Enzymolysis Reaction Kinetics and Liquid Chromatography High-Resolution Mass Spectrometry Analysis of Ovalbumin Glycated with Microwave Radiation.

Microwave radiation was adopted to accelerate glycation between ovalbumin (OVA) and d-glucose. We evaluated the digestibility of glycated OVA from the perspective of kinetics, using pepsin and trypsin as model enzymes. Hydrolysed protein concentrations, enzymolysis kinetics, and activation energy (Ea) were investigated. The results showed that, under the conditions of simulating human digestion, the hydrolysis rate of OVA by pepsin was faster than that by trypsin, but for digestive enzymes, the digestion efficiency of OVA hydrolyzed by trypsin was higher. It was found that the rate constant of enzymatic hydrolysis of OVA was independent of the initial concentration of OVA but related to the type of protease and temperature. The reaction rate constants of glycated OVAs were significantly higher than that of native OVA during enzymolysis. Ea required for glycated OVA enzymatic hydrolysis by pepsin decreased, while that required by trypsin enzymatic hydrolysis nearly doubled. Liquid chromatography high-resolution mass spectrometry revealed that sample 1 had three glycated sites (R111, K227, and K264), sample 2 had two glycated sites (K207 and K323), sample 3 had five glycated sites (R127, R159, K227, R340, and K370), sample 4 had three glycated sites (R85, R143, and K323), and sample 5 had two glycated sites (R51 and R59). These sites increased Ea required for enzymatic hydrolysis of glycated OVA by trypsin.

Post-translational modifications of protein in response to ionizing radiation.

Based on central dogma of genetics, protein is the embodiment and executor of genetic function, post-translational modifications (PTMs) of protein are particularly important and involved in almost all aspects of cell biology and pathogenesis. Studies have shown that ionizing radiation (IR) alters gene expression much more profoundly and a broad variety of cell-process pathways, lots of proteins are modified and activated. Our understanding of the protein in response to ionizing radiation is steadily increasing. Among the various biological processes known to induce radioresistance, PTMs have attracted marked attention in recent years. The present review summarizes the latest knowledge about how PTMs response to ionizing radiation and pathway analysis were conducted. The data provided insights into biological effects of IR and contributing to the development of novel IR-based strategies.

Nanomechanical characterization of time-dependent deformation/recovery on human dentin caused by radiation-induced glycation.

An increase in non-enzymatic collagen matrix cross-links, such as advanced glycation end-products (AGEs), is known to be a major complication in human mineralized tissues, often causing abnormal fractures. However, degradation of mechanical properties in relation to AGEs has not been fully elucidated at the material level. Here, we report nanoscale time-dependent deformation and dimensional recovery of human tooth dentin that has undergone glycation induced by x-ray irradiation. The reduction in enzymatic collagen cross-linking and the increased level of AGEs with concomitant growth of disordered collagen matrix diminished creep deformation recovery in the lower mineralized target region. However, the elevated AGEs level alone did not cause a reduction in time-dependent deformation and its recovery in the higher mineralized target region. In addition to the elevated AGEs level, the degradation of the mechanical properties of mineralized tissues should be assessed with care in respect to multiple parameters in the collagen matrix at the molecular level.

The effect of simulated space radiation on the N-glycosylation of human immunoglobulin G1.

On a roundtrip to Mars, astronauts are expectedly exposed to an approximate amount of radiation that exceeds the lifetime limits on Earth. This elevated radiation dose is mainly due to Galactic Cosmic Rays and Solar Particle Events. Specific patterns of the N-glycosylation of human Igs have already been associated with various ailments such as autoimmune diseases, malignant transformation, chronic inflammation, and ageing. The focus of our work was to investigate the effect of low-energy proton irradiation on the IgG N-glycosylation profile with the goal if disease associated changes could be detected during space travel and not altered by space radiation. Two ionization sources were used during the experiments, a Van de Graaff generator for the irradiation of solidified hIgG samples in vacuum, and a Tandetron accelerator to irradiate hIgG samples in aqueous solution form. Structural carbohydrate analysis was accomplished by CE with laser induced fluorescent detection to determine the effects of simulated space radiation on N-glycosylation of hIgG1 samples. Our results revealed that even several thousand times higher radiation doses that of astronauts can suffer during long duration missions beyond the shielding environment of Low Earth Orbit, no changes were observed in hIgG1 N-glycosylation. Consequently, changes in N-linked carbohydrate profile of IgG1 can be used as molecular diagnostic tools in space.

Glycation of ovalbumin after high-intensity ultrasound pretreatment: effects on conformation, immunoglobulin (Ig)G/IgE binding ability and antioxidant activity.

BACKGROUND: Ovalbumin (OVA), a protein with excellent nutritional and processing properties, is the major allergen of hen egg white. High-intensity ultrasound treatment increases the immunoglobulin (Ig)G and IgE binding abilities by unfolding the conformational structure of OVA. This may allow a modification of the IgG and IgE binding of OVA by combining high-intensity ultrasound with other methods, such as glycation, thus representing a promising method for the improvement of protein properties. RESULTS: Glycation with mannose (M) after ultrasound pretreatment at 0-600 W significantly reduced the IgG and IgE binding abilities and dramatically enhanced the antioxidant activity of OVA-M conjugates, with the lowest values of IgG and IgE binding and highest values of antioxidant capacity observed at 600 W. Polyacrylamide gel electrophoresis showed that the molecular weight of OVA-M conjugates with ultrasound pretreatment increased more than non-pretreatment sample, implying that ultrasound pretreatment promoted glycation. The α-helix content and ultraviolet absorption of OVA were observably increased, whereas ß-sheet content, intrinsic fluorescence and surface hydrophobicity were notably decreased, indicating that the tertiary and secondary structures of OVA were markedly changed. CONCLUSION: High-intensity ultrasound pretreatment can be conducive to reducing the binding abilities of IgG and IgE and enhancing the antioxidant activity of OVA-M conjugates. Therefore, glycation combined with high-intensity ultrasound pretreatment might be a promising method for producing hypo-allergenic and high-antioxidant OVA products. © 2018 Society of Chemical Industry.

Changes of protein glycosylation in the course of radiotherapy.

This is the first study of changes in protein glycosylation due to exposure of human subjects to ionizing radiation. Site specific glycosylation patterns of 7 major plasma proteins were analyzed; 171 glycoforms were identified; and the abundance of 99 of these was followed in the course of cancer radiotherapy in 10 individual patients. It was found that glycosylation of plasma proteins does change in response to partial body irradiation (∼ 60 Gy), and the effects last during follow-up; the abundance of some glycoforms changed more than twofold. Both the degree of changes and their time-evolution showed large inter-individual variability.

Mass spectrometry-based proteomics: basic principles and emerging technologies and directions.

As the main catalytic and structural molecules within living systems, proteins are the most likely biomolecules to be affected by radiation exposure. Proteomics, the comprehensive characterization of proteins within complex biological samples, is therefore a research approach ideally suited to assess the effects of radiation exposure on cells and tissues. For comprehensive characterization of proteomes, an analytical platform capable of quantifying protein abundance, identifying post-translation modifications and revealing members of protein complexes on a system-wide level is necessary. Mass spectrometry (MS), coupled with technologies for sample fractionation and automated data analysis, provides such a versatile and powerful platform. In this chapter we offer a view on the current state of MS-proteomics, and focus on emerging technologies within three areas: (1) New instrumental methods; (2) New computational methods for peptide identification; and (3) Label-free quantification. These emerging technologies should be valuable for researchers seeking to better understand biological effects of radiation on living systems.

Glycosylation of stress glycoprotein GP62 in cells exposed to heat-shock and subculturing.

GP62 is a member of the stress glycoprotein family that was proposed to have a chaperone-like function in the heat-shock response. Using lectin blotting we have studied glycosylation of GP62 and determined that in addition to heat-shock, even simple subculturing of cells is a sufficient stimulus to provoke induction of GP62. Interestingly, both kinetics of induction and glycosylation of GP62 induced by subculturing were different than when GP62 was induced by heat-shock. While GP62 induced by heat-shock was recognized by SNA, DSA and PHA-E lectins, and not by BSA I, Con A, RCA I, SJA, UEA I, VVA, and WGA lectins, GP62 induced by subculturing was also recognized by RCA I and WGA lectins.

UVA irradiation of human lens proteins produces residual oxidation of ascorbic acid even in the presence of high levels of glutathione.

The oxidation products of ascorbic acid (AscH-) can rapidly glycate and crosslink lens proteins in vitro, producing fluorophores and browning products similar to those present in cataractous lenses. The accumulation of AscH- oxidation products, however, would largely be prevented by the millimolar levels of glutathione (GSH) present in human lens. Here we investigate whether protein aggregation could allow the oxidation of AscH- by UVA-induced reactive oxygen species in the presence of physiological levels of GSH. The metal-catalyzed oxidation of 1.0 mM AscH- by 50 microM Cu(II) was almost complete after 1 h, but no oxidation was seen in the presence of GSH concentrations as low as 0.5 mM. UVA irradiation of protein aggregates from human lens, which accumulated more than 2.0 mM singlet oxygen after 1 h, caused a 50-60% oxidation of 1.0 mM AscH-. The addition of 204 mM GSH, however, decreased AscH- oxidation by less than half, and 30% of the AscH- was oxidized even in the presence of 15 mM GSH. This diminished protection may be due, in part, to the ability of AscH-, but not GSH, to penetrate to the sites of singlet oxygen generation located within the protein. Consistent with this hypothesis, greater GSH protection was seen when a proteolytic digest of the human proteins was subjected to the same irradiation or when singlet oxygen was chemically generated from 3-(4-methyl-1-naphthyl)propionic acid endoperoxide (MNPAE) at 37 degrees C in the medium. The addition of 50 microM Cu(II) had no effect on the rate of degradation of dehydroascorbic acid (DHA). Singlet oxygen, either UVA- or MNPAE-generated, increased the rate of DHA loss. This secondary oxidation of DHA by singlet oxygen would allow the accumulation of AscH- oxidation products was not reducible by GSH. Therefore, the data presented here argue that the protein aggregation seen in older human lenses may permit oxidized AscH--induced crosslinking to occur even at physiological GSH levels.

Altered N-glycosylation of glucose transporter-1 associated with radiation-induced tumorigenesis of human cell hybrids.

Studies on human cell hybrids between a cervical carcinoma cell line, HeLa, and normal fibroblasts have indicated that their tumorigenicity is under the control of a putative tumor suppressor on chromosome 11. We have previously demonstrated that a tumorigenic cell hybrid CGL4 expresses a larger glucose transporter, GLUT1, due to altered glycosylation when compared to the nontumorigenic counterpart CGL1. In this study, we demonstrated this glycosylation change in GLUT1 in gamma-ray-induced tumorigenic mutants (GIMs) isolated from CGL1 cells as expressing a tumor-associated surface antigen, intestinal alkaline phosphatase. In contrast, GLUT1 in the gamma-irradiated nontumorigenic control cells (CONs) did not show this alteration. In accordance with this glycosylation change, affinity to 2-deoxyglucose in these GIM clones was increased by about twofold when compared to the nontumorigenic CONs. These results suggest a close correlation between the glycosylation change in GLUT1 with increased affinity to D-glucose and tumorigenicity of these human cell hybrids.