By using chemically synthesized homogeneous glycoprotein in both correctly folded form and misfolded form, the UGGT recognition pattern as well as glycoprotein quality control system in detail was systematically investigated. in 2018, the Kajihara group synthesized four glycoform of EPO in both correctly folded and misfolded form.[34] The chemical synthesis is achieved by SPPS toward both glycopeptides and non-glycosylated peptides, followed with sequential NCL to afford full-length EPO bearing three M9 high-mannose type N-glycan at Asn24, 38 and 83. The in vitro folding under dialysis and redox condition afford both correctly folded and heterogeneous misfolded EPO 110. The four pairs of folded/misfolded EPO glycoforms were used for UGGT recognition assay. (Fig 13, top right) However, unexpectedly, all these glycoproteins are recognized by UGGT to give G1M9 glucosylated product 112. The authors hypothesized that UGGT might recognize the hydrophobic area on protein surface as the misfolded characteristic nature.

In order to better understand how the refolding of misfolded glycoprotein proceeded in glycoprotein quality control (GQC) system, the synthetic misfolded glycoprotein EPO 110 and IL-8 111 was treated with isolated ER lysate contains all GQC enzyme and chaperons. (Fig13, bottom right) These assays elucidated that a fast glucosylation / deglucosylation process exist during GQC process and the folding intermediate G1M9-misfolded glycoprotein was efficiently converted to native M9 IL-8 113. This research demonstrated that synthetic homogeneous misfolded glycoprotein can be used as a powerful tool for investigating GQC process in detail.[34]

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