Tumor necrosis factor-stimulated gene-6 (TSG-6) is a hyaluronan (HA)-binding protein that plays important roles in inflammation and ovulation. TSG-6-mediated cross-linking of HA has been proposed as a functional mechanism (e.g. for regulating leukocyte adhesion), but direct evidence for cross-linking is lacking, and we know very little about its impact on HA ultrastructure. Here we used films of polymeric and oligomeric HA chains, end-grafted to a solid support, and a combination of surface-sensitive biophysical techniques to quantify the binding of TSG-6 into HA films and to correlate binding to morphological changes. We find that full-length TSG-6 binds with pronounced positive cooperativity and demonstrate that it can cross-link HA at physiologically relevant concentrations. Our data indicate that cooperative binding of full-length TSG-6 arises from HA-induced protein oligomerization and that the TSG-6 oligomers act as cross-linkers. In contrast, the HA-binding domain of TSG-6 (the Link module) alone binds without positive cooperativity and weaker than the full-length protein.Both the Link module and full-length TSG-6 condensed and rigidified HA films, and the degree of condensation scaled with the affinity between the TSG-6 constructs and HA. We propose that condensation is the result of protein-mediated HA crosslinking. Our findings firmly establish that TSG-6 is a potent HA cross-linking agent and might hence have important implications for the mechanistic understanding of the biological function of TSG-6 (e.g. in inflammation). Hyaluronan (HA)3 is a structurally simple and linear polysaccharide. It is ubiquitous in the extracellular matrix of vertebrates and plays important roles in numerous physiological and pathological processes, such as inflammation, fertilization, embryogenesis, tumor development, osteoarthritis, and atherosclerosis (1, 2). HA is considered a "pericellular cue" (3) (i.e. it serves as a versatile scaffold within which other molecules are organized and regulated). A number of proteins, called hyaladherins (4), can bind to the flexible HA chains and engender self-assembly into large and hydrated multimolecular complexes (5-7).The secreted product of tumor necrosis factor-stimulated gene-6 (TSG-6) (8, 9) is of particular importance for the formation and remodeling of HA-rich pericellular coats (10, 11) and extracellular matrices (12). There is little or no constitutive expression of TSG-6 in most adult tissues (with the exception of bone marrow (13) and epidermis (14)). Expression is elevated in response to stimulation with proinflammatory mediators or certain growth factors (8, 9, 15-18), and TSG-6 is detected in the context of many inflammatory diseases (19,20) and in inflammation-like processes, such as ovulation (21,22). TSG-6 is composed mainly of two contiguous domains, a Link module and a CUB module (8,17,23,24). The Link module is conserved among members of the hyaladherin family (4) and is essential for binding to HA (23). Administration of recombinant human TSG-6 Link module (L...

Background: Inflammation/ovulation-associated protein TSG-6 performs multiple functions in hyaluronan (HA)-rich extracellular matrices.Results: Inter-α-inhibitor (IαI) affects HA-TSG-6 interactions and enhancement of cell adhesion while promoting covalent complex formation between IαI heavy chains and HA.Conclusion: IαI dictates TSG-6 activity and remodels HA matrix properties.Significance: These results provide novel insights into the regulation of HA-protein interactions and assembly of biologically important extracellular matrices.

Background: The proteins pentraxin 3 (PTX3) and TNF-stimulated gene-6 (TSG-6) and the proteoglycan inter-α-inhibitor (IαI) are known to be involved in the stabilization of hyaluronan (HA)-rich extracellular matrices.Results: PTX3 incorporation into HA matrices is tightly regulated.Conclusion: PTX3, TSG-6, and IαI are sufficient to cross-link HA matrices.Significance: The results provide mechanistic insights into the regulation of HA-protein interactions.

Conventional wisdom has it that proteins fold and assemble into definite structures, and that this defines their function. Glycosaminoglycans (GAGs) are different. In most cases the structures they form have a low degree of order, even when interacting with proteins. Here, we discuss how physical features common to all GAGs-hydrophilicity, charge, linearity and semi-flexibility-underpin the overall properties of GAG-rich matrices. By integrating soft matter physics concepts (e.g. polymer brushes and phase separation) with our molecular understanding of GAG-protein interactions, we can better comprehend how GAG-rich matrices assemble, what their properties are, and how they function. Taking perineuronal nets (PNNs)-a GAG-rich matrix enveloping neurons-as a relevant example, we propose that microphase separation determines the holey PNN anatomy that is pivotal to PNN functions.

Most bacteria accomplish cell division with the help of a dynamic protein complex called the divisome, which spans the cell envelope in the plane of division. Assembly and activation of this machinery is coordinated by the tubulin-related GTPase FtsZ, which was found to form treadmilling filaments on supported bilayers in vitro 1 and in live cells where they circle around the cell division site 2,3. Treadmilling of FtsZ is thought to actively move proteins around the cell thereby distributing peptidoglycan synthesis and coordinating the inward growth of the septum to form the new poles of the daughter cells 4. However, the molecular mechanisms underlying this function are largely unknown. Here, to study how FtsZ polymerization dynamics are coupled to downstream proteins, we reconstituted part of the bacterial cell division machinery using its purified components FtsZ, FtsA and truncated transmembrane proteins essential for cell division. We found that the membrane-bound cytosolic peptides of FtsN and FtsQ co-migrated with Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: