Synthetic Cellular Chemistry Laboratory

Outline

Our laboratory is working on the interface of synthetic chemistry and glycobiology. Glycoconjugates are involved in a variety of biological events such as cell-cell recognition, malignant transformation, cell differentiation and signal transduction. In fact, a majority of proteins produced in eukaryotic cells are glycoproteins. Although syntheses of these classes of molecules are a significant challenge, they are expected to provide useful molecular probes to clarify the biological functions of glycoconjugates. Our current research activities are directed at (1) development of novel synthetic methodologies and their application to glycoconjugate-related molecular probes, (2) synthesis studies toward delineating biological functions of glycoconjugates and discovery of novel bioactive compounds, (3) synthetic approaches to glycoprotein processing and protein quality control, and (4) analysis of carbohydrate binding molecules.

Recent Research Topic

Clarifying endoplasmic reticulum events through organic synthesis

Fig. 1 Glycoprotein Specific Folding Machinery in the ER

A number of carbohydrate recognizing molecules are involved in the quality control system of glycoprotein. Our study has been conducted to gain precise understanding of this process by using synthetic substrates. Quantitative analysis of Calnexin/Calreticulin, UGGT and Glucosidase II has been achieved.

Most proteins are functional only when they are correctly folded. The process that regulates and maximizes protein folding is called “protein quality control.” In the endoplasmic reticulum (ER) of eukaryotic cells, many molecules play roles in this process.

High-mannose-type oligosaccharides, which are cotranslationally introduced to nascent polypeptides during N-glycosylation, play critical roles in protein quality control. Involved in this process are a number of intracellular carbohydrate-recognizing proteins or carbohydrate processing enzymes. Although all of these proteins seem to recognize high-mannose glycans, their precise specificities have yet to be clarified. In order to conduct quantitative evaluation of the activity and specificity of these proteins, a comprehensive set of high-mannose-type glycans and their variously functionalized derivatives were synthesized and used to analyze enzymes involved in glycoprotein quality control system.

At the initial phase of the study, a dodecasaccharide, which is a proposed ligand of ER-residing chaperones, calnexin and calreticulin, was synthesized using our original methodologies and their specificity confirmed. Subsequently, we addressed the property of other key players, UDP-glucose: glycoprotein glucosyltransferase (UGGT) and glucosidase II (G-II). Our study developed a fully synthetic, non-proteinic substrate of these enzymes and successfully clarified their properties.

An updated summary of these works was published recently [Y. Takeda, et al. Curr. Opin. Chem. Biol. 2009, 13, 582].

Fig. 2 Systematic synthesis of glycan probes

We established a unified method to synthesize “high-mannose-type” glycans and derivatize them to introduce various probes.

Fig. 3 Synthetic substrates clarified specificity of UGGT

A synthetic substrate of UGGT (M9-MTX) was developed. Its activity was comparable with glycoprotein substrate (Tg-denatured), which has been used as a benchmark substrate of this enzyme. This discovery resulted in the clarification of glycan specificity of UGGT.

Fig. 2, 3

Reproduced, with permission, from Y. Takeda, K. Totani, I. Matsuo, Y. Ito, Chemical approaches toward understanding glycan-mediated protein quality control, Curr. Opin. Chem. Biol. 2009, 13, 582. © (2012) Elsevier