival. Cells cooled first to as low as 5°C and then subjected to lysozyme while cool did not lose their ability to form colonies subsequent to rewarming. However, 70% of the cells cooled first to 0°C and subjected to lysozyme lost their colony-forming ability. Cell lysis also began when treated near 5°C, but even when treated at 0°C about 50% of the cells maintained their rod shape in the presence of lysozyme. These results are discussed in terms of a possible phase transition in a portion of the cell envelope and/or a transient osmotic swelling as a results of metabolic pumps failing at the low temperatures.     

Sweet proteins lysozyme and thaumatin are protein-type agonists for the calcium-sensing receptor

In addition to the maintenance of Ca²⁺ homeostasis, the calcium-sensing receptor (CaSR) is involved in many diverse physiological functions in the mammalian body. The receptor works as a kokumi taste receptor in taste buds and as a nutrient sensor in the gut, where it regulates the secretion of glycemic response and appetite-related hormones. To identify novel human CaSR (hCaSR) activators from food ingredients, we conducted a screening using a cell-based hCaSR assay. Hen egg-white lysozyme, which is a sweet protein, was found to be a novel orthosteric agonist of hCaSR with an EC₅₀ value of 592 μM. Lysozyme hydrolysate was not able to activate hCaSR, thus suggesting that the protein structure of lysozyme is necessary for hCaSR activation. Thaumatin, which is another sweet protein, also activated hCaSR with an EC₅₀ value of 71 μM. This is the first report that shows hCaSR activation by proteins with molecular weights exceeding 10,000 Da. These results provide a new avenue for the development of hCaSR activators, which could be applicable in food or drugs that modulate taste perception, appetite, or glucose tolerance, in addition to Ca²⁺ homeostasis.     

Modulation of lysozyme function and degradation after nitration with peroxynitrite

Background

Peroxynitrite (PN) is formed from superoxide and nitric oxide, both of which are increased during hepatic ethanol metabolism. Peroxynitrite forms adducts with proteins, causing structural and functional alterations. Here, we investigated PN-induced alterations in lysozyme structure and function, and whether they altered the protein's susceptibility to proteasome-catalyzed degradation.

Methods

Hen egg lysozyme was nitrated using varying amounts of either PN or the PN donor, 3-morpholinosydnonimine (SIN-1). The activity, nitration status and the susceptibility of lysozyme to proteasome-catalyzed degradation were assessed.

Results

Lysozyme nitration by PN or SIN-1 caused dose-dependent formation of 3-nitrotyrosine-lysozyme adducts, causing decreased catalytic activity, and enhanced susceptibility to degradation by the 20S proteasome. Kinetic analyses revealed an increased affinity by the 20S proteasome toward nitrated lysozyme compared with the native protein.

Conclusion

Lysozyme nitration enhances the affinity of the modified enzyme for degradation by the proteasome, thereby increasing its susceptibility to proteolysis.

General significance

Increased levels of peroxynitrite have been detected in tissues of ethanol-fed animals. The damaging effects from excessive peroxynitrite in the cell increase hepatotoxicity and cellular death by protein modification due to nitration. Cellular defenses against such changes include enhanced proteolysis by the proteasome in order to maintain protein quality control.     

Interactions of superoxide anion with enzyme radicals: Kinetics of reaction with lysozyme tryptophan radicals and corresponding effects on tyrosine electron transfer

The kinetics of O^·-₂ reaction with semi-oxidized tryptophan radicals in lysozyme, Trp^·(Lyz) have been investigated at various pHs and conformational states by pulse radiolysis. The Trp^·(Lyz) radicals were formed by Br^·-₂ oxidation of the 3–4 exposed Trp residues in the protein. At pH lower than 6.2, the apparent bimolecular rate is about 2 × 10⁸M^-1s^-1; but drops to 8 × 10⁷M^-1s^-1 or less above pH 6.3 and in CTAC micelles. Similarly, the apparent bimolecular rate constant for the intermolecular Trp^·(Lyz) + Trp^·(Lyz) recombination reaction is about (4-7 × 10⁶M^-1s^-1) at/or below pH 6.2 then drops to 1.3-1.6 × 10⁶M^-1s^-1 at higher pH or in micelles. This behavior suggests important conformational and/or microenvironmental rearrangement with pH, leading to less accessible semioxidized Trp^· residues upon Br^·-₂ reaction. The kinetics of Trp^·(Lyz) with ascorbate, a reducing species rather larger than O^·-₂ have been measured for comparison. The well-established long range intramolecular electron transfer from Tyr residues to Trp radicals-leading to the repair of the semi-oxidized Trp^·(Lyz) and formation of the tyrosyl phenoxyl radical is inhibited by the Trp^·(Lyz)+O^·-₂ reaction, as is most of the Trp^·(Lyz)+Trp^·(Lyz) reaction. However, the kinetic behavior of Trp^·(Lyz) suggests that not all oxidized Trp residues are involved in the intermolecular recombination or reaction with O^·-₂. As the kinetics are found to be quite pH sensitive, this study demonstrates the effect of the protein conformation on O^·-₂ reactivity. To our knowledge, this is the first report on the kinetics of a protein-O^·-₂ reaction not involving the detection of change in the redox state of a prosthetic group to probe the reactivity of the superoxide anion.     

Lysozyme activities and immunoglobulin concentrations in seminal plasma and spermatozoa of different teleost species and indications on its significance for sperm function

The occurrence of lysozyme and immunoglobulin (Ig) in semen of different teleost species (brown trout—Salmo trutta, perch—Perca fluviatilis, burbot—Lota lota) was studied. In all investigated species lysozyme activities (1.13-1.45 U ml⁻¹) and Ig concentrations (T-Ig: 1.11-1.61 μg ml⁻¹, IgG [measured only in brown trout]: 1.49 μg ml⁻¹) were detected in seminal plasma. Ig was also found in spermatozoa (T-Ig: 0.234-0.357 μg/g protein, IgG: 0.198 μg ml⁻¹) while spermatozoal lysozyme activities were low and fluctuating (0.093-0.164 U/g protein). In Salmo trutta lysozyme activities and immunoglobulin levels were compared between semen samples with high and low sperm motility as motility is an indicator for sperm fertility. Lysozyme activities were higher in seminal plasma of samples with high motility than in those with low motility while seminal plasma and spermatozoal immunoglobulin concentrations (T-Ig, IgG) were increased in samples with low motility in comparison to samples with high motility. Seminal plasma and spermatozoal IgG concentrations and seminal plasma lysozyme activities showed significant correlations with the sperm motility rate and swimming velocity. Moreover, lysozyme improved the viability of spermatozoa in in vitro experiments. Possible physiological meanings of these results are discussed.     

Localization and release of lysozyme from ferret trachea: Effects of adrenergic and cholinergic drugs

Summary Lysozyme is a bacteriolytic enzyme found in respiratory tract fluid. In this study, immunocytochemistry was used to determine the cells of origin of tracheal lysozyme in the ferret. Lysozyme was found in secretory granules of serous but not mucous cells in the submucosal glands, and was absent from the surface epithelium, cartilage, and connective tissue. The exclusive presence of lysozyme in serous gland cells renders it useful as a biochemical marker of that cell type.Measurements of lysozyme assayed from the incubating medium indicated that bethanechol stimulated lysozyme release by 260±80.9% (mean ±SE), phenylephrine by 80±16.4%, and terbutaline by 25±10.2%. Electron-microscopic and immunocytochemical analysis of incubated tissues revealed loss of serous granules and lysozyme immuno-reactivity in response to the drugs. Atropine, propranolol, and phentolamine blocked the stimulatory effects of bethanechol, terbutaline, and phenylephrine, respectively.These findings establish the usefulness of lysozyme as a serous-cell marker and demonstrate that secretory responses of different magnitude are evoked by equimolar concentrations of alpha- and beta-adrenergic and cholinergic drugs.     

Pilot-scale separation of lysozyme from hen egg white by integrating aqueous two-phase partitioning and membrane separation processes

A novel, cost-effective method of lysozyme separation from hen egg white was studied. This method integrates aqueous two-phase partitioning in the system EO₅₀PO₅₀/phosphates with membrane separation processes. The experiments were carried out in a pilot-scale on crude hen egg white.Initially, by forming an aqueous two-phase system, lysozyme was selectively extracted to the upper, polymer-rich phase while the other egg white proteins partitioned to the lower, phosphate-rich phase. Then, in order to recover lysozyme, thermoseparation of polymer-rich phase was applied. A novel approach for the simultaneous thermoseparation of the polymer-rich phase as well as for the recovery of the lysozyme was proposed, using a cross-flow microfiltration. Additionally, recovery of proteins by ultrafiltration from lower, phosphate-rich phase was also investigated.Lysozyme could be obtained after the thermal phase separation by means of microfiltration at a total recovery over the extraction steps of 47.5 and the purification factor of 10.5. The specific activity of lysozyme preparations was 34 188 U/mg of protein. Using cross-flow membrane techniques, it was found that the recovery of the polymer by microfiltration from the top phase was 83.9.     

Quantitative studies of the non-productive binding of lysozyme to partially N-acetylated chitosans: Binding of large ligands to a one-dimensional binary lattice studied by a modified McGhee and von Hippel model

This paper considers the non-productive (inhibitory) binding of chitosans to lysozyme from chicken egg white. Chitosans are linear, binary heteropolysaccharides consisting of 2-acetamido-2-deoxy-β-d-glucose (GlcNAc; A-unit) and 2-amino-2-deoxy-β-d-glucose (GlcN, D-unit). The active site cleft of lysozyme can bind six consecutive sugar residues in subsites named A–F, and specific binding of chitosan sequences to lysozyme occurs with A-units in subsite C. Chitosans with different fractions of A-units (F_A) induced nearly identical changes in the ¹H NMR spectrum of lysozyme upon binding, and the concentration of bound lysozyme could be determined. The data were analysed using a modified version of the McGhee and von Hippel model for binding of large ligands to one-dimensional homogeneous lattices. The average value of the dissociation constant for different sequences that may bind to lysozyme (K^ave_D) was estimated, as well as the number of chitosan units covered by lysozyme upon binding. K^ave_D decreased with increasing F_A-values at pH* 3 and 4.5, while the opposite was true at pH* 5.5. Contributions from different hexamer sequences to K^ave_D of the chitosans were considered, and the data revealed interesting features with respect to binding of lysozyme to partially N-acetylated chitosans. The relevance of the present data with respect to understanding lysozyme degradation kinetics of chitosans is discussed.     

Raman spectra of chemically modified lysozyme. Oxindole derivatives.

The Raman spectra of oxidation products of lysozyme have been investigated. The protein was oxidized by N-bromosuccinimide and dimethyl sulfoxide/HCl. Depending on the experimental conditions one to six tryptophan residues are oxidized to oxindole. The most prominent difference between the spectra of lysozyme and its oxindole derivatives is the strong band at 1017 cm^?1 which displaces the tryptophan peak at 1010 cm^?1. Other tryptophan bands are also weakened corresponding to the number of the tryptophan side chains destroyed. Shifts are observed in the amide I and in the amide III regions sensitive to conformational changes. These shifts indicate conformational differences in the higher oxidized species and in the native enzyme, although the amide III maxima overlap with a strong oxindole band. Similar effects are observed in the range of the C-C stretching vibrations of the peptide backbone. If more than one tryptophan side chain is oxidized changes have also been found in the S-S stretching range. The evaluation of this effect is difficult because of the strong oxindole vibration appearing in this region. In species oxidized by great excess of N-bromosuccinimide the tyrosine vibrations can no longer be detected, indicating the modification of this amino acid too.     

A novel thermophilic lysozyme from bacteriophage phiIN93

The lysozyme of bacteriophage φIN93 was purified to apparent homogeneity with Carboxymethyl Sepharose and Hydroxyapatie columns from lysates of the phage grown on Thermus aquaticus TZ2. The enzyme is a single polypeptide chain with a molecular weight of 33,000. From the determined N-terminal amio acids of the enzyme, the locus of the gene was specified on a φIN93 genome. The enzyme was not similar to egg white lysozyme, T4 phage lysozyme, or lambda phage lysozyme. The enzyme, φIN93 lysozyme, was found to be a novel type of thermophilic lysozyme, which lyses specifically Thermus sp. cells, and exhibited conspicuous thermal stability at 95 °C for 1 h in the presence of β-mercaptoethanol.     

Synthesis of thermo-sensitive polyacrylamide derivatives for affinity precipitation and its application in purification of lysozyme

The thermo-sensitive N-alkyl substituted polyacrylamide polymer was synthesized by radical polymerization and its lower critical solution temperature (LCST) was controlled to be 28 °C. The thermo-sensitive recovery of polymer was over 95% in the presence of 0.05 M NaClO₄. Cibacron Blue F3GA was covalently immobilized onto the polymer via the nucleophilic reaction between the active chlorine atom of its triazine ring and the hydroxyl group of the polymer. The ligands density was 30 μmol g⁻¹ polymer. The adsorption capacity of lysozyme on the polymer was 3.4 mg g⁻¹polymer in affinity precipitation process. And over 90% of adsorbed lysozyme was eluted by 0.5 M KSCN at pH 8.0. When the affinity polymer was applied in the purification of lysozyme from egg white, the purification factor was 28 and lysozyme yield was 80% or so.     

Inhibitory effects of lysozyme on endothelial protein C 1receptor shedding in vitro and in vivo

Lysozyme protects us from the ever-present danger of bacterial infection and binds to bacterial lipopolysaccharide (LPS) with high affinity. Beyond its role in the activation of protein C, the endothelial cell protein C receptor (EPCR) plays an important role in the cytoprotective pathway. EPCR can be shed from the cell surface, which is mediated by tumor necrosis factor-α converting enzyme (TACE). However, little is known about the effects of lysozyme on EPCR shedding. We investigated this issue by monitoring the effects of lysozyme on phorbol-12-myristate 13-acetate (PMA)-, tumor necrosis factor (TNF)-α-, interleukin (IL)-1βand cecal ligation and puncture (CLP)-mediated EPCR shedding and underlying mechanism. Data demonstrate that lysozyme induced potent inhibition of PMA-, TNF-α-, IL-1β-, and CLP-induced EPCR shedding. Lysozyme also inhibited the expression and activity of PMA-induced TACE in endothelial cells. These results demonstrate the potential of lysozyme as an anti-EPCR shedding reagent against PMA-mediated and CLP-mediated EPCR shedding. [BMB Reports 2015; 48(11): 624-629]     

Cell wall degradation ofStaphylococcus aureus by lysozyme

In contrast to former findings lysozyme was able to attack the cell walls ofStaphylococcus aureus under acid conditions. However, experiments with¹⁴C-labelled cell walls and ribonuclease indicated that, under these conditions, lysozyme acted less as an muralytic enzyme but more as an activator of pre-existing autolytic wall enzymes. Electron microscopic studies showed that under these acid conditions the cell walls were degraded by a new mechanism (i.e. attack from the inside). This attack on the cell wall started asymmetrically within the region of the cross wall and induced the formation of periodically arranged lytic sites between the cytoplasmic membrane and the cell wall proper. Subsequently, a gap between the cell wall and the cytoplasmic membrane resulted and large cell wall segments became detached and suspended in the medium. The sequence of lytic events corresponded to processes known to take place during wall regeneration and wall formation. In the final stage of lysozyme action at pH 5 no cell debris but stabilized protoplasts were to be seen without detectable alterations of the primary shape of the cells. At the same time long extended ribbon-like structures appeared outside the bacteria. The origin as well as the chemical nature of this material is discussed. Furthermore, immunological implications are considered.     

Induction of lysozymelike activity in the hemolymph and hemocytes of an insect, Spodoptera eridania

Lysozymelike activity is present in the hemocytes and cell-free hemolymph of Spodoptera eridania. Its level remains essentially constant during larval development and can be induced by injection of various foreign materials. Serum bacteriolytic activity rises 24 hr after injection of saline, BSA, bacteria, bacterial endotoxin (LPS), latex particles, or sham injection. However, the magnitude and subsequent duration of the response depends on the nature of the injected material. The response is transient following sham injection or injection of soluble substances, such as saline and BSA, as compared to treatment with latex or bacteria. Both soluble and insoluble fractions of bacterial LPS preparations stimulated the lysozyme response. The response to a single injection of E. coli LPS was dose dependent and persisted for at least 5 days; however, additional injections had no effect on serum lysozyme level. The basal intracellular lysozyme level was significantly increased by E. coli LPS injection. Lysozyme release by hemocytes was proportional to intracellular concentration and did not increase after phagocytic stimulation of hemocytes.     

Interaction of lysozyme with f2 bacteriophage

Lysozyme precipitates f2 bacteriophage at a concentration of about one molecule of lysozyme per molecule of capsid subunit. This activity of lysozyme depends on its conformation but not upon the catalytic activity of the enzyme. The precipitation is thought to be a disruption of the solvation of the phage particle by adsorption of the enzyme to the negatively charged outer surface which has been postulated for this virus previously. A patch of six to nine positive charges on the lysozyme molecule is probably involved. Hemoglobin, RNase, α lactalbumin bovine serum albumin, and trypsin do not precipitate the phage but histone, protamine and spermine do. Consistent with the proposed mechanism, lysozyme is 10⁵ times more effective than spermine on a molar basis.     

Action of lysozyme on oligosaccharides from peptidoglycan N-unacetylated at glucosamine residues

On N-acetylmuramyl-L-alanine-amidase treatment followed by lysozyme digestion, peptidoglycan N-unacetylated partially at glucosamine residues yielded three oligosaccharides with N-unacetylated glucosamine residues, GlcN-MurNAc-GlcNAc-MurNAc¹, GlcN-MurNAc-GlcN-MurNAc-GlcNAc-MurNAc, and GlcNAc-MurNAc-GlcN-MurNAc-GlcNAc-MurNAc. Lysozyme can not hydrolyze the former two saccharides, whereas it cleaves the latter one into GlcNAc-MurNAc and GlcN-MurNAc-GlcNAc-MurNAc. This confirms requirement for the acetamido group of the N-acetylglucosamine in the interaction with subsite C of lysozyme.     

Production of human lysozyme in biofilm reactor and optimization of growth parameters of Kluyveromyces lactis K7

Lysozyme (1,4-β-N-acetylmuramidase) is a lytic enzyme, which degrades the bacterial cell wall. Lysozyme has been of interest in medicine, cosmetics, and food industries because of its anti-bactericidal effect. Kluyveromyces lactis K7 is a genetically modified organism that expresses human lysozyme. There is a need to improve the human lysozyme production by K. lactis K7 to make the human lysozyme more affordable. Biofilm reactor provides high biomass by including a solid support, which microorganisms grow around and within. Therefore, the aim of this study was to produce the human lysozyme in biofilm reactor and optimize the growth conditions of K. lactis K7 for the human lysozyme production in biofilm reactor with plastic composite support (PCS). The PCS, which includes polypropylene, soybean hull, soybean flour, bovine albumin, and salts, was selected based on biofilm formation on PCS (CFU/g), human lysozyme production (U/ml), and absorption of lysozyme inside the support. To find the optimum combination of growth parameters, a three-factor Box–Behnken design of response surface method was used. The results suggested that the optimum conditions for biomass and lysozyme productions were different (27 °C, pH 6, 1.33 vvm for biomass production; 25 °C, pH 4, no aeration for lysozyme production). Then, different pH and aeration shift strategies were tested to increase the biomass at the first step and then secrete the lysozyme after the shift. As a result, the lysozyme production amount (141 U/ml) at 25 °C without pH and aeration control was significantly higher than the lysozyme amount at evaluated pH and aeration shift conditions (p?<?0.05).