Natural Glycoforms of Human Interleukin 6 Show Atypical Plasma Clearance

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.

Keywords: glycopeptides; glycoproteins; native chemical ligation; oligosaccharides; serum clearance.

Scheme 1

a) Retrosynthesis of IL‐6 glycoforms; b) N‐glycans detected in natural human IL‐6 (values in parentheses give percentage of total N‐glycans and were deduced from ref. [2]); c) structures of N‐glycans envisioned for systematically varied library of hIL‐6 glycoforms. The IL‐6 glycoforms marked in bold (Gn and G2) were available from previous work. ^[5]

Scheme 2

a) Chemical and enzymatic synthesis of IL‐6 glycopeptides B2–B9; b) glycosylamines G2, G3, G4, G7,and G8 employed for coupling with 3.

Scheme 3

a) Recombinant expression of fusion protein F and conversion to disulfide‐protected fragment C; b) native chemical ligation of segments A and B3–B9 to IL‐6 (1–48) hydrazides D3–D9 and conversion to thioesters E3–E9; c) native chemical ligation of thioesters E3–E9 with segment C followed by a two‐step refolding and oxidation of the full‐length glycopeptides H3–H9 to the IL‐6 glycoforms IL‐6³–IL‐6⁹.

Scheme 4

a) RP‐HPLC‐ESI‐TOF‐MS of glycoforms IL‐6³–IL‐6⁹ using acetonitrile/water + 0.1 % HCOOH gradients. b) direct injections of desalted IL‐6³–IL‐6⁹ (plain water) into ESI‐TOF mass spectrometer show gaussian distribution of charge states; c) overlay of the CD‐spectra of glycosylated IL‐6³–IL‐6⁹, d) SDS‐PAGE of glycoforms IL‐6¹–IL‐6⁹ (here termed 1–9).

Scheme 5

a) Structure superposition of glycosylated IL‐6¹ (PDB code 7NXZ, green) and non‐glycosylated IL‐6 (PDB code 1ALU, gray); b) Cα‐atom RMSD plot between both forms, showing the main deviations around the glycosylation site (Asn 44); c) enlargement of the Asn 44 glycosylation site containing helix A (Tyr 31 to Asn 44) showing the gradually increasing deviation towards and beyond the glycosylation site.

Scheme 6

Proliferation assay of IL‐6 glycoforms IL‐6¹–IL‐6⁹ using an IL‐6‐dependent Ba/F3 cell line.

Scheme 7

Normalized percentage of hIL‐6 glycoforms detected in rat serum after IV injection. The 10 % of max. values were chosen arbitrarily for a ranking of the plasma clearance.