Design of Glycoengineered IL-4 Antagonists Employing Chemical and Biosynthetic Glycosylation.

Interleukin-4 (IL-4) plays a key role in atopic diseases. It coordinates T-helper cell differentiation to subtype 2, thereby directing defense toward humoral immunity. Together with Interleukin-13, IL-4 further induces immunoglobulin class switch to IgE. Antibodies of this type activate mast cells and basophilic and eosinophilic granulocytes, which release pro-inflammatory mediators accounting for the typical symptoms of atopic diseases. IL-4 and IL-13 are thus major targets for pharmaceutical intervention strategies to treat atopic diseases. Besides neutralizing antibodies against IL-4, IL-13, or its receptors, IL-4 antagonists can present valuable alternatives. Pitrakinra, an Escherichia coli-derived IL-4 antagonist, has been evaluated in clinical trials for asthma treatment in the past; however, deficits such as short serum lifetime and potential immunogenicity among others stopped further development. To overcome such deficits, PEGylation of therapeutically important proteins has been used to increase the lifetime and proteolytic stability. As an alternative, glycoengineering is an emerging strategy used to improve pharmacokinetics of protein therapeutics. In this study, we have established different strategies to attach glycan moieties to defined positions in IL-4. Different chemical attachment strategies employing thiol chemistry were used to attach a glucose molecule at amino acid position 121, thereby converting IL-4 into a highly effective antagonist. To enhance the proteolytic stability of this IL-4 antagonist, additional glycan structures were introduced by glycoengineering utilizing eucaryotic expression. IL-4 antagonists with a combination of chemical and biosynthetic glycoengineering could be useful as therapeutic alternatives to IL-4 neutralizing antibodies already used to treat atopic diseases.

Site-Directed Immobilization of BMP-2: Two Approaches for the Production of Innovative Osteoinductive Scaffolds.

The regenerative potential of bone is strongly impaired in pathological conditions, such as nonunion fractures. To support bone regeneration various scaffolds have been developed in the past, which have been functionalized with osteogenic growth factors such as bone morphogenetic proteins (BMPs). However, most of them required supra-physiological levels of these proteins leading to burst releases, thereby causing severe side effects. Site-specific, covalent coupling of BMP2 to implant materials might be an optimal strategy in order to overcome these problems. Therefore, we created a BMP-2 variant (BMP2-K3Plk) containing a noncanonical amino acid (propargyl-l-lysine) substitution introduced by genetic code expansion that allows for site-specific and covalent immobilization onto polymeric scaffold materials. To directly compare different coupling strategies, we also produced a BMP2 variant containing an additional cysteine residue (BMP2-A2C) allowing covalent coupling by thioether formation. The BMP2-K3Plk mutant was coupled to functionalized beads by a copper-catalyzed azide-alkyne cycloaddition (CuAAC) either directly or via a short biotin-PEG linker both with high specificity. After exposing the BMP-coated beads to C2C12 cells, ALP expression appeared locally restricted in close proximity to these beads, showing that both coupled BMP2 variants trigger cell differentiation. The advantage of our approach over non-site-directed immobilization techniques is the ability to produce fully defined osteogenic surfaces, allowing for lower BMP2 loads and concomitant higher bioactivities, for example, due to controlled orientation toward BMP2 receptors. Such products might provide superior bone healing capabilities with potential safety advantages as of homogeneous product outcome.

Interleukin-4-Clicked Surfaces Drive M2 Macrophage Polarization.

Driving macrophage (Mϕ) polarization into the M2 phenotype provides potential against inflammatory diseases. Interleukin-4 (IL-4) promotes polarization into the M2-Mϕ phenotype, but its systemic use is constrained by dose-limiting toxicity. Consequently, we developed IL-4-decorated surfaces aiming at sustained and localized activity. IL-4 muteins were generated by genetic code expansion; Lys42 was replaced by unnatural amino acids (uAAs). Both muteins showed cell-stimulation ability and binding affinity to IL4Rα similar to those of wt-IL-4. Copper-catalyzed (CuAAC) and copper-free strain-promoted (SPAAC) 1,3-dipolar azide-alkyne cycloadditions were used to site-selectively anchor IL-4 to agarose surfaces. These surfaces had sustained IL-4 activity, as demonstrated by TF-1 cell proliferation and M2, but not M1, polarization of M-CSF-generated human Mϕ. The approach provides a blueprint for the engineering of cytokine-activated surfaces profiled for sustained and spatially controlled activity.

Dynamin-dependent endocytosis of Bone Morphogenetic Protein2 (BMP2) and its receptors is dispensable for the initiation of Smad signaling.

Bone Morphogenetic Protein (BMP) signal transduction via the canonical Smad158 pathway has previously been linked to dynamin-dependent endocytosis, since the application of chemical inhibitors of clathrin or dynamin in functional cell culture based assays negatively affects initiation and propagation of the Smad response. More recent studies, however, demonstrated efficient Smad signaling by non-internalizable BMP2. The role of endocytosis in BMP signal transduction thus remained controversial. In our study we aimed to refine cell biological assays and to apply novel tools, including a new site-directed fluorescently labeled BMP2 ligand, to revisit key steps in BMP Smad signaling. We found that dynamin2 function was required for BMP2 uptake but was dispensable for C-terminal phosphorylation, nuclear translocation and transcriptional activity of BMP-dependent Smads. Furthermore, we demonstrated a role of dynamin2 in the regulation of steady-state and surface BMP receptor levels, as well as an impact on Smad1 protein level. Thus, dynamin2 allows for modulation of basal and ligand-dependent Smad signaling capacity. High levels of functional dynamin2 enhanced the myogenic differentiation of precursor cells. From our study we conclude that dynamin-dependent endocytosis serves as a regulatory mechanism to fine-tune Smad signaling, but it is not a prerequisite for signal initiation and propagation. Our findings contribute to the understanding of fundamental mechanisms of BMP signaling and thus provide important information for future consideration in the context of therapeutic applications of BMPs.

The clip-segment of the von Willebrand domain 1 of the BMP modulator protein Crossveinless 2 is preformed.

Bone Morphogenetic Proteins (BMPs) are secreted protein hormones that act as morphogens and exert essential roles during embryonic development of tissues and organs. Signaling by BMPs occurs via hetero-oligomerization of two types of serine/threonine kinase transmembrane receptors. Due to the small number of available receptors for a large number of BMP ligands ligand-receptor promiscuity presents an evident problem requiring additional regulatory mechanisms for ligand-specific signaling. Such additional regulation is achieved through a plethora of extracellular antagonists, among them members of the Chordin superfamily, that modulate BMP signaling activity by binding. The key-element in Chordin-related antagonists for interacting with BMPs is the von Willebrand type C (VWC) module, which is a small domain of about 50 to 60 residues occurring in many different proteins. Although a structure of the VWC domain of the Chordin-member Crossveinless 2 (CV2) bound to BMP-2 has been determined by X-ray crystallography, the molecular mechanism by which the VWC domain binds BMPs has remained unclear. Here we present the NMR structure of the Danio rerio CV2 VWC1 domain in its unbound state showing that the key features for high affinity binding to BMP-2 is a pre-oriented peptide loop.

Catalytically active filaments - pyruvate decarboxylase from Neurospora crassa. pH-controlled oligomer structure and catalytic function.

Pyruvate decarboxylase is a key enzyme in organisms whose energy metabolism is based on alcoholic fermentation. The enzyme catalyses the nonoxidative decarboxylation of 2-oxo acids in the presence of the cofactors thiamine diphosphate and magnesium ions. Pyruvate decarboxylase species from yeasts and plant seeds studied to date are allosterically activated by their substrate pyruvate. However, detailed kinetic studies on the enzyme from Neurospora crassa demonstrate for the first time the lack of substrate activation for a yeast pyruvate decarboxylase species. The quaternary structure of this enzyme species is also peculiar because it forms filamentous structures. The complex enzyme structure was analysed using a number of methods, including small-angle X-ray solution scattering, transmission electron microscopy, analytical ultracentrifugation and size-exclusion chromatography. These measurements were complemented by detailed kinetic studies in dependence on the pH.

Glycosylation of Twisted Gastrulation is Required for BMP Binding and Activity during Craniofacial Development.

Twisted gastrulation (TWSG1) is a conserved, secreted glycoprotein that modulates signaling of bone morphogenetic proteins (BMPs) in the extracellular space. Deletion of exon 4 of mouse Twsg1 (mTwsg1) is associated with significant craniofacial defects. However, little is understood about the biochemical properties of the corresponding region of the protein. We have uncovered a significant role for exon 4 sequences as encoding the only two glycosylation sites of the mTWSG1 protein. Deletion of the entire exon 4 or mutation of both glycosylation sites within exon 4 abolishes glycosylation of mTWSG1. Importantly, we find that constructs with mutated glycosylation sites have significantly reduced BMP binding activity. We further show that glycosylation and activity of TWSG1 recombinant proteins vary markedly by cellular source. Non-glycosylated mTWSG1 made in E. coli has both reduced affinity for BMPs, as shown by surface plasmon resonance analysis, and reduced BMP inhibitory activity in a mandibular explant culture system compared to glycosylated proteins made in insect cells or murine myeloma cells. This study highlights an essential role for glycosylation in Twisted gastrulation action.