CXCL17 binds efficaciously to glycosaminoglycans with the potential to modulate chemokine signaling.

Introduction: CXCL17 is a mucosally secreted protein, and the most recently identified human chemokine, an assignment based on protein fold prediction and chemotactic activity for leukocytes. However, these credentials have been the subject of much recent discussion and no experimental evidence has been presented regarding the definitive structure of CXCL17. In this study, we evaluated the structural and chemoattractant credentials of CXCL17 to better characterize this molecule, and gain deeper insights into its functional role as a glycosaminoglycan (GAG) binding protein. Methods: In the absence of structural information, in silico modeling techniques assessed the likelihood of CXCL17 adopting a chemokine fold. Recombinant CXCL17 was synthesized in mammalian and prokaryotic systems. Modified Boyden chamber and real-time chemotaxis assays assessed the ability of CXCL17 to promote chemotaxis of murine splenocytes, human neutrophils, and CXCR1 transfectants. The efficacy of CXCL17 binding to GAGs was quantified with solid-phase assays and bio-layer interferometry techniques. Results: All modeling efforts failed to support classification of CXCL17 as a chemokine based on its predicted conformation. Recombinant CXCL17 was observed to dimerize as a function of concentration, a characteristic of several chemokines. Contrary to a previous report, CXCL17 was not chemotactic for murine splenocytes, although it was a low-potency chemoattractant for human neutrophils at micromolar concentrations, several orders of magnitude higher than those required for CXCL8. As anticipated owing to its highly basic nature, CXCL17 bound to GAGs robustly, with key C-terminal motifs implicated in this process. While inactive via CXCR1, CXCL17 was found to inhibit CXCR1-mediated chemotaxis of transfectants to CXCL8 in a dose-dependent manner. Discussion: In summary, despite finding little evidence for chemokine-like structure and function, CXCL17 readily bound GAGs, and could modulate chemotactic responses to another chemokine in vitro. We postulate that such modulation is a consequence of superior GAG binding, and that C-terminal fragments of CXCL17 may serve as prototypic inhibitors of chemokine function.

Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans.

Chemokine-driven leukocyte recruitment is a key component of the immune response and of various diseases. Therapeutically targeting the chemokine system in inflammatory disease has been unsuccessful, which has been attributed to redundancy. We investigated why chemokines instead have specific, specialized functions, as demonstrated by multiple studies. We analyzed the expression of genes encoding chemokines and their receptors across species, tissues, and diseases. This analysis revealed complex expression patterns such that genes encoding multiple chemokines that mediated recruitment of the same leukocyte type were expressed in the same context, such as the genes encoding the CXCR3 ligands CXCL9, CXCL10, and CXCL11. Through biophysical approaches, we showed that these chemokines differentially interacted with extracellular matrix glycosaminoglycans (ECM GAGs), which was enhanced by sulfation of specific GAGs. Last, in vivo approaches demonstrated that GAG binding was critical for the CXCL9-dependent recruitment of specific T cell subsets but not of others, irrespective of CXCR3 expression. Our data demonstrate that interactions with ECM GAGs regulated whether chemokines were presented on cell surfaces or remained more soluble, thereby affecting chemokine availability and ensuring specificity of chemokine action. Our findings provide a mechanistic understanding of chemokine-mediated immune cell recruitment and identify strategies to target specific chemokines during inflammatory disease.

Chemokine CXCL4 interactions with extracellular matrix proteoglycans mediate widespread immune cell recruitment independent of chemokine receptors.

Leukocyte recruitment from the vasculature into tissues is a crucial component of the immune system but is also key to inflammatory disease. Chemokines are central to this process but have yet to be therapeutically targeted during inflammation due to a lack of mechanistic understanding. Specifically, CXCL4 (Platelet Factor 4, PF4) has no established receptor that explains its function. Here, we use biophysical, in vitro, and in vivo techniques to determine the mechanism underlying CXCL4-mediated leukocyte recruitment. We demonstrate that CXCL4 binds to glycosaminoglycan (GAG) sugars on proteoglycans within the endothelial extracellular matrix, resulting in increased adhesion of leukocytes to the vasculature, increased vascular permeability, and non-specific recruitment of a range of leukocytes. Furthermore, GAG sulfation confers selectivity onto chemokine localization. These findings present mechanistic insights into chemokine biology and provide future therapeutic targets.

Interaction standards for biophysics: anti-lysozyme nanobodies.

There is a significant demand in the molecular biophysics community for robust standard samples. They are required by researchers, instrument developers and pharmaceutical companies for instrumental quality control, methodological development and in the design and validation of devices, diagnostics and instrumentation. To-date there has been no clear consensus on the need and type of standards that should be available and different research groups and instrument manufacturers use different standard systems which significantly hinders comparative analysis. One of the major objectives of the Association of Resources for Biophysical Research in Europe (ARBRE) is to establish a common set of standard samples that can be used throughout the biophysics community and instrument developers. A survey was circulated among ARBRE members to ascertain the requirements of laboratories when using standard systems and the results are documented in this article. In summary, the major requirements are protein samples which are cheap, relatively small, stable and have different binding strengths. We have developed a panel of sdAb's or 'nanobodies' against hen-egg white lysozyme with different binding strengths and suitable stability characteristics. Here we show the results of the survey, the selection procedure, validation and final selection of a panel of nanobody interaction standards.

Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D): Preparing Functionalized Lipid Layers for the Study of Complex Protein-Ligand Interactions.

Quartz crystal microbalance with dissipation monitoring (QCM-D) is one of the most widely used techniques for the deposition of lipid layers and provides a useful tool for protein-ligand analysis. By using functionalized lipids, for example, with nitrilotriacetic acid (NTA) or biotin, one can couple a molecule to the surface to investigate ligand interactions. Using lipid layers in this way allows for the analysis of complex binding events such as conformational changes, fibrillation, and hierarchical clustering on the surface, which is difficult to interpret with conventional surface sensor techniques. Deposition of lipids and subsequent molecular interactions are easily monitored using both the frequency and the dissipation, which have distinct features in bilayer formation and make QCM-D the ideal technique to use. Here we describe the formation of biotinylated lipid bilayers using two different types of lipids and the subsequent addition of avidin, which can then be used as a basis for linking biotinylated molecules to the surface. These protocols can be adapted to use other lipid moieties and linking chemistries.

Inter-α-inhibitor heavy chain-1 has an integrin-like 3D structure mediating immune regulatory activities and matrix stabilization during ovulation.

Inter-α-inhibitor is a proteoglycan essential for mammalian reproduction and also plays a less well-characterized role in inflammation. It comprises two homologous "heavy chains" (HC1 and HC2) covalently attached to chondroitin sulfate on the bikunin core protein. Before ovulation, HCs are transferred onto the polysaccharide hyaluronan (HA) to form covalent HC·HA complexes, thereby stabilizing an extracellular matrix around the oocyte required for fertilization. Additionally, such complexes form during inflammatory processes and mediate leukocyte adhesion in the synovial fluids of arthritis patients and protect against sepsis. Here using X-ray crystallography, we show that human HC1 has a structure similar to integrin ß-chains, with a von Willebrand factor A domain containing a functional metal ion-dependent adhesion site (MIDAS) and an associated hybrid domain. A comparison of the WT protein and a variant with an impaired MIDAS (but otherwise structurally identical) by small-angle X-ray scattering and analytical ultracentrifugation revealed that HC1 self-associates in a cation-dependent manner, providing a mechanism for HC·HA cross-linking and matrix stabilization. Surprisingly, unlike integrins, HC1 interacted with RGD-containing ligands, such as fibronectin, vitronectin, and the latency-associated peptides of transforming growth factor ß, in a MIDAS/cation-independent manner. However, HC1 utilizes its MIDAS motif to bind to and inhibit the cleavage of complement C3, and small-angle X-ray scattering-based modeling indicates that this occurs through the inhibition of the alternative pathway C3 convertase. These findings provide detailed structural and functional insights into HC1 as a regulator of innate immunity and further elucidate the role of HC·HA complexes in inflammation and ovulation.

Enhanced avidin binding to lipid bilayers using PDP-PE lipids with PEG-biotin linkers.

Two of the most important aspects of lipid bilayers that have increased their popularity in the field of nanotechnology and biosensors are their fluid nature, which is highly beneficial in ensuring the spatial organization of attached molecules, and the relative ease in which they can be manipulated to change the surface chemistry. Here we have used two different types of functionalized lipids to study the interaction of avidin, which is a common approach to attach further ligands for study. We have tested the commonly used Biotinyl-Cap-PE lipids at different molar percentages and reveal that avidin is not evenly distributed, but forms what looks like clusters even at low percentage occupancy which hampers the level of avidin that can be associated with the surface. We have then successfully employed the novel strategy of using PDP-PE lipids which contain a reducible disulphide to which we added maleamide-PEG-biotin spacers of different lengths. There is a more even distribution of avidin on these layers and thereby increasing the amount and efficiency of avidin association. The reduced levels of avidin that was being associated with the Biotinyl-Cap-PE layers as compared to the PDP-PE lipids could be analysed with QCM-D and interferometry approaches, but it was only with SEEC microscopy that the reason for the reduced occupancy was resolved.

Metal Ion-dependent Heavy Chain Transfer Activity of TSG-6 Mediates Assembly of the Cumulus-Oocyte Matrix.

The matrix polysaccharide hyaluronan (HA) has a critical role in the expansion of the cumulus cell-oocyte complex (COC), a process that is necessary for ovulation and fertilization in most mammals. Hyaluronan is organized into a cross-linked network by the cooperative action of three proteins, inter-α-inhibitor (IαI), pentraxin-3, and TNF-stimulated gene-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. Although it is known that matrix stabilization involves the TSG-6-mediated transfer of IαI heavy chains (HCs) onto hyaluronan (to form covalent HC·HA complexes that are cross-linked by pentraxin-3) and that this occurs via the formation of covalent HC·TSG-6 intermediates, the underlying molecular mechanisms are not well understood. Here, we have determined the tertiary structure of the CUB module from human TSG-6, identifying a calcium ion-binding site and chelating glutamic acid residue that mediate the formation of HC·TSG-6. This occurs via an initial metal ion-dependent, non-covalent, interaction between TSG-6 and HCs that also requires the presence of an HC-associated magnesium ion. In addition, we have found that the well characterized hyaluronan-binding site in the TSG-6 Link module is not used for recognition during transfer of HCs onto HA. Analysis of TSG-6 mutants (with impaired transferase and/or hyaluronan-binding functions) revealed that although the TSG-6-mediated formation of HC·HA complexes is essential for the expansion of mouse COCs in vitro, the hyaluronan-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix.

Comparative surface antimicrobial properties of synthetic biocides and novel human apolipoprotein E derived antimicrobial peptides.

Medical device infection remains a major clinical concern. Biocidal compounds have been incorporated into medical device materials ideally to inhibit bacterial colonisation whilst exhibiting relatively low cytotoxicity. We compared the antibacterial activity, anti-biofilm efficacy and cytotoxicity of a novel peptide derivative of human apolipoprotein E (apoEdpL-W) to that of commonly used biocides, before and after coating onto a range of standard polymers. Since the antimicrobial function of most biocides frequently involves associations with cellular membranes, we have also studied the detailed interactions of the test antimicrobials with phospholipid bilayers, using the quartz crystal microbalance device combined with dual-polarisation interferometry. ApoEdpL-W displayed broad-spectrum antibacterial activity and marked efficacy against nascent Staphylococcus aureus biofilms. Compounds showed better antimicrobial activity when combined with hydrogel materials than with non-porous materials. The membrane interactions of apoEdpL-W were most similar to that of PHMB, with both agents appearing to readily bind and insert into lipid bilayers, possibly forming pores. However apoEdpL-W showed lower cytotoxicity than PHMB, its efficacy was less affected by the presence of serum, and it demonstrated the highest level of biocompatibility of all the biocides, as indicated by our measurement of its antimicrobial biocompatibility index. This work shows the potential of apoEdpL-W as an effective antiseptic coating agent.