We chemically synthesize high mannose-type glycans and their derivatives,
and using them, we studies the intracellular functions of N-linked glycans in the quality control system in the ER.
Glycans linked to proteins and lipids regulate their biochemical properties. Recently, it has become clear that glycans serve as not only hydrophilic moieties but also informative molecules. In most cases, molecules receiving glycan-specific signals are proteins. Elucidation of the molecular mechanism of interaction between glycans and proteins will lead to better understanding of biological phenomena. However, the greatest hindrance in the analysis of glycan–protein recognition has been difficult for large-scale preparation of defined and homogeneous oligosaccharides that can be subjected to various analyses. We will address the large-scale synthesis of high-mannose-type oligosaccharides and analyze carbohydrate–protein interactions.
We chemically synthesize glycoproteins bearing human-type N-linked glycans, and study the effects of glycans on protein function through
chemical approach. These studies will lead to the elucidation of glycan
functions and the exploration of glycoprotein medicines.
Synthesis of homogeneous glycoproteins bearing high-mannose or complex type biantennary oligosaccharides
Protein glycosylation is one of the most important post-translational modification on protein surface and the presence of carbohydrates on proteins is associated with a number of biological events. In nature glycoproteins are found in complex heterogeneous mixture, which complicates analyses of their characterisation and function. For understanding the role of each carbohydrate substituent on the glycoprotein, it is therefore essential to have access to homogeneous glycoproteins. Using small model protein we are going to synthesize homogeneous glycoproteins bearing high-mannose type or asialo complex type oligosaccharides in order to investigate the functions of N-glycans on the protein structure. In order to prepare homogeneous glycoproteins we used both peptide solid phase synthesis and Native Chemical Ligation (NCL). The full length amino acid sequence is divided into several segments that have been prepared using Fmoc or Boc chemistry and later on are combined using NCL. With this methodology we prepared both homogeneously folded and misfolded proteins bearing high mannose type oligosaccharides.
In general, folded proteins exhibit a hydrophilic nature, while misfolded proteins show more hydrophobic behavior. (UDP)-Glucose: glycoprotein glucosyltransferase (UGGT) is an ER-resident enzyme which recognizes misfolded glycoproteins and is responsible for transfering a glucose unit to the specific nonreducing terminal of high-mannose glycans of misfolded glycoproteins. Recent studies on UGGT show that this enzyme recognizes subtle acceptor substrate such as methotrexate-M9 (MTX-M9), where the MTX is apparently mimicking the hydrophobic property of misfolded proteins. Therefore homogeneously misfolded glycoproteins bearing high-mannose type oligosaccharides will be useful probes to investigate the complex process governing glycoprotein folding in the endoplasmic reticulum.
We will analyze tertiary structures of glycoproteins and biosynthetic process
of glycosphingolipids in the Golge apparatus, by means of organic chemistry,
analytical chemistory and biochemistry.
Elucidating a complex glycan structure is the subject of prime importance. Investigation of the individual structure of glycoforms presented at different positions of glycoproteins is the current focal area. Amounts and compositions of glycans on the plasma membrane presented as glycoproteins and glycolipids fluctuate spatially and temporally depending on an environment of the cell. No method has been provided to address spatio-temporal, structural, and quantitative issues. In the meanwhile, although structural information of proteins is being accumulated and available in the drag-design, a majority of proteins is glycosylated and awaits to be solved. The group will make efforts to fulfill the missing link in glycobiology based on chemistry, analytical chemistry, and cell biology.