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.
Subject(s)
Glycopeptides/metabolism , Interleukin-6/metabolism , Animals , Cell Line , Cell Proliferation/drug effects , Chromatography, High Pressure Liquid , Galactose/metabolism , Glycopeptides/blood , Glycopeptides/genetics , Glycosylation , Humans , Interleukin-6/blood , Interleukin-6/genetics , Interleukin-6/pharmacology , Mice , N-Acetylneuraminic Acid/metabolism , Rats , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/blood , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/pharmacology , Spectrometry, Mass, Electrospray IonizationABSTRACT
The main glycoforms of the hydrophobic lysosomal glycoprotein saposinâ D (SapD) were synthesized by native chemical ligation. An approach for the challenging solid-phase synthesis of the fragments was developed. Three SapD glycoforms were obtained following a general and robust refolding and purification protocol. A crystal structure of one glycoform confirmed its native structure and disulfide pattern. Functional assays revealed that the lipid-binding properties of three SapD glycoforms are highly affected by the single sugar moiety of SapD showing a dependency of the size and the type of N-glycan.
Subject(s)
Carbohydrates/chemistry , Saposins/chemical synthesis , Saposins/metabolism , Carbohydrate Conformation , Glycosylation , Humans , Hydrophobic and Hydrophilic Interactions , Particle Size , Saposins/chemistryABSTRACT
Human interleukinâ 6 (IL-6) is a potent cytokine with immunomodulatory properties. As the influence of N-glycosylation on the inâ 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 Asp-Pro 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.
