Deamidation Reaction Research Articles

The Fundamental Neurobiological Mechanism of Oxidative Stress-Related 4E-BP2 Protein Deamidation.

Memory impairment is caused by the absence of the 4E-BP2 protein in the brain. This protein undergoes deamidation spontaneously in the neurons. 4E-BP2 deamidation significantly alters protein synthesis in the neurons and affects the balance of protein production required for a healthy nervous system. Any imbalance in protein production in the nervous system causes neurodegenerative diseases. Discovering what causes 4E-BP2 deamidation will make it possible to control this balance of protein production and develop effective treatments against neurodegenerative diseases such as Alzheimer's and Parkinson's. The purpose of this work is to discover the neurobiological mechanism that causes the deamidation reaction in the 4E-BP2 protein by performing immunoblotting in the retinal ganglia, the optic nerve, the dorsal root ganglia, the sciatic nerve, and the whole brain, extracted via dissection from 2-month-old, Wild-type male mice. The results show that axons and their unique properties cause neuron-specific 4E-BP2 deamidation in the nervous system, confirming conclusively that axons are the critical factors behind the fundamental neurobiological mechanism of 4E-BP2 protein deamidation.

Preparation and characterization of polymeric cellulose wood adhesive with excellent bonding properties and water resistance

Developing the most abundant cellulose resources in nature as wood adhesives is a challenging but significant work. In this study, an oxidized cellulose-hexamethylene diamine-urea (OCHU) resin adhesive, of high bonding performance, and excellent water resistance has been prepared. The cellulose was firstly enzymatically hydrolyzed to increase its solubility and expose more hydroxyl groups, then oxidized by sodium periodate (NaIO4) to generate a biological compound being rich of aldehyde groups. The OCHU adhesive was prepared by crosslinking oxidized cellulose (OC) with a synthetic reactive polyurea (HU) polymer, which was formed through the deamidation reaction between hexamethylenediamine (H) and urea (U). This adhesive exhibited a 24 h cold water soaking strength of 1.61 MPa, 3 h hot water (63 °C) strength of 1.05 MPa and a dry strength of 1.71 MPa. Compared to oxidized cellulose-hexamethylenediamine (OCH) adhesive, a significant increase of 60 % in this adhesive's wet strength. The Schiff base and addition reactions in the preparation were confirmed by XPS, solid-state NMR, and FTIR. This cellulose-based wood adhesive has great market competitiveness for mass production and application in the plywood industry, due to its excellent bonding properties.

Changes in physicochemical, structural and functional properties, and lysinoalanine formation during the unfolding and refolding of pH-shifted black soldier fly larvae albumin

The changes of physicochemical, structural and functional properties and the lysinoalanine (LAL) formation during the unfolding and refolding of black soldier fly larvae albumin (BSFLA) induced by acid/alkaline pH shift were explored. The results showed that acid/alkaline conditions induced unfolding of BSFLA structure, but also accompanied by the formation of some large aggregates due to the hydrophobic interactions, hydrogen bonds, and disulfide bonds. Compared with control or pH1.5 shift, pH12 shift treatment significantly increased the electrostatic repulsion, surface hydrophobicity, free sulfhydryl group, and deamidation reactions, but reduced the fluorescence intensity of BSFLA, and these change in protein conformation contributed to increase in solubility, emulsion activity, and emulsion stability. But the content of LAL in BSFLA was increased by 93.39 % by pH 12 shift treatment. In addition, pH1.5 shift modified BSFLA tended to form β-sheet structure through unfolding and refolding, resulting in the formation of aggregates with larger particle sizes, and reducing the solubility and the LAL content by 7.93 % and 65.53 %, respectively. SDS-PAGE profile showed that pH12/1.5 shifting did not cause irreversible denaturation of protein molecules. Therefore, pH12-shift is good way to improve the functional properties of BSFLA, but the content of LAL should be reduced to make it better used in food.

Asparagine-Glucose Amadori Compounds: Formation, Characterization, and Analysis in Dry Jujube Fruit.

Amadori rearrangement products of asparagine with glucose (Asn-Glc-ARP) were first prepared through Maillard model reactions and identified via liquid chromatography-mass spectroscopy. With the study on the effect of the reaction temperature, pH values, and reaction time, the ideal reaction condition for accumulation of Asn-Glc-ARP was determined at 100 °C for 40 min under pH 7. Asparagine (Asn) was prone to degrade from Asn-Glc-ARP in alkaline pH values within a lower temperature range, while in an acidic environment with high temperatures, deamidation of Asn-Glc-ARP to Asp-Glc-ARP (Amadori rearrangement products of aspartic acid with glucose) was displayed as the dominant pathway. The deamidation reaction on the side chain of the amide group took place at Asn-Glc-ARP and transferred it into the hydroxyl group, forming Asp-Glc-ARP at the end. Considering that lyophilization as pretreatment led to limited water activity, a single aspartic acid was not deamidated from Asn directly nor did it degrade from Asp-Glc-ARP even at 120 °C. The degradation of Asn-Glc-ARP through tandem mass spectrometry (MS/MS) analysis showed the obvious fragment ion at m/z 211, indicating that the stable oxonium ion formed during fragmentation. The structure of Asn-Glc-ARP was proposed as 1-deoxy-1-l-asparagino-d-fructose after separation and purification. Also, the content of Asn-Glc-ARP within dry jujube fruit (HeTianYuZao) was quantitated as high as 8.1 ± 0.5 mg/g.

Molecular Modeling of the Deamidation Reaction in Solution: A Theoretical-Computational Study.

In this work, a theoretical-computational method is applied to study the deamidation reaction, a critical post-translational modification in proteins, using a simple model molecule in solution. The method allows one to comprehensively address the environmental effect, thereby enabling one to accurately derive the kinetic rate constants for the three main steps of the deamidation process. The results presented, in rather good agreement with the available experimental data, underline the necessity for a rigorous treatment of environmental factors and a precise kinetic model to correctly assess the overall kinetics of the deamidation reaction.

Gasification of uric acid in supercritical water: Reaction characteristics, nitrogen transformation mechanisms, and kinetics analysis

Supercritical water gasification (SCWG) of biomass waste as a new clean waste conversion technology is increasingly valued worldwide attributable to the unique properties of supercritical water (water: T > 374 °C, P > 22.1 MPa). However, the fate of conventionally contaminated elements such as nitrogen in this process still warrants further research to control potential contamination. In this work, uric acid was used as the model compound of nitrogenous components in chicken manure to investigate the transformation mechanism of nitrogen during the SCWG process. The effects of different temperatures (550–650 °C) and residence times (2–30 min) on the gasification characteristic of uric acid and the behaviour of nitrogen were experimentally explored. The results indicated that the higher temperatures and longer residence time significantly facilitated the uric acid gasification and nitrogen transformation. The maximum nitrogen conversion efficiency was 84.83%, and the maximum yield of NH3, H2, and CO2 reached 20.20 mol/kg, 23.78 mol/kg, and 20.10 mol/kg at 650 °C and 30 min, respectively. Subsequently, the reaction pathways of nitrogen in uric acid were proposed in detail according to the product analysis. Finally, a kinetic model was developed to describe the SCWG process, especially the reaction kinetic of nitrogenous products. The results revealed that the hydrolysis reactions of uric acid led to a rapid increase in NH3 yield in the initial stage of the SCWG process. As the process continued, the increase in NH3 was mainly due to the deamidation reaction of small-molecule nitrogenous intermediates. Notably, NOx, NO2−, and NO3− were not detected in products, validating the inhibitory effects of the reducing environment of supercritical water on the generation of nitrogen oxides.

Characterization of high internal phase emulsions stabilized by protein glutaminase-deamidated wheat gluten

In the present study, effect of deamidation by protein glutaminase (PG; EC 3.5.1.44) on structure of wheat gluten (WG) and its possible to construct high internal phase emulsions (HIPEs) system were investigated. The deamidation reaction was performed for different durations (0–48 h). The deamidation degree (DD, 0–70.5%) of WG was increased with an increase of reaction time (0–48 h). After the specific deamidation by PG, the solubility of WG was significantly improved at neutral pH while maintaining its molecular weight. Unlike native WG, deamidated WG (DWG) had the ability to form stable HIPEs. Moreover, HIPEs prepared using DWGs with a moderate DD (28.7–40.5%) exhibited remarkable characteristics in terms of their network structure, rheological behavior and friction coefficient. This study demonstrated the application of PG-modified WG for HIPE preparation and characterized their structural and functional relationship, providing a promising approach for the production of stabilized emulsions in food systems.

Conformational Control of Fast Asparagine Deamidation in a Norovirus Capsid Protein.

Accelerated spontaneous deamidation of asparagine 373 and subsequent conversion into an isoaspartate has been shown to attenuate the binding of histo blood group antigens (HBGAs) to the protruding domain (P-domain) of the capsid protein of a prevalent norovirus strain (GII.4). Here, we link an unusual backbone conformation of asparagine 373 to its fast site-specific deamidation. NMR spectroscopy and ion exchange chromatography have been used to monitor the deamidation reaction of P-domains of two closely related GII.4 norovirus strains, specific point mutants, and control peptides. MD simulations over several microseconds have been instrumental to rationalize the experimental findings. While conventional descriptors such as available surface area, root-mean-square fluctuations, or nucleophilic attack distance fail as explanations, the population of a rare syn-backbone conformation distinguishes asparagine 373 from all other asparagine residues. We suggest that stabilization of this unusual conformation enhances the nucleophilicity of the backbone nitrogen of aspartate 374, in turn accelerating the deamidation of asparagine 373. This finding should be relevant to the development of reliable prediction algorithms for sites of rapid asparagine deamidation in proteins.

Computing the Differences between Asn-X and Gln-X Deamidation and Their Impact on Pharmaceutical and Physiological Proteins: A Theoretical Investigation Using Model Dipeptides

Protein deamidation is a degradation mechanism that significantly impacts both pharmaceutical and physiological proteins. Deamidation impacts two amino acids, Asn and Gln, where the net neutral residues are converted into their acidic forms. While there are multiple similarities between the reaction mechanisms of the two residues, the impact of Gln deamidation has been noted to be most significant on physiological proteins while Asn deamidation has been linked to both pharmaceutical and physiological proteins. For this purpose, we sought to analyze the thermochemical and kinetic properties of the different reactions of Gln deamidation relative to Asn deamidation. In this study, we mapped the deamidation of Gln-X dipeptides into Glu-X dipeptides using density functional theory (DFT). Full network mapping facilitated the prediction of reaction selectivity between the two primary pathways, as well as between the two products of Gln-X deamidation as a function of solvent dielectric. To achieve this analysis, we studied a total of 77 dipeptide reactions per solvent dielectric (308 total reactions). Modeled at a neutral pH and using quantum chemical and statistical thermodynamic methods, we computed the following values: enthalpy of reaction (ΔHRXN), entropy (ΔSRXN), Gibbs free energy of reaction (ΔGRXN), activation energy (EA), and the Arrhenius preexponential factor (log(A)) for each dipeptide. Additionally, using chemical reaction principles, we generated a database of computed rate coefficients for all possible N-terminus Gln-X deamidation reactions at a neutral pH, predicted the most likely deamidation reaction mechanism for each dipeptide reaction, analyzed our results against our prior study on Asn-X deamidation, and matched our results against qualitative trends previously noted by experimental literature.

Standard-Free Absolute Quantitation of Antibody Deamidation Degradation and Host Cell Proteins by Coulometric Mass Spectrometry.

Proteomic absolute quantitation strategies mainly rely on the use of synthetic stable isotope-labeled peptides or proteins as internal standards, which are highly costly and time-consuming to synthesize. To circumvent this limitation, we recently developed a coulometric mass spectrometry (CMS) approach for absolute quantitation of proteins without the use of standards, based on the electrochemical oxidation of oxidizable surrogate peptides, followed by mass spectrometry measurement of the peptide oxidation yield. Previously, CMS was only applied for single-protein quantitation. In this study, first, we demonstrated absolute quantitation of multiple proteins in a mixture (e.g., β-lactoglobulin B, α-lactalbumin, and carbonic anhydrase) by CMS in one run, without using any standards. The CMS quantitation result was validated with a traditional isotope dilution method. Second, CMS can be used for absolute quantitation of a low-level target protein in a mixture; for instance, 500 ppm of PLBL2, a problematic host cell protein (HCP), in the presence of a highly abundant monoclonal antibody (mAb) was successfully quantified by CMS with no use of standards. Third, taking one step further, this study demonstrated the unprecedented quantitative analysis of deamidated peptide products arising from the mAb heavy chain deamidation reaction. In particular, absolute quantitation of the deamidation succinimide intermediate which had not been performed before due to the lack of standard was conducted by CMS, for the first time. Overall, our data suggest that CMS has potential utilities for quantitative proteomics and biotherapeutic drug discovery.

Deamidation reaction network mapping of pharmacologic and related proteins: impact of solvation dielectric on the degradation energetics of asparagine dipeptides

Asn-X deamidation pathways in the FV region of the monoclonal antibody (mAb).

Effect of solvent polarity on the formation of flexible zein nanoparticles and their environmental adaptability

Zein possesses strong self-assembly ability due to its amphiphilicity. To investigate the effect of solvent polarity on both the flexibility of zein and the formation of flexible zein nanoparticles, various ethanol aqueous solutions were chosen. Alkaline modification was applied to improve the flexibility of the zein and a liquid-liquid method was used to produce zein nanoparticles. Solvent polarity plays a positive role in improving the flexibility of zein. Furthermore, native and flexible zein nanoparticles were formed from native zein and flexible zein, respectively. However, nanoparticles could not form from highly flexible zein due to their strong adaptability to changes in solvent. Compared with the emulsifying activity index (EAI) and emulsifying stability index (ESI) of native zein nanoparticles, a higher EAI and ESI of flexible zein nanoparticles were acquired. These results may be attributed to the strong adaptability of flexible zein to the medium changing through changing their secondary structure. Furthermore, the adaptability of flexible zein nanoparticles significantly depended on the initial polarity of the solvent. The deamidation reaction of zein was responsible for the change in zein flexibility. These findings thus provide clear evidence of the relationship between the solvent polarity and structural flexibility of modified zein.

Effect of heat-induced aggregation of soy protein isolate on protein-glutaminase deamidation and the emulsifying properties of deamidated products

The effects of heat-induced soy protein isolate (SPI) aggregation on enzymatic deamidation by protein-glutaminase (PG) and the emulsifying properties of deamidated products were investigated. The particle size distribution indicated that different aggregates were obtained by preheating SPI at three different temperatures (70 °C, 100 °C, 121 °C) for 10 min. The time-dependent degree of deamidation showed that aggregates formed by preheating treatment had a different influence on the PG deamidation reaction. Preheating treatment at 70 °C could accelerate the PG deamidation reaction and preheating treatment at 121 °C restrained this reaction. The molecular weight distribution revealed that PG deamidation caused the formation of larger aggregates. Electrophoresis analysis and the structural properties indicated that the aggregation caused by PG deamidation was probably a result of hydrophobic interaction and the formation of disulfide bonds. PG deamidation of native or preheated SPI can improve the emulsifying activity, especially in the sample preheated at 70 °C. For SPI samples preheated at 70 °C and 100 °C, the emulsifying stability increased by PG deamidation. In conclusion, preheating can influence the PG deamidation reaction and the emulsifying properties of deamidated products.

Efficient degradation of minocycline by natural bornite-activated hydrogen peroxide and persulfate: kinetics and mechanisms.

Natural bornite (NBo), a sulfide mineral of copper and iron, is one of the main mineral raw materials for copper extraction. In this study, NBo-activated hydrogen peroxide (H2O2) and persulfate processes (PS) for the degradation of minocycline (MNC) in aqueous solution were systemically investigated and compared. The MNC removal rates with the NBo/PS and NBo/H2O2 processes were 86.40% and 87.50%, respectively. The mineralization rate of NBo/PS (33.96%) was higher than that of NBo/H2O2 (29.94%) after reaction for 180 min. The effects of oxidant and activator dosage, pH, common inorganic anions (i.e., Cl-, NO3-, and HCO3-), and water composition on MNC degradation were systematically evaluated. In addition, the degradation of MNC in natural water matrix and toxicity evaluation was also studied to better evaluate the feasibility of practical application of those two processes. The results of free radical quenching experiments and electron paramagnetic resonance spectroscopy (EPR) showed that HO· was the main activated species in the NBo/H2O2 system, while SO4·- and HO· were the main activated species in the NBo/PS system. The degradation of MNC in the NBo/PS system was achieved through electron transfer, while the degradation of MNC in the NBo/H2O2 system was mainly achieved through free radical addition. The degradation pathway mainly included deamidation reactions, C-C bond breakage and hydroxylation. Reusability of NBo showed that NBo was considerably stable in activating PS and H2O2 for degradation of MNC, which was cost-effective activator. This work provides a new perspective on the degradation mechanism of pollutants by Fe-Cu bimetallic sulfide activation of PS and H2O2.

N-Linked Glycosylation Prevents Deamidation of Glycopeptide and Glycoprotein.

Deamidation has been recognized as a common spontaneous pathway of protein degradation and a prevalent concern in the pharmaceutical industry; deamidation caused the reduction of protein/peptide drug efficacy and shelf life in several cases. More importantly, deamidation of physiological proteins is related to several human diseases and considered a "timer" for the diseases. N-linked glycosylation has a variety of significant biological functions, and it interestingly occurs right on the deamidation site-asparagine. It has been perceived that N-glycosylation could prevent deamidation, but experimental support is still lacking for clearly understanding the role of N-glycosylation on deamidation. Our results presented that deamidation is prevented by naturally occurring N-linked glycosylation. Glycopeptides and corresponding nonglycosylated peptides were used to compare their deamidation rates. All the nonglycosylated peptides have different half-lives ranging from one to 20 days, for the corresponding glycosylated peptides; all the results showed that the deamidation reaction was significantly reduced by the introduction of N-linked glycosylation. A glycoprotein, RNase B, also showed a significantly elongated deamidation half-life compared to nonglycosylated protein RNase A. At last, N-linked glycosylation on INGAP-P, a therapeutic peptide, increased the deamidation half-life of INGAP-P as well as its therapeutic potency.

Enhanced molecular flexibility of α-zein in different polar solvents

α-Zein is the most abundant prolamin of zein; a specified molecular model of its structure has been proposed. α-Zein was modified by alkali and heat treatment in 70% ethanol aqueous solutions and ultrapure water to characterize the improvement in its amphiphilic property. The globular-structured aggregates formed by native α-zein were broken up after alkali and heat treatment. The size of the resulting particles was significantly reduced. The improved emulsifying stability and emulsifying activity of the modified α-zein were observed. The best emulsifying stability and emulsifying activity were observed for α-zein modified by alkali and heat treatment in ultrapure water. The enhanced amphiphilic property of α-zein resulted from the structure inversion ability achieved during treatment, which was confirmed by the recovery and unfolding of the α-helix secondary structure as the solvent polarity changed. The deamidation reaction improved the α-zein molecular flexibility, which was the fundamental reason for its structural inversion ability. Finally, a solvent-responsive pathway model was developed for alkali-heat modified α-zein.

Insulin Solution Stability and Biocompatibility with Materials Used for an Implantable Insulin Delivery Device Using Reverse Phase HPLC Methods

Insulin (Humulin® R IU500) has been delivered from an implantable artificial pancreas in diabetic rats and pigs. The artificial pancreas which was implanted in the peritoneum was fabricated from several biocompatible materials such as polycarbonate, stainless steel, polyurethane, titanium and a polyurethane resin. The device also contains a glucose responsive smart gel which controls the diffusion of insulin dependent on the surrounding glucose environment. As the insulin reservoir is refillable and in contact with the device materials, assessing its biocompatibility with these various device component materials was conducted. Insulin can undergo chemical degradation mainly via a deamidation reaction on glutamine and asparagine residues rendering its biological hormone functionality. Two Reverse Phase High Performance Liquid Chromatography (RP-HPLC) methods were developed and validated for detection of insulin and degradant Asn A21 desamido insulin (method A) and insulin and degradant Asn B3 desamido insulin (method B). Material biocompatibility studies show that stainless steel and titanium are suitable for an implantable insulin delivery device design over a 31-day period. The use of polycarbonate and polyurethane could be considered if the insulin reservoir in the device was only to remain in the device for less than 11 days after which time there is a loss in cresol which acts in a protective capacity for insulin stability.

Ion spectroscopy and guided ion beam studies of protonated asparaginyl-threonine decomposition: Influence of a hydroxyl containing C-Terminal residue on deamidation processes

The spontaneous deamidation of asparagine residues plays a significant role in various biological functions and degenerative, aging diseases. Here, we present a full description of the deamidation reaction (as well as other key dissociation processes) of protonated asparaginyl-threonine, [AsnThr + H]+, via complementary infrared multiple photon dissociation (IRMPD) ion spectroscopy, threshold collision-induced dissociation (TCID), and theoretical studies. IRMPD spectra allow for the clear identification of precursor and product ion structural conformations when compared to theoretically calculated spectra for likely structures. Analysis of kinetic energy dependent cross sections measured via TCID with xenon using a guided ion beam tandem mass spectrometer allows for characterization of the energies involved in the decomposition processes of interest. Threshold energies are compared to relative single point energies of major reaction species calculated at B3LYP, B3LYP-GD3BJ, B3P86, and MP2(full) levels of theory, thereby determining important rate-limiting steps involved in [AsnThr + H]+ decomposition. Our studies confirm the formation of a succinimide intermediate via deamidation of [AsnThr + H]+, an observation consistent with condensed-phase deamidation analyses. Interestingly, our spectroscopic results suggest deamidation does not produce furanone isomers even though theoretical results indicate this pathway (exhibited in gas-phase analyses of similar dipeptide systems) should be competitive. Dehydration of [AsnThr + H]+ is also observed, where theory suggests that oxazolone and oxazoline formation are competitive at threshold energies, but IRMPD analyses conclusively confirm the formation of the oxazoline structure. The comprehensive results presented (in addition to complementary studies discussed herein) allow for a valuable analysis of C-terminal residue side-chain effects on the deamidation process.

Computational Studies on the Nonenzymatic Deamidation Mechanisms of Glutamine Residues.

The nonenzymatic deamidation reactions of asparagine (Asn) and glutamine (Gln) residues in proteins are associated with protein turnover and age-related diseases. The reactions are also believed to provide a molecular clock for biological processes. Although Gln deamidation is assumed to occur through the glutarimide intermediate, the mechanisms for this are unclear because under normal physiological conditions, Gln deamidation occurs relatively less frequently and at a lower rate than Asn deamidation. We investigate the mechanisms underlying glutarimide formation from Gln residues, which proceeds in two steps (cyclization and deammoniation) catalyzed by phosphate and carbonate. We also compare these reactions with noncatalytic mechanisms and water-catalyzed mechanisms. The calculations were performed on the model compound Ace–Gln–Nme (Ace = acetyl, Nme = methylamino) using the density functional theory with the B3LYP/6-31+G(d,p) level of theory. Our results suggest that all the catalysts used in our study can mediate the proton relays required for glutarimide formation. We further determined that the calculated activation barriers of the reactions catalyzed by phosphate ions (115 kJ mol–1) and carbonate ions (112 kJ mol–1) are sufficiently low for the reactions to occur under normal physiological conditions. We also show that nucleophilic enhancement of Nme nitrogen is essential for the cyclization of Gln residues.

Deamidation of Amino Acids on the Surface of Adeno-Associated Virus Capsids Leads to Charge Heterogeneity and Altered Vector Function.

Post-translational modification of the adeno-associated virus capsids is a poorly understood factor in the development of these viral vectors into pharmaceutical products. Here we report the extensive capsid deamidation of adeno-associated virus serotype 8 and seven other diverse adeno-associated virus serotypes, with supporting evidence from structural, biochemical, and mass spectrometry approaches. The extent of deamidation at each site depended on the vector's age and multiple primary-sequence and three-dimensional structural factors. However, the extent of deamidation was largely independent of the vector recovery and purification conditions. We demonstrate the potential for deamidation to impact transduction activity and, moreover, correlate an early time point loss in vector activity to rapidly progressing spontaneous deamidation at several adeno-associated virus 8 asparagines. We explore mutational strategies that stabilize side-chain amides, improving vector transduction and reducing the lot-to-lot molecular variability that presents a key concern in biologics manufacturing. This study illuminates a previously unknown aspect of adeno-associated virus capsid heterogeneity and highlights its importance in the development of these vectors for gene therapy.