Abstract
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
Highlights
Many of the biological effects of the human cytokine interleukin 6 (IL-6) have been studied in detail,[1] little is known about the influence of the glycans present on this glycoprotein
In summary the chemoenzymatic semisynthesis of a systematic library of glycoforms of hIL-6 representing the most abundant as well as the minor N-glycans found on natural interleukin 6 (IL6) was accomplished
The sequential ligations followed by a two-step refolding/purification protocol was applicable to all glycoforms
Summary
Many of the biological effects of the human cytokine interleukin 6 (IL-6) have been studied in detail,[1] little is known about the influence of the glycans present on this glycoprotein. Based on the glycan structures identified earlier[2] we set out to provide a comprehensive library of IL-6 glycoforms including complex-type and oligomannosidic N-glycans for systematic studies. Angewandte Chemie International Edition published by Wiley-VCH GmbH. The low abundance triantennary N-glycan was not structurally defined and may be branched within the a1,3- or the a1,6-arm. To consider both possibilities and to generally investigate the effect of additional N-glycan branches on IL-6 we decided to incorporate tetraantennary N-glycans with terminal GlcNAc (Gn4), Gal (G4) or 2,6-linked sialic acid residues (G4S4) as a surrogate to maximize potential steric and multivalency effects in combination with sialylation/desialylation (Scheme 1 c)
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