ABSTRACT
Naturally occurring (native) sugars and carbohydrates contain numerous hydroxyl groups of similar reactivity1,2. Chemists, therefore, rely typically on laborious, multi-step protecting-group strategies3 to convert these renewable feedstocks into reagents (glycosyl donors) to make glycans. The direct transformation of native sugars to complex saccharides remains a notable challenge. Here we describe a photoinduced approach to achieve site- and stereoselective chemical glycosylation from widely available native sugar building blocks, which through homolytic (one-electron) chemistry bypasses unnecessary hydroxyl group masking and manipulation. This process is reminiscent of nature in its regiocontrolled generation of a transient glycosyl donor, followed by radical-based cross-coupling with electrophiles on activation with light. Through selective anomeric functionalization of mono- and oligosaccharides, this protecting-group-free 'cap and glycosylate' approach offers straightforward access to a wide array of metabolically robust glycosyl compounds. Owing to its biocompatibility, the method was extended to the direct post-translational glycosylation of proteins.
Subject(s)
Chemistry Techniques, Synthetic , Oligosaccharides , Sugars , Free Radicals/chemistry , Free Radicals/metabolism , Glycosylation/radiation effects , Indicators and Reagents/chemistry , Light , Oligosaccharides/chemical synthesis , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Oligosaccharides/radiation effects , Stereoisomerism , Sugars/chemical synthesis , Sugars/chemistry , Sugars/metabolism , Sugars/radiation effectsABSTRACT
Mycosporine-like amino acids (MAAs) are promising natural antioxidative compounds with cosmetic applications for the prevention of skin aging. In this study, we evaluated the protective effects of natural resources-derived MAA-containing emulsions on mouse ear tissue exposed to UV irradiation. DBA/2CrSlc male mice were irradiated by UV light at 120 mJ/cm2/day for 9 days. MAA-containing emulsions were prepared using mycosporine-2-glycine (M2G), shinorine (SHI), or porphyra-334 (P334) and applied to mice ears at a dose of 50 mg/ear/day. After that, collected ear skin tissues were subjected to the observation of melanocytes, investigation for antioxidative stress markers, and measurement of advanced glycation-end products (AGEs). In addition, the antiglycative effects of MAAs were investigated in vitro. MAA-containing emulsions prepared in this study upregulated the activities of total superoxide dismutase (SOD) and catalase (CAT) in mouse ear tissue exposed to UV irradiation. Increased accumulation of copper/zinc (Cu/Zn) -SOD and/or CAT was also found in mouse ear tissue on which M2G- or P334-containing emulsion had been applied. Furthermore, P334 exhibited an antiglycative effect on elastin in vitro. Although MAA-containing emulsions have antioxidative effects as well as in vitro antiglycation, a protective effect by the accumulation of AGEs in mice ears exposed to UV was not observed. Thus, application of MAA-containing emulsions stimulated or protected the expression of antioxidant-associated proteins, thereby leading to upregulation of antioxidative activities in mouse ear skin samples tissues under UV irradiation. Additional optimization of MAA-containing emulsions, including composition, process, and dosage should be considered for further improvement of efficacy.
Subject(s)
Antioxidants/pharmacology , Emulsions/chemistry , Skin/drug effects , Ultraviolet Rays , Animals , Antioxidants/chemistry , Catalase/metabolism , Cyclohexanols/chemistry , Cyclohexanols/pharmacology , Cyclohexanones/chemistry , Cyclohexanones/pharmacology , Cyclohexylamines/chemistry , Cyclohexylamines/pharmacology , Glycine/analogs & derivatives , Glycine/chemistry , Glycine/pharmacology , Glycosylation/drug effects , Glycosylation/radiation effects , Male , Mice , Mice, Inbred DBA , Skin/radiation effects , Superoxide Dismutase/metabolism , Up-Regulation/drug effects , Up-Regulation/radiation effectsABSTRACT
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.
Subject(s)
Ovalbumin/chemistry , Trypsin/chemistry , Amino Acid Motifs , Biocatalysis , Chromatography, Liquid , Digestion , Glycosylation/radiation effects , Humans , Hydrolysis , Kinetics , Mass Spectrometry , Microwaves , Protein ConformationABSTRACT
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.
Subject(s)
Protein Processing, Post-Translational/radiation effects , Proteins/radiation effects , Radiation, Ionizing , Amino Acid Motifs , DNA Damage/radiation effects , Genome, Human/radiation effects , Glycosylation/radiation effects , Humans , Methylation/radiation effects , Neoplasms/radiotherapy , Phosphorylation/radiation effects , Signal Transduction/radiation effects , Ubiquitination/radiation effectsABSTRACT
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.
Subject(s)
Dentin/metabolism , Dentin/radiation effects , Mechanical Phenomena/radiation effects , Nanotechnology , Adolescent , Adult , Biomechanical Phenomena/radiation effects , Collagen/metabolism , Glycosylation/radiation effects , Humans , Kinetics , Materials Testing , Young AdultABSTRACT
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.
Subject(s)
Cosmic Radiation/adverse effects , Glycosylation/radiation effects , Immunoglobulin G , Space Flight , Astronauts , Electrophoresis, Capillary , Humans , Immunoglobulin G/analysis , Immunoglobulin G/chemistry , Immunoglobulin G/radiation effects , Models, TheoreticalABSTRACT
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.
Subject(s)
Allergens/chemistry , Antioxidants/chemistry , Egg White/radiation effects , Food Handling/methods , Immunoglobulin E/chemistry , Immunoglobulin G/chemistry , Ovalbumin/chemistry , Ultrasonics/methods , Allergens/immunology , Animals , Chickens , Egg Hypersensitivity/immunology , Egg White/chemistry , Electrophoresis, Polyacrylamide Gel , Glycosylation/radiation effects , Hydrophobic and Hydrophilic Interactions/radiation effects , Immunoglobulin E/immunology , Immunoglobulin G/immunology , Ovalbumin/immunology , Protein Binding/radiation effects , Protein Conformation/radiation effectsABSTRACT
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 (â¼ 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.
Subject(s)
Blood Proteins/metabolism , Blood Proteins/radiation effects , Head and Neck Neoplasms/blood , Head and Neck Neoplasms/radiotherapy , Aged , Aged, 80 and over , Amino Acid Sequence , Blood Proteins/genetics , Female , Glycosylation/radiation effects , Head and Neck Neoplasms/genetics , Humans , Male , Middle Aged , Molecular Sequence DataABSTRACT
BACKGROUND: Biglycan (BGN) is a proteoglycan composed of a 42-kDa core protein and two glycosaminoglycan (GAG) chains, and known to be involved in structural, space-filling functions and many physiological regulations in the skin. OBJECTIVE: To investigate ultraviolet (UV) irradiation-induced changes of BGN protein and its GAG chain synthesis in cultured human dermal fibroblasts. METHODS: UV irradiation-induced or xylosyltransferase (XYLT) 1 siRNA-mediated smaller-sized protein bands detected by Western blot using BGN antibodies were identified as monoglycosylated forms of BGN, using BGN siRNA-mediated knockdown and chondroitinase ABC (ChABC). Differential activity of XYLT1 and 2 on BGN core protein was investigated by size shift of S42A- and S47A-BGN mutants to core protein size caused by XYLT1 siRNA transfection or UV irradiation. RESULTS: After UV irradiation, intact form of BGN protein (I-BGN) and core protein form were reduced in cultured fibroblasts, but other smaller-sized bands were observed to be increased. These smaller-sized ones were reduced by transfection of BGN siRNA, and shifted to the core protein size by treatment with ChABC, suggesting that they are defectively-glycosylated forms of BGN (D-BGN) protein. UV irradiation also decreased mRNA expression levels of XYLT1 and 2, which are responsible for initiation of GAG chain synthesis. UV-mediated reduction of XYLT1 expression was much stronger than that of XYLT2. Furthermore, siRNA-mediated down-regulation of XYLT1 resulted in the increase of D-BGN and the decrease of I-BGN, while down-regulation of XYLT2 resulted in no change of D-BGN and I-BGN, suggesting that the XYLT1 may react with both GAG-attaching serine sites of BGN; however, XYLT2 may prefer to react one of them. Another dermatan sulfate (DS) proteoglycan, decorin, showed no or a little change of its molecular weight by UV irradiation or XYLT1 siRNA transfection, suggesting that DS synthesis may not be a critical factor in formation of D-BGN. Co-transfection with XYLT1, 2 siRNAs and wild-type or mutant forms of BGN overexpression vectors revealed that S42A-BGN showed size reduction to core protein size by XYLT1 downregulation, but S47A-BGN did not, suggesting that XYLT2 can react only with S42 on BGN core protein. With UV irradiation, both S42A-BGN and S47A-BGN showed size reduction, which is probably because UV-caused downregulation of both XYLTs and overexpression condition resulted in incomplete glycosylation and secretion. CONCLUSIONS: UV irradiation-induced increase of BGN monoglycosylated forms in cultured human dermal fibroblasts is resulted from dominance of XYLT2 activity, which acts only at S42 on BGN core protein, caused by UV-mediated stronger reduction of XYLT1.
Subject(s)
Biglycan/biosynthesis , Biglycan/genetics , Glycosaminoglycans/biosynthesis , Pentosyltransferases/metabolism , Ultraviolet Rays , Cells, Cultured , Decorin/metabolism , Down-Regulation/radiation effects , Fibroblasts/metabolism , Fibroblasts/radiation effects , Gene Silencing , Glycosaminoglycans/radiation effects , Glycosylation/radiation effects , Humans , Molecular Weight , Pentosyltransferases/genetics , Pentosyltransferases/radiation effects , Protein Biosynthesis/radiation effects , RNA, Messenger/metabolism , Skin Physiological Phenomena/radiation effects , Xylose/metabolism , UDP Xylose-Protein XylosyltransferaseABSTRACT
The application of mild hypothermic conditions to cell culture is a routine industrial practice used to improve recombinant protein production. However, a thorough understanding of the regulation of dynamic cellular processes at lower temperatures is necessary to enhance bioprocess design and optimization. In this study, we investigated the impact of mild hypothermia on protein glycosylation. Chinese hamster ovary (CHO) cells expressing a monoclonal antibody (mAb) were cultured at 36.5°C and with a temperature shift to 32°C during late exponential/early stationary phase. Experimental results showed higher cell viability with decreased metabolic rates. The specific antibody productivity increased by 25% at 32°C and was accompanied by a reduction in intracellular nucleotide sugar donor (NSD) concentrations and a decreased proportion of the more processed glycan structures on the mAb constant region. To better understand CHO cell metabolism at 32°C, flux balance analysis (FBA) was carried out and constrained with exometabolite data from stationary phase of cultures with or without a temperature shift. Estimated fluxomes suggested reduced fluxes of carbon species towards nucleotide and NSD synthesis and more energy was used for product formation. Expression of the glycosyltransferases that are responsible for N-linked glycan branching and elongation were significantly lower at 32°C. As a result of mild hypothermia, mAb glycosylation was shown to be affected by both NSD availability and glycosyltransferase expression. The combined experimental/FBA approach generated insight as to how product glycosylation can be impacted by changes in culture temperature. Better feeding strategies can be developed based on the understanding of the metabolic flux distribution.
Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Cell Culture Techniques/methods , Cold Temperature , Glycosylation/radiation effects , Protein Processing, Post-Translational/radiation effects , Animals , CHO Cells , Carbon/metabolism , Cricetulus , Gene Expression , Glycosyltransferases/analysis , Metabolic Flux Analysis , Polysaccharides/analysis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
BACKGROUND: Protein glycation refers to the spontaneous reaction of reducing sugars with proteins and the subsequent formation of stable advanced glycation end products (AGEs). Glycation is linked with oxidative stress, and this association is called "glycoxidation". Glycoxidation alters the protein structure and function and causes tissue aging, as seen in human skin. Therefore, research on substances inhibiting glycoxidation appears to be crucial in the prevention of skin aging. With this aim, several plant extracts have been screened for antiglycation activity, and the results of the best candidates are presented in this article. METHODS: Glycation was studied on human skin proteins (collagen, elastin, and albumin) and on a model of reconstructed skin. Oxidative stress has been addressed by testing the copper-induced low-density lipoprotein oxidation, ultraviolet irradiation of glycated dermis, and carbonyl activation of human dermal fibroblasts. A clinical test evaluated the extent of oxidative stress induced by ultraviolet A irradiation. RESULTS: Among the tested products, several plant extracts have decreased the glycation effects on skin proteins collagen, elastin, and albumin. In addition, a plant extract has significantly inhibited the different forms of oxidative stress associated with protein glycation. CONCLUSIONS: We have demonstrated that plant extracts can relieve the deleterious effects of glycation on human skin. Moreover, a plant extract rich in antioxidant molecules has also significantly preserved the human skin from glycoxidation attacks.
Subject(s)
Oxidative Stress , Skin/metabolism , Albumins/chemistry , Albumins/metabolism , Collagen/chemistry , Collagen/metabolism , Copper/chemistry , Copper/pharmacology , Elastin/chemistry , Elastin/metabolism , Fibroblasts/cytology , Glycosylation/drug effects , Glycosylation/radiation effects , Glyoxal/pharmacology , Humans , Lipoproteins, LDL/metabolism , Manilkara/chemistry , Manilkara/metabolism , Models, Biological , Oxidative Stress/drug effects , Oxidative Stress/radiation effects , Plant Extracts/chemistry , Plant Extracts/pharmacology , Ultraviolet RaysABSTRACT
The human host cell line, F2N78, is a new somatic hybrid cell line designed for therapeutic antibody production. To verify its potential as a human host cell line, recombinant F2N78 cells that produce antibody against rabies virus (rF2N78) were cultivated at different culture pH (6.8, 7.0, 7.2, 7.4, and 7.6) and temperatures (33.0 °C and 37.0 °C). Regardless of the culture temperature, the highest specific growth rate was obtained at a pH of 7.0-7.4. Lowering the culture temperature from 37.0 °C to 33.0 °C suppressed cell growth while allowing maintenance of high cell viability for a longer period. However, it did not enhance antibody production because specific antibody productivity did not increase at 33.0 °C. The highest maximum antibody concentration was obtained at 37.0 °C and pH 6.8. The N-linked glycosylation of the antibody was affected by the culture pH rather than the temperature. Nevertheless, G1F was dominant and G2F occupied a larger portion than G0F in all culture conditions. Compared to the same antibody produced from recombinant CHO cells, the antibody produced from rF2N78 cells has more galactose capping and was more similar to human plasma IgG. Taken together, the results obtained here demonstrate the potential of F2N78 as an alternative human host cell line for therapeutic antibody production.
Subject(s)
Antibodies, Viral/metabolism , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , Culture Media/chemistry , Metabolism/drug effects , Metabolism/radiation effects , Antibodies, Viral/genetics , Cell Culture Techniques , Cell Line , Glycosylation/drug effects , Glycosylation/radiation effects , Humans , Hydrogen-Ion Concentration , Rabies virus/immunology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Suspensions , Technology, Pharmaceutical/methods , TemperatureABSTRACT
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.
Subject(s)
Mass Spectrometry/trends , Peptides/analysis , Protein Processing, Post-Translational/radiation effects , Proteome/analysis , Proteomics/trends , Automation, Laboratory , Chemical Fractionation , Chromatography, Liquid , Glycosylation/radiation effects , Humans , Mass Spectrometry/instrumentation , Mass Spectrometry/methods , Phosphorylation/radiation effects , Proteomics/instrumentation , Proteomics/methods , Radiation, Ionizing , Signal Processing, Computer-AssistedABSTRACT
Phosphatidylethanolamines (PE) are one of the major components of cells membranes, namely in skin and in retina, that are continuously exposed to solar UV radiation being major targets of photooxidation damage. In addition, due to the presence of the free amine group, PE can also undergo glycation, in hyperglycemic conditions which may increase the susceptibility to oxidation. The aim of this study is to develop a model, based on mass spectrometry (MS) analysis, to identify photooxidative degradation of selected PE (POPE: PE 16:0/18:1, PLPE: PE 16:0/18:2, PAPE: PE 16:0/20:4) and glycated PEs due to UV irradiation. Photooxidation products were analysed by electrospray ionization MS (ESI-MS) and tandem MS (ESI-MS/MS) in positive and negative mode. Emphasis is placed in the influence of glycation in the generation of distinct photooxidation products. ESI-MS spectra of PE after UV photo-irradiation showed mainly hydroperoxy derivatives, due to oxidation of unsaturated fatty acyl chains. Glycated PE gave rise to several new photooxidation products formed due to oxidative cleavages of the glucose moiety, namely between C1 and C2, C2 and C3, and C5 and C6 of this sugar unit. These new products were identified by ESI-MS/MS in positive mode showing distinct neutral loss depending on the different structure of the polar head group. These new identified advanced glycated photooxidation products may have a deleterious role in the etiology of diabetic retinopathy and in diabetic retinal microvascular complications.
Subject(s)
Phosphatidylethanolamines/chemistry , Phosphatidylethanolamines/radiation effects , Spectrometry, Mass, Electrospray Ionization/methods , Tandem Mass Spectrometry/methods , Glycosylation/radiation effects , Hydrogen Peroxide/analysis , Hydrogen Peroxide/metabolism , Lipid Peroxides/analysis , Lipid Peroxides/metabolism , Oxidation-Reduction/radiation effects , Photochemical Processes , Ultraviolet RaysABSTRACT
Although glycoproteins possess a variety of functional and structural roles in intracellular and intercellular activities, the effect of ionizing radiation (IR) on glycosylation is largely unknown. To explore this effect, we established a sandwich assay in which PHA-L, a phytohaemagglutinin that agglutinates leukocytes, was used as a coating layer to capture glycoproteins containing complex oligosaccharides; the bound glycoproteins were then measured. C57BL/6 mice were exposed to 0, 3, 6, or 10 Gy, and the plasma was collected at 6, 12, 18, 24, 48, 72, or 168 h and then analyzed for galactose/N-acetylgalactosamine (Gal/GalNAc) containing proteins. We found that (1) the sandwich assay accurately measured the level of glycoproteins, (2) 6-12 h after IR, the amount of glycoproteins containing GalNAc increased, and (3) at 72 and 168 h, 10 Gy was associated with a decrease in Gal/GalNAc. These IR-induced alterations might relate to the release of glycoproteins into the blood and the damage of the proteins and genes that are related to the glycosylation process.
Subject(s)
Acetylgalactosamine/blood , Galactose/blood , Glycoproteins/blood , Glycosylation/radiation effects , Mannose/blood , Whole-Body Irradiation , Acetylgalactosamine/analogs & derivatives , Animals , Enzyme-Linked Immunosorbent Assay , Male , Mice , Mice, Inbred C57BL , Phytohemagglutinins/metabolismABSTRACT
Common de-N-glycosylation protocols usually require a lengthy incubation time. Although pressure cycling technology or scientific microwave reactors can accelerate this enzyme reaction, they may not be easily accessible. In this brief report, we employed an alternative strategy using a standard domestic microwave oven to perform the de-N-glycosylation. Model glycoproteins (bovine RNase B, bovine fetuin, and human IgG) and a complex mixture from human plasma were fully deglycosylated in 20 min, without any apparent adverse affects on the glycans or protein backbones. This new method provides a simple and inexpensive solution to achieve rapid de-N-glycosylation.
Subject(s)
Glycosylation/radiation effects , Microwaves , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/chemistry , Animals , Catalysis , Cattle , Fetuins/analysis , Glycoproteins/chemistry , Humans , Immunoglobulin G/analysis , Polysaccharides/chemistry , Ribonuclease, Pancreatic/analysisABSTRACT
A parent strain Aspergillus niger LW-1 was mutated by the compound mutagenesis of vacuum microwave (VMW) and ethyl methane sulfonate (EMS). A mutant strain, designated as A. niger E-30, with high- and stable-yield ß-mannanase was obtained through a series of screening. The ß-mannanase activity of the mutant strain E-30, cultivated on the basic fermentation medium at 32°C for 96 h, reached 36,675 U/g dried koji, being 1.98-fold higher than that (18,50 1U/g dried koji) of the parent strain LW-1. The purified E-30 ß-mannanase, a glycoprotein with a carbohydrate content of 19.6%, had an apparent molecular weight of about 42.0 kDa by SDS-PAGE. Its optimal pH and temperature were 3.5 and 65°C, respectively. It was highly stable at a pH range of 3.5-7.0 and at a temperature of 60°C and below. The kinetic parameters K(m) and V(max), toward locust bean gum and at pH 4.8 and 50°C, were 3.68 mg/mL and 1067.5 U/mg, respectively. The ß-mannanase activity was not significantly affected by an array of metal ions and EDTA, but strongly inhibited by Ag(+) and Hg(2+). In addition, the hydrolytic conditions of konjak glucomannan using the purified E-30 ß-mannanase were optimized as follows: konjak gum solution 240 g/L (dissolved in deionized water), hydrolytic temperature 50°C, ß-mannanase dosage 120 U/g konjak gum, and hydrolytic time 8 h.
Subject(s)
Aspergillus niger/genetics , Aspergillus niger/metabolism , Fermentation/genetics , Genetic Engineering/methods , Mutation/drug effects , Mutation/radiation effects , beta-Mannosidase/biosynthesis , Aspergillus niger/drug effects , Aspergillus niger/radiation effects , Ethyl Methanesulfonate/pharmacology , Fermentation/drug effects , Fermentation/radiation effects , Glycosylation/drug effects , Glycosylation/radiation effects , Hydrolysis/drug effects , Hydrolysis/radiation effects , Mannans/metabolism , Microwaves , Mutagenesis/drug effects , Mutagenesis/radiation effects , beta-Mannosidase/genetics , beta-Mannosidase/metabolismABSTRACT
The number of different anthocyanin molecules potentially produced by Arabidopsis thaliana and which anthocyanin molecule is the first product of anthocyanidin modification remain unknown. To accelerate the understanding of these questions, we investigated anthocyanin biosynthesis in rosette leaves of both pap1-D and wild-type (WT) A. thaliana plants grown in nine growth conditions, which were composed of three light intensities (low light, middle light, and high light) and three media derived from MS medium (medium-1, 2, and 3). These nine growth conditions differentially affected the levels of anthocyanins and pigmentation patterns of rosette leaves, which were closely related to the diversification levels of cyanin structures. The combined growth conditions of high light and either medium-2 or medium-1 induced the most molecular diversity of anthocyanin structures in rosette leaves of pap1-D plants. Twenty cyanin molecules, including five that were previously unknown, were characterized by HPLC-ESI-MS and HPLC-TOF-MS analyses. We detected that the A. thaliana anthocyanin molecule A11 was most likely the first cyanin derived from the multiple modification steps of cyanidin. In addition, in the same growth condition, rosette leaves of pap1-D plants produced much higher levels and more diverse molecular profiling of cyanins than those of WT plants. The transcript levels of PAP1, PAL1, CHS, DFR, and ANS cDNAs were much higher in pap1-D rosette leaves than in WT ones. Furthermore, on the same agar-solidified medium, an enhancement of light intensity increased levels and molecular diversity of cyanins in both pap1-D and WT rosette leaves. In the same light intensity condition, the responses of anthocyanin levels and profiling to medium alternation were different between pap1-D and WT plants.
Subject(s)
Anthocyanins/biosynthesis , Arabidopsis/growth & development , Arabidopsis/radiation effects , Culture Media/pharmacology , Genes, Dominant/genetics , Light , Transcription Factors/metabolism , Anthocyanins/chemistry , Anthocyanins/metabolism , Arabidopsis/cytology , Arabidopsis/drug effects , Arabidopsis Proteins , Biological Transport/drug effects , Biological Transport/radiation effects , Chromatography, High Pressure Liquid , Gene Expression Regulation, Plant/drug effects , Gene Expression Regulation, Plant/radiation effects , Genes, Plant/genetics , Glycosylation/drug effects , Glycosylation/radiation effects , Mass Spectrometry , Metabolic Networks and Pathways/drug effects , Metabolic Networks and Pathways/radiation effects , Pancreatitis-Associated Proteins , Pigmentation/drug effects , Pigmentation/genetics , Pigmentation/radiation effects , Plant Leaves/drug effects , Plant Leaves/metabolism , Plant Leaves/radiation effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Factors/geneticsABSTRACT
The effects of microwave irradiation (2.45 GHz, 200 W) on glycosylation promoted by a solid super acid in supercritical carbon dioxide was investigated with particular attention paid to the structure of the acceptor substrate. Because of the symmetrical structure and high diffusive property of supercritical carbon dioxide, microwave irradiation did not alter the temperature of the reaction solution, but enhanced reaction yield when aliphatic acceptors are employed. Interestingly, the use of a phenolic acceptor under the same reaction conditions did not show these promoting effects due to microwave irradiation. In the case of aliphatic diol acceptors, the yield seemed to be dependent on the symmetrical properties of the acceptors. The results suggest that microwave irradiation do not affect the reactivity of the donor nor promoter independently. We conclude that the effect of acceptor structure on glycosylation yield is due to electric delocalization of hydroxyl group and dielectrically symmetric structure of whole molecule.
Subject(s)
Acids/chemistry , Carbon Dioxide/chemistry , Microwaves , Glycosylation/radiation effects , Hydroxyl Radical/chemistryABSTRACT
Corticotropin releasing factor receptor type 1 (CRF1), a coordinator of the body responses to stress, is also expressed in human skin, where it undergoes alternative splicing. Since the epidermis is continuously exposed to the environmental stress, human keratinocytes were chosen to study the biological role of CRF1 alternative splicing. The expression pattern of CRF1 isoforms depended on cell density, presence or absence of serum, and exposure to ultraviolet irradiation (UVR). Only two isoforms alpha and c were predominantly localized to the cell membrane, with only CRF1alpha being efficient in stimulating cAMP responding element (CRE). CRF1d, f and g had intracellular localization, showing no or very low (g) activation of CRE. The co-expression of CRF1alpha with d, f or g resulted in intracellular retention of both isoforms suggesting dimerization confirmed by detection of high molecular weight complexes. The soluble isoforms e and h were diffusely distributed in the cytoplasm or localized to the ER, respectively, and additionally found in culture medium. These findings suggest that alternatively spliced CRF1 isoforms can interact and modify CRF1alpha subcellular localization, thus affecting its activity. We suggest that alternative splicing of CRF1 may play an important role in the regulation of skin cell phenotype with potential implications in pathology.