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Biomedical subjects

Anthony J Day

Publications and source records attributed to Anthony J Day.

At least 19 recordsLinked to original sources
PubMed2007

Fourier transform mass spectrometry to monitor hyaluronan-protein interactions: use of hydrogen/deuterium amide exchange.

The use of Fourier transform mass spectrometry (FTMS) to monitor noncovalent complex formation in the gas phase under native conditions between the Link module from human tumor necrosis factor stimulated gene-6 (Link_TSG6) and hyaluronan (HA) oligosaccharides is reported. In particular, a titration experiment with increasing concentrations of octasaccharide (HA(8)) to protein produced a noncovalent complex with 1:1 stoichiometry when the oligosaccharide was in molar excess. However, in the presence of a molar excess of tetrasaccharide (HA(4)) nearly all proteins and oligosaccharides were observed in their unbound charge states. These results are consistent with solution-phase properties for this interaction in which HA(8), but not HA(4), supports high affinity Link_TSG6 binding. Hydrogen/deuterium amide exchange mass spectrometry (H/D-EX MS) was also utilized to investigate the level of global deuterium incorporation, over time, for Link_TSG6 in both the absence and presence of HA(8). After dilution into quenching conditions, deuterium incorporation reached limiting asymptotic values of 37 and 26 deuterons for the free and bound protein at 240 and 480 min, respectively, indicating that the oligosaccharide interferes with amide exchange on binding. To detect sequence-specific deuterium incorporation, pepsin digestion of Link_TSG6 in both the absence and presence of HA(8) was performed. A level of deuterium incorporation of 10-30% was observed for peptides analyzed in free Link_TSG6. Interestingly, HA(8) blocked some sites of proteolysis in Link_TSG6 compared to the free protein. Molecular modeling indicated that amino acids proximal to the ligand correlated with regions of the protein that were resistant to enzymatic digestion. Of the peptides that could be analyzed by H/D-EX MS in the presence of the ligand, a 30-60% reduction in deuterium incorporation, relative to the free protein, was observed, even for those sequences not directly involved in HA binding. These results support the utility of FTMS as a method for the characterization of protein-carbohydrate interactions.

Binding Sites↗
PubMed2006

Versican-thrombospondin-1 binding in vitro and colocalization in microfibrils induced by inflammation on vascular smooth muscle cells.

We identified a specific interaction between two secreted proteins, thrombospondin-1 and versican, that is induced during a toll-like receptor-3-dependent inflammatory response in vascular smooth muscle cells. Thrombospondin-1 binding to versican is modulated by divalent cations. This interaction is mediated by interaction of the G1 domain of versican with the N-module of thrombospondin-1 but only weakly with the corresponding N-terminal region of thrombospondin-2. The G1 domain of versican contains two Link modules, which are known to mediate TNFalpha-stimulated gene-6 protein binding to thrombospondin-1, and the related G1 domain of aggrecan is also recognized by thrombospondin-1. Therefore, thrombospondin-1 interacts with three members of the Link-containing hyaladherin family. On the surface of poly-I:C-stimulated vascular smooth muscle cells, versican organizes into fibrillar structures that contain elastin but are largely distinct from those formed by hyaluronan. Endogenous and exogenously added thrombospondin-1 incorporates into these structures. Binding of exogenous thrombospondin-1 to these structures, to purified versican and to its G1 domain is potently inhibited by heparin. At higher concentrations, exogenous thrombospondin-1 delays the poly-I:C induced formation of structures containing versican and elastin, suggesting that thrombospondin-1 negatively modulates this component of a vascular smooth muscle inflammatory response.

Aggrecans↗
PubMed2006

TSG-6 potentiates the antitissue kallikrein activity of inter-alpha-inhibitor through bikunin release.

TSG-6 (the protein product of TNF-stimulated gene-6), an inflammation-associated protein, forms covalent complexes with heavy chains (HCs) from inter-alpha-inhibitor and pre-alpha-inhibitor and associates noncovalently with their common bikunin chain, potentiating the antiplasmin activity of this serine protease inhibitor. We show that TSG-6 and TSG-6.HC complexes are present in bronchoalveolar lavage fluid from patients with asthma and increase after allergen challenge. Immunodetection demonstrated elevated TSG-6 in the airway tissue and secretions of smokers. Experiments conducted in vitro with purified components revealed that bikunin.HC complexes (byproducts of TSG-6.HC formation) release bikunin. Immunoprecipitation revealed that bikunin accounts for a significant proportion of tissue kallikrein inhibition in bronchoalveolar lavage after allergen challenge but not in baseline conditions, confirming that bikunin in its free state, but not when associated with HCs, is a relevant protease inhibitor in airway secretions. In primary cultures of differentiated human airway epithelial and submucosal gland cells, TSG-6 is induced by TNF-alpha and IL-1beta, which suggests that these cells are responsible for TSG-6 release in vivo. Bikunin and HC3 (i.e., pre-alpha-inhibitor) were also induced by TNF-alpha in primary cultures. Our results suggest that TSG-6 may play an important protective role in bronchial epithelium by increasing the antiprotease screen on the airway lumen.

Adolescent↗
PubMed2006

His-384 allotypic variant of factor H associated with age-related macular degeneration has different heparin binding properties from the non-disease-associated form.

A polymorphism in complement factor H has recently been associated with age-related macular degeneration (AMD), the leading cause of blindness in the elderly. A histidine rather than a tyrosine at residue position 384 in the mature protein increases the risk of AMD. Here, using a recombinant construct, we show that amino acid 384 is adjacent to a heparin-binding site in CCP7 of factor H and demonstrate that the allotypic variants differentially recognize heparin. This functional alteration may affect binding of factor H to polyanionic patterns on host surfaces, potentially influencing complement activation, immune complex clearance, and inflammation in the macula of AMD patients.

Alleles↗
PubMed2006

Overexpression of hyaluronan synthase 2 alters hyaluronan distribution and function in proximal tubular epithelial cells.

The functional consequences of increased renal cortical hyaluronan that is associated with both acute injury and progressive scarring are unclear. The aim of this study was to characterize hyaluronan synthase-2 (HAS2)-driven HA synthesis and determine its effect on renal proximal tubular epithelial cell (PTC) function, because this is known to be the inducible form of HA synthase in this cell type. Overexpression of HAS2 mRNA increased HA generation, which in the supernatant predominantly was HA of large molecular weight, whereas there was an increase in low molecular weight HA in cell-associated fractions. This was associated with increased expression of hyaluronidases, inhibition of HA cable formation concurrent with reduction in HA-dependent monocyte binding, and increased pericellular HA matrix. Overexpression of HAS2 led to enhanced cell migration. HA can be modified by the covalent attachment of heavy chains that are derived from the serum protein inter-alpha-inhibitor (IalphaI), a process that is known to be catalyzed by TNF-alpha-stimulated gene 6 (TSG-6; an inflammation-associated protein). Enhanced migration was abrogated by blocking antibodies to either IalphaI or TSG-6. Addition of recombinant full-length TSG-6 (TSG-6Q) or TSG-6Q_Y94F, a mutant variant with impaired HA binding, increased cell migration. Both of these proteins were able to mediate the covalent transfer of heavy chains, from IalphaI and pre-alpha-inhibitor, onto HA. Addition of the isolated TSG-6-Link module (Link_TSG-6), which binds HA but is unable to form covalent complexes with IalphaI/pre-alpha-inhibitor, had no effect on migration, suggesting that TSG-6-mediated formation of heavy chain-HA complexes is critical in the formation of a pericellular HA matrix.

Catalysis↗
PubMed2005

Hyaluronan cross-linking: a protective mechanism in inflammation?

Production of the glycosaminoglycan hyaluronan is increased at sites of inflammation, often correlating with the accumulation of leukocytes. Mounting evidence suggests that this polysaccharide can be organized into a wide variety of molecular architectures by its association with specific binding proteins, leading to the formation of fibrils and cable-like structures involving a large number of hyaluronan chains. We propose that hyaluronan cross-linking is part of a protective mechanism, promoting adhesion of leukocytes to the hyaluronan complexes rather than enabling contact with inflammation-promoting receptors on the underlying tissues. Leukocytes are thus maintained in a non-activated state by appropriate receptor clustering or receptor co-engagement. Additionally, hyaluronan networks might serve as scaffolds to prevent the loss of extracellular matrix components during inflammation and to sequester proinflammatory mediators.

Animals↗
PubMed2005

The N-terminal module of thrombospondin-1 interacts with the link domain of TSG-6 and enhances its covalent association with the heavy chains of inter-alpha-trypsin inhibitor.

We recently found that leukocytes from thrombospondin-1 (TSP1)-deficient mice exhibit significant reductions in cell surface CD44 relative to those from wild type mice. Because TSG-6 modulates CD44-mediated cellular interactions with hyaluronan, we examined the possibility that TSP1 interacts with TSG-6. We showed that recombinant full-length human TSG-6 (TSG-6Q) and the Link module of TSG-6 (Link_TSG6) bind 125I-TSP1 with comparable affinities. Trimeric recombinant constructs containing the N-modules of TSP1 or TSP2 inhibit binding of TSP1 to TSG-6Q and Link_TSG6, but other recombinant regions of TSP1 do not. Therefore, the N-modules of both TSP1 and TSP2 specifically recognize the Link module of TSG-6. Heparin, which binds to these domains of both proteins, strongly inhibits binding of TSP1 to Link_TSG6 and TSG-6Q, but hyaluronan does not. Inhibition by heparin results from its binding to TSP1, because heparin also inhibits TSP1 binding to Link_TSG6 mutants deficient in heparin binding. Removal of bound Ca2+ from TSP1 reduces its binding to full-length TSG-6. Binding of TSP1 to Link_TSG6, however, is enhanced by chelating divalent cations. In contrast, divalent cations do not influence binding of the N-terminal region of TSP1 to TSG-6Q. This implies that divalent cation dependence is due to conformational effects of calcium-binding to the C-terminal domains of TSP1. TSP1 enhances covalent modification of the inter-alpha-trypsin inhibitor by TSG-6 and transfer of its heavy chains to hyaluronan, suggesting a physiological function of TSP1 binding to TSG-6 in regulation of hyaluronan metabolism at sites of inflammation.

Alpha-Globulins↗
PubMed2005

Characterization of the interaction between tumor necrosis factor-stimulated gene-6 and heparin: implications for the inhibition of plasmin in extracellular matrix microenvironments.

TSG-6, the secreted product of tumor necrosis factor-stimulated gene-6, is not constitutively expressed but is up-regulated in various cell-types during inflammatory and inflammation-like processes. The mature protein is comprised largely of contiguous Link and CUB modules, the former binding several matrix components such as hyaluronan (HA) and aggrecan. Here we show that this domain can also associate with the glycosaminoglycan heparin/heparan sulfate. Docking predictions and site-directed mutagenesis demonstrate that this occurs at a site distinct from the HA binding surface and is likely to involve extensive electrostatic contacts. Despite these glycosaminoglycans binding to non-overlapping sites on the Link module, the interaction of heparin can inhibit subsequent binding to HA, and it is possible that this occurs via an allosteric mechanism. We also show that heparin can modify another property of the Link module, i.e. its potentiation of the anti-plasmin activity of inter-alpha-inhibitor (IalphaI). Experiments using the purified components of IalphaI indicate that TSG-6 only binds to the bikunin chain and that this is at a site on the Link module that overlaps the HA binding surface. The association of heparin with the Link module significantly increases the anti-plasmin activity of the TSG-6.IalphaI complex. Changes in plasmin activity have been observed previously at sites of TSG-6 expression, and the results presented here suggest that TSG-6 is likely to contribute to matrix remodeling, at least in part, through down-regulation of the protease network, especially in locations containing heparin/heparan sulfate proteoglycans. The differential effects of HA and heparin on TSG-6 function provide a mechanism for its regulation and functional partitioning in particular tissue microenvironments.

Alpha-Globulins↗
PubMed2005

Characterization of complexes formed between TSG-6 and inter-alpha-inhibitor that act as intermediates in the covalent transfer of heavy chains onto hyaluronan.

The high molecular mass glycosaminoglycan hyaluronan (HA) can become modified by the covalent attachment of heavy chains (HCs) derived from the serum protein inter-alpha-inhibitor (IalphaI), which is composed of three subunits (HC1, HC2 and bikunin) linked together via a chondroitin sulfate moiety. The formation of HC.HA is likely to play an important role in the stabilization of HA-rich extracellular matrices in the context of inflammatory disease (e.g. arthritis) and ovulation. Here, we have characterized the complexes formed in vitro between purified human IalphaI and recombinant human TSG-6 (an inflammation-associated protein implicated previously in this process) and show that these complexes (i.e. TSG-6 x HC1 and TSG-6 x HC2) act as intermediates in the formation of HC x HA. This is likely to involve two transesterification reactions in which an ester bond linking an HC to chondroitin sulfate in intact IalphaI is transferred first onto TSG-6 and then onto HA. The formation of TSG-6 x HC1 and TSG-6 x C2 complexes was accompanied by the production of bikunin x HC2 and bikunin x HC1 by-products, respectively, which were observed to break down, releasing free bikunin and HCs. Both TSG-6 x HC formation and the subsequent HC transfer are metal ion-dependent processes; these reactions have a requirement for either Mg2+ or Mn2+ and are inhibited by Co2+. TSG-6, which is released upon the transfer of HCs from TSG-6 onto HA, was shown to combine with IalphaI to form new TSG-6 x HC complexes and thus be recycled. The finding that TSG-6 acts as cofactor and catalyst in the production of HC x HA complexes has important implications for our understanding of inflammatory and inflammation-like processes.

Alpha-Globulins↗
PubMed2005

Towards a structure for a TSG-6.hyaluronan complex by modeling and NMR spectroscopy: insights into other members of the link module superfamily.

The Link module from human TSG-6, a hyaladherin with roles in ovulation and inflammation, has a hyaluronan (HA)-binding groove containing two adjacent tyrosine residues that are likely to form CH-pi stacking interactions with sequential rings in the sugar. We have used this observation to construct a model of a protein.HA complex, which was then tested against existing experimental information and by acquisition of new NMR data sets of [(13)C, (15)N]HA (8-mer) complexed with unlabeled protein. A major finding of this analysis was that acetamido side chains of two GlcNAc rings fit into hydrophobic pockets on either side of the adjacent tyrosines, providing a selectivity mechanism of HA over other polysaccharides. Furthermore, two basic residues have a separation that matches that of glucuronic acids in the sugar, consistent with the formation of salt bridges; NMR experiments at a range of pH values identified protein groups that titrate due to their proximity to a free carboxylate in HA. Sequence alignment and construction of homology models for all human Link modules in their HA-bound states revealed that many of these features are conserved across the superfamily, thus allowing the prediction of functionally important residues. In the case of cartilage link protein, its two Link modules were docked together (using bound HA as a guide), identifying hydrophobic residues likely to form an intra-Link module interface as well as amino acids that could be involved in supporting intermolecular interactions between link proteins and chondroitin sulfate proteoglycans. Here, we propose a mechanism for ternary complex formation that generates higher order helical structures, as may exist in cartilage aggregates.

Acetylglucosamine↗
PubMed2004

Expression and purification of functionally active hyaluronan-binding domains from human cartilage link protein, aggrecan and versican: formation of ternary complexes with defined hyaluronan oligosaccharides.

The chondroitin sulfate proteoglycan aggrecan forms link protein-stabilized complexes with hyaluronan (HA), via its N-terminal G1-domain, that provide cartilage with its load bearing properties. Similar aggregates (potentially containing new members of the link protein family), in which other chondroitin sulfate proteoglycans (i.e. versican, brevican, and neurocan) substitute for aggrecan, may contribute to the structural integrity of many other tissues including skin and brain. In this study, cartilage link protein (cLP) and the G1-domains of aggrecan (AG1) and versican (VG1) were expressed in Drosophila S2 cells. The recombinant human proteins were found to have properties similar to those described for the native molecules (e.g. cLP was able to form oligomers, and HA decasaccharides were the minimum size that could compete effectively for their binding to polymeric HA). Gel filtration and protein cross-linking/matrix-assisted laser desorption ionization time-of-flight peptide fingerprinting showed that cLP and AG1 interact in the absence or presence of HA. Conversely, cLP and VG1 did not bind directly to each other in solution yet formed ternary complexes with HA24. N-linked glycosylation of AG1 and VG1 was demonstrated to be unnecessary for either HA binding or the formation of ternary complexes. Surprisingly, the length of HA required to accommodate two G1-domains was found to be significantly larger for aggrecan than versican, which may reflect differences in the conformation of HA stabilized on binding these proteins.

Aggrecans↗
PubMed2004

Preparation and application of biologically active fluorescent hyaluronan oligosaccharides.

We report the production of biologically active hyaluronan (HA) oligosaccharides labeled with the fluorophore 2-aminobenzoic acid (2AA). Oligosaccharides from 4 to 40 residues in length were purified to homogeneity by ion exchange chromatography using a logarithmic gradient. Molecular weight and purity characterization of HA oligosaccharides is facilitated by 2AA derivatization because it enhances signals in MALDI-TOF MS and improves FACE (fluorophore-assisted carbohydrate electrophoresis) analysis by avoiding the inverted parabolic migration characteristic of 2-aminoacridone (AMAC)-labeled sugars. The small size and shape of the fluorophore maintains the biological activity of the derivatized oligosaccharides, as demonstrated by their ability to compete for polymeric HA binding to the G1-domain of human recombinant versican (VG1). An electrophoretic mobility shift assay was used to study VG1 binding to labeled HA 8-, 10-, 20-, 30-, and 40-mers, and although no stable VG1 binding was observed to labeled 8-mers, the equilibrium dissociation constant (100 nM) for VG1 with HA(10) was estimated from densitometry analysis of the free oligosaccharide. Interactions involving HA 20-, 30-, and 40-mers (proposed to be multivalent) could also be studied using this protocol. Oligosaccharides labeled with 2AA therefore show excellent potential as probes in fluorescence-based assays that investigate protein-carbohydrate interactions.

Animals↗
PubMed2004

A method for the non-covalent immobilization of heparin to surfaces.

The interaction of heparan sulfate (HS) with specific proteins facilitates a wide range of fundamental biological processes including cellular proliferation and differentiation, tissue homeostasis, and viral pathogenesis. This multiplicity of function arises through sequence diversity within the HS chain. Heparin, which is very similar in structure to the sulfated regions of HS, is an excellent model for studying HS-protein interactions. The development of high-throughput enzyme-linked immunosorbent-like assays using surface-immobilized heparin has been hindered by the inability of this glycosaminoglycan to adhere to microtiter surfaces. Here we report the passive noncovalent adsorption of heparin onto microtiter wells following their treatment by plasma polymerization; there was no detectable binding of functional heparin onto untreated plates. Heparin immobilized in this way was able to interact with four different heparin-binding proteins tested, i.e., TSG-6, chemokines IL-8 and KC, and complement factor H. Heparin preparations ranging in size from high molecular weight to a defined decasaccharide could be adsorbed onto these plates in a functionally active form. Since plasma polymerization is possible for virtually any surface, this technique is likely to be of general use in the identification and characterization of heparin/HS-binding proteins in a wide range of applications.

Allylamine↗
PubMed2004

Use of 15N-NMR to resolve molecular details in isotopically-enriched carbohydrates: sequence-specific observations in hyaluronan oligomers up to decasaccharides.

The glycosaminoglycan hyaluronan is a vital structural component of extracellular matrices with diverse biological functions, a molecular understanding of which requires a detailed description of secondary and tertiary solution structures. Various models of these structures have been proposed on the basis of 1H and 13C natural-abundance nuclear magnetic resonance (NMR) experiments, but resonance overlap limits further progress with these techniques. We have therefore produced 15N- and 13C- isotopically-labeled hyaluronan oligosaccharides and applied triple-resonance and 3D experiments to overcome this restriction. Spectra recorded on oligosaccharides (of lengths 4, 6, 8, 10, and 12 sugar rings), reveal that the 15N nucleus allows resolution of the amide groups in a decamer at high magnetic field, whereas 13C natural-abundance NMR can only resolve internal groups up to hexamers. Complete 13N sequence- specific assignments of these oligosaccharides indicate that the chemical shift dispersion can be explained by end-effects, which are seen even in the middle of octamers. Triple- resonance and 15N-edited 3D experiments, among the first of their kind in oligosaccharides, have been used to achieve resolution of ring 1H and 13C nuclei where not possible previously. The subtle chemical shift perturbations resolved suggest that different conformations and dynamics occur at the ends, which may contribute to the range of biological activities displayed by varying lengths of hyaluronan. 15N-NMR in carbohydrates has not received much attention before, however, this study demonstrates it has clear advantages for achieving resolution and assessing dynamic motion. These conclusions are likely to be applicable to the study of the structure and dynamics of other nitrogen-containing carbohydrates.

Carbohydrate Conformation↗
PubMed2004

TSG-6 modulates the interaction between hyaluronan and cell surface CD44.

Interactions between CD44 and hyaluronan are implicated in the primary adhesion of lymphocytes to endothelium at inflammatory locations. Here we show that preincubation of hyaluronan with full-length recombinant TSG-6 or its Link module domain (Link_TSG6) enhances or induces the binding of hyaluronan to cell surface CD44 on constitutive and inducible cell backgrounds, respectively. These effects are blocked by CD44-specific antibodies and are absent in CD44-negative cells. Enhancement of CD44-mediated interactions of lymphoid cells with hyaluronan by TSG-6 proteins was seen under conditions of flow at shear forces that occur in post-capillary venules. Increases in the number of rolling cells were observed on substrates comprising TSG-6-hyaluronan complexes as compared with a substrate containing hyaluronan alone. In ligand competition experiments, cell surface-bound TSG-6-hyaluronan complexes were more potent than hyaluronan alone in inhibiting cell adhesion to immobilized hyaluronan. Link_TSG6 mutants with impaired hyaluronan binding function had a reduced ability to modulate ligand binding by cell surface CD44. However, some mutants that exhibited close to wild-type hyaluronan binding were found to have either reduced or increased activity, suggesting that some amino acid residues outside of the hyaluronan binding site might be involved in protein self-association, potentially leading to the formation of cross-linked hyaluronan fibers. In turn, cross-linked hyaluronan could increase the binding avidity of CD44 by inducing receptor clustering. The ability of TSG-6 to modulate the interaction of hyaluronan with CD44 has important implications for CD44-mediated cell activity at sites of inflammation, where TSG-6 is expressed.

Animals↗
PubMed2004

PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization.

PTX3 is a prototypic long pentraxin that plays a non-redundant role in innate immunity against selected pathogens and in female fertility. Here, we report that the infertility of Ptx3(-/-) mice is associated with severe abnormalities of the cumulus oophorus and failure of in vivo, but not in vitro, oocyte fertilization. PTX3 is produced by mouse cumulus cells during cumulus expansion and localizes in the matrix. PTX3 is expressed in the human cumulus oophorus as well. Cumuli from Ptx3(-/-) mice synthesize normal amounts of hyaluronan (HA), but are unable to organize it in a stable matrix. Exogenous PTX3 restores a normal cumulus phenotype. Incorporation in the matrix of inter-alpha-trypsin inhibitor is normal in Ptx3(-/-) cumuli. PTX3 does not interact directly with HA, but it binds the cumulus matrix hyaladherin tumor necrosis factor alpha-induced protein 6 (TNFAIP6, also known as TSG6) and thereby may form multimolecular complexes that can cross-link HA chains. Thus, PTX3 is a structural constituent of the cumulus oophorus extracellular matrix essential for female fertility.

Animals↗