Natural Glycoforms of Human Interleukin 6 Show Atypical Plasma Clearance.

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.

A Single Route to Mammalian N-Glycans Substituted with Core Fucose and Bisecting GlcNAc.

The occurrence of α1,6-linked core fucose on the N-glycans of mammalian glycoproteins is involved in tumor progression and reduces the bioactivity of antibodies in antibody-dependent cell-mediated cytotoxicity (ADCC). Since core-fucosylated N-glycans are difficult to isolate from natural sources, only chemical or enzymatic synthesis can provide the desired compounds for biological studies. A general drawback of chemical α-fucosylation is that the chemical assembly of α1,6-linked fucosides is not stereospecific. A robust and general method for the α-selective fucosylation of acceptors with primary hydroxy groups in α/ß ratios exceeding 99:1 was developed. The high selectivities result from the interplay of an optimized protecting group pattern of the fucosyl donors in combination with the activation principle and the reaction conditions. Selective deprotection yielded versatile azides of all mammalian complex-type core-fucosylated N-glycans with 2-4 antennae and optional bisecting GlcNAc.

Structure of the Y. pseudotuberculosis adhesin InvasinE.

Enteropathogenic Yersinia expresses several invasins that are fundamental virulence factors required for adherence and colonization of tissues in the host. Within the invasin-family of Yersinia adhesins, to date only Invasin has been extensively studied at both structural and functional levels. In this work, we structurally characterize the recently identified inverse autotransporter InvasinE from Yersinia pseudotuberculosis (formerly InvasinD from Yersinia pseudotuberculosis strain IP31758) that belongs to the invasin-family of proteins. The sequence of the C-terminal adhesion domain of InvasinE differs significantly from that of other members of the Yersinia invasin-family and its detailed cellular and molecular function remains elusive. In this work, we present the 1.7 Å crystal structure of the adhesion domain of InvasinE along with two Immunoglobulin-like domains. The structure reveals a rod shaped architecture, confirmed by small angle X-ray scattering in solution. The adhesion domain exhibits strong structural similarities to the C-type lectin-like domain of Yersinia pseudotuberculosis Invasin and enteropathogenic/enterohemorrhagic E. coli Intimin. However, despite the overall structural similarity, the C-type lectin-like domain in InvasinE lacks motifs required for Ca2+ /carbohydrate binding as well as sequence or structural features critical for Tir binding in Intimin and ß1 -integrin binding in Invasin, suggesting that InvasinE targets a distinct, yet unidentified molecule on the host-cell surface. Although the biological role and target molecule of InvasinE remain to be elucidated, our structural data provide novel insights into the architecture of invasin-family proteins and a platform for further studies towards unraveling the function of InvasinE in the context of infection and host colonization.

Synthetic Glycoforms Reveal Carbohydrate-Dependent Bioactivity of Human Saposin†D.

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.

Highly Efficient Synthesis of Multiantennary Bisected N-glycans Based on Imidates.

The occurrence of N-glycans with a bisecting GlcNAc modification on glycoproteins has many implications in developmental and immune biology. However, these particular N-glycans are difficult to obtain either from nature or through synthesis. We have developed a flexible and general method for synthesizing bisected N-glycans of the complex type by employing modular TFAc-protected donors for all antennae. The TFAc-protected N-glycans are suitable for the late introduction of a bisecting GlcNAc. This integrated strategy permits for the first time the use of a single approach for multiantennary N-glycans as well as their bisected derivatives via imidates, with unprecedented yields even in a one-pot double glycosylation. With this new method, rare N-glycans of the bisected type can be obtained readily, thereby providing defined tools to decipher the biological roles of bisecting GlcNAc modifications.