Deamidation Rates Research Articles

SARS-CoV-2 Omicron subvariant spike N405 unlikely to rapidly deamidate

The RGD motif on the SARS-CoV-2 spike protein has been suggested to interact with RGD-binding integrins αVβ3 and α5β1 to enhance viral cell entry and alter downstream signaling cascades. The D405N mutation on the Omicron subvariant spike proteins, resulting in an RGN motif, has recently been shown to inhibit binding to integrin αVβ3. Deamidation of asparagines in protein ligand RGN motifs has been demonstrated to generate RGD and RGisoD motifs that permit binding to RGD-binding integrins. Two asparagines, N481 and N501, on the Wild-type spike receptor-binding domain have been previously shown to have deamidation half-lives of 16.5 and 123 days, respectively, which may occur during the viral life cycle. Deamidation of Omicron subvariant N405 may recover the ability to interact with RGD-binding integrins. Thus, herein, all-atom molecular dynamics simulations of the Wild-type and Omicron subvariant spike protein receptor-binding domains were conducted to investigate the potential for asparagines, the Omicron subvariant N405 in particular, to assume the optimized geometry for deamidation to occur. In summary, the Omicron subvariant N405 was primarily found to be stabilized in a state unfavourable for deamidation after hydrogen bonding with downstream E406. Nevertheless, a small number of RGD or RGisoD motifs on the Omicron subvariant spike proteins may restore the ability to interact with RGD-binding integrins. The simulations also provided structural clarification regarding the deamidation rates of Wild-type N481 and N501 and highlighted the utility of tertiary structure dynamics information in predicting asparagine deamidation. Further work is needed to characterize the effects of deamidation on spike-integrin interactions.

1D continuous gel electrophoresis composition for the separation of deamidated proteins.

Deamidation is a spontaneous modification of peptides and proteins that has potent repercussions on their activity and stability in vivo and in vitro. Being able to implement easy techniques to detect and quantify protein deamidation is a major goal in this field. Here we focus on electrophoretic methods that can be deployed to assess protein deamidation. We provide an update on the use of Taurine/Glycinate as trailing ions to assist the detection of several examples of deamidated proteins, namely the small GTPases RhoA, Rac1 and Cdc42, but also the oncogene Bcl-xL and calcium-binding Calmodulin. We also report on the use of imidazole as a counter ion to improve the focusing of deamidated bands. Finally, we provide examples of how these gels proved useful to compare on full-length proteins the effect of ions and pH on the catalytic rates of spontaneous deamidation. Taken together, the electrophoretic method introduced here proves useful to screen at once the effect of various conditions of pH, ionic strength and buffer ions on protein stability. Direct applications can be foreseen to tailor buffer formulations to control the stability of proteins drug products.

Nonenzymatic Deamidation Mechanism on a Glutamine Residue with a C-Terminal Adjacent Glycine Residue: A Computational Mechanistic Study

The deamidation of glutamine (Gln) residues, which occurs non-enzymatically under physiological conditions, triggers protein denaturation and aggregation. Gln residues are deamidated via the cyclic glutarimide intermediates to l-α-, d-α-, l-β-, and d-β-glutamate residues. The production of these biologically uncommon amino acid residues is implicated in the pathogenesis of autoimmune diseases. The reaction rate of Gln deamidation is influenced by the C-terminal adjacent (N +1) residue and is highest in the Gln-glycine (Gly) sequence. Here, we investigated the effect of the (N + 1) Gly on the mechanism of Gln deamidation and the activation barrier using quantum chemical calculations. Energy-minima and transition-state geometries were optimized by the B3LYP density functional theory, and MP2 calculations were used to obtain the single-point energy. The calculated activation barrier (85.4 kJ mol−1) was sufficiently low for the reactions occurring under physiological conditions. Furthermore, the hydrogen bond formation between the catalytic ion and the main chain of Gly on the C-terminal side was suggested to accelerate Gln deamidation by stabilizing the transition state.

Assessing the degradation of ancient milk proteins\xa0through site-specific deamidation patterns

The origins, prevalence and nature of dairying have been long debated by archaeologists. Within the last decade, new advances in high-resolution mass spectrometry have allowed for the direct detection of milk proteins from archaeological remains, including ceramic residues, dental calculus, and preserved dairy products. Proteins recovered from archaeological remains are susceptible to post-excavation and laboratory contamination, a particular concern for ancient dairying studies as milk proteins such as beta-lactoglobulin (BLG) and caseins are potential laboratory contaminants. Here, we examine how site-specific rates of deamidation (i.e., deamidation occurring in specific positions in the protein chain) can be used to elucidate patterns of peptide degradation, and authenticate ancient milk proteins. First, we characterize site-specific deamidation patterns in modern milk products and experimental samples, confirming that deamidation occurs primarily at low half-time sites. We then compare this to previously published palaeoproteomic data from six studies reporting ancient milk peptides. We confirm that site-specific deamidation rates, on average, are more advanced in BLG recovered from ancient dental calculus and pottery residues. Nevertheless, deamidation rates displayed a high degree of variability, making it challenging to authenticate samples with relatively few milk peptides. We demonstrate that site-specific deamidation is a useful tool for identifying modern contamination but highlight the need for multiple lines of evidence to authenticate ancient protein data.

Effect of pH and heat treatment on physicochemical and functional properties of spray-dried whey protein concentrate powder

Effects of pH and heating on deamidation of whey protein concentrate (WPC) solution and functional properties of resultant spray-dried WPC powder were investigated. Temperature and heating time affected deamidation rates with the highest reactivities for WPC solutions heated at 120 °C for 15 min and 145 °C for 120 s. Deamidation sites were pH dependent: pH 3 induced more glutamine deamidation; pH 10 induced more asparagine deamidation. The functional properties of spray-dried WPC powders were also pH dependent. WPC solution adjusted to pH 3 and heated at 145 °C for 120 s (prior to spray drying) exhibited a reduction in solubility and foamability, but markedly improved foam stability of the resultant powders; meanwhile, the properties of powders were not significantly impacted by pH adjustment to 10.0 and heating at 145 °C for 120 s. However, pH 3 and 10 with and without heating significantly improved emulsifying properties of spray-dried WPC.

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.

Prediction Machines: Applied Machine Learning for Therapeutic Protein Design and Development.

The rapid growth in technological advances and quantity of scientific data over the past decade has led to several challenges including data storage and analysis. Accurate models of complex datasets were previously difficult to develop and interpret. However, improvements in machine learning algorithms have since enabled unparalleled classification and prediction capabilities. The application of machine learning can be seen throughout diverse industries due to their ease of use and interpretability. In this review, we describe popular machine learning algorithms and highlight their application in pharmaceutical protein development. Machine learning models have now been applied to better understand the nonlinear concentration dependent viscosity of protein solutions, predict protein oxidation and deamidation rates, classify sub-visible particles and compare the physical stability of proteins. We also applied several machine learning algorithms using previously published data and describe models with improved predictions and classification. The authors hope that this review can be used as a resource to others and encourage continued application of machine learning algorithms to problems in pharmaceutical protein development.

Mechanisms of Deamidation of Asparagine Residues and Effects of Main-Chain Conformation on Activation Energy.

Deamidation of asparagine (Asn) residues is a nonenzymatic post-translational modification of proteins. Asn deamidation is associated with pathogenesis of age-related diseases and hypofunction of monoclonal antibodies. Deamidation rate is known to be affected by the residue following Asn on the carboxyl side and by secondary structure. Information about main-chain conformation of Asn residues is necessary to accurately predict deamidation rate. In this study, the effect of main-chain conformation of Asn residues on deamidation rate was computationally investigated using molecular dynamics (MD) simulations and quantum chemical calculations. The results of MD simulations for γS-crystallin suggested that frequently deamidated Asn residues have common main-chain conformations on the N-terminal side. Based on the simulated structure, initial structures for the quantum chemical calculations were constructed and optimized geometries were obtained using the B3LYP density functional method. Structures that were frequently deamidated had a lower activation energy barrier than that of the little deamidated structure. We also showed that dihydrogen phosphate and bicarbonate ions are important catalysts for deamidation of Asn residues.

NGR-TNF Engineering with an N-Terminal Serine Reduces Degradation and Post-Translational Modifications and Improves Its Tumor-Targeting Activity.

The therapeutic index of cytokines in cancer therapy can be increased by targeting strategies based on protein engineering with peptides containing the CNGRC (NGR) motif, a ligand that recognizes CD13-positive tumor vessels. We show here that the targeting domain of recombinant CNGRC-cytokine fusion proteins, such as NGR-TNF (a CNGRC-tumor necrosis factor-α (TNF) conjugate used in clinical studies) and NGR-EMAP-II, undergoes various post-translational modification and degradation reactions that lead to the formation of markedly heterogeneous products. These modifications include N-terminal cysteine acetylation or the formation of various asparagine degradation products, the latter owing to intramolecular interactions of the cysteine α-amino group with asparagine and/or its succinimide derivative. Blocking the cysteine α-amino group with a serine (SCNGRC) reduced both post-translational and degradation reactions. Furthermore, the serine residue reduced the asparagine deamidation rate to isoaspartate (another degradation product) and improved the affinity of NGR for CD13. Accordingly, genetic engineering of NGR-TNF with the N-terminal serine produced a more stable and homogeneous drug (called S-NGR-TNF) with improved antitumor activity in tumor-bearing mice, either when used alone or in combination with chemotherapy. In conclusion, the targeting domain of NGR-cytokine conjugates can undergo various untoward modification and degradation reactions, which can be markedly reduced by fusing a serine to the N-terminus. The SCNGRC peptide may represent a ligand for cytokine delivery to tumors more robust than conventional CNGRC. The S-NGR-TNF conjugate (more stable, homogeneous, and active than NGR-TNF) could be rapidly developed for clinical trials.

DeamiDATE 1.0: Site-specific deamidation as a tool to assess authenticity of members of ancient proteomes

Contamination is a potential problem in the study of ancient proteins, either from prior handling of the sample, laboratory consumables, or cross-sample carryover from mass spectrometers. Recently, deamidation of glutamine has been proposed as a measure for assessing the degradation of ancient proteins. Here, we present deamiDATE 1.0, a method for the authentication of ancient proteins using measure of site-specific deamidation rates. We test this approach on shotgun proteomic data derived from bone collagen from modern, archaeological and extinct taxa. We further demonstrate how this method may be used to differentiate between modern contaminants and authentic ancient proteins using a case study from Neolithic dental calculus.

Machine Learning Enables Accurate Prediction of Asparagine Deamidation Probability and Rate

The spontaneous conversion of asparagine residues to aspartic acid or iso-aspartic acid, via deamidation, is a major pathway of protein degradation and is often seriously disruptive to biological systems. Deamidation has been shown to negatively affect both in vitro stability and in vivo biological function of diverse classes of proteins. During protein therapeutics development, deamidation liabilities that are overlooked necessitate expensive and time-consuming remediation strategies, sometimes leading to termination of the project. In this paper, we apply machine learning to a large (n = 776) liquid chromatography-tandem mass spectrometry (LC-MS/MS) dataset of monoclonal antibody peptides to create computational models for the post-translational modification asparagine deamidation, using the random decision forest method. We show that our categorical model predicts antibody deamidation with nearly 5% increased accuracy and 0.2 MCC over the best currently available models. Surprisingly, our model also paces or outperforms advanced and conventional models on an independent non-antibody dataset. In addition to deamidation probability, we are able to accurately predict deamidation rate (R2 = 0.963 and Q2 = 0.822), a capability with no peer in current models. This method should enable significant improvement in protein candidate selection, especially in biopharmaceutical development, and can be applied with similar accuracy to enzymes, monoclonal antibodies, next-generation formats, vaccine component antigens, and gene therapy vectors such as adeno-associated virus.

Rapid Prediction of Deamidation Rates of Proteins to Assess Their Long-Term Stability Using Hydrogen Exchange–Mass Spectrometry

Deamidation is an important degradation pathway for proteins. Estimating deamidation propensities is essential for predicting their long-term stability. However, predicting deamidation rates in folded proteins is challenging because higher-order structure has a significant and unpredictable effect on deamidation. Here, we investigated the correlation between amide hydrogen exchange (HX) and deamidation to assess the potential of using hydrogen exchange–mass spectrometry (HX-MS) to rapidly predict deamidation propensity. Maltose-binding protein and a structurally less stable mutant, W169G, were stored in the dark at pH 7.0 at 23 ± 2°C for 1 year. Deamidation at each asparagine site was measured using liquid chromatography–mass spectrometry after trypsin digestion. Deamidation rates at each deamidation site were determined based on first-order kinetics. HX rates at the deamidation sites were determined before storage using the shortest peptic peptide containing each site using conventional bottom-up HX-MS at pD 7.0 at 25°C. We observed a power law correlation between deamidation half-life and HX half-life for the NG sites with measurable kinetics. For NA sites, slow deamidation was only observed at 2 sites located in rapidly exchanging regions. Our findings demonstrate that HX-MS can be used to reliably and rapidly rank deamidation propensity in folded proteins.

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.

Asparagine Deamidation in a Complementarity Determining Region of a Recombinant Monoclonal Antibody in Complex with Antigen.

Asparagine deamidation in the complementarity determining regions of recombinant monoclonal antibodies has been extensively studied and shown to have a negative impact on antigen binding. Those asparagine residues are typically exposed and thus have a higher tendency toward deamidation, depending also on local structure and environmental factors such as temperature and pH. Deamidation rates and products of a susceptible asparagine residue in the complementarity determining regions of a recombinant monoclonal antibody free in solution or in the antibody-antigen complex were studied. The results demonstrated that incubation of the antibody or its antigen complex generated a similar amount of aspartate. The expected amount of isoaspartate product was detected in free antibody, but it was completely lacking in the antibody-antigen complex.

Bcl-xL deamidation is regulated by multiple ion transporters and is intramolecularly catalyzed

In susceptible tumor cells, DNA-damaging antineoplastic agents induce an increase in intracellular pH during the premitochondrial stage of apoptosis. The rate of nonenzymatic deamidation of two asparagines in the anti-apoptotic protein Bcl-xL is accelerated by this increase in pH. Deamidation of these asparagines is a signal for the degradation of Bcl-xL, which is a component of the apoptotic response to DNA damage. It has previously been shown that the increase in pH is mediated by the ion transporter Na+/H+ exchanger 1 in some cells. Here we demonstrate that one or more additional ion transporters also have a role in the regulation of Bcl-xL deamidation in at least some tumor cell lines and fibroblasts. As a second, independent finding, we report that there are histidines in close proximity to the Bcl-xL deamidation sites that are highly conserved in land-dwelling species and we present evidence that deamidation of human Bcl-xL is intramolecularly catalyzed in a manner that is dependent upon these histidines. Further, we present evidence that these histidines act as a pH-sensitive switch that enhances the effect of the increase in pH on the rate of Bcl-xL deamidation. The conservation of such histidines implies that human Bcl-xL is in essence “designed” to be deamidated, which provides further evidence that deamidation serves as a bona fide regulatory post-translational modification of Bcl-xL.

Mutation-Induced Deamidation of Corneal Dystrophy-Related Transforming Growth Factor β-Induced Protein.

Mutations in the transforming growth factor β-induced protein (TGFBIp) cause phenotypically diverse corneal dystrophies, where protein aggregation in the cornea leads to severe visual impairment. Previous studies have shown a relationship between mutant-specific corneal dystrophy phenotypes and the thermodynamic stability of TGFBIp. Using liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance (NMR), we investigated correlations between the structural integrity of disease-related mutants of the fourth FAS1 domain (FAS1-4) and deamidation of TGFBIp residue Asn622. We observed a high rate of Asn622 deamidation in the A546D and A546D/P551Q FAS1-4 mutants that were both largely unstructured as determined by NMR. Conversely, the more structurally organized A546T and V624M FAS1-4 mutants had reduced deamidation rates, suggesting that a folded and stable FAS1-4 domain precludes Asn622 deamidation. Wild-type, R555Q, and R555W FAS1-4 mutants displayed very slow deamidation, which agrees with their similar and ordered NMR structures, where Asn622 is in a locked conformation. We confirmed the FAS1-4 mutational effect on deamidation rates in full-length TGFBIp mutants and found a similar ranking compared to that of the FAS1-4 domain alone. Consequently, the deamidation rate of Asn622 can be used to predict the structural effect of the many destabilizing and/or stabilizing mutations reported for TGFBIp. In addition, the deamidation of Asn622 may influence the pathophysiology of TGFBIp-induced corneal dystrophies.

A chimeric IgE that mimics IgE from patients allergic to acid-hydrolyzed wheat proteins is a novel tool for in vitro allergenicity assessment of functionalized glutens.

BackgroundAcid-hydrolyzed wheat proteins (acid-HWPs) have been shown to provoke severe allergic reactions in Europe and Japan that are distinct from classical wheat allergies. Acid-HWPs were shown to contain neo-epitopes induced by the deamidation of gluten proteins. However, products with variable rates of deamidation can be found.ObjectivesIn this work, we studied the effect of the extent of wheat proteins deamidation on its allergenicity. A recombinant chimeric IgE was produced and compared to patients’ IgE for its capacity to assess the IgE-mediated triggering potential of acid-HWPs.MethodsSera from acid-HWP allergic patients were analyzed via ELISA and a functional basophil assay for their IgE reactivity to wheat proteins with different deamidation levels. A chimeric mouse/human IgE (chIgE-DG1) specific for the main neo-epitope, QPEEPFPE, involved in allergy to acid-HWPs was characterized with respect to its functionality and its reactivity compared to that of patients’ IgE.ResultsAcid-HWPs with medium (30%) and high (50–60%) deamidation levels displayed a markedly stronger IgE binding and capacity to activate basophils than those of samples with weak (15%) deamidation levels. The monoclonal chIgE-DG1 allowed basophil degranulation in the presence of deamidated wheat proteins. ChIgE-DG1 was found to mimic patients’ IgE reactivity and displayed the same ability to rank acid-HWP products in a degranulation assay.ConclusionIncreasing the deamidation level of products from 15% to 60% resulted in an approximately 2-fold increase in their antigenicity and a 100-fold increase in their eliciting potential. The chimeric ChIgE-DG1 may be a useful tool to evaluate functionalized glutens for their allergenic potential. By mimicking patient sera reactivity, chIgE-DG1 also provided data on the patients' IgE repertoire and on the functionality of certain repeated epitopes in gluten proteins.

Sequence and Solution Effects on the Prevalence of d-Isomers Produced by Deamidation.

Deamidation of asparagine is a spontaneous and irreversible post-translational modification associated with a growing list of human diseases. While pervasive, deamidation is often overlooked because it represents a relatively minor chemical change. Structural and functional characterization of this modification is complicated because deamidation of asparagine yields four isomeric forms of Asp. Herein, radical directed dissociation (RDD), in conjunction with mass spectrometry, is used to identify and quantify all four isomers in a series of model peptides that were subjected to various deamidation conditions. Although primary sequence significantly influences the rate of deamidation, it has little impact on the relative proportions of the product isomers. Furthermore, the addition of ammonia can be used to increase the rate of deamidation without significantly perturbing isomer populations. Conversely, external factors such as buffer conditions and temperature alter product distributions but exhibit less dramatic effects on the deamidation rate. Strikingly, the common laboratory and biologically significant bicarbonate buffer is found to strongly promote racemization, yielding increased amounts of d-Asp and d-isoAsp. These outcomes following deamidation have broad implications in human aging and should be considered during the development of protein-based therapeutics.

Effect of Water on the Chemical Stability of Amorphous Pharmaceuticals: 2. Deamidation of Peptides and Proteins

Role of water in chemical (in)stability is revisited, with focus on deamidation in freeze-dried amorphous proteins and peptides. Two distinct patterns for deamidation versus water have been reported, that is, a consistent increase in rate constant with water, and a “hockey stick”–type behavior. For the latter, deamidation is essentially independent of water at lower water contents and accelerates when water content increases above a threshold value. Two simple kinetic models are developed to analyze literature-reported relationships between water content and deamidation rate constants. One model is based on catalytic role of water clusters in enabling proton transfer, which is a critical reaction step. Water clusters are formed when water content increases above a threshold value, while unclustered (and less catalytically-active) water molecules are predominant at lower water levels. The second model considers the dual role of water, as both a destabilizer via catalysis and a stabilizer of protein native structure. Considering that both models emphasize the importance of local structure and that local structure is intrinsically related to fast (and non-cooperative) relaxation modes, it is plausible to expect correlations between local mobility, such as beta-relaxation, and amorphous chemical instability.

Rate of Asparagine Deamidation in a Monoclonal Antibody Correlating with Hydrogen Exchange Rate at Adjacent Downstream Residues.

Antibodies are an important class of drugs, comprising more than half of all new FDA approvals. Therapeutic antibodies must be chemically stable both in storage and in vivo, following administration to patients. Deamidation is a major degradation pathway for all natural and therapeutic proteins circulating in blood. Here, the linkage between deamidation propensity and structural dynamics is investigated by examining two antibodies with differing specificities. While both antibodies share a canonical asparagine-glycine (NG) motif in a structural loop, this is prone to deamidation in one of the antibodies but not the other. We found that the hydrogen-exchange rate at the adjacent two amides, often the autocatalytic nucleophiles in deamidation, correlated with the rate of degradation. This previously unreported observation was confirmed upon mutation to stabilize the deamidation lability via a generally applicable orthogonal engineering strategy presented here. We anticipate that the structural insight into chemical degradation in full-length monoclonal antibodies and the high-resolution hydrogen-exchange methodology used will have broad application across biochemical study and drug discovery and development.