Ryo Okamoto

© 2019 FCCA (Forum: Carbohydrates Coming of Age). Recent advances in total chemical synthesis of proteins enable us to prepare complex glycoproteins. Herein, the total chemical synthesis of antifreeze glycoprotein, which is a mucin type O-glycoprotein, is introduced as an example of the latest total chemical synthesis of glycoproteins. The functional analysis of the structurally-defined form of the synthetic O-glycoproteins revealed a unique function of O-GalNAcylation of a protein.

© Springer Nature Singapore Pte Ltd. 2018. This chapter describes the folding of synthetic homogeneous glycosylpolypeptides into glycoproteins depending on the position and number of glycosylation sites and oligosaccharide structures. To evaluate the role of oligosaccharides in protein folding, we synthesized small glycoprotein models, homogeneous misfolded glycoproteins, and erythropoietins. In addition to these chemical syntheses, this chapter introduces a unique method for 15N-labeling of synthetic glycoproteins to enable structural analysis. Based on experimental results, it can be suggested that N-glycans stabilize the structure of glycoproteins.

Oligosaccharides of glycoprotein are known to be heterogeneous. These diverse oligosaccharide profiles have been a hindrance to elucidating oligosaccharide functions in many biological events. In order to elucidate oligosaccharide functions, glycoproteins having homogeneous oligosaccharides that can be varied as much as chemists like are requisite. Chemical synthesis of glycoproteins recently emerged and provides homogeneous glycoproteins such as erythropoietin. For this purpose, an efficient preparation of complex-type oligosaccharides, solid-phase glycopeptide synthesis, glycopeptide segment coupling, and glycopeptide folding procedure are essential. This chapter introduces typical procedure for the chemical synthesis of homogeneous glycoprotein.

Herein, we describe a new semisynthetic strategy of a post-translationally modified protein in which the middle region is glycosylated. We designed a single-plasmid coding for a fusion polypeptide, which can provide both an N-terminal -thioester and a C-terminal cysteine peptide of a target glycoprotein by using chemical-cleavage and activation methods. The use of these resultant peptide derivatives resulted in the successful synthesis of N-glycosylated-interleukin13.

We have been developing the method for the total chemical synthesis of glycoprotein. Recently, we found a new synthetic method of peptide-alpha-thioester that is a key component of the modern chemical protein synthesis. In addition to this, we have succeeded the structural analysis of a chemically synthesized homogeneous glycosyl-chemokine (CCL1) by quasi-racemate X-ray crystallography. In this presentation, we would like to discuss the details of these results. [1] We have pursued a new synthetic method of peptide-alpha-thioester by using an un-protected peptide as a starting material. We designed a three-step conversion sequence consists of S-thiocarbonylation of the C-terminal Cys residue (Figure 2 (i)), treatment of the afforded S-thiocarbonyl peptide with N-acetylguanidine (Figure 2 (ii)) and thiolysis reaction (Figure 2 (iii)). S-Thiocarbonyl group could induce an activation of alpha-nitrogen on the Cys residue in the presence of N-acetylguanidine. This provided a cleavage of peptide bond at AA-Cys sequence and afforded a peptide having N-acetylguanidine at C-terminal. We found this compound was easily converted to a corresponding peptide-alpha-thioester under neutral buffer solution. We also successfully obtained a desired glycopeptide-thioester by this new methodology (Figure 3). [2] There are well known difficulties in X-ray diffraction structural analysis of glycoproteins, which can be hard to crystallize because of the heterogeneity of the oligosaccharide moiety. Recently, racemic protein crystallization has reported. This method is facile and straightforward crystallization method by using racemic protein which is a 1 : 1 mixture of native protein consist of L-amino acids (L-protein) and mirror image protein consist of D-amino acids (D-protein). We envisioned quasi-racemic protein consists of a 1 : 1 mixture of native glycoprotein and D-protein would also provide facile crystallization. Then, we carried out convergent chemical syntheses of CCL1 derivatives and successfully obtained three distinct protein molecules: glycosyl-L-CCL1, L-CCL1 and D-CCL1 made from D-amino acids. Crystallization of the homogeneous L-glycoprotein was still difficult. In contrast, we could get a single crystal by means of quasi-racemic crystallization, from a mixture of glycosyl-L-CCL1 and D-CCLI as well as racemic crystallization, from a mixture of L-CCL1 and D-CCL1. Diffraction data from the quasi-racemic crystal and the racemic crystal have been acquired to 2.08 Angstroms and 2.0 Angstroms, respectively. Structural analyses revealed there are no significant structural differences between glycosyl-CCL1 and CCL1, and we could confirm the completion of the chemical synthesis of the glycoprotein.

Oligosaccharides in protein play important roles in several biological events. In order to investigate the functions of oligosaccharides of protein, glycoproteins having homogeneous oligosaccharides should be prepared. For this purpose, preparation methods of diverse complex-type oligosaccharides as well as synthetic methods of glycopeptides are essential. This report describes the recent progress in the synthesis of glycopeptides having homogeneous complex-type sialyloligosaccharides.

Protein cysteines (cysteinyl residues) play critical roles in biological processes. In the course of protein evolution under oxidizing atmosphere of the Earth, organisms have utilized highly reactive cysteines in many proteins essential for maintenance of life, i.e. enzymes, transcriptional factors, cytoskeletons, and receptors. In some enzymes, sophistical cysteine modification characterizes each catalytic mechanism. In vivo modification of protein cysteines with natural chemical compounds modulates protein functions as a molecular switch. Oxidation/reduction, thiol-disulfide exchange, nitrosylation, sulfuration, thiolation, acylation and prenylation are involved. Some protein cysteines coordinate metals or metal cofactors such as a heme or an iron sulfur cluster to form metalloproteins, serving as sensor proteins, metalloenzymes or transcriptional factors. Information on the in vitro chemical modifications and their reaction specificities of protein cysteines are essential for the investigation of the mechanisms and functions of in vivo protein cysteine modifications. In this review, we also mention historically important knowledge other than recent results on protein cysteine modification and modulation of protein function to fertilize medical proteomics.

Oligosaccharide chains on the protein play important roles for several biological events. However, the oligosaccharide exhibits heterogeneous structure, so called glycoform. Therefore synthesis of glycoprotein having intact and homogeneous oligosaccharide chains have been paid an attention in order to investigate what oligosaccharide structure on protein is essential for individual biological events. We have examined synthesis of glycoprotein having homogeneous human oligosaccharide and we presented the first chemical synthesis of glycoprotein, CC-Chemokine in the last of this meeting. Our synthetic strategy employed solid phase peptide synthesis and repetitive native chemical ligation method. We have examined synthesis of several small glycoproteins such as Ovomucoid and Crambin. In addition, we examined to evaluate an effect of oligosaccharide for protein folding process and protein conformation. We selected additional two targets, Ovomucoide and Crambin. Ovomucoid consists of 58 amino acids and it has three disulfide bonds. Crambin consists of 46 amino acids and it has also three disulfide bonds. We succeeded the synthesis of these glycosylated Ovomucoid and Crambin by use of our strategy. For glycosylated Ovomucoide, oligosaccharide was found to disturb protein folding process, but there was no interference during folding process in the case of glycosylated Crambin. We also succeeded measurement of the first TOCSY spectrum of glycoprotein synthesized. In this presentation, we would like to explain these interesting results in detail.