Since the establishment of site-specific mutagenesis of single amino acids to interrogate protein function in the 1970s, biochemists have sought to tailor protein structure in the native cell ...environment. Fine-tuning the chemical properties of proteins is an indispensable way to address fundamental mechanistic questions. Unnatural amino acids (UAAs) offer the possibility to expand beyond the 20 naturally occurring amino acids in most species and install new and useful chemical functions. Here, we review the literature about advances in UAA incorporation technology from chemoenzymatic aminoacylation of modified tRNAs to in vitro translation systems to genetic encoding of UAAs in the native cell environment and whole organisms. We discuss innovative applications of the UAA technology to challenges in bioengineering and medicine.
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.
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.
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.
The role of O-linked N-acetylglucosamine (O-GlcNAc) modification in the cell cycle has been enigmatic. Previously, both O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) disruptions have been shown to ...derail the mitotic centrosome numbers, suggesting that mitotic O-GlcNAc oscillation needs to be in concert with mitotic progression to account for centrosome integrity. Here, using both chemical approaches and biological assays with HeLa cells, we attempted to address the underlying molecular mechanism and observed that incubation of the cells with the OGA inhibitor Thiamet-G strikingly elevates centrosomal distances, suggestive of premature centrosome disjunction. These aberrations could be overcome by inhibiting Polo-like kinase 1 (PLK1), a mitotic master kinase. PLK1 inactivation is modulated by the myosin phosphatase targeting subunit 1 (MYPT1)–protein phosphatase 1cβ (PP1cβ) complex. Interestingly, MYPT1 has been shown to be abundantly O-GlcNAcylated, and the modified residues have been detected in a recent O-GlcNAc–profiling screen utilizing chemoenzymatic labeling and bioorthogonal conjugation. We demonstrate here that MYPT1 is O-GlcNAcylated at Thr-577, Ser-585, Ser-589, and Ser-601, which antagonizes CDK1-dependent phosphorylation at Ser-473 and attenuates the association between MYPT1 and PLK1, thereby promoting PLK1 activity. We conclude that under high O-GlcNAc levels, PLK1 is untimely activated, conducive to inopportune centrosome separation and disruption of the cell cycle. We propose that too much O-GlcNAc is equally deleterious as too little O-GlcNAc, and a fine balance between the OGT/OGA duo is indispensable for successful mitotic divisions.
Plant cell-wall polysaccharides represent a vast source of food in nature. To depolymerize polysaccharides to soluble sugars, many organisms use multifunctional enzyme mixtures consisting of ...glycoside hydrolases, lytic polysaccharide mono-oxygenases, polysaccharide lyases, and carbohydrate esterases, as well as accessory, redox-active enzymes for lignin depolymerization. Many of these enzymes that degrade lignocellulose are multimodular with carbohydrate-binding modules (CBMs) and catalytic domains connected by flexible, glycosylated linkers. These linkers have long been thought to simply serve as a tether between structured domains or to act in an inchworm-like fashion during catalytic action. To examine linker function, we performed molecular dynamics (MD) simulations of the Trichoderma reesei Family 6 and Family 7 cellobiohydrolases ( Tr Cel6A and Tr Cel7A, respectively) bound to cellulose. During these simulations, the glycosylated linkers bind directly to cellulose, suggesting a previously unknown role in enzyme action. The prediction from the MD simulations was examined experimentally by measuring the binding affinity of the Cel7A CBM and the natively glycosylated Cel7A CBM-linker. On crystalline cellulose, the glycosylated linker enhances the binding affinity over the CBM alone by an order of magnitude. The MD simulations before and after binding of the linker also suggest that the bound linker may affect enzyme action due to significant damping in the enzyme fluctuations. Together, these results suggest that glycosylated linkers in carbohydrate-active enzymes, which are intrinsically disordered proteins in solution, aid in dynamic binding during the enzymatic deconstruction of plant cell walls.
In nature, many microbes secrete mixtures of glycoside hydrolases, oxidoreductases, and accessory enzymes to deconstruct polysaccharides and lignin in plants. These enzymes are often decorated with ...N- and O-glycosylation, the roles of which have been broadly attributed to protection from proteolysis, as the extracellular milieu is an aggressive environment. Glycosylation has been shown to sometimes affect activity, but these effects are not fully understood. Here, we examine N- and O-glycosylation on a model, multimodular glycoside hydrolase family 7 cellobiohydrolase (Cel7A), which exhibits an O-glycosylated carbohydrate-binding module (CBM) and an O-glycosylated linker connected to an N- and O-glycosylated catalytic domain (CD)—a domain architecture common to many biomass-degrading enzymes. We report consensus maps for Cel7A glycosylation that include glycan sites and motifs. Additionally, we examine the roles of glycans on activity, substrate binding, and thermal and proteolytic stability. N-glycan knockouts on the CD demonstrate that N-glycosylation has little impact on cellulose conversion or binding, but does have major stability impacts. O-glycans on the CBM have little impact on binding, proteolysis, or activity in the whole-enzyme context. However, linker O-glycans greatly impact cellulose conversion via their contribution to proteolysis resistance. Molecular simulations predict an additional role for linker O-glycans, namely that they are responsible for maintaining separation between ordered domains when Cel7A is engaged on cellulose, as models predict α-helix formation and decreased cellulose interaction for the nonglycosylated linker. Overall, this study reveals key roles for N- and O-glycosylation that are likely broadly applicable to other plant cell-wall–degrading enzymes.
Proteins are made from 19 aa and, curiously, one N-alkylamino acid ("imino acid"), proline (Pro). Pro is thought to be incorporated by the translation apparatus at the same rate as the 19 aa, even ...though the alkyl group in Pro resides directly on the nitrogen nucleophile involved in peptide bond formation. Here, by combining quench-flow kinetics and charging of tRNAs with cognate and noncognate amino acids, we find that Pro incorporates in translation significantly more slowly than Phe or Ala and that other N-alkylamino acids incorporate much more slowly. Our results show that the slowest step in incorporation of N-alkylamino acids is accommodation/peptidyl transfer after GTP hydrolysis on EF-Tu. The relative incorporation rates correlate with expectations from organic chemistry, suggesting that amino acid sterics and basicities affect translation rates at the peptidyl transfer step. Cognate isoacceptor tRNAs speed Pro incorporation to rates compatible with in vivo, although still 3-6 times slower than Phe incorporation from Phe-tRNAPhe depending on the Pro codon. Results suggest that Pro is the only N-alkylamino acid in the genetic code because it has a privileged cyclic structure that is more reactive than other N-alkylamino acids. Our data on the variation of the rate of incorporation of Pro from native Pro-tRNAPro isoacceptors at 4 different Pro codons help explain codon bias not accounted for by the "tRNA abundance" hypothesis.
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.
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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