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Glycosylation is a valuable tool for modulating protein solubility; however, the lack of reliable research strategies has impeded efficient progress in understanding and applying this modification. This study aimed to bridge this gap by investigating the solubility of a model glycoprotein molecule, the carbohydrate-binding module (CBM), through a two-stage process. In the first stage, an approach involving chemical synthesis, comparative analysis, and molecular dynamics simulations of a library of glycoforms was employed to elucidate the effect of different glycosylation patterns on solubility and the key factors responsible for the effect. In the second stage, a predictive mathematical formula, innovatively harnessing machine learning algorithms, was derived to relate solubility to the identified key factors and accurately predict the solubility of the newly designed glycoforms. Demonstrating feasibility and effectiveness, this two-stage approach offers a valuable strategy for advancing glycosylation research, especially for the discovery of glycoforms with increased solubility.
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Aprendizado de Máquina , Simulação de Dinâmica Molecular , Solubilidade , Glicosilação , Glicoproteínas/químicaRESUMO
Acute sleep deprivation is a common condition in modern life and increases anxiety symptoms in healthy individuals. The neuroinflammatory response induced by microglial activation could be an important contributing factor, but its underlying molecular mechanisms are still unclear. In the present study, we first found that acute paradoxical sleep deprivation (PSD) induced by the modified multiple platform method (MMPM) for 6 h led to anxiety-like behavior in mice, as verified by the open field test, elevated plus maze test, light-dark box test, and marble burying test. In addition, bioinformatic analysis suggested an important relationship between acute sleep deprivation and brain inflammatory signaling pathways. Key genes enriched in the TNF signaling pathway were confirmed to be altered during acute PSD by qPCR and Western blot analyses, including the upregulation of the prostaglandin-endoperoxide synthase 2 (Ptgs2) and suppressor of cytokine signaling 3 protein (Socs3) genes and the downregulation of the cysteine-aspartic acid protease 3 (Casp3) gene. Furthermore, we found that microglial cells in the prefrontal cortex (PFC) were activated with significant branch structure changes and that the cell body area was increased in the PSD model. Finally, we found that minocycline, a tetracycline with anti-inflammatory properties, may ameliorate the anxiogenic effect and microglial activation. Our study reveals significant correlations of anxiety-like behavior, microglial activation, and inflammation during acute PSD.
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Microglia , Privação do Sono , Animais , Ansiedade/metabolismo , Camundongos , Microglia/metabolismo , Córtex Pré-Frontal/metabolismo , Transdução de Sinais , Privação do Sono/complicações , Privação do Sono/metabolismo , Sono REMRESUMO
Therapeutic proteins have unique advantages over small-molecule drugs in the treatment of various diseases, such as higher target specificity, stronger pharmacological efficacy and relatively low side effects. These advantages make them increasingly valued in drug development and clinical practice. However, although highly valued, the intrinsic limitations in their physical, chemical and pharmacological properties often restrict their wider applications. As one of the most important post-translational modifications, glycosylation has been shown to exert positive effects on many properties of proteins, including molecular stability, and pharmacodynamic and pharmacokinetic characteristics. Glycoengineering, which involves changing the glycosylation patterns of proteins, is therefore expected to be an effective means of overcoming the problems of therapeutic proteins. In this review, we summarize recent efforts and advances in the glycoengineering of erythropoietin and IgG monoclonal antibodies, with the goals of illustrating the importance of this strategy in improving the performance of therapeutic proteins and providing a brief overview of how glycoengineering is applied to protein-based drugs.
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Anticorpos Monoclonais , Engenharia de Proteínas , Glicosilação , Anticorpos Monoclonais/metabolismo , Processamento de Proteína Pós-Traducional , Imunoglobulina G/química , Polissacarídeos/metabolismoRESUMO
Protein glycosylation is one of the most common post-translational modifications and can influence many properties of proteins. Abnormal protein glycosylation can lead to protein malfunction and serious disease. While appreciation of glycosylation's importance is growing in the scientific community, especially in recent years, a lack of homogeneous glycoproteins with well-defined glycan structures has made it difficult to understand the correlation between the structure of glycoproteins and their properties at a quantitative level. This has been a significant limitation on rational applications of glycosylation and on optimizing glycoprotein properties. Through the extraordinary efforts of chemists, it is now feasible to use chemical synthesis to produce collections of homogeneous glycoforms with systematic variations in amino acid sequence, glycosidic linkage, anomeric configuration, and glycan structure. Such a technical advance has greatly facilitated the study and application of protein glycosylation. This Perspective highlights some representative work in this research area, with the goal of inspiring and encouraging more scientists to pursue the glycosciences.
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Glicoproteínas/síntese química , Engenharia de Proteínas/métodos , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Previsões , Glicopeptídeos/síntese química , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutagênese Sítio-Dirigida , Polissacarídeos/química , Conformação Proteica , Estabilidade ProteicaRESUMO
Many human proteins have the potential to be developed as therapeutic agents. However, side effects caused by direct administration of natural proteins have significantly slowed expansion of protein therapeutics into the clinic. Post-translational modifications (PTMs) can improve protein properties, but because of significant knowledge gaps, we are considerably limited in our ability to apply PTMs to generate better protein therapeutics. Here, we seek to fill the gaps by studying the PTMs of a small representative chemotactic cytokine, RANTES. RANTES can inhibit HIV-1 infection by competing with it for binding to receptor CCR5 and stimulating CCR5 endocytosis. Unfortunately, RANTES can induce strong signaling, leading to severe inflammatory side effects. We apply a chemical biology approach to explore the potential of post-translationally modified RANTES as safe inhibitors of HIV-1 infection. We synthesized and systematically tested a library of RANTES isoforms for their ability to inhibit inflammatory signaling and prevent HIV-1 infection of primary human cells. Through this research, we revealed that most of the glycosylated variants have decreased inflammation-associated properties and identified one particular glyco variant, a truncated RANTES containing a Galß1-3GalNAc disaccharide α-linked to Ser4, which stands out as having the best overall properties: relatively high HIV-1 inhibition potency but also weak inflammatory properties. Moreover, our results provided a structural basis for the observed changes in the properties of RANTES. Taken together, this work highlights the potential importance of glycosylation as an alternative strategy for developing CCR5 inhibitors to treat HIV-1 infection and, more generally, for reducing or eliminating unwanted properties of therapeutic proteins.
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Quimiocina CCL5/química , Quimiocina CCL5/farmacologia , Inibidores da Fusão de HIV/química , Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Acilação , Biopolímeros , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Quimiocina CCL5/efeitos adversos , Quimiocina CCL5/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Glicosaminoglicanos/metabolismo , Glicosilação , Inibidores da Fusão de HIV/efeitos adversos , Inibidores da Fusão de HIV/metabolismo , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Humanos , Espectroscopia de Prótons por Ressonância Magnética , Receptores CCR5/metabolismo , Células THP-1RESUMO
Protein O-glycosylation is a diverse, common, and important post-translational modification of both proteins inside the cell and those that are secreted or membrane-bound. Much work has shown that O-glycosylation can alter the structure, function, and physical properties of the proteins to which it is attached. One gap remaining in our understanding of O-glycoproteins is how O-glycans might affect the folding of proteins. Here, we took advantage of synthetic, homogeneous O-glycopeptides to show that certain glycosylation patterns have an intrinsic effect, independent of any cellular folding machinery, on the folding pathway of a model O-glycoprotein, a carbohydrate binding module (CBM) derived from the Trichoderma reesei cellulase TrCel7A. The strongest effect, a 6-fold increase in overall folding rate, was observed when a single O-mannose was the glycan, and the glycosylation site was near the N-terminus of the peptide sequence. We were also able to show that glycosylation patterns affected the kinetics of each step in unique ways, which may help to explain the observations made here. This work is a first step toward quantitative understanding of how O-glycosylation might control, through intrinsic means, the folding of O-glycoproteins. Such an understanding is expected to facilitate future investigations into the effects of glycosylation on more biological processes related to protein folding.
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Celulase/metabolismo , Proteínas Fúngicas/metabolismo , Glicoproteínas/metabolismo , Polissacarídeos/metabolismo , Dobramento de Proteína , Trichoderma/enzimologia , Celulase/química , Celulase/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glicoproteínas/química , Glicoproteínas/genética , Polissacarídeos/química , Polissacarídeos/genética , Trichoderma/genéticaRESUMO
Prolyl thioesters have shown significantly lower reactivities in native chemical ligation (NCL) in comparison to that of the alanyl thioester. This report describes a mild and efficient internal activation protocol of peptidyl prolyl thioesters in NCL without using any thiol-based additives, where the introduction of a 4-mercaptan substituent on the C-terminal proline significantly improves the reactivity of prolyl thioesters via the formation of a bicyclic thiolactone intermediate. The kinetic data indicate that the reaction rate is comparable to that of the reported data of alanyl thioesters, and the mechanistic studies suggest that the ligation of two peptide segments proceeds through an NCL-like pathway instead of a direct aminolysis, which ensures the chemoselectivity and compatibility of various amino acid side chains. This 4-mercaptoprolyl thioester-based protocol also allows an efficient one-pot ligation-desulfurization procedure. The utility of this method has been further demonstrated in the synthesis of a proline-rich region of Wilms tumor protein 1.
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Peptídeos/química , Prolina/química , Compostos de Sulfidrila/química , Aminoácidos/química , Cinética , Proteínas WT1/químicaRESUMO
The role of glycosylation in the binding of glycoproteins to carbohydrate substrates has not been well understood. The present study addresses this knowledge gap by elucidating the links between the glycosylation patterns of a model glycoprotein, a Family 1 carbohydrate-binding module (TrCBM1), and the thermodynamic and structural properties of its binding to different carbohydrate substrates using isothermal titration calorimetry and computational simulation. The variations in glycosylation patterns cause a gradual transition of the binding to soluble cellohexaose from an entropy-driven process to an enthalpy-driven one, a trend closely correlated with the glycan-induced shift of the predominant binding force from hydrophobic interactions to hydrogen bonding. However, when binding to a large surface of solid cellulose, glycans on TrCBM1 have a more dispersed distribution and thus have less adverse impact on the hydrophobic interaction forces, leading to overall improved binding. Unexpectedly, our simulation results also suggest an evolutionary role of O-mannosylation in transforming the substrate binding features of TrCBM1 from those of type A CBMs to those of type B CBMs. Taken together, these findings provide new fundamental insights into the molecular basis of the role of glycosylation in protein-carbohydrate interactions and are expected to better facilitate further studies in this area.
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Celulose , Polissacarídeos , Glicosilação , Celulose/química , Simulação por Computador , Termodinâmica , Ligação Proteica , Sítios de LigaçãoRESUMO
There is an increasing interest in using S-glycosylation as a replacement for the more commonly occurring O-glycosylation, aiming to enhance the resistance of glycans against chemical hydrolysis and enzymatic degradation. However, previous studies have demonstrated that these two types of glycosylation exert distinct effects on protein properties and functions. In order to elucidate the structural basis behind the observed differences, we conducted a systematic and comparative analysis of 6 differently glycosylated forms of a model glycoprotein, CBM, using NMR spectroscopy and molecular dynamic simulations. Our findings revealed that the different stabilizing effects of S- and O-glycosylation could be attributed to altered hydrogen-bonding capability between the glycan and the polypeptide chain, and their diverse impacts on binding affinity could be elucidated by examining the interactions and motion dynamics of glycans in substrate-bound states. Overall, this study underscores the pivotal role of the glycosidic linkage in shaping the function of glycosylation and advises caution when switching glycosylation types in protein glycoengineering.
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Glicoproteínas , Polissacarídeos , Glicosilação , Glicoproteínas/química , Polissacarídeos/metabolismo , Peptídeos/química , Espectroscopia de Ressonância MagnéticaRESUMO
The development and application of commercially available automated peptide synthesizers has played an essential role in almost all areas of peptide and protein research. Recent advances in peptide synthesis method and solid-phase chemistry provide new opportunities for optimizing synthetic efficiency of peptide synthesizers. The efforts in this direction have led to the successful preparation of peptides up to more than 150 amino acid residues in length. Such success is particularly useful for addressing the challenges associated with the chemical synthesis of glycoproteins. The purpose of this review is to provide a brief overview of the evolution of peptide synthesizer and glycoprotein synthesis. The discussions in this article include the principles underlying the representative synthesizers, the strengths and weaknesses of different synthesizers in light of their principles, and how to further improve the applicability of peptide synthesizers in glycoprotein synthesis.
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Glycoproteins obtained from cell culture supernatants or lysates generally exist as mixtures of over 100 differently glycosylated protein forms (glycoforms). The study of glycosylation is significantly impeded because of the heterogeneous nature of glycoproteins. To overcome this challenge, we developed and optimized a glycoform library-based strategy to investigate the role of protein glycosylation. In this strategy, chemical synthesis was used to prepare individual homogeneous glycoforms and the role of glycosylation was determined by comparing a series of glycoforms with systematic differences in their glycosylation patterns.
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Glicoproteínas , Glicoproteínas/metabolismo , GlicosilaçãoRESUMO
Schizophrenia is a severe mental disorder characterized by positive, negative, and cognitive symptoms. Cognitive symptoms are a kind of symptoms with high incidence and great impact on patients. There is no effective treatment in clinical practice. N-methyl-d-aspartic acid (NMDA) receptor hypofunction may be an important cause of cognitive symptoms. MK-801 (also named Dizocilpine), a noncompetitive antagonist of NMDA receptor, is often used to construct a model of NMDA receptor dysfunction. In terms of treatment, environmental enrichment (EE) as an environmental intervention can effectively improve the symptoms of cognitive impairment in rodents. In this paper, we first briefly introduce the background of cognitive symptoms and EE in schizophrenia, and then investigate the manifestations of MK-801 induced cognitive impairment, the improvement of EE on these cognitive impairments based on the MK-801 induced schizophrenia rodent models, and the possible mechanism of EE in improving cognitive symptoms. This article reviews the literature in recent years, which provides an important reference for MK-801 to construct a cognitive symptom model of schizophrenia and the mechanism of EE in improving cognitive symptoms of schizophrenia.
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Introduction: Schizophrenia is a severe psychiatric disorder with a high prevalence worldwide, however, its pathogenesis remains poorly understood. Methods and results: In this study, we used the non-competitive NMDA receptor antagonist MK-801 to induce schizophrenia-like behaviors and confirmed that mice exhibited stereotypic rotational behavior and hyperlocomotion, social interaction defects and cognitive dysfunction, similar to the clinical symptoms in patients. Here, the anterior cingulate cortex (ACC) and basolateral amygdala (BLA) were involved in the schizophrenia-like behaviors induced by MK-801. Furthermore, we confirmed BLA sent glutamatergic projection to the ACC. Chemogenetic and optogenetic regulation of BLA-ACC projecting neurons affected social and cognitive deficits but not stereotypic rotational behavior in MK-801-treated mice. Discussion: Overall, our study revealed that the BLA-ACC circuit plays a major role and may be a potential target for treating schizophrenia-related symptoms.
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Self-grooming is an innate, cephalo-caudal progression of body cleaning behaviors that are found in normal rodents but exhibit repetitive and stereotyped patterns in several mouse models, such as autism spectrum disorders (ASDs). It is also recognized as a marker of stress and anxiety. Mice with Shank3B gene knockout (KO) exhibit typical ASD-like behavioral abnormalities, including stereotyped self-grooming and increased levels of anxiety. However, the exact relationship between anxiety and stereotyped self-grooming in certain types of animal models is not clear. We selected three animal models with high anxiety to compare their self-grooming parameters. First, we confirmed that Shank3B KO mice (ASD model), acute restraint stress mouse model (stress model), and chronic inflammatory pain mouse model (pain model) all showed increased anxiety levels in the open field test (OFT) and elevated plus maze (EPM). We found that only the ASD model and the stress model produced increased total grooming duration. The pain model only exhibited an increasing trend of mean self-grooming duration. We used the grooming analysis algorithm to examine the self-grooming microstructure and assess the cephalo-caudal progression of grooming behavior. The results showed distinct self-grooming microstructures in these three models. The anxiolytic drug diazepam relieved the anxiety level and the total time of grooming in the ASD and stress models. The grooming microstructure was not restored in Shank3B KO mice but was partially relieved in the stress model, which suggested that anxiety aggravated stereotyped self-grooming duration but not the grooming microstructure in the ASD mouse model. Our results indicated that stereotyped behavior and anxiety may be shared by separate, but interacting, neural circuits in distinct disease models, which may be useful to understand the mechanisms and develop potential treatments for stereotyped behaviors and anxiety.
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The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a high number of deaths in the world. To combat it, it is necessary to develop a better understanding of how the virus infects host cells. Infection normally starts with the attachment of the virus to cell-surface glycans like heparan sulfate (HS) and sialic acid-containing glycolipids/glycoproteins. In this study, we examined and compared the binding of the subunits and spike (S) proteins of SARS-CoV-2, SARS-CoV, and Middle East respiratory disease (MERS)-CoV to these glycans. Our results revealed that the S proteins and subunits can bind to HS in a sulfation-dependent manner and no binding with sialic acid residues was detected. Overall, this work suggests that HS binding may be a general mechanism for the attachment of these coronaviruses to host cells, and supports the potential importance of HS in infection and in the development of antiviral agents against these viruses.
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Natural proteins are an important source of therapeutic agents and industrial enzymes. While many of them have the potential to be used as highly effective medical treatments for a wide range of diseases or as catalysts for conversion of a range of molecules into important product types required by modern society, problems associated with poor biophysical and biological properties have limited their applications. Engineering proteins with reduced side-effects and/or improved biophysical and biological properties is therefore of great importance. As a common protein modification, glycosylation has the capacity to greatly influence these properties. Over the past three decades, research from many disciplines has established the importance of glycoengineering in overcoming the limitations of proteins. In this review, we will summarize the methods that have been used to glycoengineer proteins and briefly discuss some representative examples of these methods, with the goal of providing a general overview of this research area.
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Improved understanding of the effect of protein glycosylation is expected to provide the foundation for the design of protein glycoengineering strategies. In this study, we examine the impact of O-glycosylation on the binding selectivity of a model Family 1 carbohydrate-binding module (CBM), which has been shown to be one of the primary sub-domains responsible for non-productive lignin binding in multi-modular cellulases. Specifically, we examine the relationship between glycan structure and the binding specificity of the CBM to cellulose and lignin substrates. We find that the glycosylation pattern of the CBM exhibits a strong influence on the binding affinity and the selectivity between both cellulose and lignin. In addition, the large set of binding data collected allows us to examine the relationship between binding affinity and the correlation in motion between pairs of glycosylation sites. Our results suggest that glycoforms displaying highly correlated motion in their glycosylation sites tend to bind cellulose with high affinity and lignin with low affinity. Taken together, this work helps lay the groundwork for future exploitation of glycoengineering as a tool to improve the performance of industrial enzymes.
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Recent advances have demonstrated the feasibility and robustness of chemical synthesis for the production of homogeneously glycosylated protein forms (glycoforms). By taking advantage of the unmatchable flexibility and precision provided by chemical synthesis, the quantitative effects of glycosylation were obtained using chemical glycobiology approaches. These findings greatly advanced our fundamental knowledge of glycosylation. More importantly, analysis of these findings has led to the development of glycoengineering guidelines for rationally improving the properties of peptides and proteins. In this chapter, we present the key experimental steps for chemical biology studies of protein glycosylation, with the aim of facilitating and promoting research in this important but significantly underexplored area of biology.
Assuntos
Glicopeptídeos/síntese química , Glicoproteínas/síntese química , Animais , Técnicas de Química Sintética/métodos , Glicopeptídeos/química , Glicoproteínas/química , Glicosilação , Humanos , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Biologia Sintética/métodosRESUMO
Protein glycosylation is a diverse post-translational modification that serves myriad biological functions. O-linked glycans in particular vary widely in extent and chemistry in eukaryotes, with secreted proteins from fungi and yeast commonly exhibiting O-mannosylation in intrinsically disordered regions of proteins, likely for proteolysis protection, among other functions. However, it is not well understood why mannose is often the preferred glycan, and more generally, if the neighboring protein sequence and glycan have coevolved to protect against proteolysis in glycosylated intrinsically disordered proteins (IDPs). Here, we synthesized variants of a model IDP, specifically a natively O-mannosylated linker from a fungal enzyme, with α-O-linked mannose, glucose, and galactose moieties, along with a non-glycosylated linker. Upon exposure to thermolysin, O-mannosylation, by far, provides the highest extent of proteolysis protection. To explain this observation, extensive molecular dynamics simulations were conducted, revealing that the axial configuration of the C2-hydroxyl group (2-OH) of α-mannose adjacent to the glycan-peptide bond strongly influences the conformational features of the linker. Specifically, α-mannose restricts the torsions of the IDP main chain more than other glycans whose equatorial 2-OH groups exhibit interactions that favor perpendicular glycan-protein backbone orientation. We suggest that IDP stiffening due to O-mannosylation impairs protease action, with contributions from protein-glycan interactions, protein flexibility, and protein stability. Our results further imply that resistance to proteolysis is an important driving force for evolutionary selection of α-mannose in eukaryotic IDPs, and more broadly, that glycan motifs for proteolysis protection likely coevolve with the protein sequence to which they attach.
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Diabetes is a leading cause of death worldwide and results in over 3 million annual deaths. While insulin manages the disease well, many patients fail to comply with injection schedules, and despite significant investment, a more convenient oral formulation of insulin is still unavailable. Studies suggest that glycosylation may stabilize peptides for oral delivery, but the demanding production of homogeneously glycosylated peptides has hampered transition into the clinic. We report here the first total synthesis of homogeneously glycosylated insulin. After characterizing a series of insulin glycoforms with systematically varied O-glycosylation sites and structures, we demonstrate that O-mannosylation of insulin B-chain Thr27 reduces the peptide's susceptibility to proteases and self-association, both critical properties for oral dosing, while maintaining full activity. This work illustrates the promise of glycosylation as a general mechanism for regulating peptide activity and expanding its therapeutic use.