Generation and Characterization of a Polyclonal Human Reference Antibody to Measure Anti-Drug Antibody Titers in Patients with Fabry Disease.

Male patients with Fabry disease (FD) are at high risk for the formation of antibodies to recombinant α-galactosidase A (AGAL), used for enzyme replacement therapy. Due to the rapid disease progression, the identification of patients at risk is highly warranted. However, currently suitable references and standardized protocols for anti-drug antibodies (ADA) determination do not exist. Here we generate a comprehensive patient-derived antibody mixture as a reference, allowing ELISA-based quantification of antibody titers from individual blood samples. Serum samples of 22 male patients with FD and ADAs against AGAL were pooled and purified by immune adsorption. ADA-affinities against agalsidase-α, agalsidase-ß and Moss-AGAL were measured by quartz crystal microbalance with dissipation monitoring (QCM-D). AGAL-specific immune adsorption generated a polyclonal ADA mixture showing a concentration-dependent binding and inhibition of AGAL. Titers in raw sera and from purified total IgGs (r2 = 0.9063 and r2 = 0.8952, both p < 0.0001) correlated with the individual inhibitory capacities of ADAs. QCM-D measurements demonstrated comparable affinities of the reference antibody for agalsidase-α, agalsidase-ß and Moss-AGAL (KD: 1.94 ± 0.11 µM, 2.46 ± 0.21 µM, and 1.33 ± 0.09 µM, respectively). The reference antibody allows the ELISA-based ADA titer determination and quantification of absolute concentrations. Furthermore, ADAs from patients with FD have comparable affinities to agalsidase-α, agalsidase-ß and Moss-AGAL.

Dynamic Light-Induced Protein Patterns at Model Membranes.

The precise localization and activation of proteins at the cell membrane at a certain time gives rise to many cellular processes, including cell polarization, migration, and division. Thus, methods to recruit proteins to model membranes with subcellular resolution and high temporal control are essential when reproducing and controlling such processes in synthetic cells. Here, a method is described for fabricating light-regulated reversible protein patterns at lipid membranes with high spatiotemporal precision. For this purpose, we immobilize the photoswitchable protein iLID (improved light-inducible dimer) on supported lipid bilayers (SLBs) and on the outer membrane of giant unilamellar vesicles (GUVs). Upon local blue light illumination, iLID binds to its partner Nano (wild-type SspB) and allows the recruitment of any protein of interest (POI) fused to Nano from the solution to the illuminated area on the membrane. This binding is reversible in the dark, which provides dynamic binding and release of the POI. Overall, this is a flexible and versatile method for regulating the localization of proteins with high precision in space and time using blue light.

A disordered tether to iLID improves photoswitchable protein patterning on model membranes.

Reversible protein patterning on model membranes is important to reproduce spatiotemporal protein dynamics in vitro. An engineered version of iLID, disiLID, with a disordered domain as a membrane tether improves the recruitment of Nano under blue light and the reversibility in the dark, which enables protein patterning on membranes with higher spatiotemporal precision.

NTA-Cholesterol Analogue for the Nongenetic Liquid-Ordered Phase-Specific Functionalization of Lipid Membranes with Proteins.

The nongenetic modification of cell membranes with proteins is a straightforward way of cellular engineering. In these processes, it is important to specifically address the proteins to liquid-ordered (Lo) or liquid-disordered (Ld) domains as this can largely affect their biological functions. Herein, we report a cholesterol analogue (CHIM) with a nitrilotriacetic acid (NTA) headgroup, named CHIM-NTA. CHIM-NTA integrates into lipid membranes similar to the widely used phospholipid-derived DGS-NTA and, when loaded with Ni2+, allows for specific membrane immobilization of any polyhistidine-tagged proteins of choice. Yet, unlike DGS-NTA, it localizes to the Lo phase in phase-separated giant unilamellar vesicles (GUVs) and allows addressing His-tagged proteins to Lo domains. Furthermore, CHIM-NTA readily integrates into the membranes of live cells and thus enables the nongenetic modification of the cell surface with proteins. Overall, CHIM-NTA provides a facile and flexible way to modify biological membranes, in particular Lo domains, with His-tagged proteins and can serve as a broadly applicable molecular tool for cell surface engineering.

Drosophila hedgehog signaling range and robustness depend on direct and sustained heparan sulfate interactions.

Morphogens determine cellular differentiation in many developing tissues in a concentration dependent manner. As a central model for gradient formation during animal development, Hedgehog (Hh) morphogens spread away from their source to direct growth and pattern formation in the Drosophila wing disc. Although heparan sulfate (HS) expression in the disc is essential for this process, it is not known whether HS regulates Hh signaling and spread in a direct or in an indirect manner. To answer this question, we systematically screened two composite Hh binding areas for HS in vitro and expressed mutated proteins in the Drosophila wing disc. We found that selectively impaired HS binding of the second site reduced Hh signaling close to the source and caused striking wing mispatterning phenotypes more distant from the source. These observations suggest that HS constrains Hh to the wing disc epithelium in a direct manner, and that interfering with this constriction converts Hh into freely diffusing forms with altered signaling ranges and impaired gradient robustness.

Hedgehog is relayed through dynamic heparan sulfate interactions to shape its gradient.

Cellular differentiation is directly determined by concentration gradients of morphogens. As a central model for gradient formation during development, Hedgehog (Hh) morphogens spread away from their source to direct growth and pattern formation in Drosophila wing and eye discs. What is not known is how extracellular Hh spread is achieved and how it translates into precise gradients. Here we show that two separate binding areas located on opposite sides of the Hh molecule can interact directly and simultaneously with two heparan sulfate (HS) chains to temporarily cross-link the chains. Mutated Hh lacking one fully functional binding site still binds HS but shows reduced HS cross-linking. This, in turn, impairs Hhs ability to switch between both chains in vitro and results in striking Hh gradient hypomorphs in vivo. The speed and propensity of direct Hh switching between HS therefore shapes the Hh gradient, revealing a scalable design principle in morphogen-patterned tissues.

Towards applications of synthetic cells in nanotechnology.

Synthetic cells, which are assembled anew from well-defined molecular parts, open-up new possibilities for nanotechnological applications due to their reduced complexity and high functionality. In this review, we discuss how synthetic cells are being implemented in different fields ranging from biomedicine to material science. On one hand, synthetic cells can serve as microreactors that house metabolic networks and as therapeutic carriers that directly communicate with living cells. On the other hand, synthetic cells can become active components in a new-generation of materials that process inputs and result in autonomous and adaptive behavior. These early examples highlight the potential impact that synthetic cells will have in future applications.

Multivalent Noncovalent Interfacing and Cross-Linking of Supramolecular Tubes.

Natural supramolecular filaments have the ability to cross-link with each other and to interface with the cellular membrane via biomolecular noncovalent interactions. This behavior allows them to form complex networks within as well as outside the cell, i.e., the cytoskeleton and the extracellular matrix, respectively. The potential of artificial supramolecular polymers to interact through specific noncovalent interactions has so far only seen limited exploration due to the dynamic nature of supramolecular interactions. Here, a system of synthetic supramolecular tubes that cross-link forming supramolecular networks, and at the same time bind to biomimetic surfaces by the aid of noncovalent streptavidin-biotin linkages, is demonstrated. The architecture of the networks can be engineered by controlling the density of the biotin moiety at the exterior of the tubes as well as by the concentration of the streptavidin. The presented strategy provides a pathway for designing adjustable artificial supramolecular superstructures, which can potentially yield more complex biomimetic adaptive materials.

Surface Modification with Control over Ligand Density for the Study of Multivalent Biological Systems.

In the study of multivalent interactions at interfaces, as occur for example at cell membranes, the density of the ligands or receptors displayed at the interface plays a pivotal role, affecting both the overall binding affinities and the valencies involved in the interactions. In order to control the ligand density at the interface, several approaches have been developed, and they concern the functionalization of a wide range of materials. Here, different methods employed in the modification of surfaces with controlled densities of ligands are being reviewed. Examples of such methods encompass the formation of self-assembled monolayers (SAMs), supported lipid bilayers (SLBs) and polymeric layers on surfaces. Particular emphasis is given to the methods employed in the study of different types of multivalent biological interactions occurring at the functionalized surfaces and their working principles.

Recruitment of receptors at supported lipid bilayers promoted by the multivalent binding of ligand-modified unilamellar vesicles.

The development of model systems that mimic biological interactions and allow the control of both receptor and ligand densities, is essential for a better understanding of biomolecular processes, such as the recruitment of receptors at interfaces, at the molecular level. Here we report a model system based on supported lipid bilayers (SLBs) for the investigation of the clustering of receptors at their interface. Biotinylated SLBs, used as cell membrane mimics, were functionalized with streptavidin (SAv), used here as receptor. Subsequently, biotinylated small (SUVs) and giant (GUVs) unilamellar vesicles were bound to the SAv-functionalized SLBs by multivalent interactions and found to induce the recruitment of both SAv on the SLB surface and the biotin moieties in the vesicles. The recruitment of receptors was investigated with quartz crystal microbalance with dissipation monitoring (QCM-D), which allowed the identification of the biotin and SAv densities necessary to obtain receptor recruitment. At approx. 0.6% of biotin in the vesicles, a transition between dense and low vesicle packing was observed, which coincided with the transitions between recruitment in the vesicles vs. recruitment in the SLB and between full and partial use of the biotin moieties in the vesicle. Direct optical visualization of the clustering at the interface of individual GUVs with the SLB platform was achieved with fluorescence microscopy, showing recruitment of SAv at the contact area as well as the deformation of the vesicles upon binding. Different vesicle binding regimes were observed for lower and higher biotin densities in the vesicles and at the SLBs. A more quantitative analysis of the molecular parameters implied in the interaction, indicated that approx. 10% of the vesicle area constitutes the contact area. Moreover, the SUV binding and recruitment appeared to be fast on the analysis time scale, whereas the binding of GUVs is slower due to the larger SLB area over which SAv recruitment needs to occur. The mechanisms revealed in this study may provide insight in biological processes in which recruitment occurs.

Hierarchical Multivalent Effects Control Influenza Host Specificity.

Understanding how emerging influenza viruses recognize host cells is critical in evaluating their zoonotic potential, pathogenicity, and transmissibility between humans. The surface of the influenza virus is covered with hemagglutinin (HA) proteins that can form multiple interactions with sialic acid-terminated glycans on the host cell surface. This multivalent binding affects the selectivity of the virus in ways that cannot be predicted from the individual receptor-ligand interactions alone. Here, we show that the intrinsic structural and energetic differences between the interactions of avian- or human-type receptors with influenza HA translate from individual site affinity and orientation through receptor length and density on the surface into virus avidity and specificity. We introduce a method to measure virus avidity using receptor density gradients. We found that influenza viruses attached stably to a surface at receptor densities that correspond to a minimum number of approximately 8 HA-glycan interactions, but more interactions were required if the receptors were short and human-type. Thus, the avidity and specificity of influenza viruses for a host cell depend not on the sialic acid linkage alone but on a combination of linkage and the length and density of receptors on the cell surface. Our findings suggest that threshold receptor densities play a key role in virus tropism, which is a predicting factor for both their virulence and zoonotic potential.

Influenza-induced thrombocytopenia is dependent on the subtype and sialoglycan receptor and increases with virus pathogenicity.

Thrombocytopenia is a common complication of influenza virus infection, and its severity predicts the clinical outcome of critically ill patients. The underlying cause(s) remain incompletely understood. In this study, in patients with an influenza A/H1N1 virus infection, viral load and platelet count correlated inversely during the acute infection phase. We confirmed this finding in a ferret model of influenza virus infection. In these animals, platelet count decreased with the degree of virus pathogenicity varying from 0% in animals infected with the influenza A/H3N2 virus, to 22% in those with the pandemic influenza A/H1N1 virus, up to 62% in animals with a highly pathogenic A/H5N1 virus infection. This thrombocytopenia is associated with virus-containing platelets that circulate in the blood. Uptake of influenza virus particles by platelets requires binding to sialoglycans and results in the removal of sialic acids by the virus neuraminidase, a trigger for hepatic clearance of platelets. We propose the clearance of influenza virus by platelets as a paradigm. These insights clarify the pathophysiology of influenza virus infection and show how severe respiratory infections, including COVID-19, may propagate thrombocytopenia and/or thromboembolic complications.

Clickable poly-l-lysine for the formation of biorecognition surfaces.

Biomolecules are immobilized onto surfaces employing the fast and stable adsorption of poly-l-lysine (PLL) polymers and the versatile copper-free click chemistry reactions. This method provides the combined advantages of versatile surface adsorption with density control using polyelectrolytes and of the covalent and orthogonal immobilization of biomolecules with higher reaction rates and improved yields of click chemistry. Using DNA attachment as a proof of concept, control over the DNA probe density and applicability in electrochemical detection are presented.

Weak Multivalent Binding of Influenza Hemagglutinin Nanoparticles at a Sialoglycan-Functionalized Supported Lipid Bilayer.

Quantification of the multivalent interactions of influenza viruses binding at interfaces may provide ways to tackle key biological questions regarding influenza virulence and zoonoses. Yet, the deconvolution of the contributions of molecular and interfacial parameters, such as valency, interaction area, and receptor density, to the binding of whole viruses is hindered by difficulties in the direct determination of these parameters. We report here a chemical platform technology to study the binding of multivalent recombinant hemagglutinin (rHA) nanoparticles at artificial sialoglycan cell receptor-presenting interfaces in which all these parameters can be derived, thus allowing the desired full and quantitative binding analysis. SiO2 substrates were functionalized with supported lipid bilayers containing a targeted and tunable fraction of a biotinylated lipid, followed by the adsorption of streptavidin and biotinylated polyvalent 2,3- or 2,6-sialyl lactosamine (SLN). rHA nanoparticles were used as a virus mimic to provide a good prediction of the number of interactions involved in binding. Low nanomolar affinities and selectivities for binding at the 2,6-SLN platforms were observed for rHA particles from three different virus variants. When fitting the data to a multivalency model, the nanomolar overall affinity appears to be achieved by 6-9 HA-sugar molecular interaction pairs, which individually present a rapid association/dissociation behavior. This dynamic behavior may be an essential biological attribute in the functioning of the influenza virus.

"Plug-n-Play" Polymer Substrates: Surface Patterning with Reactive-Group-Appended Poly-l-lysine for Biomolecule Adhesion.

The immobilization of biomolecules onto polymeric surfaces employed in the fabrication of biomedical and biosensing devices is generally a challenging issue, as the absence of functional groups in such materials does not allow the use of common surface chemistries. Here we report the use of modified poly-l-lysine (PLL) as an effective method for the selective modification of polymeric materials with biomolecules. Cyclic olefin polymer (COP), Ormostamp, and polydimethylsiloxane (PDMS) surfaces were patterned with modified PLLs displaying either biotin or maleimide functional groups. Different patterning techniques were found to provide faithful microscale pattern formation, including micromolding in capillaries (MIMIC) and a hydrogel-based stamping device with micropores. The surface modification and pattern stability were tested with fluorescence microscopy, contact angle and X-ray photoelectron spectroscopy (XPS), showing an effective functionalization of substrates stable for over 20 days. By exploiting the strong biotin-streptavidin interaction or the thiol-maleimide coupling, DNA and PNA probes were displayed successfully on the surface of the materials, and these probes maintained the capability to specifically recognize complementary DNA sequences from solution. The printing of three different PNA-thiol probe molecules in a microarray fashion allowed selective DNA detection from a mixture of DNA analytes, demonstrating that the modified PLL methodology can potentially be used for multiplexed detection of DNA sequences.