Semisynthesis of Biologically Active Glycoforms of the Human Cytokine Interleukin 6

Human interleukin 6 (IL-6) is a potent cytokine with immunomodulatory properties. As the influence of N-glycosylation on the in vivo activities of IL-6 could not be elucidated so far, a semisynthesis of homogeneous glycoforms of IL-6 was established by sequential native chemical ligation. The four cysteines of IL-6 are convenient for ligations and require only the short synthetic glycopeptide 43–48. The Cys-peptide 49–183 could be obtained recombinantly by cleavage of a SUMO tag. The fragment 1–42 was accessible by the simultaneous cleavage of two inteins, leading to the 1–42 thioester with the native N-terminus. Ligation and refolding studies showed that the inherently labile AspPro bond 139–140 was detrimental for the sequential C- to N-terminal ligation. A reversed ligation sequence using glycopeptide hydrazides gave full-length IL-6 glycoproteins, which showed full bioactivity after efficient refolding and purification.