GQC effectively uses chaperone-type enzymes and lectins such as UDP-glucose:glycoprotein glucosyltransferase (UGGT), calnexin/calreticulin, and glucosidases to generate natively folded glycoproteins from nascent glycosylpolypeptides. However, the individual processes of the GQC system at the molecular level are still not well understood. We chemically synthesized a series of several homogeneous glycoproteins bearing M9-high-mannose type oligosaccharides and their misfolded forms. We then used these glycoprotein probes to better understand the GQC folding processes8-12). Monitoring of their folding processes in the presence of GQC-enzymes and chaperones showed that the synthetic misfolded glycoproteins refolded to their native forms. Experiments using these glycoprotein-probes also demonstrated that the GQC fraction isolated from rat liver acts as a catalytic cycle regulated by the fast crosstalk of glucosylation/deglucosylation in order to accelerate the refolding of misfolded glycoproteins. In order to understand the role of N-sialyglycans, we selected EPO and designed an efficient synthesis strategy for the preparation of five homogeneous EPO glycoforms varying glycosylation positions (N24, N38 and N83) and their number with a homogeneous human-type biantennary sialyloligosaccharide3,5). All EPO glycoforms synthesized showed biological activity in vivo depending on glycosylation number and their position. On the other hand, EPOs lacking the 38 or 83 N-glycans were found to lose biological activity in vivo. We speculated that the hydrophobic protein surface found around 38 and 83 positions should be covered by a hydrophilic N-glycan3).Therefore, we synthesized EPO with a homogeneous triantennary sialyglycan13) at the 83 positions and then used it for biological assays. The EPO with a triantennary N-sialylglycan indeed showed very potent biological activity in vivo14). We have noticed that three N-glycans were found at the interface between EPO and EPO receptor when these made a complex. We hypothesized that these N-glycan regulate the binding processes of EPO and its receptor in the water layer. This presentation also introduces an interesting finding about how N-glycans behave in water.References 1) Y. Kajihara, et al. Prompt chemo-enzymatic synthesis of diverse complex-type oligosaccharides and its application to the solid-phase synthesis of a 2) N. Yamamoto, et al. Chemical Synthesis of a Glycoprotein Having an Intact Human Complex-Type Sialyloligosaccharide under the Boc and Fmoc 3) M. Murakami, et al. Chemical synthesis of erythropoietin glycoforms for insights into the relationship between glycosylation pattern and bioactivity. 4) I.Sakamoto, et al. Chemical Synthesis of homogeneous glycosyl-interferon-β that exhibits potent antitumor activity in vivo. J. Am. Chem. Soc. 2012, 134, 5) M. Murakami, et al. Chemical Synthesis of an Erythropoietin Glycoform Containing a Complex-type Disialyloligosaccharide. Angew. Chem. Int. Ed. 6) R. Okamoto; K. Iritani; Y. Amazaki; D. Zhao; C. Chandrashekar; Y. Maki; Y. Kanemitsu; T. Kaino; Y. Kajihara,Semisynthesis of a Homogeneous Glycoprotein Using Chemical Transformation of Peptides to Thioester Surrogates, J. Org. Chem. (2022), 87, 114-124. https://doi.org/10.1021/acs.joc.1c02031. 7) Nomura, K.; Maki, Y.; Okamoto, R.; Satoh, A.; Kajihara, Y. Glycoprotein Semisynthesis by Chemical Insertion of Glycosyl Asparagine Using a 8) M. Izumi, et al. Substrate recognition of glycoprotein folding sensor UGGT analyzed by site-specifically 15N-labeled glycopeptide and small 9) M. Izumi, et al. Synthesis of Glc1Man9-glycoprotein probes by a unique misfolding/enzymatic glucosylation/intentional misfolding approach. Angew. glycopeptide with Asn-linked sialyl-undeca- and asialo-nona-saccharides. Chem. Eur. J. 2004, 10, 971-985. doi.org/10.1002/chem.200305115.Synthetic Strategies. J. Am. Chem. Soc. 2008, 130, 501-510. doi.org/10.1021/ja072543f. Sci. Adv. 2016, 2:e1500678, doi.org/10.1126/sciadv.1500678.5428-5431. doi.org/10.1021/ja2109079.2012, 51, 3567-3572. doi.org/10.1002/anie.201109034. Bifunctional Thioacid-Mediated Strategy, J. Am. Chem. Soc. 2021, 143 (27), 10157-10167. https://doi.org/10.1021/jacs.1c02601glycopeptide library prepared by parallel native chemical ligation. J. Am. Chem. Soc. 2017, 139, 11421-11426. doi.org/10.1021/jacs.7b03277. Chem. Int. Ed. 2016, 128, 4036-4039. Doi.org/10.1002/anie.201511491.10) S. Dedola, et al. Folding of Synthetic Homogeneous Glycoproteins in the Presence of a Glycoprotein Folding Sensor Enzyme. Angew. Chem. Int. Ed. 2014, 53, 2883-2887. doi.org/10.1002/anie.201309665.11) M. Izumi, et al. Chemical Synthesis of intentionally Misfolded homogeneous Glycoprotein: a unique approach for the study of glycoprotein quality control. J. Am. Chem. Soc. 2012, 134, 7238-7241. doi.org/10.1021/ja3013177.12) T. Kiuchi, et al. Monitoring of glycoprotein quality control system with a series of chemically synthesized homogeneous native and misfolded glycoproteins. J. Am. Chem. Soc. 2018, 140, 17499–17507. doi.org/10.1021/jacs.8b0865313) Y. Maki, et al. Semisynthesis of intact complex-type triantennary oligosaccharides from a biantennary oligosaccharide isolated from a natural source by selective chemical and enzymatic glycosylation. J. Am. Chem. Soc. 2016, 138, 3461-3468. doi.org/10.1021/jacs.5b13098. 14) Y. Maki, et al. 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. J. Am. Chem. Soc. 2020, 142, 20671–20679. doi.org/10.1021/jacs.0c0871923Special LectureYasuhiro Kajihara
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