Hen Ovalbumin Research Articles

Production of bovine beta-lactoglobulin and hen egg ovalbumin by Trichoderma reesei using precision fermentation technology and testing of their techno-functional properties

The food protein ingredient market is dominated by dairy and egg proteins. Both milk whey and egg proteins are challenging proteins to replace, e.g. with plant proteins, due to the unique structural features of the animal proteins that render them highly functional. Thus, to provide a non-animal source of these important proteins the fungal host Trichoderma reesei was utilized for the biotechnical production of recombinant hen ovalbumin (TrOVA) and bovine beta lactoglobulin (TrBLG). These food proteins were investigated using two different promoter systems to test the concept of effectively expressing them in a fungal host. Both proteins were successfully produced in 24 well plate and bioreactor scale. The production level of TrBLG and TrOVA were 1 g/L and 2 g/L, respectively. Both proteins were further purified and characterized, and their functional properties were tested. TrBLG and TrOVA secondary structures determined by circular dichroism corresponded to the proteins of bovine and hen. The T. reesei produced proteins were found to be N-glycosylated, mostly with Man 5. TrBLG had emulsification properties matching to corresponding bovine protein. TrOVA showed excellent foaming characteristics and heat-induced gelation, although the strength of the gel was somewhat lower than with hen ovalbumin, possibly due to the partial degradation of TrOVA or presence of other host proteins. Biotechnical production of whey and egg proteins using precision fermentation technology offers an innovative way to increase the sustainability of the conventional food industry, without further reliance on animal farming.Industrial relevance: The food protein ingredient market is dominated by dairy (largely whey proteins) and egg proteins. Whey proteins are valuable and versatile food ingredients due to their functional and nutritional quality. They are largely used in meat and milk products, low fat products, bakery, confectionary, infant formulas and sports nutrition. Similarly, egg white protein ovalbumin is a highly functional protein ingredient that facilitates structure formation and high nutritional quality in most food products. Together they comprise 40–70% of the revenue in the animal protein ingredients market. Both whey and egg proteins are extremely challenging proteins to replace, e.g., by plant proteins due to their unique structural features that render them with high functionality. Biotechnical production of whey and egg proteins using precision fermentation technology offers an innovative way to increase the sustainability of the conventional food industry, without further reliance on animal farming.

Raising antibodies against epoxyscillirosidine, the toxic principle contained in Moraea pallida Bak. (Iridaceae), in rabbits

Moraea pallida Bak. (yellow tulp) poisoning is the most important plant cardiac glycoside toxicosis in South Africa. The toxic principle, a bufadienolide, is 1α, 2α-epoxyscillirosidine. The aim was to investigate the potential to develop a vaccine against epoxyscillirosidine. Epoxyscillirosidine, proscillaridin and bufalin, were successfully conjugated to hen ovalbumin (OVA), bovine serum albumin (BSA) and keyhole limpet haemocyanin (KLH).There was a low immune response following vaccination of adult male New Zealand White rabbits with epoxyscillirosidine-OVA (n = 3) and OVA (n = 3) using Freund's adjuvant in Trial (T) 1. The immune response improved significantly in T2 following doubling of the dose to 0.8 mg/rabbit and changing the adjuvant to Montanide. In T3, the rabbits (n = 15), allocated into 5 equal groups, vaccinated with proscillaridin-BSA, bufalin-BSA, epoxyscillirosidine-KLH, epoxyscillirosidine-BSA and BSA respectively, using Montanide adjuvant, developed antibodies against the administered immunogens, with epoxyscillirosidine-KLH inducing the highest immune response. Proscillaridin and bufalin antibodies cross-reacted with epoxyscillirosidine in an enzyme linked immunosorbent assay.The conjugation methodology will be adjusted in the future to target optimal conjugation efficiency. Additional vaccination will be conducted in search of neutralizing antibodies against the yellow tulp toxin. The cross-reactivity of proscillaridin and bufalin antibodies with epoxyscillirosidine could be studied in future to explore the potential to prevent yellow tulp poisoning.

Heat-Induced Aggregation of Hen Ovalbumin Suggests a Key Factor Responsible for Serpin Polymerization.

Although ovalbumin (OVA), a main component of hen egg white and a non-inhibitory serpin superfamily protein, has been reported to form fibrillar aggregates, its relationship with amyloid fibrils associated with various degenerative diseases is unclear. We studied the heat-induced aggregation of intact OVA using an amyloid-specific thioflavin T assay with a fluorometer or direct imaging with a light-emitting diode lamp and several physicochemical approaches, and the results confirmed that intact OVA forms aggregates with a small part of amyloid cores and dominantly amorphous aggregates. We isolated the amyloidogenic core peptide by proteolysis with trypsin. The isolated 23-residue peptide, pOVA, with marked amyloidogenicity, corresponded to one (β-strand 3A) of the key regions involved in serpin latency transition and domain-swap polymerization leading to serpinopathies. Although the strong amyloidogenicity of pOVA was suppressed in a mixture of tryptic digests, it was observed under acidic conditions in the presence of various salts, with which pOVA has a positive charge. Cytotoxicity measurements suggested that, although heat-treated OVA aggregates exhibited the strongest toxicity, it was attributed to a general property of amorphous aggregates rather than amyloid toxicity. Predictions indicated that the high amyloidogenicity of the β-strand 3A region is common to various serpins. This suggests that the high amyloidogenicity of β-strand 3A that is important for serpin latency transition and domain-swap polymerization is retained in OVA and constitutes β-spine amyloid cores upon heat aggregation.

Participation of cysteine 30 residue in the folding process of ovalbumin evaluated in a refolding experiment using cysteine mutants

To understand the role of cysteine (SH) residues in the folding of hen ovalbumin (OVA), SH-mutated OVAs, in which each SH residue was replaced by alanine (C11A, C30A, C367A, and C382A), were prepared. SDS-PAGE analysis under non-reducing conditions showed that the C11A and C30A mutants produced a disulfide (SS) isomer in addition to a protein with a native SS bond (Cys73–Cys120). The susceptibility to elastase digestion suggested that the Cys73 residue in the SS isomer participates as a counterpart of the SS bond. Upon refolding of the SH-mutated OVAs under the denatured and SS-reduced states, only C30A failed to refold into an intact form. This indicated that the Cys30 residue plays an important role in correct refolding. To confirm this, each of the four SH-mutated OVAs, in which the original SS-forming sites (C11/73/120A, C30/73/120A, C73/120/367A, and C73/120/382A) were deleted, was constructed and expressed. The C11/73/120A and C30/73/120A mutants formed no SS form, in contrast to C73/120A as a control. Thus, we concluded that Cys30 participates in the correct folding of OVA, and that its SS bond (Cys11–Cys30) is transiently generated during the early folding stage to avoid misfolding, and then the native SS form of OVA is regenerated through SH–SS exchanges.

Regulatory T Cells Are Not Required for Prevention of RBC-Specific Autoantibody Generation

Introduction: Loss of humoral tolerance to red blood cell (RBC) antigens may lead to the generation of pathogenic autoantibodies and result in autoimmune hemolytic anemia (AIHA), a severe and potentially fatal disease. Failure of tolerance to RBC antigens occurs with considerable frequency (1-3 cases/1,000 adults) and prevalence of AIHA is as high as 30% in persons with compromised B and/or T cell tolerance mechanisms. However, RBC-specific tolerance mechanisms are poorly understood. To elucidate the immune tolerances to RBC autoantigens, we utilized HOD mice. The HOD mouse expresses an RBC-specific transgene consisting of hen egg lysozyme (HEL), ovalbumin (OVA), and Duffy. Using the HOD model, we previously demonstrated B cell tolerance to RBC-specific HOD antigen is incomplete; however, T cell tolerance is stringent. HOD mice have similar detectable frequencies of HOD-specific CD4+ T cells compared to B6 mice. Although present, autoreactive HOD-specific CD4+ T cells are non-functional. Circumventing T cell tolerance by adoptive transfer, HOD mice make high titer anti-HOD autoantibodies in vivo. Thus, despite the presence of autoreactive B cells, no HOD-reactive antibodies are detectable unless CD4+ T cells are given, indicating T cell tolerance is a stopgap to autoimmunity. Methods: Leukocytes from C57BL/6 (B6) and HOD mice were harvested and OVA-specific CD4+ T cell responses were assessed by tetramer-pulldown assays with pooled tetramers I-Ab-OVA 329-337/326-334. Isolated cells were stained for surface and intracellular markers and analyzed via flow cytometry. For in vivo analysis, mice were treated with 300ug anti-CD25 (clone PC-61) depleting antibody or isotype control; a subset of antibody-treated mice was immunized with OVA/CFA. Antibodies bound to HOD RBCs were determined by direct antibody test. Anti-HOD antibodies were quantified by indirect immunofluorescence using HOD RBCs as targets. Results: Tetramer pull-down assays revealed similar numbers of OVA-reactive CD4+ T cells from HOD and B6 mice (mean 56 and 40, respectively, p = 0.3). However, cell surface and intracellular marker staining demonstrated that HOD mice had higher numbers of OVA-tetramer reactive CD4+ T cells that express regulatory markers CD25 and FoxP3, and exhaustion marker PD1 as compared to control B6 mice. Inhibitory CTLA4 expression was not detectable on OVA-reactive CD4+ T cells from HOD or B6 mice. To test whether regulatory T cells were required for RBC-specific immune tolerance, HOD and B6 mice were treated with CD25 depleting antibody or isotype control antibody. Anti-CD25 antibody treated mice had a significant reduction of CD25+ cells 4 days post treatment (p < 0.001, 2 independent experiments). Similarly, there was a significant reduction in FoxP3+CD25+CD4+ T cells (Tregs) in anti-CD25 treated mice (p < 0.001), compared to isotype. Mice received weekly injections of anti-CD25 or isotype antibody to maintain depletion for one month. A subset of mice received an OVA/CFA immunization. Sustained CD25+ depletion did not result in anti-HOD autoantibody generation. Further, there was no change in the endogenous frequency of OVA-reactive CD4+ T cells between HOD and B6 mice, regardless of antibody treatment. Similarly, HOD mice treated with depletion (or isotype) antibody and immunized with OVA/CFA did not make detectable anti-HOD autoantibodies. Consistent with lack of detectable autoantibodies, no expansion of OVA-tetramer reactive CD4+ T cells was observed in HOD mice. In contrast, B6 mice (treated with anti-CD25 or isotype antibody) had a detectable expansion of OVA-specific CD4+ T cells as a result of immunization. Conclusions: The data demonstrate a phenotypic difference between the OVA-reactive CD4+ T cells from HOD and B6 mice, with an increase in number of Tregs detectable in HOD mice. Administration of anti-CD25 antibody significantly reduced the number of overall CD25+ cells and Tregs. Prolonged depletion of these cellular subsets did not elicit autoantibodies in HOD mice. Further, immunization of CD25 depleted mice with a strong immune stimulus (OVA/CFA, known to expand OVA-reactive T cells in B6 mice), did not induce anti-HOD autoantibodies nor did it expand OVA-specific autoreactive CD4+ T cells in HOD mice. Together, these data demonstrate that CD25+ cells are not required for the maintenance of RBC-specific T cell tolerance and suggest a role for other regulatory mechanisms. Disclosures No relevant conflicts of interest to declare.

Functional properties of ovalbumin glycosylated with carboxymethyl cellulose of different substitution degree

The glycation of hen ovalbumin (OVA) with carboxymethyl cellulose (CMC) was studied in an attempt to improve its functionality. OVA was dry-heated with CMC with three different degrees of substitution (0.69, 0.81, 0.89) at 60 °C and 79% relative humidity for 7 days. The emulsifying properties of OVA–CMC conjugates were strengthened remarkably with increasing substitution degree of CMC. The foam stability of OVA–CMC conjugates was improved strongly with the extension of incubation time while the foaming ability decreased. The rheological behaviors of all the three grafted products were similar. With the incubation time extension, it took more time for the formation of the protein gel network, and the thermal denaturation temperature of protein became higher. The glycation also produced OVA–CMC conjugates with higher surface hydrophobicity and lower content of lysine and arginine.

O4-S2.03 An anti-adhesive approach to prevention of C trachomatis infection

BackgroundWe have reported that the major outer membrane of C trachomatis is glycosylated and the glycan is a high mannose oligosaccharide. Cumulative studies have demonstrated that the glycan is important...

Multiple T cell epitope peptides suppress allergic responses in an egg allergy mouse model by the elicitation of forkhead box transcription factor 3‐ and transforming growth factor‐β‐associated mechanisms

Peptide-based immunotherapy (PIT) represents an attractive approach for targeted interventions in immunological disorders, but has not been widely explored in the context of food allergy. In this study, we built on the information obtained from the recent identification of three immunodominant T cell epitopes of hen ovalbumin (OVA), a major egg allergen, to assess the therapeutic potential of PIT for food allergy, using the BALB/c mouse model. Groups of mice were sensitized to OVA by repeated oral gavages, and subsequently administered with single or multiple synthetic peptides containing OVA T cell epitopes. Following the peptide administration period, all mice were orally challenged with high doses of OVA to elicit active anaphylaxis. Serum, spleen, and intestinal tissues were collected for the determination of immunoglobulin levels, cytokine secretions, and intestinal gene expression. Significantly lower anaphylactic scores were exhibited by mice that received multiple epitope-containing peptides, accompanied by lower serum histamine and OVA-specific IgE levels, compared with placebo-treated mice. Mechanistically, the quantification of cytokine secretions in splenocyte cultures revealed a T helper type 1-biased response (IFN-gamma) in all peptide-treated mice to the detriment of a T helper type 2-response (IL-4). Interestingly, a similar cytokine expression profile was determined in intestinal tissues, accompanied by a pronounced mRNA expression of regulatory molecules TGF-beta and forkhead box transcription factor 3 (FOXP3). These data suggest the activation of local repressive mechanisms mediated by subsets of regulatory T cells. We demonstrated the therapeutic potential of PIT in a mouse model of food allergy model and provided evidence that mechanistic pathways entailing regulatory molecules TGF-beta and FOXP3, stand as promising trails for the further understanding of peptide-based strategies for food allergy.

Mass spectrometric analysis of carbohydrates labeled with a biotinylated tag

A derivatization method for mass spectrometric analysis of oligosaccharides is presented. Small saccharides, complex, high-mannose-type oligosaccharides and oligosaccharides released from hen ovalbumin were converted into their biotin derivatives by incubating them with biotinamidocaproyl hydrazide (BACH). Improved sensitivity of mass spectrometric analysis of labeled glycans in comparison with their natural counterparts was achieved after derivatization. The labeling reagent contains a biotin handle at one end and a hydrazide group at the other. Hence, the key feature of biotinylated sugars is that in addition to their usefulness in functional studies (e.g. analysis of the interaction between lectins and biotin-derivatized oligosaccharides) they might be utilized also for structural analysis of oligosaccharides. Mass spectrometric studies were performed by matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization mass spectrometry.

Novel T-cell epitopes of ovalbumin in BALB/c mouse: Potential for peptide-immunotherapy

The identification of food allergen T-cell epitopes provides a platform for the development of novel immunotherapies. Despite extensive knowledge of the physicochemical properties of hen ovalbumin (OVA), a major egg allergen, the complete T-cell epitope map of OVA has surprisingly not been defined in the commonly used BALB/c mouse model. In this study, spleen cells obtained from OVA-sensitized mice were incubated in the presence of 12-mer overlapping synthetic peptides, constructed using the SPOTS ® synthesis method. Proliferative activity was assessed by 72-h in vitro assays with use of the tetrazolium salt WST-1 and led to identification of four mitogenic sequences, i.e., A39R50, S147R158, K263E274, and A329E340. ELISA analyses of interferon (IFN)-γ and interleukin (IL)-4 productions in cell culture supernatants upon stimulation with increasing concentrations of peptides confirmed their immunogenicity. Knowledge of the complete T-cell epitope map of OVA opens the way to a number of experimental investigations, including the exploration of peptide-based immunotherapy.

Simvastatin impairs humoral and cell‐mediated immunity in mice by inhibiting lymphocyte homing, T‐cell activation and antigen cross‐presentation

Statins block the activity of HMG-CoA reductase, which catalyses the production of mevalonate, an intermediate in cholesterol biosynthesis, which is also a precursor of isoprenoids. In addition to lowering circulating cholesterol, these drugs display anti-inflammatory and immunomodulatory activities in vitro; however, their effects on the development of adaptive immune responses in vivo, as well as the underlying mechanisms, are as yet largely unknown. Here we investigated the outcome of simvastatin treatment on a number of processes, which together orchestrate adaptive immunity to specific antigen. Simvastatin treatment resulted in a marked reduction of T and B cells in spleen, lymph nodes and peripheral blood in mice. This effect could be ascribed principally to an impairment of lymphocyte homing to secondary lymphoid organs. In addition, simvastatin was found to strongly inhibit T-cell responses to the MHCI restricted hen ovalbumin peptide antigen SIINFEKL and to impair ovalbumin uptake and cross-presentation by MHCI. Simvastatin also suppressed antibody responses to immunization with ovalbumin and delayed-type hypersensitivity to allergens. These activities could be largely accounted for by the simvastatin-dependent inhibition of HMG-CoA reductase. The data provide novel mechanistic insight into the activities of simvastatin in the highly complex context of the immune response.

Crystal structure of VC1805, a conserved hypothetical protein from a Vibrio cholerae pathogenicity island, reveals homology to human p32

The severe diarrhoeal disease cholera is caused by the gram-negative bacterium Vibrio cholerae and continues to be a major cause of morbidity and mortality. Like many Vibrio species V. cholerae inhabits an aquatic ecosystem, and most V. cholerae isolates do not possess the ability to cause cholera. Of more than 200 known serotypes, only O1 and O139 serogroups are highly pathogenic and acknowledged to cause epidemic disease.1 The O1 serogroup can be divided into two groups: classical and El Tor, with the first cholera pandemic, beginning in Asia in 1817, and the subsequent five pandemics probably caused by the classical biotype. The seventh and present pandemic began in 1961 caused by the El Tor biotype.2 In 1992 a novel O-serogroup, O-139, emerged to cause epidemic cholera.3 All V. cholerae O1 and O139 serogroups encode the major virulence factors cholera toxin and toxin coregulated pilus, the latter being encoded on a pathogenicity island, named Vibrio Pathogenicity Island-I (VPI-1).4 Several other genomic regions have been identified that occur mainly among epidemic O1 and O139 serogroup isolates, including the so-called Vibrio seventh pandemic island-I (VSP-I) encoding ORFs VC0175 to VC0185, VSP-II encoding ORFs VC0490 to VC0516 and VPI-2 encoding ORFs VC1758 to VC1809.5–7 A microarray analysis of V. cholerae El Tor isolates was used to identify VSP-I and VSP-II encompassing genes that are possibly responsible for the unique characteristics of the seventh pandemic (El Tor) strains.5 An evolutionary genetic analysis of clinical and environmental isolates suggested that pandemic strains arose from a common O1 serogroup progenitor through the successive acquisition of new virulence regions.8 VPI-2 is a 57.3 kb region which consists of 52 ORFs present in all toxigenic O1 and O139 serogroup isolates, but lacking in non-O1 and non-O139 nontoxigenic isolates.6 VPI-2 encodes a type-I restriction modification system, a nan-nag region of genes involved in sialic acid metabolism that may play a nutritional role,9 a sialidase/neuraminidase known to convert intestinal higher-order gangliosides to GM1,10 and a region with homology to Mu phage. In addition, VPI-2 contains several genes that code for hypothetical proteins. One of these proteins, VC1805, is a 148 amino acid protein with no function revealed so far through sequence analysis. VC1805 is also encoded in the reduced 20 kb VPI-2 region present among most O139 serogroup isolates that are missing ORFs VC1761 to VC1788.6 A paralogue of VC1805 exists within VSP-II, VC0508 a 147 residue protein that shares 59% amino acid sequence identity with VC1805. A search of the sequence database using PSI-BLAST reveals homologues of VC1805 in several Vibrio species: V. vulnificus, V. splendidus, V. alginolyticus and V. fischeri, and orthologues in Altermonas macleodii, Aeromonas hydrophilia, and some Shewanella species. In addition, PSI-BLAST reveals that the adjacent hypothetical protein VC1804, with 104 residues, is a homologue of VC1805 sharing 26% sequence identity. Similarly the hypothetical protein VC0509 is a homologue of the adjacent protein VC0508. The four proteins VC0508, VC0509, VC1804, and VC1805 are therefore likely to share the same protein fold and be functionally related. As part of a structural genomics approach to understanding the function of hypothetical proteins within V. cholerae genomic islands we have determined the crystal structure of VC1805 to a resolution of 2.1 A using heavy atom isomorphous replacement. The structure reveals a similarity to the human mitochondrial protein p32 that is known to have several binding partners, including the human complement system protein C1q. This study shows that VC1805 does bind C1q, suggesting potential biological roles for the protein and its homologues.

Electrophoresis of Protein Charge Ladders: A Comparison of Experiment with Various Continuum Primitive Models

Detailed modeling of the free solution electrophoresis of five proteins (bovine R-lactalbumin, hen egg white lysozyme, bovine superoxide dismutase, human carbonic anhydrase II, and hen ovalbumin) is carried out within the framework of the continuum primitive model. Protein crystal structures and translational diffusion constants are used to design and parametrize the models. The modeling results are compared with experimental mobilities of protein charge ladders, collections of protein derivatives where the number of charge groups is varied by partial acylation of lysine residues or by amidation of glutamic and aspartic acid residues. The simplest model considered is the Yoon and Kim model of a prolate/oblate ellipsoid with uniform surface potential, electrostatics treated at the level of the linear Poisson -Boltzmann equation, and distortion of the ion atmosphere from equilibrium (ion relaxation) ignored (Yoon, B. J.; Kim, S. J. Colloid Interface Sci. 1989, 128, 275). This model provides good agreement with experiment but only if the net absolute protein charge is low or the average absolute surface, or œ, potential is less than 25 mV. Boundary element (BE) modeling is also carried out in which detailed surface models are employed and the electrostatics are solved at the level of the nonlinear Poisson-Boltzmann equation. Ion relaxation is also included in some of the BE studies. All of the experimental mobilities are in good (human carbonic anhydrase II and hen ovalbumin) to excellent (bovine R-lactalbumin, hen egg white lysozyme, and bovine superoxide dismutase) agreement with BE modeling that includes ion relaxation. We believe that these results taken as a whole serve to confirm the ability of the continuum primitive model to predict, with quantitative accuracy, the free solution electrophoretic mobilities of proteins, provided the underlying models are sufficiently realistic. When a discrepancy occurs, it may be due to error in modeling either the protein charge or the solution conformation. The models described in this work also provide a useful approach for determining values of ¢Z, the change in net charge of proteins due to the chemical modification of charged groups. Knowledge of ¢Z is essential for the use of protein charge ladders in the quantitative description of the electrostatic properties and interactions of proteins. This paper supports the view that the continuum primitive model may be more appropriate for the modeling of electrokinetics than for electrostatics. The main challenge to the accurate predictions of electrophoretic mobilities may lie primarily in the modeling of electrostatics, not electrokinetics. I. Introduction The prediction of electrophoretic mobilities of proteins from their sequences and structures is challenging because such predictions require accurate models of both the electrostatic and the electrokinetic properties of proteins. Electrostatic models are used to predict the distribution of charges on the protein due to the reversible binding of protons to ionizable groups and also the electrostatic potential in the vicinity of the model protein. Electrokinetic models take as input a particular distribution of charge and electrostatic potential and predict the electrophoretic mobility. Both types of models are generally based on the continuum primitive model. The combination of capillary electrophoresis (CE) and protein charge ladders is a useful experimental system for addressing this challenge, as it provides values of electrophoretic mobilities for a collection of

Matrix-assisted laser desorption/ionization tandem mass spectrometry and post-source decay fragmentation study of phenylhydrazones of N-linked oligosaccharides from ovalbumin

N-linked oligosaccharides were released from hen ovalbumin by PNGase F and derivatized with phenylhydrazine. They were then examined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Phenylhydrazones of N-glycans under MALDI-tandem mass spectrometry (MS/MS) and post-source decay (PSD) conditions produced relatively similar fragmentation patterns; however, more cross-ring cleavages and fragment ions corresponding to low abundance isomeric structures were detected by MS/MS and not in PSD. Most fragment ions corresponded to glycosidic cleavages with preferential loss of residues from the chitobiose core and the 3-antenna. Sialylated phenylhydrazone- N-glycans, characterized here for the first time in ovalbumin by tandem mass spectrometry, underwent losses of sialic acid residues followed the same fragmentation pathways observed with neutral derivatized glycans. The relative abundances of some fragment ions indicated the linkage position of sialic acid and provided information on the number of residues attached to the 6-antenna. Also, new structures of ovalbumin glycans were observed as part of this study and are reported here.

Modification of ovalbumin with a rare ketohexose through the Maillard reaction: effect on protein structure and gel properties.

The effect of nonenzymatic glycation on the structural changes and gelling properties of hen ovalbumin (OVA) through the Maillard reaction was studied. OVA was incubated at the dry state with a rare ketohexose (D-psicose, Psi) and two alimentary sugars (D-fructose, Fru; D-glucose, Glc) at 55 degrees C and 65% relative humidity. To evaluate the modification of OVA by different reducing sugars during the glycation process, the extent of the Maillard reaction, aggregation processes, structural changes, and gelling behaviors were investigated. Reactivity of Psi with the protein amino groups was much lower than that of both Fru and Glc, whereas Psi induced production of browning and fluorescent substances more strongly than the two alimentary sugars did. Furthermore, OVA showed an increased tendency toward multimeric aggregation upon modifying with Psi through covalent bond. The modified OVAs with reducing sugar were similar to nonglycated control sample in Fourier transform infrared (FT-IR) characteristics, but significantly decreased in intensity of tryptophan-related fluorescence. The results indicate that although glycation brought about similar changes in the secondary structure without great disruption of native structure, its influence on the side chains of protein in tertiary structure could be different. Breaking strength of heat-induced glycated OVA gels with Psi was markedly enhanced by the Maillard reaction. These results suggest that Psi had a strong cross-linking activity with OVA; consequently, the glycated OVA with Psi could improve gelling properties under certain controlled conditions.

Role of the Carbohydrate Chain and Two Phosphate Moieties in the Heat-Induced Aggregation of Hen Ovalbumin

We investigated the effect of the carbohydrate chain and two phosphate moieties on heat-induced aggregation of hen ovalbumin. The dephosphorylated form of ovalbumin was obtained by treating the original protein with acid phosphatase. The single carbohydrate chain was removed by digestion of heat-denatured ovalbumin with glycopeptidase F, and the resulting polypeptide without this carbohydrate chain was correctly refolded to acquire protease-resistance. Thermal unfolding can be approximated by a mechanism involving a two-state transition between the folded and unfolded states with a midpoint temperature of 76 degrees C for the original form, of 74 degrees C for the dephosphorylated form, and of 71 degrees C for the carbohydrate-free form. The conformational stability of the original form was higher than that of the carbohydrate-free form. When the three forms of ovalbumin were heated to 80 degrees C and then cooled rapidly in an ice bath, the polypeptide chains were compactly collapsed to metastable intermediates with secondary structures whose properties were indistinguishable. Upon incubation at 60 degrees C, renaturation was possible for a large portion of the intermediates of the original form, but for only a small portion of those of the carbohydrate-free form. Light scattering experiments showed that in the presence of sulfate anions, the intermediates of the carbohydrate-free form aggregated to a greater extent than did those of the original form. The intermediates of the carbohydrate-free form bound to the chaperonin GroEL with about 10-fold higher affinity than those of the original form. It follows that the carbohydrate chain and the two phosphate moieties do not affect hydrophobic collapse in the kinetic refolding of hen ovalbumin but play an important role in the slow rearrangement. They block the off-pathway reaction that competes with correct refolding by effectively decreasing surface hydrophobicity.

Profiling of N-linked oligosaccharides using phenylhydrazine derivatization and mass spectrometry

N-linked oligosaccharide standards obtained from commercial sources were derivatized with phenylhydrazine (PHN) and analyzed by on-line reversed-phase high performance liquid chromatography (HPLC)/electrospray ionization mass spectrometry (ESI-MS). This procedure was then applied to mixtures of N-glycans enzymatically released from hen ovalbumin. Under ESI-MS conditions, phenylhydrazones of asialylated oligosaccharide standards and ovalbumin glycans produced mainly [M+2H]2+ molecular ions at low cone voltage values, while minimal fragmentation was observed. Reversed-phase HPLC/ESI-MS total and selected ion chromatograms obtained for derivatized N-glycans from ovalbumin showed partial but useful separation. Overall glycan profiles obtained by ESI-MS were compared with results obtained by matrix-assisted laser desorption/ionization (MALDI)-MS. Qualitatively, profiles were similar from one technique to the other in terms of relative abundance of glycans versus composition. Post-source decay (PSD) analysis of the [M+Na]+ ions of PHN–glycans showed dominant B, C and internal B/Y, C/Y cleavages. These patterns were helpful in relating fragmentation to proposed structures. Cross-ring cleavage fragment ions (A-type) were also observed in most cases. The PHN derivatization method is fast and simple. It produces abundant parent ions in both MALDI-MS and ESI-MS, while avoiding the presence of salt contaminants during the labeling procedure.

Labelling saccharides with phenylhydrazine for electrospray and matrix-assisted laser desorption–ionization mass spectrometry

A well-known reaction of carbonyl compounds with phenylhydrazine has been applied to saccharides, providing increased sensitivity for mass spectrometric (MS) and ultraviolet (UV) detection during high-performance liquid chromatographic (HPLC) separations. After a simple derivatization procedure for 1 h at 70 °C and purification of the reaction mixture from excess reagent by extraction, the sugar derivatives were characterized by direct injection or on-line HPLC/electrospray ionization (ESI) and by matrix-assisted laser desorption/ionization (MALDI) MS. Because no salts are used or produced upon reaction, this procedure is very simple and suitable for the tagging of saccharides. The reaction allows for on-target derivatization and products are very stable. The derivatization procedure has been applied to commercially-obtained small saccharides and standard N-linked oligosaccharides. Lastly, hen ovalbumin N-glycans were detached enzymatically and characterized by MALDI–MS as their phenylhydrazone derivatives.

Heat-induced gels of egg white/ovalbumins from five avian species: thermal aggregation, molecular forces involved, and rheological properties.

Product processing (heating, pH change, etc.) usually alters protein structure, improves rheological properties, and gives a unique texture to foods. The thermal aggregation and structural properties of ovalbumins from five avian species were studied at different pH values by polyacrylamide gel electrophoresis (PAGE) and determinations of sulfhydryl group content and surface hydrophobicity. The results showed that sulfhydryl group content changed insignificantly in heat-denatured ovalbumins other than hen ovalbumin (pH-independent), and surface hydrophobicity markedly increased (pH-dependent) after heating, with a significant difference among species. Furthermore, it was demonstrated that the hydrophobic interaction and sulfhydryl-disulfide interchange reaction were necessary in the aggregation and cross-linking of gel networks. Creep tests were also used to characterize the gel network structures of various egg white/ovalbumins upon heating. The viscoelastic behavior of the ovalbumins of all species was dependent on pH values, and changed significantly with the phylogeny of these species. With increases in pH value (7.0-8.5), the heat-induced gels of ovalbumins gradually changed from turbid to translucent, the instantaneous modulus (E(0)) increased slightly and reached a nearly constant value, and the Newtonian modulus (etaN) increased significantly in each sample. The heated egg white from these five avian species also formed highly viscoelastic gels, with a good correlation of viscoelastic behavior between ovalbumin and egg white in corresponding species.

Selective Organic Precipitation/Extraction of Released N-Glycans Following Large-Scale Enzymatic Deglycosylation of Glycoproteins

A major difficulty with isolating enzymatically or chemically released oligosaccharides from large-scale glycoprotein deglycosylation reactions is the time-consuming chromatography, desalting, and concentration steps required to prepare a glycan fraction of manageable proportions. To overcome these time and preparative chromatography equipment requirements, we have developed a rapid organic solvent precipitation/extraction procedure that allows sequential isolation of endo-β-N-acetylglucosaminidase H (EC 3.2.1.96)-released high-mannose and hybrid, peptide-N4-(N-acetyl-β-glucosaminyl) Asn amidase (EC 3.5.1.52)-released complex, and β-eliminated O-linked glycans without the need for intermediate chromatography, desalting, or concentration steps. The method involves precipitation of protein and released glycans at −20°C in 80% acetone and extraction of the glycans from the pellet with 60% aqueous methanol after each deglycosylation step. Three pools of essentially salt- and detergent-free oligosaccharides (high-mannose/hybrid, complex, and O-linked) can be isolated in a high yield in 4 days with this protocol, which has been extensively tested using bovine RNase B, human bile salt-stimulated lipase expressed in Pichia pastoris, hen ovalbumin, bovine fetuin, bovine thyroglobulin, and several invertase preparations from wild-type and mutant yeast strains.