Natural Glycoforms of Human Interleukin 6 Show Atypical Plasma Clearance

A library of glycoforms of human interleukin 6 (IL‐6) comprising complex and mannosidic N‐glycans was generated by semisynthesis. The three segments were connected by sequential native chemical ligation followed by two‐step refolding. The central glycopeptide segments were assembled by pseudoproline‐assisted Lansbury aspartylation and subsequent enzymatic elongation of complex N‐glycans. Nine IL‐6 glycoforms were synthesized, seven of which were evaluated for in vivo plasma clearance in rats and compared to non‐glycosylated recombinant IL‐6 from E. coli. Each IL‐6 glycoform was tested in three animals and reproducibly showed individual serum clearances depending on the structure of the N‐glycan. The clearance rates were atypical, since the 2,6‐sialylated glycoforms of IL‐6 cleared faster than the corresponding asialo IL‐6 with terminal galactoses. Compared to non‐glycosylated IL‐6 the plasma clearance of IL‐6 glycoforms was delayed in the presence of larger and multibranched N‐glycans in most cases Sugars affect plasma lifetime: The main glycoforms of human interleukin 6 (IL‐6) were synthesized by native chemical ligation combining chemical, enzymatic, and recombinant methods. The native fold was evident from CD spectra, mass spectrometry, crystallography, and receptor binding. In vivo assays showed a different plasma clearance for each glycoform with an unexpected correlation to the glycan structures.