Natural Garlic Organosulfur Compounds Prevent Metabolic Disorder of Lipid and Glucose by Increasing Gut Commensal Bacteroides acidifaciens.

A number of reports of the effects of garlic on gut microbiota revealed that the active garlic organosulfur compounds (OSCs) are destabilized by the action of alliinase during garlic preparation. In this study, garlic alliinase was deactivated to obtain stable garlic OSCs. Experiments with C57BL/6J mice fed with lipid and glucose metabolic disorder-inducing Western diet (WD) revealed that stable garlic OSCs prevented the disorder by increasing the relative abundance of gut Bacteroides acidifaciens. Molecular analysis indicated that garlic OSCs inhibited dyslipidemia and fatty liver by increasing taurine and subsequently promoting hepatic fatty acid ß-oxidation. In parallel, garlic OSCs could meliorate glucose homeostasis by inhibiting dipeptidyl peptidase-4 (DPP-4) and hepatic gluconeogenesis. In vitro bacterial culture experiments revealed that garlic OSCs directly increased the growth of gut Bacteroides acidifaciens. The results of this study demonstrate that the molecular mechanism of the preventive effect of garlic OSCs on the WD-induced metabolic disorder is attributed to the enhanced growth of Bacteroides acidifaciens and the consequent increase in taurine.

Highly efficient protein expression of Plasmodium vivax surface antigen, Pvs25, by silkworm and its biochemical analysis.

Plasmodium vivax ookinete surface protein, Pvs25, is a candidate for a transmission-blocking vaccine (TBV) for malaria. Pvs25 has four EGF-like domains containing 22 cysteine residues forming 11 intramolecular disulfide bonds, a structural feature that makes its recombinant protein expression difficult. In this study, we report the high expression of recombinant Pvs25 as a soluble form in silkworm, Bombyx mori. The Pvs25 protein was purified from hemolymphs of larvae and pupae by affinity chromatography. In the Pvs25 expressed by silkworm, no isoforms with inappropriate disulfide bonds were found, requiring no further purification step, which is necessary in the case of Pichia pastoris-based expression systems. The Pvs25 from silkworm was confirmed to be molecularly uniform by sodium dodecyl sulfate gel electrophoresis and size-exclusion chromatography. To examine the immunogenicity, the Pvs25 from B. mori was administered to BALB/c mice subcutaneously with oil adjuvant. The Pvs25 produced by silkworm induced potent and robust immune responses, and the induced antisera correctly recognized P. vivax ookinetes in vitro, demonstrating the potency of Pvs25 from silkworm as a candidate for a malaria TBV. To the best of our knowledge, this is the first study to construct a system for mass-producing malaria TBV antigens using silkworm.

Potential antioxidant bioactive peptides from camel milk proteins.

Camel milk is traditionally considered to have medicinal characteristics that it has potential health benefits and could help to treat several illnesses. Particularly, it is closest to human breast milk and has high levels of nutrients and bioactive components. The aim of this study was to explore the antioxidant peptides derived from protein fractions of camel milk. Camel milk proteins (CMP) were fractionated into camel casein protein (CCP) and camel whey protein (CWP), which were hydrolyzed with pepsin to produce peptic digests P-CCP and P-CWP, respectively. RP-HPLC was used for fractionation of the peptides from the P-CCP and P-CWP. The antioxidant activities were evaluated using superoxide anion generating system of xanthine oxidase (XOD) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay. Active peptides were analyzed using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) whereas a number of antioxidant peptides, with masses ranging from 913 to 2,951 Da, derived mainly from alpha-casein, lactophorin and lactoferrin, were identified. When yeast cells are used as a system for modeling mitochondrial disease, the peptides in caseins and whey fractions significantly enhanced the tolerance of yeast cells against peroxide-induced oxidative stress. The results show that both caseins and whey proteins of camel milk possess bioactive peptides with significant radical-scavenging activities and thus herald a fascinating opportunity for their potential as nutraceuticals or therapeutic peptides for prevention and treatment of oxidative stress-associated diseases.

Novel peptide motifs from lysozyme suppress pro-inflammatory cytokines in macrophages by antagonizing toll-like receptor and LPS-scavenging action.

Lysozyme is commonly found in spots where bacterial infections are most likely to enter the body. Earlier we found that lysozyme possesses five antimicrobial peptide motifs in its N-terminal region which can be generated by newborn pepsin. In this study, we explore the role of these peptides in the anti-inflammatory activity of lysozyme. The five peptides, helix1 (H1), helix2 (H2), H1 and H2 connected with a loop (HLH), H2 extended with either 2 ß-strands (H2-S12) or 3 ß-strands (H2-S13), were synthesized and examined for anti-inflammatory action. The five peptides dose-dependently decreased, to different degrees, expression of pro-inflammatory cytokines, TNF-α, IL-6 and IL-1ß, in lipopolysaccharide (LPS)- or interferon-gamma (INF-γ)-stimulated mouse macrophage cells (RAW264.7). The HLH peptide and its individual helices (H1 and H2) were markedly the most potent anti-inflammatory. When macrophage cells were stimulated with live bacteria (E. coli), H1 peptide was the most powerful suppressor of TNF-α and IL-6 expression, providing evidence that the peptide is able to antagonize the pathogen-induced inflammatory response. Receptor binding assay and docking simulation provided evidence that H1 peptide bind specifically to the pocket for endotoxin binding of the toll-like receptor 4 (TLR-4) of macrophage. The results demonstrate, for the first time, the molecular basis of anti-inflammatory action of lysozyme that N-terminal helical peptides are the main contributors. This exciting finding offers new classes of therapeutic peptides with potential in the treatment of infection-induced inflammatory diseases.

Novel angiotensin-converting enzyme inhibitory peptides from caseins and whey proteins of goat milk.

Angiotensin-converting enzyme (ACE) plays a central role in blood pressure regulation by producing the vasoconstrictor angiotensin II. The inhibition of ACE with natural inhibitors, as alternatives to avoid the side effect of synthetic drugs, is a major target in the prevention of hypertension. In this study, we examined the separated caseins and whey proteins of goat milk for the presence of ACE inhibitory peptides. Digestion of isolated whey proteins and caseins of goat milk by gastric pepsin generated soluble hydrolysates exhibiting significant inhibition of ACE compared to weak inhibition by undigested proteins. The hydrolysates were fractionated by size exclusion chromatography, Sephacryl S-100 column, into four fractions (F1-F4). The late-eluting fraction (F4) of either whey or caseins exhibited greater ACE inhibition. Peptides in both F4 fractions, isolated by RP-HPLC, exhibited variable ACE inhibitory activities with the hydrophobic peptide peaks being the most potent ACE inhibitors. MALDI-TOF MS/MS resulted in identification of three potent ACE inhibitory peptides: PEQSLACQCL from ß-lactoglobulin (residues 113-122), QSLVYPFTGPI from ß-casein (residues 56-66), and ARHPHPHLSFM from κ-casein (residues 96-106). The peptides from whey and caseins exert significant ACE inhibitory activities comparable to that of captopril, an antihypertensive drug, exhibiting IC50 values of 4.45 µM and 4.27 µM, respectively. The results introduce, for the first time, new potent ACE-inhibitory peptides that can be released by gastric pepsin of goat milk whey and caseins and thus may pave the way for their candidacy as anti-hypertensive bioactive peptides and prevention of associated disorders.

Identification of TENP as the Gene Encoding Chicken Egg White Ovoglobulin G2 and Demonstration of Its High Genetic Variability in Chickens.

Ovoglobulin G2 (G2) has long been known as a major protein constituent of chicken egg white. However, little is known about the biochemical properties and biological functions of G2 because the gene encoding G2 has not been identified. Therefore, the identification of the gene encoding G2 and an analysis of its genetic variability is an important step toward the goal of understanding the biological functions of the G2 protein and its utility in poultry production. To identify and characterize the gene encoding G2, we separated G2 from egg white using electrophoresis on a non-denaturing polyacrylamide gel. Two polymorphic forms of G2 protein (G2A and G2B), with different mobilities (fast and slow respectively), were detected by staining. The protein band corresponding to G2B was electro-eluted from the native gel, re-electrophoresed under denaturing conditions and its N-terminal sequence was determined by Edman degradation following transfer onto a membrane. Sequencing of the 47 kDa G2B band revealed it to be identical to TENP (transiently expressed in neural precursors), also known as BPI fold-containing family B, member 2 (BPIFB2), a protein with strong homology to a bacterial permeability-increasing protein family (BPI) in mammals. Full-length chicken TENP cDNA sequences were determined for 78 individuals across 29 chicken breeds, lines, and populations, and consequently eleven non-synonymous substitutions were detected in the coding region. Of the eleven non-synonymous substitutions, A329G leading to Arg110Gln was completely associated with the noted differential electrophoretic mobility of G2. Specifically G2B, with a slower mobility is encoded by A329 (Arg110), whereas G2A, with a faster mobility, is encoded by G329 (Gln110). The sequence data, derived from the coding region, also revealed that the gene encoding G2 demonstrates significant genetic variability across different chicken breeds/lines/populations. These variants, and how they correlate with egg white properties, may allow us to understand further G2's functions.

Chicken Ovotransferrin Variants OTFB and OTFC Harboring Substitution of GAT (Asp) to AAT (Asn) in the Codon 500 and their Antimicrobial Activity.

Chicken egg white ovotransferrin (OTF) has been reported to exist in three electrophoretic variants (OTFA, OTFB and OTFC). In this report, we identified a causal polymorphism between the OTFB and OTFC variants in Japanese and Taiwanese native chickens and compared the antibacterial activity between these two variants. The cDNA sequence analyses from Satsumadori oviducts revealed that three non-synonymous SNPs T1809G (Ser52Ala), A2258G (Ile96Val) and G7823A (Asp500Asn) corresponded to the OTF electrophoretic phenotypes. Of the three SNPs, the G7823A mutation perfectly corresponded to the electrophoretic phenotypes OTFB (G/G, Asp500Asp), OTFB/C (G/A, Asp500Asn) and OTFC (A/A, Asn500Asn) in three chicken populations. The variants OTFB and OTFC exhibited similar antibacterial potency against Gram-positive and Gram-negative bacteria. This study provides, for the first time, molecular information on polymorphism of OTFB and OTFC variants of chicken ovotransferrin and its effect on the antimicrobial activity of the respective variants.

Identification of potent antioxidant bioactive peptides from goat milk proteins.

Goat milk proteins have gained increasing attention especially the bioactive peptides released from the parent proteins by digestive enzymes. Specifically, the interest in bioactives of goat milk is intensifying due to its reduced allergenicity compared to bovine milk. In this study, proteins of goat milk were fractionated into caseins (GCP) and whey proteins (GWP), hydrolyzed by pepsin and the generated peptides were examined for radical scavenging activities. The hydrolysates of whey (P-GWP) and casein (P-GCP) proteins exhibited potent superoxide anion (O2ï½¥-) scavenging activity in a dose-dependent manner, as investigated using the natural xanthine/xanthine oxidase (X/XOD) system. The P-GWP and P-GCP dramatically quenched the O2ï½¥- flux but had negligible effect on the catalytic function of the enzyme, indicating specificity to scavenge O2ï½¥- but not oxidase inhibition. Further, both P-GWP and P-GCP were able to remarkably quench the chemical DPPH radical. Fractionation of hydrolysates by size-exclusion chromatography produced four fractions (F1-F4) from both hydrolysates, with variable O2ï½¥- scavenging activities. However, the slow eluting fractions (F4) of both hydrolysates and fast eluting fraction (F2) of P-GCP contained peptides with the highest scavenging activities. Peptides in the active fractions of P-GWP and P-GCP, isolated by reversed phase-HPLC, exhibited significantly strong O2ï½¥- scavenging activities. MALDI-TOF-MS allowed the identification of several antioxidant peptides derived from both caseins and whey proteins, with ß-casein and ß-lactoglobulin being the major contributors, respectively. The results demonstrate that digestion with pepsin generates multiple soluble peptides from goat milk protein fractions with remarkable ability to scavenge superoxide radicals and thus providing a fascinating opportunity for their potential candidacy as antioxidant bioactive peptides.

A novel antibiotic-delivery system by using ovotransferrin as targeting molecule.

Synthetic antibiotics and antimicrobial agents, such as sulfonamide and triclosan (TCS), have provided new avenues in the treatment of bacterial infections, as they target lethal intracellular pathways. Sulfonamide antibiotics block synthesis of folic acid by inhibiting dihydrofolate reductase (DHFR) while TCS block fatty acid synthesis through inhibition of enoyl-ACP reductase (FabI). They are water-insoluble agents and high doses are toxic, limiting their therapeutic efficiency. In this study, an antibiotic drug-targeting strategy based on utilizing ovotransferrin (OTf) as a carrier to allow specific targeting of the drug to microbial or mammalian cells via the transferrin receptor (TfR) is explored, with potential to alleviate insolubility and toxicity problems. Complexation, through non-covalent interaction, with OTf turned sulfa antibiotics or TCS into completely soluble in aqueous solution. OTf complexes showed superior bactericidal activity against several bacterial strains compared to the activity of free agents. Strikingly, a multi-drug resistant Salmonella strain become susceptible to antibiotics-OTf complexes while a tolC-knockout mutant strain become susceptible to OTf and more sensitive to the complexes. The antibiotic bound to OTf was, thus exported through the multi-drug efflux pump TolC in Salmonella wild-type strain. Further, antibiotics-OTf complexes were able to efficiently kill intracellular pathogens after infecting human colon carcinoma cells (HCT-116). The results demonstrate, for the first time, that the TfR mediated endocytosis of OTf can be utilized to specifically target drugs directly to pathogens or intracellularly infected cells and highlights the potency of the antibiotic-OTf complex for the treatment of infectious diseases.

Expression of ovotransferrin enhances tolerance of yeast cells toward oxidative stress.

Recently, we found that ovotransferrin (OTf) undergoes distinct self-cleavage in a redox-dependent process and exhibited in vitro superoxide dismutase (SOD)-like activity. In this study, we explore that the expression of OTf confers high tolerance to oxidative stress in yeast cells. The OTf gene was cloned into the vector pPICZB and was successfully expressed in methylotrophic yeast, Pichia pastoris KM71H. There was no growth difference between the non-transformed strain and recombinant strains harboring a mock vector (pPICZB) or the OTf gene carrying a vector (OTf-pPICZB). Intracellularly expressed OTf was found to undergo self-cleavage, producing a major fragment of 15 kDa, which corresponded to the disulfide kringle domain of the N-terminal lobe. The yeast OTf transformants exhibited strong tolerance to oxidative stress induced by either hydrogen peroxide (H2O2) or diethyl maleate (DEM). Further, OTf transformants showed higher intracellular reducing capacity and enhanced cytosolic reductase activity. This study is the first to describe the ability of OTf to confer in vivo antioxidative stress function within a complicated milieu of eukaryotic cells and provide novel insights for the potential of the OTf gene for molecular breeding of industrial yeast strains with high tolerance to oxidative stress.

Effect of polymorphism in egg white lysozyme on muramidase and antibacterial activities as well as hatchability in the Japanese quail (Coturnix japonica).

Lysozyme is one of the best characterized antimicrobial proteins in egg white. Three phenotypes of egg white lysozyme in Japanese quail, Coturnix japonica, (namely fast; slow; and the combination, FS) were observed by acid polyacrylamide gel electrophoresis. The fast phenotype showed faster mobility on Acid-PAGE than the slow phenotype. Comparison of the coding sequences for lysozyme derived from the slow and fast phenotypes revealed a nonsynonymous SNP at nucleotide position 115 from the translation initiation site, which alters AA sequence of lysozyme. This nonsynonymous SNP converted glutamine (Q) in the slow phenotype to lysine (K) in the fast phenotype at AA residue 21 of mature lysozyme (Q21K). Here, we investigated the effect of these phenotypes on muramidase activity, antibacterial activity, and hatchability. Muramidase activity toward isolated cell walls of Micrococcus lysodeikticus was in the order: fast allozyme > slow allozyme > chicken (Gallus gallus), but no significant difference was found among the 3 (P > 0.05). Antibacterial activity against live Staphylococcus aureus cells was significantly greater for the fast allozyme than the slow allozyme from 20 h after incubation (P < 0.05). For the antibacterial effects against live Escherichia coli cells, the activity of fast was significantly higher than that of slow at 16 h after incubation (P < 0.05). Hatchability was estimated for reciprocal crosses of Japanese quail with the FF (fast) and SS (slow) genotypes. Hatchability was 92.5% in FF male × SS female crosses and 87.2% in SS male × FF female crosses. A Cochran-Mantel-Haenszel test revealed a significant difference between the crosses (P < 0.05) and indicated that the female-derived slow phenotype led to improved rates of hatching. Our results suggest that the nonsynonymous SNP in Japanese quail lysozyme influences the electrophoretic migration, muramidase activity, and antibacterial activity of the protein, in addition to the hatchability of the eggs. These results demonstrate, for the first time, a significant difference in antibacterial activity and hatchability between 2 lysozyme phenotypes in Japanese quail.

Human lysozyme possesses novel antimicrobial peptides within its N-terminal domain that target bacterial respiration.

Human milk lysozyme is thought to be a key defense factor in protecting the gastrointestinal tract of newborns against bacterial infection. Recently, evidence was found that pepsin, under conditions relevant to the newborn stomach, cleaves chicken lysozyme (cLZ) at specific loops to generate five antimicrobial peptide motifs. This study explores the antimicrobial role of the corresponding peptides of human lysozyme (hLZ), the actual protein in breast milk. Five peptide motifs of hLZ, one helix-loop-helix (HLH), its two helices (H1 and H2), and two helix-sheet motifs, H2-ß-strands 1-2 (H2-S12) or H2-ß-strands 1-3 (H2-S13), were synthesized and examined for antimicrobial action. The five peptides of hLZ exhibit microbicidal activity to various degrees against several bacterial strains. The HLH peptide and its N-terminal helix (H1) were significantly the most potent bactericidal to Gram-positive and Gram-negative bacteria and the fungus Candida albicans . Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its N-terminal helix (H1) kill bacteria by crossing the outer membrane of Gram-negative bacteria via self-promoted uptake and are able to dissipate the membrane potential-dependent respiration of Gram-positive bacteria. This finding is the first to describe that hLZ possesses multiple antimicrobial peptide motifs within its N-terminal domain, providing insight into new classes of antibiotic peptides with potential use in the treatment of infectious diseases.

Potent antimicrobial action of triclosan-lysozyme complex against skin pathogens mediated through drug-targeted delivery mechanism.

Triclosan (TCS), an antimicrobial agent that inhibits bacterial fatty acid synthesis by blocking the active site of enoyl-ACP reductase (FabI), is a water-insoluble agent that limits its therapeutic candidacy. We have recently shown that the water solubility and antimicrobial activity of TCS were greatly enhanced when complexed to lysozyme (LZ). This study is to examine the therapeutic potential of triclosan-lysozyme (T-LZ) complex against common skin pathogens expressing different levels of FabI, and to delineate the drug-targeting mechanism by lysozyme. The T-LZ exhibited superior antimicrobial activity against two bacterial skin pathogens, Propionibacterium acnes and Corynebacterium minutissimum, while yeast pathogens, Candida albicans and Malassezia furfur lacking FabI enzyme were insensitive to the complex. Unlike free TCS or LZ, the T-LZ complex exhibited a potent antibacterial activity under a wide range of pH condition and salt concentration. Interestingly, P. acnes expressing greater amount of FabI was more susceptible to the T-LZ complex than C. minutissimum that produces lesser amount of the enzyme. A sensitive assay of FabI activity revealed that P. acnes and C. minutissimum treated with the complex exhibited significant inhibition of the intracellular FabI activity than cells treated with free TCS, indicating the efficiency of lysozyme to specifically deliver TCS to its target (FabI) in the cytoplasm of bacterial cells. These results demonstrate, for the first time, that lysozyme is a potential drug carrier that allows specific targeting to the microbial cells of the water-insoluble triclosan and highlights the potency of the complex for the treatment of skin bacterial infections.

New drug-targeting strategy from beneath the shell of egg.

IMPORTANCE OF THE FIELD: Antibiotic resistance is a serious problem that continues to challenge the healthcare sectors and has become increasingly alarming in the past few years. To face this emerging global crisis, there is a need to find a new class of antibiotics that act on new microbial targets and/or harness existing antibiotics by developing new drug-targeting strategies. AREAS COVERED IN THIS REVIEW: This review: explores an innovative drug-delivery strategy of using hen egg lysozyme as a carrier to enable water solubilization and to allow specific targeting to the microbial cells of a water-insoluble antimicrobial agent with a powerful killing action; addresses potentials for lysozyme in antibiotics drug targeting; and provides insight for the future direction of this highly prospective technology. WHAT THE READER WILL GAIN: The unique features and advantages of lysozyme-based drug delivery system are highlighted. The efficiency of lysozyme in solubilization and delivery of lipophilic antibiotics, to reformulate drugs that may fail clinical trials owing to low solubility, is emphasized. TAKE HOME MESSAGE: Fewer pharmaceutical companies are inventing new antibiotics because of long development times and high failure rates. Combining lysozyme with a powerful old antibiotic may open doors to revolutionizing medicine, particularly in the treatment of deadly infections.

Novel anticancer activity of the autocleaved ovotransferrin against human colon and breast cancer cells.

Proteins of avian egg albumin have been suggested to play various biological roles during the development of chick embryo to confer protection. Recently, we have shown that ovotransferrin (OTf), the second major protein in egg albumin, undergoes thiol-linked autocleavage at distinct sites upon reduction. This study explores the physiological significance of OTf autocleavage by examining the effect of the reduced autocleaved OTf (termed rac-OTf) on modulation of cell proliferation, lethality, and apoptosis in two human cancer cell lines, colon cancer (HCT-116) and breast cancer (MCF-7). The rac-OTf was prepared by reduction of OTf with a non-thiol reductant (TCEP), to avoid reductive alkylation and produce highly soluble fragments. Unlike OTf, rac-OTf remarkably inhibited the proliferation of cancerous MCF-7 and HCT-116 cells in a dose-dependent manner, with the greatest effect on HCT-116, but had no effect on normal human mammary epithelial cells (HMEC). Cytofluorometric and trypan blue exclusion analyses indicated that rac-OTf exhibits cytotoxicity to HCT-116 in a dose-dependent fashion. The cytotoxic mechanism of rac-OTf against cancer cells was found to be induction of apoptosis as judged by changes in cell morphology, annexin-V binding, collapse of mitochondrial membrane potential, and caspase-9 and -6 activation, indicating the involvement of the mitochondrial pathway. This finding is the first to describe the reduction-dependent autocleaved OTf as an anticancer molecule, providing insights into a novel physiological function of OTf, suggesting its therapeutic potential in the treatment of human cancers and health benefit in nutraceuticals.

Triclosan-lysozyme complex as novel antimicrobial macromolecule: a new potential of lysozyme as phenolic drug-targeting molecule.

A novel anti-infection strategy to alleviate antibiotic-resistance problem and non-specific toxicity associated with chemotherapy is explored in this study. It is based on utilizing a bacteriolytic enzyme (lysozyme) as a carrier to allow specific targeting of a potential phenolic antimicrobial drug (triclosan) to microbial cells. Lysozyme (LZ) was complexed, via electrostatic and hydrophobic condensation at alkaline pH, to various degrees with triclosan (TCS), a negatively charged phenolic antimicrobial that inhibits bacterial fatty acid synthesis. Fluorescence and absorbance spectra analysis revealed non-covalent association of TCS with the aromatic residues at the interior of LZ molecule. The conjugation greatly promoted the lytic activity of LZ as the degree of TCS derivatization increased. The complexation with LZ turned TCS into completely soluble in aqueous solution. TCS-LZ complexes showed significantly enhanced bactericidal activity against several strains of Gram-positive and Gram-negative bacteria compared to the activity of TCS or LZ alone when tested at the same molar basis. Strikingly, TCS-LZ complex, but not LZ or TCS alone, exhibited unique specificity to scavenge superoxide radicals, generated by the natural xanthine/xanthine oxidase coupling system, without affecting the catalytic function of oxidase. This finding is the first to describe that the membrane disrupting function of lysozyme can be utilized to specifically target antimicrobial drug(s) to pathogen cells and heralding a fascinating opportunity for the potential candidacy of TCS-LZ as novel antimicrobial strategy for human therapy.

Ovotransferrin possesses SOD-like superoxide anion scavenging activity that is promoted by copper and manganese binding.

The embryo of oviparous species is confronted by a highly oxidative stress generating as it grows and must rely on effective antioxidant system for protection. Proteins of avian egg albumin have been suggested to play the major redox-modulatory role during embryo development. Recently, we found that ovotransferrin (OTf) undergoes distinct thiol-linked self-cleavage in a redox-dependent process. In this study, we explore that OTf is SOD mimic protein with a potent superoxide anion (O(2)(-)) scavenging activity. The O(2)(-) scavenging activity was investigated using the natural xanthine/xanthine oxidase (X/XOD) coupling system. OTf exhibited O(2)(-) scavenging activity in a dose-dependent manner and showed remarkably higher scavenging activity than the known antioxidants, ascorbate or serum albumin. The isolated half-molecules of OTf exhibited higher scavenging activity than the intact molecule, whereas the N-lobe showed much greater activity. OTf dramatically quenched the O(2)(-) flux but had no effect on the urate production in the X/XOD system, indicating its unique specificity to scavenge O(2)(-) but not oxidase inhibition. Strikingly, metal-bound OTf exhibited greater O(2)(-) dismutation capacity than the apo-protein, ranging from moderate (Zn(2+)-OTf and Fe(2+)-OTf) to high (Mn(2+)-OTf and Cu(2+)-OTf) activity with the Cu(2+)-OTf being the most potent scavenger. In a highly sensitive fluorogenic assay, the metal-bound OTf exhibited significant increase in the rate of H(2)O(2) production in the X/XOD reaction than the apo-OTf, providing evidence that Zn(2+)-, Mn(2+)- and Cu(2+)-OTf possess SOD mimic activity. This finding is the first to describe that OTf is an O(2)(-) scavenging molecule, providing insight into its novel SOD-like biological function, and heralding a fascinating opportunity for its potential candidacy as antioxidant drug.

Glycation and phosphorylation of beta-lactoglobulin by dry-heating: effect on protein structure and some properties.

Beta-lactoglobulin (beta-Lg) was glycated with maltopentaose and subsequently phosphorylated by dry-heating in the presence of pyrophosphate to investigate the structural and functional properties of phosphorylated beta-Lg. The circular dichroism spectra showed that the change of the secondary structure in the beta-Lg molecule by glycation and subsequent phosphorylation was small. The differential scanning calorimetry thermograms of beta-Lg showed that the denaturation temperature of the most stable domain was only slightly affected, whereas the retinol-binding activity of beta-Lg was somewhat reduced by glycation and subsequent phosphorylation. These results indicated that the conformational changes of the beta-Lg molecule by glycation and subsequent phosphorylation were mild. The anti-beta-Lg antibody response was somewhat reduced by glycation, but significant changes were not observed by phosphorylation. Although the stability of beta-Lg against heat-induced insolubility was improved by glycation alone, it was further enhanced by phosphorylation. The calcium phosphate solubilizing ability of beta-Lg was enhanced by phosphorylation following glycation.

Processing of lysozyme at distinct loops by pepsin: a novel action for generating multiple antimicrobial peptide motifs in the newborn stomach.

C-type lysozyme (cLZ) is an antimicrobial enzyme that plays a major defense role in many human secretions. Recently, we have identified a helix-loop-helix antimicrobial peptide fragment of cLZ. This finding suggests that processing by coexisting proteases might be a relevant physiological process for generating peptides that contribute to the in vivo mucosal defense role of cLZ. In this study, we found that pepsin, under condition relevant to the newborn stomach (pH 4.0), generated various peptides from cLZ with potent bactericidal activity against several strains of Gram-negative and Gram-positive bacteria. Microsequencing and mass spectral analysis revealed that pepsin cleavage occurred at conserved loops within the alpha-domain of cLZ. We found that the bactericidal domain, which was isolated by gel filtration and reversed-phase HPLC, contains two cationic alpha-helical peptides generated from a helix-loop-helix domain (residues 1-38 of cLZ) by nicking at leucine17. A third peptide consisting of an alpha-helix (residues 18-38) and a two-stranded beta-sheet (residues 39-56) structure was also identified. These peptides share structural motifs commonly found in different innate immune defenses. Functional cellular studies with outer membrane-, cytoplasmic membrane vitality- and redox-specific fluorescence dyes revealed that the lethal effect of the isolated antimicrobial peptides is due to membrane permeabilization and inhibition of redox-driven bacterial respiration. The results provide the first demonstration that pepsin can fine-tune the antimicrobial potency of cLZ by generating multiple antimicrobial peptide motifs, delineating a new molecular switch of cLZ in the mucosal defense systems. Finally, this finding offers a new strategy for the design of antibiotic peptide drugs with potential use in the treatment of infectious diseases.

Phosphorylation of ovalbumin by dry-heating in the presence of pyrophosphate: effect on protein structure and some properties.

Ovalbumin (OVA) was phosphorylated by dry-heating in the presence of pyrophosphate at pH 4.0 and 85 degrees C for 1 and 5 days, and the physicochemical and structural properties of phosphorylated OVA were investigated. The phosphorus content of OVA increased to 1.01% by phosphorylation, and the electrophoretic mobility of PP-OVA also increased. Although the solubility of dry-heated OVA decreased, the decrease was slightly depressed by phosphorylation. The circular dichroism spectra showed that the change of the secondary structure in the OVA molecule, as measured by alpha-helix content, was mild by phosphorylation. The exchange reaction between the sulfhydryl and disulfide groups was enhanced and the surface hydrophobicity of OVA increased by phosphorylation. The tryptophan fluorescence intensity of OVA decreased by phosphorylation, suggesting that the conformational change occurred in the OVA molecule by phosphorylation. Although the differential scanning calorimetry thermograms of OVA showed a lowering of the denaturation temperature from 78.3 to 70.1 degrees C by phosphorylation, the stability of OVA against heat-induced insolubility at pH 7.0 was improved. The results indicated molten (partially unfolded) conformations of OVA formed by dry-heating in the presence of pyrophosphate.