Monitoring of Glycoprotein Quality Control System with a Series of Chemically Synthesized Homogeneous Native and Misfolded Glycoproteins.

The glycoprotein quality control (GQC) system in the endoplasmic reticulum (ER) effectively uses chaperone-type enzymes and lectins such as UDP-glucose:glycoprotein glucosyltransferase (UGGT), calnexin (CNX), calreticulin (CRT), protein disulfide bond isomerases (ERp57 or PDIs), and glucosidases to generate native-folded glycoproteins from nascent glycopolypeptides. However, the individual processes of the GQC system at the molecular level are still unclear. We chemically synthesized a series of several homogeneous glycoproteins bearing M9-high-mannose type oligosaccharides (M9-glycan), such as erythropoietin (EPO), interferon-ß (IFN-ß), and interleukin 8 (IL8) and their misfolded counterparts, and used these glycoprotein probes to better understand the GQC process. The analyses by high performance liquid chromatography and mass spectrometer clearly showed refolding processes from synthetic misfolded glycoproteins to native form through folding intermediates, allowing for the relationship between the amount of glucosylation and the refolding of the glycoprotein to be estimated. The experiment using these probes demonstrated that GQC system isolated from rat liver acts in a catalytic cycle regulated by the fast crosstalk of glucosylation/deglucosylation in order to accelerate refolding of misfolded glycoproteins.

Chemical synthesis of erythropoietin glycoforms for insights into the relationship between glycosylation pattern and bioactivity.

The role of sialyloligosaccharides on the surface of secreted glycoproteins is still unclear because of the difficulty in the preparation of sialylglycoproteins in a homogeneous form. We selected erythropoietin (EPO) as a target molecule and designed an efficient synthetic strategy for the chemical synthesis of a homogeneous form of five EPO glycoforms varying in glycosylation position and the number of human-type biantennary sialyloligosaccharides. A segment coupling strategy performed by native chemical ligation using six peptide segments including glycopeptides yielded homogeneous EPO glycopeptides, and folding experiments of these glycopeptides afforded the correctly folded EPO glycoforms. In an in vivo erythropoiesis assay in mice, all of the EPO glycoforms displayed biological activity, in particular the EPO bearing three sialyloligosaccharides, which exhibited the highest activity. Furthermore, we observed that the hydrophilicity and biological activity of the EPO glycoforms varied depending on the glycosylation pattern. This knowledge will pave the way for the development of homogeneous biologics by chemical synthesis.

Efficient synthesis of glycopeptide-α-thioesters with a high-mannose type oligosaccharide by means of tert-Boc-solid phase peptide synthesis.

High-mannose type oligosaccharides consist of nine mannose and two N-acetylglucosamine residues (Man(9)GlcNAc(2):M9) and play an important role in protein folding processes in the endoplasmic reticulum. A highly efficient preparation method of this asparaginyl-M9-oligosaccharide from hen egg yolk was established by a two-step proteolysis with commercially available proteases and subsequent purification using high performance liquid chromatography (HPLC). To avoid the hydrolysis of the desired M9-oligosaccharide during the proteolysis steps, several commercially available proteases were screened for their contamination with mannosidases. The α-amino group of the resultant H(2)N-Asn-(M9-oligosaccharide)-OH was protected with 9-fluorenylmethyloxycarbonyl (Fmoc) group for convenient separation by HPLC. The structure of Fmoc-Asn-(M9-oligosaccharide)-OH thus obtained was confirmed by ESI-MS spectrometry and several NMR experiments. Using this Fmoc-Asn-(M9-oligosaccharide)-OH, the synthesis of the M9-glycopeptide-α-thioester was demonstrated by means of tert-Boc-solid phase peptide synthesis. These tert-Boc conditions afforded the M9-glycopeptide-α-thioester in moderate yield.