Ligand binding of interleukin-8: a comparison of glycosaminoglycans and acidic peptides.

Recognition and binding of regulatory proteins to glycosaminoglycans (GAGs) from the extracellular matrix is a process of high biological importance. The interaction between negatively charged sulfate or carboxyl groups of the GAGs and clusters of basic amino acids on the protein is crucial in this binding process and it is believed that electrostatics represent the key factor for this interaction. However, given the rather undirected nature of electrostatics, it is important to achieve a clear understanding of its role in protein-GAG interactions and how specificity and selectivity in these systems can be achieved, when the classical key-lock binding motif is not applicable. Here, we compare protein binding of a highly charged heparin (HP) hexasaccharide with four de novo designed decapeptides of varying negative net charge. The charge density of these peptides was comparable to typical GAGs of the extracellular matrix. We used the regulatory protein interleukin-8 (IL-8) because its interactions with GAGs are well described. All four peptide ligands bind to the same epitope of IL-8 but show much weaker binding affinity as revealed in 1H-15N HSQC NMR titration experiments. Complementary molecular docking and molecular dynamics simulations revealed further atomistic details of the interaction mode of GAG versus peptide ligands. Overall, similar contributions to the binding energy and hydrogen bond formation are determined for HP and the highly charged peptides, suggesting that the entropic loss of the peptides upon binding likely account for the remarkably different affinity of GAG versus peptide ligands to IL-8.

Sulfation Pattern Dependent Iron(III) Mediated Interleukin-8 Glycan Binding.

Cytokines such as interleukin-8 activate the immune system during infection and interact with sulfated glycosaminoglycans with specific sulfation patterns. In some cases, these interactions are mediated by metal ion binding which can be used to tune surface-based glycan-protein interactions. We evaluated the effect of both hyaluronan sulfation degree and Fe3+ on interleukin-8 binding by electrochemical impedance spectroscopy and surface characterizations. Our results show that sulfation degree and metal ion interactions have a synergistic effect in tuning the electrochemical response of the glycated surfaces to the cytokine.

NMR and molecular modeling reveal specificity of the interactions between CXCL14 and glycosaminoglycans.

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.

A Selective Mucin/Methylcellulose Hybrid Gel with Tailored Mechanical Properties.

Mucin glycoproteins are key components of native mucus which serves as an initial barrier in the human body against microbial attack. Mucins are able to prevent bacterial adhesion and can trap viruses. However, the weak mechanical properties of mucin solutions have so far prevented their application in a physiological environment. Here, methylcellulose biopolymers are used as mechanical adjuvants to overcome this limitation and generate a thermoresponsive mucin/methylcellulose hybrid system. The hybrid material developed combines the selective permeability properties brought about by mucins with the thermal autogelation properties of methylcellulose. As a consequence, triggered by contact with body-warm surfaces, the hybrid material rapidly forms a gel at physiological conditions, and this external temperature stimulus can also be harnessed to stimulate drug release from incorporated thermosensitive liposomes. Finally, the hybrid gel selectively retards the release of embedded molecules which can be used to further control and prolong drug release from the material.

Scleral cross-linking by riboflavin and blue light application in young rabbits: damage threshold and eye growth inhibition.

BACKGROUND: Scleral cross-linking (SXL) by riboflavin and light application has been introduced as a possible treatment to increase scleral tissue stiffness and to inhibit excessive axial elongation of highly myopic eyes. We evaluated an ocular tissue damage threshold for blue light irradiation, and used SXL treatment to induce eye growth inhibition. METHODS: The sclera of 3-week-old rabbits (39 pigmented and 15 albino rabbits) were treated with different blue light intensities (450 ± 50 nm) and riboflavin. Alterations and a damage threshold were detected in ocular tissues by means of light microscopy and immunohistochemistry. The influence of SXL on the eye growth was examined in 21 young rabbits and was measured by using A-scan ultrasonography, micrometer caliper, and for selected eyes additionally by MR imaging. RESULTS: Light microscopic examinations demonstrated degenerative changes in ocular tissue after irradiation with blue light intensities above 400 mW/cm(2) (with and without riboflavin application). Therefore, that light intensity was defined as the damage threshold. Tissue alteration in retina, choroid, and sclera and activation of retinal microglia cells and Müller cells could be earlier observed at blue light intensities of 150 and 200 mW/cm(2). Albino rabbits were less sensitive to this SXL treatment. A significant reduction of the eye growth could be detected by SXL treatment with the minimal efficient blue light intensity of 15 mW/cm(2) and maintained stable for 24 weeks. CONCLUSIONS: SXL with riboflavin and blue light intensities below a defined damage threshold can induce a long lasting growth inhibitory effect on young rabbit eyes. Therefore, SXL might be a realistic approach to inhibit eye elongation in highly myopic eyes.