Hisham R. Ibrahim

Ovotransferrin antimicrobial peptide (OTAP-92) kills bacteria through a membrane damage mechanism.

Ovotransferrin antimicrobial peptide (OTAP-92) is a cationic fragment of hen ovotransferrin (OTf). OTAP-92 consists of 92 amino acid residues located within the 109-200 sequence of the N-lobe of OTf. This study was aimed to delineate the antimicrobial mechanism of OTAP-92 and to identify its interaction with bacterial membranes. OTAP-92 caused permeation of Escherichia coli outer membrane (OM) to 1-N-phenylnaphthylamine fluorescent probe in a dose-dependent manner. These results suggested that OTAP-92 crossed the bacterial OM by a self-promoted uptake. Cytoplasmic membrane of E. coli was found to be the target for OTAP-92 bactericidal activity, as assayed by the unmasking of cytoplasmic beta-galactosidase due to membrane permeabilization in a kinetic manner. Pretreatment of bacteria with uncoupler, carbonyl cyanide m-chlorophenylhydrazone, markedly enhanced permeation of cytoplasmic membrane, suggesting that the membrane permeation due to OTAP-92 is independent of the transmembrane potential. In an E. coli phospholipid liposome model, it was demonstrated that OTAP-92 has the ability to dissipate the transmembrane electrochemical potential. Intrinsic fluorescence spectra of the two tryptophan residues in OTAP-92, using liposomal membrane, have identified the lipid-binding region as a helix-sheet motif, and suggested an adjacent Ca(2+)-sensitive site within OTAP-92. These data indicated that OTAP-92 possesses a unique structural motif similar to the insect defensins. Further, this cationic antimicrobial peptide is capable of killing Gram-negative bacteria by crossing the OM by a self-promoted uptake and cause damage to the biological function of cytoplasmic membrane.

Occurrence of a sialylglycopeptide and free sialylglycans in hen's egg yolk

Free sialylglycans (FSGs) and a sialylglycopeptide (SGP) as components of hens egg yolk were found and their chemical structures were determined. SGP and FSGs were isolated from fresh egg yolk by treatment with phenol, gel filtration and successive chromatographies on columns of anion- and cation-exchangers. They were localized in the yolk plasma. The glycan moiety of SGP, which was liberated by PNGase digestion, was studied for the chemical structure by HPLC mapping with p-aminobenzoic ethylester-derivatization, sugar composition analysis, 1H nuclear magnetic resonance and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and the glycomoiety was found to be an N-linked disialyl-biantennary glycan. The amino acid sequence of the peptide moiety of SGP was determined to consist of Lys-Val-Ala-Asn-Lys-Thr, the Asn of which is modified with the disialylglycan moiety. FSGs were determined to be two free disialyl-biantennary glycans whose reducing end was either Man beta1-4GlcNAc (FSG-I) or Man beta1-4GlcNAc beta1-4GlcNAc (FSG-II). Since the molar value of SGP present in one egg yolk (2.8 micromol) is comparable to those of well-known major yolk proteins, low density lipoprotein, lipovitellins and phosvitin, it can be considered that SGP is one of the major components in hens egg yolk.

Identification of a distinct antibacterial domain within the N-lobe of ovotransferrin

We have evaluated the bactericidal activity of hen ovotransferrin (OTf), which was found to operate regardless of its iron-deprivation properties, with the objective of isolating the bactericidal domain. The amino-terminal half-molecule (N-lobe, residues 1-332) of OTf, isolated by trypsin-nicking, retained the bactericidal activity independently of iron-deprivation, but not the carboxyl-terminal half-molecule (C-lobe, residue 342-686), suggesting the presence of a bactericidal domain within the N-lobe of the molecule. Specific cleavage at the aspartyl residues of OTf, by diluted-acid procedure, yielded fairly large peptides, whereas proteolysis for 150 min produced the strongest bactericidal peptides mixture. The bactericidal domain was purified from the active hydrolysate by gel filtration and reversed-phase HPLC and showed activity against S. aureus as well as E. coli K-12. Electrophoretic analysis on tricine-SDS-PAGE revealed a bactericidal peptide with an average M(r) of 9900 Da under non-reducing conditions. In combination with the specificity of cleavage (Asp-X) and the molecular mass, its N-terminal microsequencing corresponded to a cationic peptide consisting of 92 residues located within the 109-200 sequence of the N-lobe of OTf, containing three intrachain disulfide bridges, featuring a common structural motif occurs in the N-lobes of transferrins for which the sequence is available. Two of the disulfides (C160-C174 and C171-C182) form surface exposed cringle bridges lying on the opposite side of the iron-binding site from the interdomain cleft and showing marked sequence homology to insect defensins, which are blockers of the voltage-dependent K+ channels. The peptide lost antibacterial activity when its disulfide bonds were reduced, indicating the importance of its tertiary structure for the exertion of antibiotic activity.

Improvement of heat stability and emulsifying activity of ovalbumin by conjugation with glucuronic acid through the Maillard reaction

Abstract Ovalbumin (OVA) was conjugated with glucuronic acid (GlcUA) through the Maillard reaction by incubation at 50°C and 65% relative humidity. Approximately 32 and 45% of the free amino groups of OVA were blocked with GlcUA after 1 and 2 days of incubation respectively, and these values were lower than those with glucose. Polymerization and browning color development of OVA modified with GlcUA were more marked than those with glucose. It was considered that the preferable incubation time was less than 1 day since the brown color development and polymerization of OVA proceeded quickly during incubation. OVA–GlcUA-conjugates prepared by the incubation for 0.5 or 1 days were soluble after heating the solution at 95°C for 10 min whereas most of the intact OVA was precipitated at 90°C. Emulsifying properties of OVA were improved by conjugation with GlcUA.

Phosphorylation of egg white proteins by dry-heating in the presence of phosphate.

Food proteins were phosphorylated by heating in a dry state in the presence of phosphate. When casein, whey protein isolate (WPI), and egg white proteins (EWP), which were lyophilized from their solutions in a phosphate buffer, were dry-heated at various temperatures and pH levels for 1-5 days, EWP was more highly phosphorylated than casein and WPI. Phosphorylation of EWP was promoted with a decrease of pH from 7.0 to 3.0 when the incubation temperature was raised from 55 to 100 degrees C. The phosphorus content of EWP increased from 0.08 to 0.64% by dry-heating at pH 3.0 and 85 degrees C for 5 days in the presence of phosphate. The electrophoretic mobility of EWP increased with an increase in the phosphorylation level. The heat-induced polymerization of EWP by dry-heating was not affected by the presence of phosphate. Although the solubility of EWP decreased by dry-heating at pH 3.0-5.5, the phosphorylation depressed the insolubilization at low pH. The phosphate bonds in phosphorylated EWP (P-EWP) were stable at pH 2.0-10.0 and were more acid-labile and base-stable than phosphoesters of egg riboflavin-binding protein (RfBP). (31)P NMR spectral data suggested that besides phosphoesters, phosphodiester and polyphosphate bonds were introduced in P-EWP. Heat stability of EWP was improved, and calcium phosphate-solubilizing ability of EWP was enhanced by phosphorylation.

Modification of ovalbumin with oligogalacturonic acids through the Maillard reaction

Abstract First, to examine the effect of the chain length of oligosaccharides on the advanced stage of the Maillard reaction, ovalbumin (OVA) was incubated at the dry-state with glucose or maltooligosaccharides from maltose to maltohexaose in the same molar ratio. The browning and polymerization of OVA were suppressed with an increase in the chain length of oligosaccharides. Secondly, OVA was incubated with oligogalacturonic acids prepared by hydrolysis of pectin and pectic acid with pectinase. After 3 days of incubation, 20–25 and 31% of amino groups in OVA were modified with oligogalacturonic-acids and galacturonic acid, respectively. The browning and polymerization of OVA modified with oligogalacturonic acids were markedly lower than those of OVA modified with galacturonic acid. Only small amount of precipitate was formed even when OVA modified with oligogalacturonic acid was heated at 95°C. These results suggest that the heat stability of OVA can be improved without its marked browning and polymerization by conjugation with oligogalacturonic acids through the Maillard reaction.

Enhanced secretion of hydrophobic peptide fused lysozyme by the introduction of N-glycosylation signal and the disruption of calnexin gene in Saccharomyces cerevisiae.

The insertion of a hydrophobic pentapeptide (Phe‐Phe‐Val‐Ala‐Pro) into the C‐terminus in hen egg white lysozyme by genetic modification resulted in an unstable structure which caused little secretion in a yeast expression system, although this modification is useful to enhance bactericidal action to Gram‐negative bacteria [Ibrahim et al. (1994) J. Biol. Chem. 269, 5059–5063]. To enhance the secretion of the unstable hydrophobic pentapeptide fused lysozymes (H5‐Lz), we attempted to introduce the signal sequence (Asn‐X‐Ser/Thr) of N‐linked glycosylation into lysozyme and to suppress the quality control of the unstable mutant in the yeast expression system. The polymannosyl hydrophobic fused lysozyme (H5/G49N‐Lz) having the N‐glycosylation signal sequence was expressed in the medium at 3.4 times that of unglycosylated lysozyme. Further, the secretion of the unstable mutant lysozyme was done in the Saccharomyces cerevisiae disrupted calnexin gene to avoid the degradation of the unstable mutant by the quality control. Although disruption of the calnexin gene did not lead to gross effects on the levels of growth of S. cerevisiae (W303‐1b), the secretion amount of H5/G49N‐Lz in calnexin disrupted S. cerevisiae was 2.5 times larger than that in wild type S. cerevisiae. These results suggest that the secretion of unstable glycosylated lysozyme (H5/G49N) was suppressed by the quality control function of calnexin and that the disruption of calnexin is effective to increase the secretion of unstable glycosylated protein.

Improvement of functional properties of β-lactoglobulin by conjugation with glucose-6-phosphate through the Maillard reaction

Abstract β-Lactoglobulin (β-Lg) was conjugated with glucose-6-phosphate (G6P) through the Maillard reaction by incubation at 50 °C and 65% relative humidity. It was considered that the preferable incubation time was less than 1 day because modification of amino group proceeded quickly through 1 day of incubation and then slowed down; the brown color development and polymerization of β-Lg proceeded steadily from 1 day on. The β-Lg treated with G6P for 0.5 days, of which phosphorus content was 1.06%, was estimated to conjugate 6.9 mol G6P per 1 mol β-Lg. β-Lg-G6P conjugate was completely soluble after heating its solution at 95 °C for 10 min whereas more than 90% of intact β-Lg was precipitated at 90 °C. Emulsifying properties of β-Lg were improved by conjugation of G6P. No calcium and phosphate were sedimented at 1000 g in the solution containing 30 mM calcium, 22 mM phosphate, and 10 mM citrate at pH 6.7 in the presence of 2% β-Lg-G6P conjugate, but 14.5 mM calcium and 9.1 mM phosphate were sedimented at 100 000 g. The particules of the complex of the β-Lg-G6P conjugate and calcium phosphate with 30–200 nm of diameter were observed in electron micrograph. It was concluded from the above results that modification of β-Lg with G6P was useful in improving its functional properties.

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.

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 (O2･-) 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 O2･- flux but had negligible effect on the catalytic function of the enzyme, indicating specificity to scavenge O2･- 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 O2･- 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 O2･- scavenging activities. MALDI-TOF-MS allowed the identification of several antioxidant peptides derived from both caseins and whey proteins, with β-casein and β-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.