Semaphorin-3A (Sema-3A) is a chemorepellant protein with various biological functions, including kidney development. It interacts with a protein complex consisting of the receptors neuropilin-1 (NRP-1) and plexin-A1. After acute kidney injury, Sema-3A is overexpressed and secreted, leading to a loss of kidney function. The development of peptide inhibitors is a promising approach to modulate the interaction of Sema-3A with its receptor NRP-1. Few interaction points between these binding partners are known. However, an immunoglobulin-like domain-derived peptide of Sema-3A has shown a positive effect on cell proliferation. To specify these interactions between the peptide inhibitor and the Sema-3A-NRP-1 system, the peptides were modified with the photoactivatable amino acids 4-benzoyl-l-phenylalanine or photo-l-leucine by solid-phase peptide synthesis. Activity was tested by an enzyme-linked immunosorbent-based binding assay, and crosslinking experiments were analyzed by Western blot and mass spectrometry, demonstrating a specific binding site of the peptide at Sema-3A. The observed signals for Sema-3A-peptide interaction were found in a defined area of the Sema domain, which was also demonstrated to be involved in NRP-1 binding. The presented data identified the interaction site for further development of therapeutic peptides to treat acute kidney injury by blocking the Sema-3A-NRP-1 interaction.

The maintenance of therapeutic glycoproteins within the circulatory system is associated, in large part, with the integrity of sialic acids as terminal sugars on the glycans. Glycoprotein desialylation, either by spontaneous cleavage or through host sialidases, leads to protein clearance, mainly through the liver. Thus, the installation of minimally modified sialic acids that are hydrolysis-resistant yet biologically equivalent should lead to increased circulatory half-lives and improved pharmacokinetic profiles. Here we describe the chemoenzymatic synthesis of CMP–sialic acid sugar donors bearing fluorine atoms at the 7-position, starting from the corresponding 4-deoxy-4-fluoro-N-acetylhexosamine precursors. For the derivative with natural stereochemistry we observe efficient glycosyl transfer by sialyltransferases, along with improved stability of the resultant 7-fluorosialosides toward spontaneous hydrolysis (3- to 5-fold) and toward cleavage by GH33 sialidases (40- to 250-fold). Taking advantage of the rapid transfer of 7-fluorosialic acid by sialyltransferases, we engineered the O-glycan of Interferon α-2b and the N-glycans of the therapeutic glycoprotein α1-antitrypsin. Studies of the uptake of the glyco-engineered α1-antitrypsin by HepG2 liver cells demonstrated the bioequivalence of 7-fluorosialic acid to sialic acid in suppressing interaction with liver cell lectins. In vivo pharmacokinetic studies reveal enhanced half-life of the protein decorated with 7-fluorosialic acid relative to unmodified sialic acid in the murine circulatory system. 7-Fluorosialylation therefore offers considerable promise as a means of prolonging circulatory half-lives of glycoproteins and may pave the way toward biobetters for therapeutic use.

Polysialyltransferases (polySTs) are glycosyltransferases that synthesize polymers of sialic acid found in vertebrates and some bacterial pathogens. Bacterial polySTs have utility in the modification of therapeutic proteins to improve serum half-life, and the potential for tissue engineering. PolySTs are membrane-associated proteins and as recombinant proteins suffer from inherently low solubility, low expression levels and poor thermal stability. To improve their physicochemical and biochemical properties, we applied a directed evolution approach using a FACS-based ultrahigh-throughput assay as a simple, robust and reliable screening method. We were able to enrich a large mutant library and, in combination with plate-based high-throughput secondary screening, we discovered mutants with increased enzymatic activity and improved stability compared to the wildtype enzyme. This work presents a powerful strategy for the screening of directed evolution libraries of bacterial polySTs to identify better catalysts for in vitro polysialylation of therapeutics.

CXCL14, chemokine (C-X-C motif) ligand 14, is a novel highly conserved chemokine with unique features. Despite exhibiting the typical chemokine fold, it has a very short N-terminus of just two amino acid residues responsible for chemokine receptor activation. CXCL14 actively participates in homeostatic immune surveillance of skin and mucosae, is linked to metabolic disorders and fibrotic lung diseases and possesses strong anti-angiogenic properties in early tumor development. In this work, we investigated the interaction of CXCL14 with various glycosaminoglycans (GAGs) by nuclear magnetic resonance spectroscopy, microscale thermophoresis, analytical heparin (HE) affinity chromatography and in silico approaches to understand the molecular basis of GAG-binding. We observed different GAG-binding modes specific for the GAG type used in the study. In particular, the CXCL14 epitope for HE suggests a binding pose distinguishable from the ones of the other GAGs investigated (hyaluronic acid, chondroitin sulfate-A/C, -D, dermatan sulfate). This observation is also supported by computational methods that included molecular docking, molecular dynamics and free energy calculations. Based on our results, we suggest that distinct GAG sulfation patterns confer specificity beyond simple electrostatic interactions usually considered to represent the driving forces in protein-GAG interactions. The CXCL14-GAG system represents a promising approach to investigate the specificity of GAG-protein interactions, which represents an important topic for developing the rational approaches to novel strategies in regenerative medicine.

The chemokine interleukin-8 (IL-8, CXCL8) plays an important role in inflammatory processes and consecutive wound healing. It recruits primarily neutrophils to infection sites and stimulates their degranulation and phagocytosis in effector cells. IL-8 binds glycosaminoglycans (GAGs), a class of complex linear anionic polysaccharides often organized into diversely sulfated micro-domains, that enriches the protein concentration locally and so facilitate the formation of stable concentration gradients. In this study, we applied experimental and computational techniques to investigate the binding of wild type and truncated IL-8 variants to natural and chemically modified GAGs to gain further insight into the IL-8/GAG interaction. Circular dichroism spectroscopy of IL-8 variants did not reveal major structural changes upon GAG binding. Heparin affinity chromatography clearly demonstrates that gradual truncation of the C-terminal helix leads to decreasing affinities. Similarly, surface plasmon resonance indicates participation of both IL-8 termini in GAG binding, which strength is dependent on GAG sulfation degree. Molecular modeling suggests that C-terminal truncation of IL-8 weakens the interaction with GAGs by an alteration of IL-8 GAG binding site. Our study provides more detailed understanding of the IL-8/GAG interaction and contributes to the data of potential use for the development of biomedical implications in tissue regeneration.

Polysialic acid (polySia) is a homopolymeric saccharide that is associated with some neuroinvasive pathogens and is found on selective cell types in their eukaryotic host. The presence of a polySia capsule on these bacterial pathogens helps with resistance to phagocytosis, cationic microbial peptides and bactericidal antibody production. The biosynthesis of bacterial polySia is catalysed by a single polysialyltransferase (PST) transferring sialic acid from a nucleotide-activated donor to a lipid-linked acceptor oligosaccharide. Here we present the X-ray structure of the bacterial PST from Mannheimia haemolytica serotype A2, thereby defining the architecture of this class of enzymes representing the GT38 family. The structure reveals a prominent electropositive groove between the two Rossmann-like domains forming the GT-B fold that is suitable for binding of polySia chain products. Complex structures of PST with a sugar donor analogue and an acceptor mimetic combined with kinetic studies of PST active site mutants provide insight into the principles of substrate binding and catalysis. Our results are the basis for a molecular understanding of polySia biosynthesis in bacteria and might assist the production of polysialylated therapeutic reagents and the development of novel antibiotics.

The stromal cell-derived factor 1α (CXCL12) belongs to the CXC chemokine family and plays an important role in tissue regeneration and the recruitment of stem cells. Here, a stable chemotactic gradient is essential that is formed by the interaction of CXCL12 with the extracellular matrix. Binding properties of CXCL12 to naturally occurring glycosaminoglycans (GAGs) as well as to the artificial highly sulfated hyaluronic acid (HA) are investigated by using a combination of NMR spectroscopy, molecular modeling and molecular dynamics simulations. Our results demonstrate a preferred protein binding for the sulfated GAGs heparin (HE) and highly sulfated HA. Furthermore, we could demonstrate that the orientation of the sulfate is crucial for binding. All sulfated GAGs interact with the CXCL12 GAG-binding motif (K24-H25-L26-K27-R41-K43-R47), where K27 and R41 represent the anchor points. Furthermore, differences could be observed in the second interaction interface of CXCL12: both HE and highly sulfated HA interfere with the receptor-binding motif, while chondroitin sulfate binds different amino acids in close proximity to this motif. CXCL12 does not interact with HA, which was directly demonstrated by NMR spectroscopy and molecular modeling and explained by the lack of sulfate groups of the HA molecule.

In recent years, there has been a growing interest in teichoic acids as targets for antibiotic drug design against major clinical pathogens such as Staphylococcus aureus, reflecting the disquieting increase in antibiotic resistance and the historical success of bacterial cell wall components as drug targets. It is now becoming clear that β-O-GlcNAcylation of S. aureus wall teichoic acids plays a major role in both pathogenicity and antibiotic resistance. Here we present the first structure of S. aureus TarS, the enzyme responsible for polyribitol phosphate β-O-GlcNAcylation. Using a divide and conquer strategy, we obtained crystal structures of various TarS constructs, mapping high resolution overlapping N-terminal and C-terminal structures onto a lower resolution full-length structure that resulted in a high resolution view of the entire enzyme. Using the N-terminal structure that encapsulates the catalytic domain, we furthermore captured several snapshots of TarS, including the native structure, the UDP-GlcNAc donor complex, and the UDP product complex. These structures along with structure-guided mutants allowed us to elucidate various catalytic features and identify key active site residues and catalytic loop rearrangements that provide a valuable platform for anti-MRSA drug design. We furthermore observed for the first time the presence of a trimerization domain composed of stacked carbohydrate binding modules, commonly observed in starch active enzymes, but adapted here for a poly sugar-phosphate glycosyltransferase.

Cell fate decisions in many physiological processes, including embryogenesis, stem cell niche homeostasis and wound healing, are regulated by secretion of small signaling proteins, called cytokines, from source cells to their neighbors or into the environment. Concentration level and steepness of the resulting paracrine gradients elicit different cell responses, including proliferation, differentiation or chemotaxis. For an in-depth analysis of underlying mechanisms, in vitro models are required to mimic in vivo cytokine gradients. We set up a microparticle-based system to establish short-range cytokine gradients in a three-dimensional extracellular matrix context. To provide native binding sites for cytokines, agarose microparticles were functionalized with different glycosaminoglycans (GAG). After protein was loaded onto microparticles, its slow release was quantified by confocal microscopy and fluorescence correlation spectroscopy. Besides the model protein lysozyme, SDF-1 was used as a relevant chemokine for hematopoietic stem and progenitor cell (HSPC) chemotaxis. For both proteins we found gradients ranging up to 50 μm from the microparticle surface and concentrations in the order of nM to pM in dependence on loading concentration and affinity modulation by the GAG functionalization. Directed chemotactic migration of cells from a hematopoietic cell line (FDCPmix) and primary murine HSPC (Sca-1+ CD150+ CD48−) toward the SDF-1-laden microparticles proved functional short-range gradients in a two-dimensional and three-dimensional setting over time periods of many hours. The approach has the potential to be applied to other cytokines mimicking paracrine cell–cell interactions in vitro.

Structural studies of human polysialyltransferase ST8SialII in apo form and in complex with donor sugar and sulfated glycan acceptor shed light on the substrate binding and specificity as well as the catalytic activity of this class of polysialyltransferases.

In the search for effective therapeutic strategies, protein‐based biologicals are under intense development. While monoclonal antibodies represent the majority of these drugs, other innovative approaches are exploring the use of scaffold proteins for the creation of binding molecules with tailor‐made properties. Ubiquitin is especially suited for this strategy due to several key characteristics. Ubiquitin is a natural serum protein, 100% conserved across the mammalian class and possesses high thermal, structural and proteolytic stability. Because of its small size and lack of posttranslational modifications, it can be easily produced inEscherichia coli. In this work we provide evidence that ubiquitin is safe as tested experimentallyin vivo. In contrast to previously published results, we show that, in our hands, ubiquitin does not act as a functional ligand of the chemokine receptor CXCR4. Cellular assays based on different signaling pathways of the receptor were conducted with the natural agonist SDF‐1 as a benchmark. In none of the assays could a response to ubiquitin treatment be elicited. Furthermore, intravenous application to mice at high concentrations did not induce any detectable effect on cytokine levels or hematological parameters.

Significance This paper describes the structure of Staphylococcus aureus TarM, an enzyme responsible for the glycosylation of wall teichoic acid that is important in pathological processes such as host immunity, phage binding, and antibiotic resistance in strains such as Methicillin-resistant S. aureus . The TarM structure is presented in an unusual ternary-like complex that features a polymeric acceptor substrate analogue and a trapped product of enzyme action, lending novel structural and mechanistic insight into the glycosylation of glycopolymers. More generally, the positioning of this product in the active site as well as the distorted conformation of its pyranose ring provide direct structural evidence for an internal substitution-like catalytic mechanism for retaining GT-B class enzymes.

AbstractA set of photoactivatable SDF‐1α analogues by introduction of O‐acyl isopeptides into proteins using expressed protein ligation is prepared.

Preparation of smart materials by coatings of established surfaces with biomolecules will lead to the next generation of functionalized biomaterials. Rejection of implants is still a major problem in medical applications but masking the implant material with protein coatings is a promising approach. These layers not only disguise the material but also equip it with a certain biological function. The anti-inflammatory chemokine stromal cell-derived factor 1α (SDF-1α) is well suited to take over this function, because it efficiently attracts stem cells and promotes their differentiation and proliferation. At least the initial stem cell homing requires the formation of a concentration gradient. Thus, a reliable and robust release mechanism of SDF-1α from the material is essential. Several proteases, most notably matrix metalloproteinases, are upregulated during inflammation, which, in principle, can be exploited for a tightly controlled release of SDF-1α. Herein, we present the covalent immobilization of M-[S4V]-SDF-1α on novel biodegradable polymer films, which consist of heterobifunctional poly(ethylene glycol) and oligolactide-based functionalized macromers. A peptidic linker with a trimeric matrix metalloproteinase 9 (MMP-9) cleavage site (MCS) was used as connection and the linkage between the three components was achieved by combination of expressed protein ligation and Cu(I) catalyzed azide/alkyne cycloaddition. The MCS was used for MMP-9 mediated release of M-[S4V]-SDF-1α from the biomaterial and the released SDF-1α derivative was biologically active and induced strong cell migration, which demonstrates the great potential of this system.

In order to link structural features on a molecular level to the function of chemokines, site-specific modification strategies are strongly required. These can be used to incorporate fluorescent dyes and/or physical probes to allow investigations in a wide range of biological and physical techniques, e.g., nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, fluorescence resonance energy transfer (FRET), or fluorescence correlation spectroscopy (FCS). Only a limited number of functional groups within the 20 canonical amino acids allow ligation strategies that can be helpful to introduce novel functionalities, which in turn expand the scope of chemoselective and orthogonal reactivity of (semi)synthetic chemokines. In the present chapter we mainly focus on the fabulous history of native chemical ligation (NCL) and provide a general protocol for the preparation of C-terminally modified SDF-1α including tips and tricks for practical work. We believe that this protocol can be easily adapted to other chemokines and many proteins in general.

The CXC chemokine stromal cell-derived factor-1α (SDF-1α, CXCL12) has been proven to recruit CXCR4 positive stem and progenitor cells of different sources to defected heart sites, with significant clinical benefits. However, the rapid proteolytic inactivation by inflammation-related proteases, inaccurate drug delivery or inappropriate local concentrations belong to the largest disadvantages for feasible application. Herein, we present a switchable, biased-like SDF-1α variant, AAV-[S4V]-SDF-1α, whose distinct activity is coupled to the inflammation-associated presence of dipeptidylpeptidase-4 (DPP-4), which cleaves an alanine-alanine dipeptide from the precursor. We decorated starPEG-heparin hydrogels with our novel SDF-1α variant and tested them for immobilization efficiency, time-dependent protein release as well as mobilization of early endothelial progenitor cells (eEPCs) in vitro. We found higher migration rates compared to conventional SDF-1α. In summary, we provide a conceptual work on cooperative effects of enzymatically activatable SDF-1α and starPEG-heparin hydrogels.

The interactions between glycosaminoglycans (GAGs), important components of the extracellular matrix, and proteins such as growth factors and chemokines play critical roles in cellular regulation processes. Therefore, the design of GAG derivatives for the development of innovative materials with bio-like properties in terms of their interaction with regulatory proteins is of great interest for tissue engineering and regenerative medicine. Previous work on the chemokine interleukin-8 (IL-8) has focused on its interaction with heparin and heparan sulfate, which regulate chemokine function. However, the extracellular matrix contains other GAGs, such as hyaluronic acid (HA), dermatan sulfate (DS) and chondroitin sulfate (CS), which have so far not been characterized in terms of their distinct molecular recognition properties towards IL-8 in relation to their length and sulfation patterns. NMR and molecular modeling have been in great part the methods of choice to study the structural and recognition properties of GAGs and their protein complexes. However, separately these methods have challenges to cope with the high degree of similarity and flexibility that GAGs exhibit. In this work, we combine fluorescence spectroscopy, NMR experiments, docking and molecular dynamics simulations to study the configurational and recognition properties of IL-8 towards a series of HA and CS derivatives and DS. We analyze the effects of GAG length and sulfation patterns in binding strength and specificity, and the influence of GAG binding on IL-8 dimer formation. Our results highlight the importance of combining experimental and theoretical approaches to obtain a better understanding of the molecular recognition properties of GAG–protein systems.