RESUMEN
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.
Asunto(s)
Glicopéptidos/metabolismo , Interleucina-6/metabolismo , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Galactosa/metabolismo , Glicopéptidos/sangre , Glicopéptidos/genética , Glicosilación , Humanos , Interleucina-6/sangre , Interleucina-6/genética , Interleucina-6/farmacología , Ratones , Ácido N-Acetilneuramínico/metabolismo , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/sangre , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
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.
Asunto(s)
Carbohidratos/química , Saposinas/síntesis química , Saposinas/metabolismo , Conformación de Carbohidratos , Glicosilación , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Tamaño de la Partícula , Saposinas/químicaRESUMEN
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.