RESUMO
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.
Assuntos
Aprendizado de Máquina , Simulação de Dinâmica Molecular , Solubilidade , Glicosilação , Glicoproteínas/químicaRESUMO
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.
Assuntos
Anticorpos Monoclonais , Engenharia de Proteínas , Glicosilação , Anticorpos Monoclonais/metabolismo , Processamento de Proteína Pós-Traducional , Imunoglobulina G/química , Polissacarídeos/metabolismoRESUMO
The introduction of modifications into oligonucleotides is important for a large number of applications in the nucleic acids field. However, the method of solid-phase DNA synthesis presents significant challenges for incorporating many useful modifications that are unstable to the conditions for preparing synthetic DNA. Here we report that boranephosphonate diesters undergo facile nucleophilic substitution in a stereospecific manner upon activation by iodine. We have subsequently used this reactivity to post-synthetically introduce modifications including azides and fluorophores into DNA by first synthesizing boranephosphonate-linked 2'-deoxyoligonucleotides and then treating these oligomers with iodine and various nucleophiles. In addition, we show that this reaction is an attractive method for preparing stereodefined phosphorus-modified oligonucleotides. We have also examined the mechanism of this reaction and show that it proceeds via an iodophosphate intermediate. Beyond nucleic acids synthesis, due to the ubiquity of phosphate derivatives in natural compounds and therapeutics, this stereospecific reaction has many potential applications in organophosphorus chemistry.
Assuntos
Compostos de Boro/química , DNA/química , Oligonucleotídeos/síntese química , Organofosfonatos/química , Amidas/química , Azidas/química , Técnicas de Química Sintética , DNA/síntese química , Dimerização , Ésteres/química , Etilaminas/química , Iodo/química , Espectroscopia de Ressonância Magnética , Oligonucleotídeos/química , Oxirredução , Ácidos Fosfóricos/química , EstereoisomerismoRESUMO
The power of chemical synthesis of large cysteine-free polypeptides has been significantly enhanced through the use of nonproteogenic constructs which bear strategically placed thiol groups, enabling native chemical ligation. Central to these much expanded capabilities is the specific, radical-induced, metal-free dethiolation, which can be accomplished in aqueous medium.
Assuntos
Cisteína/química , Hormônio Paratireóideo/síntese química , Sequência de Aminoácidos , Substituição de Aminoácidos , Dicroísmo Circular , Humanos , Dados de Sequência Molecular , Hormônio Paratireóideo/químicaRESUMO
The conversion of peptide and proteins from their soluble state into well-organized aggregates, together with the accompanied oxidation of methionine residue, presents a significant challenge to human health, to the manufacture of protein therapeutics, and to the synthesis of proteins and glycoproteins. Despite their fundamental importance, little is known about the molecular basis of these two side reactions and their control. Here, using chemical peptide synthesis, we further confirmed the importance of the balance between hydrophobic interactions and electrostatic repulsive forces in inducing and inhibiting aggregation and methionine oxidation. Most importantly, through extending the established principle, we are able to effectively stabilize the problematic peptide fragment through the attachment of cleavable arginine tags. Future applications of our approach are expected to facilitate the synthesis and study of difficult peptides, proteins, and glycoproteins and will provide more opportunities for the optimization of protein biopharmaceuticals and for the development of cell-permeable biomolecules.
Assuntos
Bioquímica/métodos , Proteínas/química , Sequência de Aminoácidos , Cromatografia Líquida , Eritropoetina/química , Humanos , Espectrometria de Massas , Metionina/metabolismo , Dados de Sequência Molecular , Oxirredução , Peptídeos/química , Estrutura Quaternária de ProteínaRESUMO
Spatially selective deposition of metal onto complex DNA assemblies is a promising approach for the preparation of metallic nanostructures with features that are smaller than what can be produced by top-down lithographic techniques. We have recently reported the ability of 2'-deoxyoligonucleotides containing boranephosphonate linkages (bpDNA) to reduce AuCl4(-), Ag(+), and PtCl4(2-) ions to the corresponding nanoparticles. Here we demonstrate incorporation of bpDNA oligomers into a two-dimensional DNA array comprised of tiles containing double crossover junctions. We further demonstrate the site-specific deposition of metallic silver onto this DNA structure which generates well-defined and preprogrammed arrays of silver nanoparticles. With this approach the size of the metallic features that can be produced is limited only by the underlying DNA template. These advances were enabled due to a new method for synthesizing bpDNA that uses a silyl protecting group on the DNA nucleobases during the solid-phase 2'-deoxyoligonucleotide synthesis.
Assuntos
Boranos/química , DNA/química , Nanoestruturas/química , Organofosfonatos/química , Prata/química , DNA/síntese química , Indicadores e Reagentes , Análise em Microsséries , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Conformação de Ácido Nucleico , Oxirredução , Platina/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.
Assuntos
Celulose , Polissacarídeos , Glicosilação , Celulose/química , Simulação por Computador , Termodinâmica , Ligação Proteica , Sítios de LigaçãoRESUMO
Application of native chemical ligation logic to the case of an N-terminal proline is described. Two approaches were studied. One involved incorporation of a 3R-substituted thiyl-proline derivative. Improved results were obtained from a 3R-substituted selenol function, incorporated in the context of an oxidized dimer.
Assuntos
Prolina/química , Eritropoetina/síntese química , Eritropoetina/química , Estrutura Molecular , Prolina/análogos & derivados , EstereoisomerismoRESUMO
Application of chemical synthesis to gain access to high purity hPTH as well as more stable analogues was accomplished through a menu of extended NCL followed by metal free dethiylation.
Assuntos
Hormônio Paratireóideo/síntese química , Peptídeos/síntese química , Engenharia de Proteínas , Animais , Cálcio/sangue , Cálcio/metabolismo , Células HEK293 , Humanos , Injeções Subcutâneas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Hormônio Paratireóideo/administração & dosagem , Hormônio Paratireóideo/farmacologia , Peptídeos/administração & dosagem , Peptídeos/farmacologiaRESUMO
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.
RESUMO
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.
Assuntos
Glicoproteínas , Glicoproteínas/metabolismo , GlicosilaçãoRESUMO
Native chemical ligation (NCL) is widely applicable for building proteins in the laboratory. Since the discovery of this method, many strategies have been developed to enhance its capability and efficiency. Because of the poor reactivity of proline thioesters, ligation at a C-terminal proline site is not readily accomplished. Here, we demonstrate that ligation at an N-terminal protein is feasible using the combined logic of NCL and metal-free dethiylation (MFD).
Assuntos
Prolina/química , Proteínas/síntese química , Proteínas/química , Estereoisomerismo , Enxofre/químicaRESUMO
We describe herein the assembly of hEPO(79-166), a key glycopeptide segment en route to erythropoietin, in minimally protected form. Key to the success of this synthetic endeavor was the application of our two-step cysteine-free native chemical ligation strategy, by which we achieved formal ligation at alanine and proline residues through the use of an N-terminal amino acid surrogate presenting a readily removable thiol functionality.
RESUMO
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.
RESUMO
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.
RESUMO
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.
RESUMO
Single erythropoietin (EPO) glycoforms with defined mature oligosaccharide structures and amino acid sequences are essential to elucidate the molecular mechanisms by which carbohydrates exert various physiological and metabolic functions and to explore the possible links between carbohydrates and the prevention or management of diseases. To demonstrate that it is possible to generate EPO even without recourse to cysteine-based native chemical ligation, a concise synthesis of the partially protected EPO fragment (78-166) bearing fully mature N- and O-glycans is described.