Engineering of a Biologically Active Glycosylated Glucagon-Like Peptide-1 Analogue.

Glucagon-like peptide receptor (GLP-1R) agonists (e.g., semaglutide, liraglutide, etc.) are efficient treatment options for people with type 2 diabetes and obesity. The manufacturing method to produce semaglutide, a blockbuster GLP-1 drug on the market, involves multistep synthesis. The large peptide has a hydrophobic fatty acid side chain that makes it sparingly soluble, and its handling, purification, and large-scale production difficult. The growing demand for semaglutide that the manufacturer is not capable of addressing immediately triggered a worldwide shortage. Thus, we have developed a potential alternative analogue to semaglutide by replacing the hydrophobic fatty acid with a hydrophilic human complex-type biantennary oligosaccharide. Our novel glycoGLP-1 analogue was isolated in an ∼10-fold higher yield compared with semaglutide. Importantly, our glycoGLP-1 analogue possessed a similar GLP-1R activation potency to semaglutide and was biologically active in vivo in reducing glucose levels to a similar degree as semaglutide.

Isopeptide bond formation mediated by δ-selenolysine for chemical ubiquitination.

Protein ubiquitination is involved in nearly all biological processes in Eukaryotes. To gain precise insights into the function of ubiquitination in these processes, researchers frequently employ ubiquitinated protein probes with well-defined structures. While chemical protein synthesis has afforded a variety of ubiquitinated protein probes, there remains a demand for efficient synthesis methods for complex probes, such as ubiquitinated glycoproteins and ubiquitinated cysteine-containing proteins. In this study, we introduce a new method to obtain ubiquitinated proteins through isopeptide bond formation mediated by δ-selenolysine residues. We synthesized δ-selenolysine derivatives in both L- and D-forms starting from DL-δ-hydroxy-DL-lysine, accomplished by substituting the δ-mesylate with KSeCN and by enzymatic optical resolution with L- and D-aminoacylase. We synthesized ubiquitin (46-76)-α-hydrazide with a δ-seleno-L-lysine residue at position 48, as well as ubiquitin (46-76)-α-thioester, using solid-phase peptide synthesis. Subsequently, the δ-selenolysine-mediated ligation of these peptides, followed by one-pot deselenization, provided the desired isopeptide-linked ubiquitin peptide. The new δ-selenolysine-mediated isopeptide bond formation offers an alternative method to obtain complex ubiquitin- and ubiquitin-like probes with multiple post-translational modifications. These probes hold promise for advancing our understanding of ubiquitin biology.

Convergent synthesis of proteins using peptide-aminothiazoline.

Sequential peptide coupling plays a central role in chemical protein synthesis. This paper describes a new peptide derivative, peptide-aminothiazoline (At), whereof the C-terminus is functionalized with 2-aminothiazoline. Peptide-At streamlined the sequential peptide ligation in a one-pot manner and demonstrated the convergent synthesis of a circular protein and homogeneous glycoproteins.

Semisynthesis of homogeneous misfolded glycoprotein interleukin-8.

To uncover how cells distinguish between misfolded and correctly-folded glycoproteins, homogeneous misfolded glycoproteins are needed as a probe for analysis of their structure and chemical characteristic nature. In this study, we have synthesized misfolded glycosyl interleukin-8 (IL-8) by combining E. coli expression and chemical synthesis to improve the synthetic efficiency. In order to prepare N-terminal peptide-thioester segment (1-33), we prepared an E. coli expressed peptide and then activated the C-terminal Cys by using an intramolecular N-to-S acyl shift reaction, followed by trans-thioesterification of the Cys-thioester with an external bis(2-sulfanylethyl)amine (SEA). The glycopeptide segment (34-49) was prepared by solid phase peptide synthesis and the C-terminal peptide (50-72) was prepared in E. coli. These peptide and glycopeptide segments were successfully coupled by sequential native chemical ligation. To obtain homogeneous misfolded glycoproteins by shuffling the disulfide bond pattern, folding conditions were optimized to maximize the yield of individual homogeneous misfolded glycoproteins.

Rapid Chemical Synthesis of Serine Protease Inhibitor Kazal-type 13 (SPINK13) Glycoform by a Combined Method with Glycan Insertion Strategy and Fast-Flow Fmoc SPPS.

Serine protease inhibitor Kazal type 13 (SPINK13) is a secreted protein that has been recently studied as a therapeutic drug and an interesting biomarker for cancer cells. Although SPINK13 has a consensus sequence (Pro-Asn-Val-Thr) for N-glycosylation, the existence of N-glycosylation and its functions are still unclear. In addition to this, the preparation of glycosylated SPINK 13 has not been examined by both the cell expression method and chemical synthesis. Herein we report the chemical synthesis of the scarce N-glycosylated form of SPINK13 by a rapid synthetic method combined with the chemical glycan insertion strategy and a fast-flow SPPS method. Glycosylated asparagine thioacid was designed to chemoselectively be inserted between two peptide segments where is the sterically bulky Pro-Asn(N-glycan)-Val junction by two coupling reactions which consist of diacyl disulfide coupling (DDC) and thioacid capture ligation (TCL). This insertion strategy successfully afforded the full-length polypeptide of SPINK13 within two steps from glycosylated asparagine thioacid. Because the two peptides used for this synthesis were prepared by a fast-flow SPPS, the total synthetic time of glycoprotein was considerably shortened. This synthetic concept enables us to repetitively synthesize a target glycoprotein easily. Folding experiments afforded well-folded structure confirmed by CD and disulfide bond map. Invasion assays of glycosylated SPINK13 and non-glycosylated SPINK13 with pancreatic cancer cells showed that non-glycosylated SPINK-13 was more potent than that of glycosylated SPINK13.

Glycosylation Improves the Proteolytic Stability of Exenatide.

Exenatide was the first marketed GLP-1 receptor agonist for the treatment of type 2 diabetes. Modification to the chemical structure or the formulation has the potential to increase the stability of exenatide. We introduced human complex-type sialyloligosaccharide to exenatide at the native Asn28 position. The synthesis was achieved using both solid phase peptide synthesis (SPPS) and Omniligase-1-mediated chemoenzymatic ligation. The results demonstrate that glycosylation increases the proteolytic stability of exenatide while retaining its full biological activity.

Regulating Antifreeze Activity through Water: Latent Functions of the Sugars of Antifreeze Glycoprotein Revealed by Total Chemical Synthesis.

Antifreeze glycoprotein (AFGP), which inhibits the freezing of water, is highly O-glycosylated with a disaccharide, d-Galß1-3-d-GalNAcα (GalGalNAc). To elucidate the function of the sugar residues for antifreeze activity at the molecular level, we conducted a total chemical synthesis of partially sugar deleted AFGP derivatives, and unnatural forms of AFGPs incorporating glucose (Glc)-type sugars instead of galactose (Gal)-type sugars. These elaborated AFGP derivatives demonstrated that the stereochemistry of each sugar residue on AFGPs precisely correlates with the antifreeze activity. A hydrogen-deuterium exchange experiment using synthetic AFGPs revealed a different dynamic behavior of water around sugar residues depending on the sugar structures. These results indicate that sugar residues on AFGP form a unique dynamic water phase that disturbs the absorbance of water molecules onto the ice surface, thereby inhibiting freezing.

Recent advances on the synthesis of N-linked glycoprotein for the elucidation of glycan functions.

Glycoproteins play roles in many biological events, while, the glycan structure-function relationship has remained to be studied. In order to understand glycan function, homogeneous glycoproteins have been synthesized. This review introduced recent progress of their synthetic approaches.

Squaryl group-modified UDP analogs as inhibitors of the endoplasmic reticulum-resident folding sensor enzyme UGGT.

UDP-Glc:glycoprotein glucosyltransferase (UGGT) has a central role to retain quality control of correctly folded N-glycoprotein in the endoplasmic reticulum (ER). A selective and potent inhibitor against UGGT could lead to elucidation of UGGT-related events, but such a molecule has not been identified so far. Examples of small molecules with UGGT inhibitory activity are scarce. Here, we report squaryl group-modified UDP analogs as a promising UGGT inhibitor. Among these, the compound possessing a 2'-amino group of the uridine moiety and hydroxyethyl-substituted squaramide exhibited the highest potency, suggesting its relevance as a molecule for further optimization.

Synthesis of homogeneous glycoproteins with diverse N-glycans.

In the post-genomic era, post- and co-translational modifications (P/C-TM) of proteins are known as the more essential elements for the activation of protein function. Among these protein modifications, glycosylation is one of the most abundant modifications in eukaryotic cells. The synthesis of glycoproteins with uniform glycan structures is essential for functional analysis of glycoproteins and biochemical research. For that purpose, chemical methods to synthesize glycoproteins with chemically uniform glycan structures have been developed. In this review, we highlight our recent advances in the preparation of homogeneous glycoproteins. Especially, we introduce both semi-synthesis and chemical synthesis of glycoproteins along with semi-synthesis of various complex-type N-glycans for the solid-phase synthesis of glycopeptide-thioesters.

Isolation and characterization of high-mannose type glycans containing five or six mannose residues from hen egg yolk.

High-mannose type glycans play important roles in biosynthesis of glycoproteins including glycoprotein quality control system. In the endoplasmic reticulum (ER), α1,2-mannosidases cleave several mannose (Man) residues to give small high-mannose type glycans, such as glycans containing five or six mannose residues (M5-glycan or M6-glycan). These glycans are reported to act as a signal for degradation processes of glycoproteins in the ER. In this work, we isolated the M5-glycan and the M6-glycan from delipidated egg yolk and confirmed that their structures were identical to human type glycans based on rigorous NMR experiments, suggesting the potential use for semisynthesis of glycoconjugates and glycan analysis.

Design and Synthesis of Glycosylated Cholera Toxin B Subunit as a Tracer of Glycoprotein Trafficking in Organelles of Living Cells.

Glycosylation of proteins is known to be essential for changing biological activity and stability of glycoproteins on the cell surfaces and in body fluids. Delivering of homogeneous glycoproteins into the endoplasmic reticulum (ER) and the Golgi apparatus would enable us to investigate the function of asparagine-linked (N-) glycans in the organelles. In this work, we designed and synthesized an intentionally glycosylated cholera toxin B-subunit (CTB) to be transported to the organelles of mammalian cells. The heptasaccharide, the intermediate structure of various complex-type N-glycans, was introduced to the CTB. The synthesized monomeric glycosyl-CTB successfully entered mammalian cells and was transported to the Golgi and the ER, suggesting the potential use of synthetic CTB to deliver and investigate the functions of homogeneous N-glycans in specific organelles of living cells.

Optimizing the Semisynthesis towards glycosylated interferon-ß-polypeptide by utilizing bacterial protein expression and chemical modification.

The synthesis of a sufficient amount of homogeneous glycoprotein is of great interest because natural glycoproteins show considerable heterogeneity in oligosaccharide structures, making the studies on glycan structure-function relationship difficult. Herein, we report optimized methods that can accelerate the semisynthesis of homogeneous glycoproteins based on recombinant expression and chemical conversion. Peptide thioesters and peptides with Cys residues at their N-terminals are necessary intermediates to perform native chemical ligation. We successfully performed thioesterification for a peptide prepared in E. coli via Cys-cyanylation at its C-terminal followed by hydrazinolysis and acidic thiolysis. These optimized conditions could tolerate an acid labile Thz protected Cys at the N-terminal of a peptide-hydrazide and specific cyanylation of the C-terminal Cys to yield a peptide thioester. To reduce the amount of precious oligosaccharide that is required in the conventional SPPS method, an improved liquid phase glycopeptide coupling was also optimized in a good yield (46% over four steps). Lastly, chemoselective protection of the internal cysteines and activation of the N-terminal cysteine were optimized toward a long peptide prepared in E. coli. By using these strategies, a full-length interferon-ß glycosyl polypeptide as a model was successfully obtained.

Semisynthesis of a Homogeneous Glycoprotein Using Chemical Transformation of Peptides to Thioester Surrogates.

Semisynthesis using recombinant polypeptides as building blocks is a powerful approach for the preparation of proteins with a variety of modifications such as glycosylation. The activation of the C terminus of recombinant peptides is a key step for coupling peptide building blocks and preparing a full-length polypeptide of a target protein. This article reports two chemical approaches for transformation of the C terminus of recombinant polypeptides to thioester surrogates. The first approach relies on efficient substitution of the C-terminal Cys residue with bis(2-sulfanylethyl)amine (SEA) to yield peptide-thioester surrogates. The second approach employs a native tripeptide, cysteinyl-glycyl-cysteine (CGC), to yield peptide-thioesters via a process mediated by a thioester surrogate. Both chemical transformation methods employ native peptide sequences and were thereby successfully applied to recombinant polypeptides. As a consequence, we succeeded in the semisynthesis of a glycosylated form of inducible T cell costimulator (ICOS) for the first time.

Purified EDEM3 or EDEM1 alone produces determinant oligosaccharide structures from M8B in mammalian glycoprotein ERAD.

Sequential mannose trimming of N-glycan, from M9 to M8B and then to oligosaccharides exposing the α1,6-linked mannosyl residue (M7A, M6, and M5), facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). We previously showed that EDEM2 stably disulfide-bonded to the thioredoxin domain-containing protein TXNDC11 is responsible for the first step (George et al., 2020). Here, we show that EDEM3 and EDEM1 are responsible for the second step. Incubation of pyridylamine-labeled M8B with purified EDEM3 alone produced M7 (M7A and M7C), M6, and M5. EDEM1 showed a similar tendency, although much lower amounts of M6 and M5 were produced. Thus, EDEM3 is a major α1,2-mannosidase for the second step from M8B. Both EDEM3 and EDEM1 trimmed M8B from a glycoprotein efficiently. Our confirmation of the Golgi localization of MAN1B indicates that no other α1,2-mannosidase is required for gpERAD. Accordingly, we have established the entire route of oligosaccharide processing and the enzymes responsible.

Chemical Synthesis and Characterization of a Nonfibrillating Glycoglucagon.

The current commercially available glucagon formulations for the treatment of severe hypoglycemia must be reconstituted immediately prior to use, owing to the susceptibility of glucagon to fibrillation and aggregation in an aqueous solution. This results in the inconvenience of handling, misuse, and wastage of this drug. To address these issues, we synthesized a glycosylated glucagon analogue in which the 25th residue (Trp) was replaced with a cysteine (Cys) and a Br-disialyloligosaccharide was conjugated at the Cys thiol moiety. The resulting analogue, glycoglucagon, is a highly potent full agonist at the glucagon receptor. Importantly, glycoglucagon exhibits markedly reduced propensity for fibrillation and enhanced thermal and metabolic stability. This novel analogue is thus a valuable lead for producing stable liquid glucagon formulations that will improve patient compliance and minimize wastage.

Glycoprotein Semisynthesis by Chemical Insertion of Glycosyl Asparagine Using a Bifunctional Thioacid-Mediated Strategy.

Glycosylation is a major modification of secreted and cell surface proteins, and the resultant glycans show considerable heterogeneity in their structures. To understand the biological processes arising from each glycoform, the preparation of homogeneous glycoproteins is essential for extensive biological experiments. To establish a more robust and rapid synthetic route for the synthesis of homogeneous glycoproteins, we studied several key reactions based on amino thioacids. We found that diacyl disulfide coupling (DDC) formed with glycosyl asparagine thioacid and peptide thioacid yielded glycopeptides. This efficient coupling reaction enabled us to develop a new glycoprotein synthesis method, such as the bifunctional thioacid-mediated strategy, which can couple two peptides with the N- and C-termini of glycosyl asparagine thioacid. Previous glycoprotein synthesis methods required valuable glycosyl asparagine in the early stage and subsequent multiple glycoprotein synthesis routes, whereas the developed concept can generate glycoproteins within a few steps from peptide and glycosyl asparagine thioacid. Herein, we report the characterization of the DDC of amino thioacids and the efficient ability of glycosyl asparagine thioacid to be used for robust glycoprotein semisynthesis.

Chemical Synthesis of an Erythropoietin Glycoform Having a Triantennary N-Glycan: Significant Change of Biological Activity of Glycoprotein by Addition of a Small Molecular Weight Trisaccharide.

The glycosylation of proteins contributes to the modulation of the structure and biological activity of glycoproteins. Asparagine-linked glycans (N-glycans) of glycoproteins naturally exhibit diverse antennary patterns, such as bi-, tri-, and tetra-antennary forms. However, there are no chemical or biological methods to obtain homogeneous glycoproteins via the intentional alteration of the antennary form of N-glycans. Thus, the functions of the individual antennary form of N-glycan at a molecular level remain unclear. Herein, we report the chemical synthesis of an erythropoietin (EPO) glycoform having a triantennary sialylglycan at position 83, as well as two biantennary sialylglycans at both positions 24 and 38. We demonstrated efficient liquid-phase condensation reactions to prepare a sialylglycopeptide having a triantennary N-glycan prepared by the addition of a Neu5Ac-α-2,6-Gal-ß-1,4-GlcNAc element to the biantennary glycan under semisynthetic conditions. The molecular weight of the newly added antennary element was ∼3% of the EPO glycoform, and the introduced position was the most distant from the bioactive protein. However, in vivo assays using mice revealed that the additional antennary element at position 83 dramatically increased the hematopoietic activity compared to a commercially available native EPO. These unprecedented data clearly indicate that the antennary pattern of N-glycans inherently plays a critical role in the modulation of protein functions.

Chemical-Synthesis-Based Approach to Glycoprotein Functions in the Endoplasmic Reticulum.

The introduction of Asn-linked glycans to nascent polypeptides occurs in the lumen of the endoplasmic reticulum of eukaryotic cells. After the removal of specific sugar residues, glycoproteins acquire signals in the glycoprotein quality control (GPQC) system and enter the folding cycle composed of lectin-chaperones calnexin (CNX) and calreticulin (CRT), glucosidase II (G-II), and UDP-Glc:glycoprotein glucosyltransferase (UGGT). G-II initiates glycoproteins' entry and exit from the cycle, and UGGT serves as the "folding sensor". This account summarizes our effort to analyze the properties of enzymes and lectins that play important roles in GPQC, especially those involved in the CNX/CRT cycle. To commence our study, general methods for the synthesis of high-mannose-type glycans and glycoproteins were established. Based on these, various substrates to analyze components of the GPQC were created, and properties of CRT, G-II, and UGGT have been clarified.

Chemical Synthesis of Ubiquitinated High-Mannose-Type N-Glycoprotein CCL1 in Different Folding States.

Degradation of misfolded glycoproteins by the ubiquitin-proteasome system (UPS) is a very important process for protein homeostasis. To demonstrate the accessibility toward a ubiquitinated glycoprotein probe for the study of glycoprotein degradation by UPS, we synthesized ubiquitinated glycoprotein CC motif chemokine 1 (CCL1) bearing a high-mannose-type N-glycan, starting from six peptide segments. A native isopeptide linkage was constructed using δ-thiolysine (thioLys)-mediated chemical ligation. CCL1 glycopeptide with a high-mannose-type N-glycan as well as a δ-thioLys residue was synthesized chemically. The chemical ligation between δ-thioLys-containing glycopeptide and ubiquitin-α-thioester successfully yielded a ubiquitinated glycopeptide with a native isopeptide bond after desulfurization, even in the presence of a large N-glycan. In vitro folding experiments under reduced and redox conditions gave the desired two types of ubiquitinated glycosylated CCL1s, consisting of unfolded CCL1 and folded ubiquitin, and the folded form of both CCL1 as well as ubiquitin. We achieved the chemical synthesis of a complex protein molecule that contains not only the two major post-translational modifications, ubiquitination and glycosylation, but also controlled folding states of ubiquitin and CCL1. These chemical probes could have useful applications in the study of complex ubiquitin biology and glycobiology.