The main glycoforms of the hydrophobic lysosomal glycoprotein saposin 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.

The main glycoforms of the hydrophobic lysosomal glycoprotein saposin 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 ag eneral and robust refolding and purification protocol. Acrystal 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 ad ependency of the size and the type of Nglycan.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under http://dx.

Recombinant human erythropoietin (EPO) is the main therapeutic glycoprotein for the treatment of anemia in cancer and kidney patients.T he in-vivo activity of EPO is carbohydrate-dependent with the number of sialic acid residues regulating its circulatory half-life.E PO carries three Nglycans and thus obtaining pure glycoforms provides am ajor challenge.W eh ave developed ar obust and reproducible chemoenzymatic approach to glycoforms of EPO with and without sialic acids.E PO was assembled by sequential native chemical ligation of two peptide and three glycopeptide segments.The glycopeptides were obtained by pseudoprolineassisted Lansbury aspartylation. Enzymatic introduction of the sialic acids was readily accomplished at the level of the glycopeptide segments but even more efficiently on the refolded glycoprotein. Biological recognition of the synthetic EPOs was shown by formation of 1:1c omplexes with recombinant EPO receptor.

Recombinant human erythropoietin (EPO) is the main therapeutic glycoprotein for the treatment of anemia in cancer and kidney patients.T he in-vivo activity of EPO is carbohydrate-dependent with the number of sialic acid residues regulating its circulatory half-life.E PO carries three Nglycans and thus obtaining pure glycoforms provides am ajor challenge.W eh ave developed ar obust and reproducible chemoenzymatic approach to glycoforms of EPO with and without sialic acids.E PO was assembled by sequential native chemical ligation of two peptide and three glycopeptide segments.The glycopeptides were obtained by pseudoprolineassisted Lansbury aspartylation. Enzymatic introduction of the sialic acids was readily accomplished at the level of the glycopeptide segments but even more efficiently on the refolded glycoprotein. Biological recognition of the synthetic EPOs was shown by formation of 1:1c omplexes with recombinant EPO receptor.

Alibrary 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.N ine IL-6 glycoforms were synthesized,s even of which were evaluated for in vivo plasma clearance in rats and compared to nonglycosylated 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