Uncovering a Latent Bioactive Interleukin-6 Glycoform

• Published in: Angewandte Chemie International Edition p. e202411213
• Main Authors: Kajihara, Yasuhiro, Liu, Yanbo, Maki, Yuta, Okamoto, Ryo, Satoh, Ayano, Todokoro, Yasuto, Kanemitsu, Yurie, Otani, Keito
• Format: Journal Article
• Language: English
• Published: Germany 24.10.2024
• Subjects: Glycoprotein Protein folding chemistry Protein aggregation Protein synthesis Interleukin 6
• ISSN: 1433-7851; 1521-3773; 1521-3773
• DOI: 10.1002/anie.202411213

A bioinspired semisynthesis of human-interleukin-6 bearing N-glycan at Asn143 (143glycosyl-IL-6) was performed by intentional glycosylation effects and protein folding chemistry for regioselective peptide-backbone activation. 143Glycosyl-IL-6 is a genetically coded cytokine, but isolation was difficult owing to a tiny amount. IL6-polypeptide (1-141-position) with an intentionally inserted cysteine at 142-position was expressed in E. coli. The expressed polypeptide was treated with a chemical folding process to make a specific helices bundle conformation through native two-disulfide bonds (43-49 and 72-82). Utilizing the successfully formed free-142-cysteine, sequential conversions using cyanation of 142-cysteine, hydrazinolysis, and thioesterification created a long polypeptide (1-141)-thioester. However, the resultant polypeptide-thioester caused considerable aggregation owing to a highly hydrophobic peptide sequence. After the reduction of two-disulfide bonds of polypeptide (1-141)-thioester, an unprecedented hydrophilic N-glycan tag was inserted at the resultant cysteine thiols. The N-glycan tags greatly stabilized polypeptide-thioester. The subsequent native chemical ligation and desulfurization successfully gave a whole 143glycosyl-IL-6 polypeptide (183-amino acids). Removal of four N-glycan tags and immediate one-pot in vitro folding protocol efficiently produced the folded 143glycosyl-IL-6. The folded 143glycosyl-IL-6 exhibited potent cell proliferation activity. The combined studies with molecular dynamics simulation, semisynthesis, and bioassays predict the bioactive conformation of latent 143glycosyl-IL-6.