Determinants of Superselectivity─Practical Concepts for Application in Biology and Medicine.

Multivalent interactions are common in biological systems and are also widely deployed for targeting applications in biomedicine. A unique feature of multivalent binding is "superselectivity". Superselectivity refers to the sharp discrimination of surfaces (e.g., on cells or cell compartments) by their comparative surface densities of a given receptor. This feature is different from the conventional "type" selectivity, which discriminates surfaces by their distinct receptor types. In a broader definition, a probe is superselective if it converts a gradual change in any one interaction parameter into a sharp on/off dependency in probe binding.This Account describes our systematic experimental and theoretical efforts over the past decade to analyze the determinants of superselective binding. It aims to offer chemical biologists, biophysicists, biologists, and biomedical scientists a set of guidelines for the interpretation of multivalent binding data, and design rules for tuning superselective targeting. We first provide a basic introduction that identifies multiple low-affinity interactions and combinatorial entropy as the minimal set of conditions required for superselective recognition. We then introduce the main experimental and theoretical tools and analyze how salient features of the multivalent probes (i.e., their concentration, size, ligand valency, and scaffold type), of the surface receptors (i.e., their affinity for ligands, surface density, and mobility), and of competitors and cofactors (i.e., their concentration and affinity for the ligands and/or receptors) influence the sharpness and the position of the threshold for superselective recognition.Emerging from this work are a set of relatively simple yet quantitative data analysis guidelines and superselectivity design rules that apply to a broad range of probe types and interaction systems. The key finding is the scaling variable xS which faithfully predicts the influence of the surface receptor density, probe ligand valency, receptor-ligand affinity, and competitor/cofactor concentrations and affinities on superselective recognition. The scaling variable is a simple yet versatile tool to quantitatively tune the on/off threshold of superselective probes. We exemplify its application by reviewing and reinterpreting literature data for selected biological and biomedical interaction systems where superselectivity clearly is important.Our guidelines can be deployed to generate a new mechanistic understanding of multivalent recognition events inside and outside cells and the downstream physiological/pathological implications. Moreover, the design rules can be harnessed to develop novel superselective probes for analytical purposes in the life sciences and for diagnostic/therapeutic intervention in biomedicine.

Competitive Specific Anchorage of Molecules onto Surfaces: Quantitative Control of Grafting Densities and Contamination by Free Anchors.

The formation of surfaces decorated with biomacromolecules such as proteins, glycans, or nucleic acids with well-controlled orientations and densities is of critical importance for the design of in vitro models, e.g., synthetic cell membranes and interaction assays. To this effect, ligand molecules are often functionalized with an anchor that specifically binds to a surface with a high density of binding sites, providing control over the presentation of the molecules. Here, we present a method to robustly and quantitatively control the surface density of one or several types of anchor-bearing molecules by tuning the relative concentrations of target molecules and free anchors in the incubation solution. We provide a theoretical background that relates incubation concentrations to the final surface density of the molecules of interest and present effective guidelines toward optimizing incubation conditions for the quantitative control of surface densities. Focusing on the biotin anchor, a commonly used anchor for interaction studies, as a salient example, we experimentally demonstrate surface density control over a wide range of densities and target molecule sizes. Conversely, we show how the method can be adapted to quality control the purity of end-grafted biopolymers such as biotinylated glycosaminoglycans by quantifying the amount of residual free biotin reactant in the sample solution.

Selective Colloid Transport across Planar Polymer Brushes.

Polymer brushes are attractive as surface coatings for a wide range of applications, from fundamental research to everyday life, and also play important roles in biological systems. How colloids (e.g., functional nanoparticles, proteins, viruses) bind and move across polymer brushes is an important yet under-studied problem. A mean-field theoretical approach is presented to analyze the binding and transport of colloids in planar polymer brushes. The theory explicitly considers the effect of solvent strength on brush conformation and of colloid-polymer affinity on colloid binding and transport. The position-dependent free energy of the colloid insertion into the polymer brush which controls the rate of colloid transport across the brush is derived. It is shown how the properties of the brush can be adjusted for brushes to be highly selective, effectively serving as tuneable gates with respect to colloid size and affinity to the brush-forming polymer. The most important parameter regime simultaneously allowing for high brush permeability and selectivity corresponds to a condition when the repulsive and attractive contributions to the colloid insertion free energy nearly cancel. This theory should be useful to design sensing and purification devices with enhanced selectivity and to better understand mechanisms underpinning the functions of biological polymer brushes.

Controlling Superselectivity of Multivalent Interactions with Cofactors and Competitors.

Moieties that compete with multivalent interactions or act as cofactors are common in living systems, but their effect on multivalent binding remains poorly understood. We derive a theoretical model that shows how the superselectivity of multivalent interactions is modulated by the presence of cofactors or competitors. We find that the role of these participating moieties can be fully captured by a simple rescaling of the affinity constant of the individual ligand-receptor bonds. Theoretical predictions are supported by experimental data of the membrane repair protein annexin A5 binding to anionic lipid membranes in the presence of Ca2+ cofactors and of the extracellular matrix polysaccharide hyaluronan (HA) binding to CD44 cell surface receptors in the presence of HA oligosaccharide competitors. The obtained findings should facilitate understanding of multivalent recognition in biological systems and open new routes for fine-tuning the selectivity of multivalent nanoprobes in medicinal chemistry.

Impact of Antigen Density on Recognition by Monoclonal Antibodies.

Understanding antigen-antibody interactions is important to many emerging medical and bioanalytical applications. In particular, the levels of antigen expression at the cell surface may determine antibody-mediated cell death. This parameter has a clear effect on outcome in patients undergoing immunotherapy. In this context, CD20 which is expressed in the membrane of B cells has received significant attention as target for immunotherapy of leukemia and lymphoma using the monoclonal antibody rituximab. To systematically study the impact of CD20 density on antibody recognition, we designed self-assembled monolayers that display tunable CD20 epitope densities. For this purpose, we developed in situ click chemistry to functionalize SPR sensor chips. We find that the rituximab binding affinity depends sensitively and nonmonotonously on CD20 surface density. Strongest binding, with an equilibrium dissociation constant (KD = 32 nM) close to values previously reported from in vitro analysis with B cells (apparent KD between 5 and 19 nM), was obtained for an average inter-antigen spacing of 2 nm. This distance is required for improving rituximab recognition, and in agreement with the known requirement of CD20 to form clusters to elicit a biological response. More generally, this study offers an interesting outlook in the understanding of the necessity of epitope clusters for effective mAb recognition.

Reversible Membrane Tethering by ZipA Determines FtsZ Polymerization in Two and Three Dimensions.

In most bacteria, the early step of septum formation implies the association of soluble FtsZ polymers with the cytoplasmic membrane. ZipA, together with FtsA, provides membrane tethering to FtsZ in Escherichia coli, forming a dynamic proto-ring that serves as an assembly scaffold for the remaining elements of the divisome. Despite their importance for bacterial cell division, multivalent interactions between proto-ring elements at membrane surfaces remain poorly characterized in quantitative terms. We measured the binding of FtsZ to ZipA incorporated in supported lipid bilayers at controlled densities by using a combination of biophysical surface-sensitive techniques (quartz crystal microbalance and spectroscopic ellipsometry) and analyzed how ZipA density and FtsZ concentration control the state of assembly of FtsZ. We found that ZipA attachment enables FtsZ-GMPCPP (where GMPCPP is a GTP analogue with a reduced level of hydrolysis) to assemble in several distinct ways: (i) two-dimensional polymerization at the membrane and (ii) three-dimensional polymerization from the membrane into the solution phase where this may be associated with the formation of higher-order complexes. In these processes, ZipA is required to enrich FtsZ at the surface but the FtsZ bulk concentration defines which morphology is being formed. Moreover, we report a strong effect of the nucleotide (GDP vs GMPCPP/GTP) on the kinetics of ZipA association/dissociation of FtsZ. These results provide insights into the mode of interaction of proto-ring elements in minimal membrane systems and contribute to the completion of our understanding of the initial events of bacterial division.

Multivalent Recognition at Fluid Surfaces: The Interplay of Receptor Clustering and Superselectivity.

The interaction between a biological membrane and its environment is a complex process, as it involves multivalent binding between ligand/receptor pairs, which can self-organize in patches. Any description of the specific binding of biomolecules to membranes must account for the key characteristics of multivalent binding, namely, its unique ability to discriminate sharply between high and low receptor densities (superselectivity), but also for the effect of the lateral mobility of membrane-bound receptors to cluster upon binding. Here we present an experimental model system that allows us to compare systematically the effects of multivalent interactions on fluid and immobile surfaces. A crucial feature of our model system is that it allows us to control the membrane surface chemistry, the properties of the multivalent binder, and the binding affinity. We find that multivalent probes retain their superselective binding behavior at fluid interfaces. Supported by numerical simulations, we demonstrate that, as a consequence of receptor clustering, superselective binding is enhanced and shifted to lower receptor densities at fluid interfaces. To translate our findings into a simple, predictive tool, we propose an analytical model that enables rapid predictions of how the superselective binding behavior is affected by the lateral receptor mobility as a function of the physicochemical characteristics of the multivalent probe. We believe that our model, which captures the key physical mechanisms underpinning multivalent binding to biological membranes, will greatly facilitate the rational design of nanoprobes for the superselective targeting of cells.

Single-Molecule Unbinding Forces between the Polysaccharide Hyaluronan and Its Binding Proteins.

The extracellular polysaccharide hyaluronan (HA) is ubiquitous in all vertebrate tissues, where its various functions are encoded in the supramolecular complexes and matrices that it forms with HA-binding proteins (hyaladherins). In tissues, these supramolecular architectures are frequently subjected to mechanical stress, yet how this affects the intermolecular bonding is largely unknown. Here, we used a recently developed single-molecule force spectroscopy platform to analyze and compare the mechanical strength of bonds between HA and a panel of hyaladherins from the Link module superfamily, namely the complex of the proteoglycan aggrecan and cartilage link protein, the proteoglycan versican, the inflammation-associated protein TSG-6, the HA receptor for endocytosis (stabilin-2/HARE), and the HA receptor CD44. We find that the resistance to tensile stress for these hyaladherins correlates with the size of the HA-binding domain. The lowest mean rupture forces are observed for members of the type A subgroup (i.e., with the shortest HA-binding domains; TSG-6 and HARE). In contrast, the mechanical stability of the bond formed by aggrecan in complex with cartilage link protein (two members of the type C subgroup, i.e., with the longest HA-binding domains) and HA is equal or even superior to the high affinity streptavidin⋅biotin bond. Implications for the molecular mechanism of unbinding of HA⋅hyaladherin bonds under force are discussed, which underpin the mechanical properties of HA⋅hyaladherin complexes and HA-rich extracellular matrices.

Reversible Immobilization of Proteins in Sensors and Solid-State Nanopores.

The controlled functionalization of surfaces with proteins is crucial for many analytical methods in life science research and biomedical applications. Here, a coating for silica-based surfaces is established which enables stable and selective immobilization of proteins with controlled orientation and tunable surface density. The coating is reusable, retains functionality upon long-term storage in air, and is applicable to surfaces of complex geometry. The protein anchoring method is validated on planar surfaces, and then a method is developed to measure the anchoring process in real time using silicon nitride solid-state nanopores. For surface attachment, polyhistidine tags that are site specifically introduced into recombinant proteins are exploited, and the yeast nucleoporin Nsp1 is used as model protein. Contrary to the commonly used covalent thiol chemistry, the anchoring of proteins via polyhistidine tag is reversible, permitting to take proteins off and replace them by other ones. Such switching in real time in experiments on individual nanopores is monitored using ion conductivity. Finally, it is demonstrated that silica and gold surfaces can be orthogonally functionalized to accommodate polyhistidine-tagged proteins on silica but prevent protein binding to gold, which extends the applicability of this surface functionalization method to even more complex sensor devices.

Elastohydrodynamic Lift at a Soft Wall.

We study experimentally the motion of nondeformable microbeads in a linear shear flow close to a wall bearing a thin and soft polymer layer. Combining microfluidics and 3D optical tracking, we demonstrate that the steady-state bead-to-surface distance increases with the flow strength. Moreover, such lift is shown to result from flow-induced deformations of the layer, in quantitative agreement with theoretical predictions from elastohydrodynamics. This study thus provides the first experimental evidence of "soft lubrication" at play at small scale, in a system relevant, for example, to the physics of blood microcirculation.

Structure and properties of polydisperse polyelectrolyte brushes studied by self-consistent field theory.

Two complementary self-consistent field theoretical approaches are used to analyze the equilibrium structure of binary and ternary brushes of polyions with different degrees of polymerization. Stratification in binary brushes is predicted: the shorter chains are entirely embedded in the proximal sublayer depleted of end-points of longer chains while the peripheral sublayer contains exclusively terminal segments of longer chains. The boundary between sublayers is enriched with counterions that neutralize the residual charge of the proximal sublayer. These analytical predictions for binary brushes are confirmed and extended to ternary brushes using the numerical Scheutjens-Fleer approach.

Designing multivalent probes for tunable superselective targeting.

Specific targeting is common in biology and is a key challenge in nanomedicine. It was recently demonstrated that multivalent probes can selectively target surfaces with a defined density of surface binding sites. Here we show, using a combination of experiments and simulations on multivalent polymers, that such "superselective" binding can be tuned through the design of the multivalent probe, to target a desired density of binding sites. We develop an analytical model that provides simple yet quantitative predictions to tune the polymer's superselective binding properties by its molecular characteristics such as size, valency, and affinity. This work opens up a route toward the rational design of multivalent probes with defined superselective targeting properties for practical applications, and provides mechanistic insight into the regulation of multivalent interactions in biology. To illustrate this, we show how the superselective targeting of the extracellular matrix polysaccharide hyaluronan to its main cell surface receptor CD44 is controlled by the affinity of individual CD44-hyaluronan interactions.

Controlling Multivalent Binding through Surface Chemistry: Model Study on Streptavidin.

Although multivalent binding to surfaces is an important tool in nanotechnology, quantitative information about the residual valency and orientation of surface-bound molecules is missing. To address these questions, we study streptavidin (SAv) binding to commonly used biotinylated surfaces such as supported lipid bilayers (SLBs) and self-assembled monolayers (SAMs). Stability and kinetics of SAv binding are characterized by quartz crystal microbalance with dissipation monitoring, while the residual valency of immobilized SAv is quantified using spectroscopic ellipsometry by monitoring binding of biotinylated probes. Purpose-designed SAv constructs having controlled valencies (mono-, di-, trivalent in terms of biotin-binding sites) are studied to rationalize the results obtained on regular (tetravalent) SAv. We find that divalent interaction of SAv with biotinylated surfaces is a strict requirement for stable immobilization, while monovalent attachment is reversible and, in the case of SLBs, leads to the extraction of biotinylated lipids from the bilayer. The surface density and lateral mobility of biotin, and the SAv surface coverage are all found to influence the average orientation and residual valency of SAv on a biotinylated surface. We demonstrate how the residual valency can be adjusted to one or two biotin binding sites per immobilized SAv by choosing appropriate surface chemistry. The obtained results provide means for the rational design of surface-confined supramolecular architectures involving specific biointeractions at tunable valency. This knowledge can be used for the development of well-defined bioactive coatings, biosensors and biomimetic model systems.

Micromechanical Analysis of the Hyaluronan-Rich Matrix Surrounding the Oocyte Reveals a Uniquely Soft and Elastic Composition.

The cumulus cell-oocyte complex (COC) matrix is an extended coat that forms around the oocyte a few hours before ovulation and plays vital roles in oocyte biology. Here, we analyzed the micromechanical response of mouse COC matrix by colloidal-probe atomic force microscopy. We found that the COC matrix is elastic insofar as it does not flow and its original shape is restored after force release. At the same time, the COC matrix is extremely soft. Specifically, the most compliant parts of in vivo and in vitro expanded COC matrices yielded Young's modulus values of 0.5 ± 0.1 Pa and 1.6 ± 0.3 Pa, respectively, suggesting both high porosity and a large mesh size (≥100 nm). In addition, the elastic modulus increased progressively with indentation. Furthermore, using optical microscopy to correlate these mechanical properties with ultrastructure, we discovered that the COC is surrounded by a thick matrix shell that is essentially devoid of cumulus cells and is enhanced upon COC expansion in vivo. We propose that the pronounced nonlinear elastic behavior of the COC matrix is a consequence of structural heterogeneity and serves important functions in biological processes such as oocyte transport in the oviduct and sperm penetration.

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.

Incorporation of pentraxin 3 into hyaluronan matrices is tightly regulated and promotes matrix cross-linking.

Mammalian oocytes are surrounded by a highly hydrated hyaluronan (HA)-rich extracellular matrix with embedded cumulus cells, forming the cumulus cell·oocyte complex (COC) matrix. The correct assembly, stability, and mechanical properties of this matrix, which are crucial for successful ovulation, transport of the COC to the oviduct, and its fertilization, depend on the interaction between HA and specific HA-organizing proteins. Although the proteins inter-α-inhibitor (IαI), pentraxin 3 (PTX3), and TNF-stimulated gene-6 (TSG-6) have been identified as being critical for COC matrix formation, its supramolecular organization and the molecular mechanism of COC matrix stabilization remain unknown. Here we used films of end-grafted HA as a model system to investigate the molecular interactions involved in the formation and stabilization of HA matrices containing TSG-6, IαI, and PTX3. We found that PTX3 binds neither to HA alone nor to HA films containing TSG-6. This long pentraxin also failed to bind to products of the interaction between IαI, TSG-6, and HA, among which are the covalent heavy chain (HC)·HA and HC·TSG-6 complexes, despite the fact that both IαI and TSG-6 are ligands of PTX3. Interestingly, prior encounter with IαI was required for effective incorporation of PTX3 into TSG-6-loaded HA films. Moreover, we demonstrated that this ternary protein mixture made of IαI, PTX3, and TSG-6 is sufficient to promote formation of a stable (i.e. cross-linked) yet highly hydrated HA matrix. We propose that this mechanism is essential for correct assembly of the COC matrix and may also have general implications in other inflammatory processes that are associated with HA cross-linking.

Sensor Based on Aptamer Folding to Detect Low-Molecular Weight Analytes.

Aptamers have emerged as promising biorecognition elements in the development of biosensors. The present work focuses on the application of quartz crystal microbalance with dissipation monitoring (QCM-D) for the enantioselective detection of a low molecular weight target molecule (less than 200 Da) by aptamer-based sensors. While QCM-D is a powerful technique for label-free, real-time characterization and quantification of molecular interactions at interfaces, the detection of small molecules interacting with immobilized receptors still remains a challenge. In the present study, we take advantage of the aptamer conformational changes upon the target binding that induces displacement of water acoustically coupled to the sensing layer. As a consequence, this phenomenon leads to a significant enhancement of the detection signal. The methodology is exemplified with the enantioselective recognition of a low molecular weight model compound, L-tyrosinamide (L-Tym). QCM-D monitoring of L-Tym interaction with the aptamer monolayer leads to an appreciable signal that can be further exploited for analytical purposes or thermodynamics studies. Furthermore, in situ combination of QCM-D with spectroscopic ellipsometry unambiguously demonstrates that the conformational change induces a nanometric decrease of the aptamer monolayer thickness. Since QCM-D is sensitive to the whole mass of the sensing layer including water that is acoustically coupled, a decrease in thickness of the highly hydrated aptamer layer induces a sizable release of water that can be easily detected by QCM-D.

Interaction of Hyaluronan with Cationic Nanoparticles.

The polysaccharide hyaluronan (HA) is a main component of peri- and extracellular matrix, and an attractive molecule for materials design in tissue engineering and nanomedicine. Here, we study the morphology of complexes that form upon interaction of nanometer-sized amine-coated gold particles with this anionic, linear, and regular biopolymer in solution and grafted to a surface. We find that cationic nanoparticles (NPs) have profound effects on HA morphology on the molecular and supramolecular scale. Quartz crystal microbalance (QCM-D) shows that depending on their relative abundance, cationic NPs promote either strong compaction or swelling of films of surface-grafted HA polymers (HA brushes). Transmission electron and atomic force microscopy reveal that the NPs do also give rise to complexes of distinct morphologies-compact nanoscopic spheres and extended microscopic fibers-upon interaction with HA polymers in solution. In particular, stable and hydrated spherical complexes of single HA polymers with NPs can be prepared when balancing the ionizable groups on HA and NPs. The observed self-assembly phenomena could be useful for the design of drug delivery vehicles and a better understanding of the reorganization of HA-rich synthetic or biological matrices.

Transient exposure of pulmonary surfactant to hyaluronan promotes structural and compositional transformations into a highly active state.

Pulmonary surfactant is a lipid-protein complex that lowers surface tension at the respiratory air-liquid interface, stabilizing the lungs against physical forces tending to collapse alveoli. Dysfunction of surfactant is associated with respiratory pathologies such as acute respiratory distress syndrome or meconium aspiration syndrome where naturally occurring surfactant-inhibitory agents such as serum, meconium, or cholesterol reach the lung. We analyzed the effect of hyaluronan (HA) on the structure and surface behavior of pulmonary surfactant to understand the mechanism for HA-promoted surfactant protection in the presence of inhibitory agents. In particular, we found that HA affects structural properties such as the aggregation state of surfactant membranes and the size, distribution, and order/packing of phase-segregated lipid domains. These effects do not require a direct interaction between surfactant complexes and HA and are accompanied by a compositional reorganization of large surfactant complexes that become enriched with saturated phospholipid species. HA-exposed surfactant reaches very high efficiency in terms of rapid and spontaneous adsorption of surfactant phospholipids at the air-liquid interface and shows significantly improved resistance to inactivation by serum or cholesterol. We propose that physical effects pertaining to the formation of a meshwork of interpenetrating HA polymer chains are responsible for the changes in surfactant structure and composition that enhance surfactant function and, thus, resistance to inactivation. The higher resistance of HA-exposed surfactant to inactivation persists even after removal of the polymer, suggesting that transient exposure of surfactant to polymers like HA could be a promising strategy for the production of more efficient therapeutic surfactant preparations.

Inter-α-inhibitor impairs TSG-6-induced hyaluronan cross-linking.

Under inflammatory conditions and in the matrix of the cumulus-oocyte complex, the polysaccharide hyaluronan (HA) becomes decorated covalently with heavy chains (HCs) of the serum glycoprotein inter-α-inhibitor (IαI). This alters the functional properties of the HA as well as its structural role within extracellular matrices. The covalent transfer of HCs from IαI to HA is catalyzed by TSG-6 (tumor necrosis factor-stimulated gene-6), but TSG-6 is also known as a HA cross-linker that induces condensation of the HA matrix. Here, we investigate the interplay of these two distinct functions of TSG-6 by studying the ternary interactions of IαI and TSG-6 with well defined films of end-grafted HA chains. We demonstrate that TSG-6-mediated cross-linking of HA films is impaired in the presence of IαI and that this effect suppresses the TSG-6-mediated enhancement of HA binding to CD44-positive cells. Furthermore, we find that the interaction of TSG-6 and IαI in the presence of HA gives rise to two types of complexes that independently promote the covalent transfer of heavy chains to HA. One type of complex interacts very weakly with HA and is likely to correspond to the previously reported covalent HC·TSG-6 complexes. The other type of complex is novel and binds stably but noncovalently to HA. Prolonged incubation with TSG-6 and IαI leads to HA films that contain, in addition to covalently HA-bound HCs, several tightly but noncovalently bound molecular species. These findings have important implications for understanding how the biological activities of TSG-6 are regulated, such that the presence or absence of IαI will dictate its function.