SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1999080471> ?p ?o ?g. }

Showing items 1 to 100 of ±130 with 100 items per page.

W1999080471 endingPage "12988" @default.
W1999080471 startingPage "12976" @default.
W1999080471 abstract "TSG-6 is an inflammation-associated hyaluronan (HA)-binding protein that has anti-inflammatory and protective functions in arthritis and asthma as well as a critical role in mammalian ovulation. The interaction between TSG-6 and HA is pH-dependent, with a marked reduction in affinity on increasing the pH from 6.0 to 8.0. Here we have investigated the mechanism underlying this pH dependence using a combined approach of site-directed mutagenesis, NMR, isothermal titration calorimetry and microtiter plate assays. Analysis of single-site mutants of the TSG-6 Link module indicated that the loss in affinity above pH 6.0 is mediated by the change in ionization state of a histidine residue (His4) that is not within the HA-binding site. To understand this in molecular terms, the pH-dependent folding profile and the pKa values of charged residues within the Link module were determined using NMR. These data indicated that His4 makes a salt bridge to one side-chain oxygen atom of a buried aspartate residue (Asp89), whereas the other oxygen is simultaneously hydrogen-bonded to a key HA-binding residue (Tyr12). This molecular network transmits the change in ionization state of His4 to the HA-binding site, which explains the loss of affinity at high pH. In contrast, simulations of the pH affinity curves indicate that another histidine residue, His45, is largely responsible for the gain in affinity for HA between pH 3.5 and 6.0. The pH-dependent interaction of TSG-6 with HA (and other ligands) provides a means of differentially regulating the functional activity of this protein in different tissue microenvironments. TSG-6 is an inflammation-associated hyaluronan (HA)-binding protein that has anti-inflammatory and protective functions in arthritis and asthma as well as a critical role in mammalian ovulation. The interaction between TSG-6 and HA is pH-dependent, with a marked reduction in affinity on increasing the pH from 6.0 to 8.0. Here we have investigated the mechanism underlying this pH dependence using a combined approach of site-directed mutagenesis, NMR, isothermal titration calorimetry and microtiter plate assays. Analysis of single-site mutants of the TSG-6 Link module indicated that the loss in affinity above pH 6.0 is mediated by the change in ionization state of a histidine residue (His4) that is not within the HA-binding site. To understand this in molecular terms, the pH-dependent folding profile and the pKa values of charged residues within the Link module were determined using NMR. These data indicated that His4 makes a salt bridge to one side-chain oxygen atom of a buried aspartate residue (Asp89), whereas the other oxygen is simultaneously hydrogen-bonded to a key HA-binding residue (Tyr12). This molecular network transmits the change in ionization state of His4 to the HA-binding site, which explains the loss of affinity at high pH. In contrast, simulations of the pH affinity curves indicate that another histidine residue, His45, is largely responsible for the gain in affinity for HA between pH 3.5 and 6.0. The pH-dependent interaction of TSG-6 with HA (and other ligands) provides a means of differentially regulating the functional activity of this protein in different tissue microenvironments. TSG-6, 5The abbreviations used are: TSG-6, secreted product of tumor necrosis factor-stimulated gene 6; ECM, extracellular matrix; HA, hyaluronan; HA ANn, an oligosaccharide of HA with n sugar residues and GlcNAc and GlcUA at the reducing and nonreducing terminus, respectively; HC, heavy chain; HMQC, heteronuclear multiple quantum coherence; HSQC, heteronuclear single quantum coherence; ITC, isothermal titration calorimetry; Link_TSG6, recombinant Link module from human TSG-6; MES, 4-morpholineethanesulfonic acid; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; TSP1, thrombospondin-1; WT, wild-type Link_TSG6. 5The abbreviations used are: TSG-6, secreted product of tumor necrosis factor-stimulated gene 6; ECM, extracellular matrix; HA, hyaluronan; HA ANn, an oligosaccharide of HA with n sugar residues and GlcNAc and GlcUA at the reducing and nonreducing terminus, respectively; HC, heavy chain; HMQC, heteronuclear multiple quantum coherence; HSQC, heteronuclear single quantum coherence; ITC, isothermal titration calorimetry; Link_TSG6, recombinant Link module from human TSG-6; MES, 4-morpholineethanesulfonic acid; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; TSP1, thrombospondin-1; WT, wild-type Link_TSG6. the secreted product of tumor necrosis factor-stimulated gene 6, is not usually expressed constitutively in healthy adult tissues but is made in response to various inflammatory mediators and growth factors, acting as a potent anti-inflammatory and chondroprotective agent (1Wisniewski H.G. Vilcek J. Cytokine Growth Factor Rev. 1997; 8: 143-156Crossref PubMed Scopus (165) Google Scholar, 2Milner C.M. Day A.J. J. Cell Sci. 2003; 116: 1863-1873Crossref PubMed Scopus (305) Google Scholar, 3Milner C.M. Higman V.A. Day A.J. Biochem. Soc Trans. 2006; 34: 446-450Crossref PubMed Scopus (69) Google Scholar). The TSG-6 protein is produced in inflammatory diseases, for example rheumatoid arthritis, osteoarthritis, and asthma (4Forteza R. Casalino-Matsuda S.M. Monzon M.E. Fries E. Rugg M.S. Milner C.M. Day A.J. Am. J. Respir. Cell Mol. Biol. 2007; 36: 20-31Crossref PubMed Scopus (62) Google Scholar), as well as in normal physiological processes that have inflammation-like characteristics (e.g. ovulation and cervical ripening). A number of roles for TSG-6 have been determined, including inhibition of neutrophil migration (5Wisniewski H.G. Hua J.C. Poppers D.M. Naime D. Vilcek J. Cronstein B.N. J. Immunol. 1996; 156: 1609-1615PubMed Google Scholar, 6Getting S.J. Mahoney D.J. Cao T. Rugg M.S. Fries E. Milner C.M. Perretti M. Day A.J. J. Biol. Chem. 2002; 277: 51068-51076Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 7Cao T.V. La M. Getting S.J. Day A.J. Perretti M. Microcirculation. 2004; 11: 615-624Crossref PubMed Scopus (49) Google Scholar) and down-regulation of the protease network (4Forteza R. Casalino-Matsuda S.M. Monzon M.E. Fries E. Rugg M.S. Milner C.M. Day A.J. Am. J. Respir. Cell Mol. Biol. 2007; 36: 20-31Crossref PubMed Scopus (62) Google Scholar, 5Wisniewski H.G. Hua J.C. Poppers D.M. Naime D. Vilcek J. Cronstein B.N. J. Immunol. 1996; 156: 1609-1615PubMed Google Scholar, 8Mahoney D.J. Mulloy B. Forster M.J. Blundell C.D. Fries E. Milner C.M. Day A.J. J. Biol. Chem. 2005; 280: 27044-27055Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), which could help explain its protective properties in, for example, joint tissues (9Glant T.T. Kamath R.V. Bardos T. Gal I. Szanto S. Murad Y.M. Sandy J.D. Mort J.S. Roughley P.J. Mikecz K. Arthritis Rheum. 2002; 46: 2207-2218Crossref PubMed Scopus (72) Google Scholar, 10Mindrescu C. Dias A.A. Olszewski R.J. Klein M.J. Reis L.F. Wisniewski H.G. Arthritis Rheum. 2002; 46: 2453-2464Crossref PubMed Scopus (61) Google Scholar, 11Szanto S. Bardos T. Gal I. Glant T.T. Mikecz K. Arthritis Rheum. 2004; 50: 3012-3022Crossref PubMed Scopus (75) Google Scholar). In addition, TSG-6 has been implicated in the stabilization of extracellular matrix (ECM) structure, particularly by supporting the formation of cross-linked hyaluronan (HA) networks (12Day A.J. de la Motte C.A. Trends Immunol. 2005; 26: 637-643Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). TSG-6-mediated HA cross-linking has a critical role in mammalian ovulation but also occurs in inflammatory diseases (4Forteza R. Casalino-Matsuda S.M. Monzon M.E. Fries E. Rugg M.S. Milner C.M. Day A.J. Am. J. Respir. Cell Mol. Biol. 2007; 36: 20-31Crossref PubMed Scopus (62) Google Scholar, 13Fulop C. Szanto S. Mukhopadhyay D. Bardos T. Kamath R.V. Rugg M.S. Day A.J. Salustri A. Hascall V.C. Glant T.T. Mikecz K. Development (Camb.). 2003; 130: 2253-2261Crossref PubMed Scopus (331) Google Scholar, 14Ochsner S.A. Day A.J. Rugg M.S. Breyer R.M. Gomer R.H. Richards J.S. Endocrinology. 2003; 144: 4376-4384Crossref PubMed Scopus (130) Google Scholar, 15Yingsung W. Zhuo L. Morgelin M. Yoneda M. Kida D. Watanabe H. Ishiguro N. Iwata H. Kimata K. J. Biol. Chem. 2003; 278: 32710-32718Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 16Salustri A. Garlanda C. Hirsch E. De Acetis M. Maccagno A. Bottazzi B. Doni A. Bastone A. Mantovani G. Beck Peccoz P. Salvatori G. Mahoney D.J. Day A.J. Siracusa G. Romani L. Mantovani A. Development (Camb.). 2004; 131: 1577-1586Crossref PubMed Scopus (366) Google Scholar, 17Rugg M.S. Willis A.C. Mukhopadhyay D. Hascall V.C. Fries E. Fulop C. Milner C.M. Day A.J. J. Biol. Chem. 2005; 280: 25674-25686Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). TSG-6 is composed mainly of contiguous Link and CUB modules (2Milner C.M. Day A.J. J. Cell Sci. 2003; 116: 1863-1873Crossref PubMed Scopus (305) Google Scholar). Although there are currently no known ligands for the CUB module, the Link module (expressed in Escherichia coli (18Day A.J. Aplin R.T. Willis A.C. Protein Expression Purif. 1996; 8: 1-16Crossref PubMed Scopus (47) Google Scholar, 19Kahmann J.D. Koruth R. Day A.J. Protein Expression Purif. 1997; 9: 315-318Crossref PubMed Scopus (26) Google Scholar) and termed Link_TSG6) has been shown to interact with a large number of molecules commonly found in the ECM. Not only does Link_TSG6 bind to five distinct glycosaminoglycans (chondroitin 4-sulfate, dermatan sulfate, heparan sulfate, heparin, and HA) (3Milner C.M. Higman V.A. Day A.J. Biochem. Soc Trans. 2006; 34: 446-450Crossref PubMed Scopus (69) Google Scholar, 8Mahoney D.J. Mulloy B. Forster M.J. Blundell C.D. Fries E. Milner C.M. Day A.J. J. Biol. Chem. 2005; 280: 27044-27055Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 20Parkar A.A. Day A.J. FEBS Lett. 1997; 410: 413-417Crossref PubMed Scopus (68) Google Scholar, 21Kohda D. Morton C.J. Parkar A.A. Hatanaka H. Inagaki F.M. Campbell I.D. Day A.J. Cell. 1996; 86: 767-775Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar), it also has several protein ligands, including pentraxin-3 (16Salustri A. Garlanda C. Hirsch E. De Acetis M. Maccagno A. Bottazzi B. Doni A. Bastone A. Mantovani G. Beck Peccoz P. Salvatori G. Mahoney D.J. Day A.J. Siracusa G. Romani L. Mantovani A. Development (Camb.). 2004; 131: 1577-1586Crossref PubMed Scopus (366) Google Scholar), the G1 domains of aggrecan (22Parkar A.A. Kahmann J.D. Howat S.L. Bayliss M.T. Day A.J. FEBS Lett. 1998; 428: 171-176Crossref PubMed Scopus (64) Google Scholar) and versican (23Kuznetsova S.A. Issa P. Perruccio E.M. Zeng B. Sipes J.M. Ward Y. Seyfried N.T. Fielder H.L. Day A.J. Wight T.N. Roberts D.D. J. Cell Sci. 2006; 119: 4499-4509Crossref PubMed Scopus (38) Google Scholar), thrombospondin-1 (TSP1) (24Kuznetsova S.A. Day A.J. Mahoney D.J. Rugg M.S. Mosher D.F. Roberts D.D. J. Biol. Chem. 2005; 280: 30899-30908Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), and the serine protease inhibitor inter-α-inhibitor, with which it forms both covalent and noncovalent complexes (8Mahoney D.J. Mulloy B. Forster M.J. Blundell C.D. Fries E. Milner C.M. Day A.J. J. Biol. Chem. 2005; 280: 27044-27055Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 17Rugg M.S. Willis A.C. Mukhopadhyay D. Hascall V.C. Fries E. Fulop C. Milner C.M. Day A.J. J. Biol. Chem. 2005; 280: 25674-25686Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 25Sanggaard K.W. Karring H. Valnickova Z. Thogersen I.B. Enghild J.J. J. Biol. Chem. 2005; 280: 11936-11942Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). This diverse range of ligands is probably unusual for a Link module, the primary role of which in other Link module-containing proteins appears to be binding to HA but serves to underline the multifunctional role TSG-6 plays in inflammatory processes. The best understood interaction of TSG-6 is that with HA (21Kohda D. Morton C.J. Parkar A.A. Hatanaka H. Inagaki F.M. Campbell I.D. Day A.J. Cell. 1996; 86: 767-775Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 26Kahmann J.D. O'Brien R. Werner J.M. Heinegard D. Ladbury J.E. Campbell I.D. Day A.J. Structure (Lond.). 2000; 8: 763-774Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 27Mahoney D.J. Blundell C.D. Day A.J. J. Biol. Chem. 2001; 276: 22764-22771Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 28Blundell C.D. Mahoney D.J. Almond A. DeAngelis P.L. Kahmann J.D. Teriete P. Pickford A.R. Campbell I.D. Day A.J. J. Biol. Chem. 2003; 278: 49261-49270Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 29Blundell C.D. Almond A. Mahoney D.J. DeAngelis P.L. Campbell I.D. Day A.J. J. Biol. Chem. 2005; 280: 18189-18201Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). HA is a ubiquitous high molecular weight glycosaminoglycan (up to 107 Da) composed entirely of a repeating disaccharide of d-glucuronic acid (GlcUA) and N-acetyl-d-glucosamine (GlcNAc). It is found in the ECM of most vertebrate tissues and displays diverse biological functions, including roles in embryonic development, cell migration, and ovulation, as well as being implicated in many disease processes (30Tammi M.I. Day A.J. Turley E.A. J. Biol. Chem. 2002; 277: 4581-4584Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 31Toole B.P. Wight T.N. Tammi M.I. J. Biol. Chem. 2002; 277: 4593-4596Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 32Toole B.P. Nat. Rev. Cancer. 2004; 4: 528-539Crossref PubMed Scopus (1663) Google Scholar). Although free HA is present in synovial fluid and the vitreous of the eye, in other tissues (e.g. cartilage, skin, brain) it is found mainly in complex with proteins, forming vital structural components of the ECM. The wide range of functions ascribed to HA is thought to arise mainly through interactions with HA-binding proteins that lead to the formation of protein-HA complexes with distinct architectures and functional activities (12Day A.J. de la Motte C.A. Trends Immunol. 2005; 26: 637-643Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 33Day A.J. Sheehan J.K. Curr. Opin. Struct. Biol. 2001; 11: 617-622Crossref PubMed Scopus (122) Google Scholar, 34Day A.J. Prestwich G.D. J. Biol. Chem. 2002; 277: 4585-4588Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). As such, the production of TSG-6 at sites of inflammation is expected to result in modulation of these architectures and has clear importance for ECM biology and inflammatory disease processes (3Milner C.M. Higman V.A. Day A.J. Biochem. Soc Trans. 2006; 34: 446-450Crossref PubMed Scopus (69) Google Scholar, 12Day A.J. de la Motte C.A. Trends Immunol. 2005; 26: 637-643Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). In this regard, changes in the affinity of TSG-6 for HA (and other ECM molecules) by alterations in tissue microenvironments could have a crucial role in the regulation of TSG-6 function. We have previously reported that the interaction of HA with Link_TSG6 is pH-dependent, having maximal affinity at pH 5.5-6.0 and a significant loss of function with either increasing or decreasing pH (see Fig. 1A) (22Parkar A.A. Kahmann J.D. Howat S.L. Bayliss M.T. Day A.J. FEBS Lett. 1998; 428: 171-176Crossref PubMed Scopus (64) Google Scholar). In marked contrast, the pH dependences of both Link protein and the G1 domain of aggrecan (two constitutively expressed HA-binding proteins) for HA have no drop in affinity between pH 6.0 and 8.0 (22Parkar A.A. Kahmann J.D. Howat S.L. Bayliss M.T. Day A.J. FEBS Lett. 1998; 428: 171-176Crossref PubMed Scopus (64) Google Scholar). Recently, the pH dependence of the HA-Link_TSG6 interaction has been questioned (35Wisniewski H.G. Snitkin E.S. Mindrescu C. Sweet M.H. Vilcek J. J. Biol. Chem. 2005; 280: 14476-14484Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), with suggestions that the earlier observation results from an artifact of the solid-phase assays used (i.e. where either the protein or HA was immobilized on the surface of the microtiter plate). Here we present isothermal titration calorimetry data for defined oligosaccharides of HA (i.e. where both the protein and HA are in the solution phase) that confirms the original observation. We have determined the solution structure of Link_TSG6 in the absence and presence of an HA octasaccharide (HA8AN) (28Blundell C.D. Mahoney D.J. Almond A. DeAngelis P.L. Kahmann J.D. Teriete P. Pickford A.R. Campbell I.D. Day A.J. J. Biol. Chem. 2003; 278: 49261-49270Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) and recently have used these and other data to produce a model for the HA8AN-Link_TSG6 complex (see Fig. 1B) (29Blundell C.D. Almond A. Mahoney D.J. DeAngelis P.L. Campbell I.D. Day A.J. J. Biol. Chem. 2005; 280: 18189-18201Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). 6The HA oligosaccharides used in these previous studies and those used here were made by digestion of HA with testicular hyaluronidase to give even length oligomers of n saccharides, with a GlcUA (A) at the nonreducing terminus and GlcNAc (N) at the reducing terminus (40Mahoney D.J. Aplin R.T. Calabro A. Hascall V.C. Day A.J. Glycobiology. 2001; 11: 1025-1033Crossref PubMed Scopus (96) Google Scholar), and are referred to as HA ANn (51Blundell C.D. Almond A. Anal. Biochem. 2006; 353: 236-247Crossref PubMed Scopus (22) Google Scholar). 6The HA oligosaccharides used in these previous studies and those used here were made by digestion of HA with testicular hyaluronidase to give even length oligomers of n saccharides, with a GlcUA (A) at the nonreducing terminus and GlcNAc (N) at the reducing terminus (40Mahoney D.J. Aplin R.T. Calabro A. Hascall V.C. Day A.J. Glycobiology. 2001; 11: 1025-1033Crossref PubMed Scopus (96) Google Scholar), and are referred to as HA ANn (51Blundell C.D. Almond A. Anal. Biochem. 2006; 353: 236-247Crossref PubMed Scopus (22) Google Scholar). Therefore, it should now be possible to come to a molecular understanding of the pH dependence of HA binding. To produce a curve with a rise and then fall in activity as seen in Fig. 1A, two pH-dependent factors are required. Previously it was proposed that the gain in activity up to pH 6.0 was due to the pH-dependent folding of Link_TSG6, which was observed (by a qualitative method) to fold mostly over the range of pH 4.5 to 6.0 (22Parkar A.A. Kahmann J.D. Howat S.L. Bayliss M.T. Day A.J. FEBS Lett. 1998; 428: 171-176Crossref PubMed Scopus (64) Google Scholar). Since Link_TSG6 showed no unfolding or gross perturbation to the structure between pH 6.0 and 7.5, it was concluded that a change in ionization state of one (or more) charged residues was likely to be responsible for the loss of activity above pH 6.0 (36Blundell C.D. Kahmann J.D. Perczel. A. Mahoney D.J. Cordell M.R. Teriete P. Campbell I.D. Day A.J. Kennedy J.F. Phillips G.O. Williams P.A. Hascall V.C. Hyaluronan. Vol. 1. Woodhead Publishing Ltd., Abington, Cambridge, UK2002: 161-172Crossref Google Scholar). The four histidine residues (His4, His29, His45, His96) within Link_TSG6 are reasonable candidates for this activity, as is an aspartate residue (Asp89) buried within the protein (see Fig. 1C) (21Kohda D. Morton C.J. Parkar A.A. Hatanaka H. Inagaki F.M. Campbell I.D. Day A.J. Cell. 1996; 86: 767-775Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 36Blundell C.D. Kahmann J.D. Perczel. A. Mahoney D.J. Cordell M.R. Teriete P. Campbell I.D. Day A.J. Kennedy J.F. Phillips G.O. Williams P.A. Hascall V.C. Hyaluronan. Vol. 1. Woodhead Publishing Ltd., Abington, Cambridge, UK2002: 161-172Crossref Google Scholar, 37Day A.J. Parkar A.A. Laurent T.C. Balazs E.A. The Chemistry, Biology and Medical Applications of Hyaluronan and Its Derivatives. Portland Press, London1998: 141-147Google Scholar). However, none of these residues is directly within the HA-binding site (as determined by site-directed mutagenesis, chemical shift mapping, and the HA-bound conformation of the protein) (6Getting S.J. Mahoney D.J. Cao T. Rugg M.S. Fries E. Milner C.M. Perretti M. Day A.J. J. Biol. Chem. 2002; 277: 51068-51076Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 26Kahmann J.D. O'Brien R. Werner J.M. Heinegard D. Ladbury J.E. Campbell I.D. Day A.J. Structure (Lond.). 2000; 8: 763-774Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 27Mahoney D.J. Blundell C.D. Day A.J. J. Biol. Chem. 2001; 276: 22764-22771Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 28Blundell C.D. Mahoney D.J. Almond A. DeAngelis P.L. Kahmann J.D. Teriete P. Pickford A.R. Campbell I.D. Day A.J. J. Biol. Chem. 2003; 278: 49261-49270Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 29Blundell C.D. Almond A. Mahoney D.J. DeAngelis P.L. Campbell I.D. Day A.J. J. Biol. Chem. 2005; 280: 18189-18201Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 38Lesley J. Gal I. Mahoney D.J. Cordell M.R. Rugg M.S. Hyman R. Day A.J. Mikecz K. J. Biol. Chem. 2004; 279: 25745-25754Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), i.e. they are very unlikely to make direct contact with the bound HA molecule. In this study, we have demonstrated, using isothermal titration calorimetry (ITC) that the previously reported decrease in affinity for HA between pH 6.0 and 7.5 (22Parkar A.A. Kahmann J.D. Howat S.L. Bayliss M.T. Day A.J. FEBS Lett. 1998; 428: 171-176Crossref PubMed Scopus (64) Google Scholar) is observed in the solution phase. Analysis of single-site mutants of the TSG-6 Link module (Link_TSG6) indicated that the loss in affinity above pH 6.0 is mediated by the change in ionization state of a particular histidine residue (His4). Nuclear magnetic resonance spectroscopy (NMR) experiments allowed the pH-dependent folding profile of the protein and the pKa values of all relevant residues within Link_TSG6 to be determined. Combining these functional and NMR data, we show how the change in charge state of His4 is relayed to HA-binding residues via a network involving a salt bridge and a hydrogen bond, thus providing novel insights into the molecular basis of this important pH-dependent interaction. Sources of Materials—Wild-type (WT) and mutant forms of Link_TSG6 were expressed in E. coli and purified as described previously (27Mahoney D.J. Blundell C.D. Day A.J. J. Biol. Chem. 2001; 276: 22764-22771Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In addition to the mutants H4K (6Getting S.J. Mahoney D.J. Cao T. Rugg M.S. Fries E. Milner C.M. Perretti M. Day A.J. J. Biol. Chem. 2002; 277: 51068-51076Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) (i.e. His4 replaced by Lys) and H29K (38Lesley J. Gal I. Mahoney D.J. Cordell M.R. Rugg M.S. Hyman R. Day A.J. Mikecz K. J. Biol. Chem. 2004; 279: 25745-25754Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), six new mutants (H4S, H29A, H45A, H45K, H45S, H96K) were prepared and analyzed by one-dimensional NMR and mass spectrometry as before (27Mahoney D.J. Blundell C.D. Day A.J. J. Biol. Chem. 2001; 276: 22764-22771Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar); all mutants had molecular masses within 2 Da of their theoretical values. Uniformly 15N- and 15N,13C-labeled WT Link_TSG6 were produced by expression in M9 minimal medium (26Kahmann J.D. O'Brien R. Werner J.M. Heinegard D. Ladbury J.E. Campbell I.D. Day A.J. Structure (Lond.). 2000; 8: 763-774Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Full-length TSG-6 (Q allele) was generated as detailed previously (39Nentwich H.A. Mustafa Z. Rugg M.S. Marsden B.D. Cordell M.R. Mahoney D.J. Jenkins S.C. Dowling B. Fries E. Milner C.M. Loughlin J. Day A.J. J. Biol. Chem. 2002; 277: 15354-15362Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Medical grade HA (Hylumed Medical; molecular mass, 0.5-1.5 MDa) was obtained from Genzyme. HA oligosaccharides of defined lengths (i.e. HA8AN and HA20AN, which comprise 4 and 10 disaccharide repeats, respectively) were purified from this high molecular weight HA following digestion with testicular hyaluronidase (40Mahoney D.J. Aplin R.T. Calabro A. Hascall V.C. Day A.J. Glycobiology. 2001; 11: 1025-1033Crossref PubMed Scopus (96) Google Scholar). Mono-biotinylated Link_TSG6 and biotinylated rooster comb HA were prepared as described previously (20Parkar A.A. Day A.J. FEBS Lett. 1997; 410: 413-417Crossref PubMed Scopus (68) Google Scholar, 27Mahoney D.J. Blundell C.D. Day A.J. J. Biol. Chem. 2001; 276: 22764-22771Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). ITC Analysis of the HA-Link_TSG6 Interaction—The interaction between Link_TSG6 (WT and folded Link_TSG6 mutants H4K, H29K, H45S, H96K) and HA oligosaccharides HA 8AN and HA20AN was investigated on a Microcal VP-ITC instrument at 25 °C in 5 mm MES (pH 6.0 or 7.5), using 28 injections for each measurement, as described previously (6Getting S.J. Mahoney D.J. Cao T. Rugg M.S. Fries E. Milner C.M. Perretti M. Day A.J. J. Biol. Chem. 2002; 277: 51068-51076Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 27Mahoney D.J. Blundell C.D. Day A.J. J. Biol. Chem. 2001; 276: 22764-22771Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 28Blundell C.D. Mahoney D.J. Almond A. DeAngelis P.L. Kahmann J.D. Teriete P. Pickford A.R. Campbell I.D. Day A.J. J. Biol. Chem. 2003; 278: 49261-49270Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar); the sugar (HA8AN, 0.2 mm;HA20AN, 0.1 mm) was added in 5-μl injections to the protein solution (0.015-0.017 and 0.012-0.017 mm for the 8-mer and 20-mer, respectively) in the 1.4-ml calorimeter cell. Single-stock solutions of HA8AN and HA20AN, for which the concentrations were determined as described previously (26Kahmann J.D. O'Brien R. Werner J.M. Heinegard D. Ladbury J.E. Campbell I.D. Day A.J. Structure (Lond.). 2000; 8: 763-774Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), were used throughout all experiments to reduce errors. Data were fitted to a one-site model by nonlinear least squares regression after subtracting heats resulting from the addition of oligosaccharides into buffer alone. The binding constants for each interaction were determined by averaging the results from three experiments. Microtiter Plate-based HA Binding Assays—The HA binding activities of Link_TSG6 (WT and mutants H4K, H29K, H45S, H96K) and full-length TSG-6 were determined using the microtiter plate assay described previously (27Mahoney D.J. Blundell C.D. Day A.J. J. Biol. Chem. 2001; 276: 22764-22771Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). WT and mutant Link_TSG6 were coated overnight onto Maxisorb microtiter plates at 25 pmol/well (27Mahoney D.J. Blundell C.D. Day A.J. J. Biol. Chem. 2001; 276: 22764-22771Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and full-length TSG-6 was coated at 8 pmol/well (39Nentwich H.A. Mustafa Z. Rugg M.S. Marsden B.D. Cordell M.R. Mahoney D.J. Jenkins S.C. Dowling B. Fries E. Milner C.M. Loughlin J. Day A.J. J. Biol. Chem. 2002; 277: 15354-15362Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar); previously we showed that these concentrations of Link_TSG6 and full-length TSG-6 give equivalent signals in HA-binding assays (39Nentwich H.A. Mustafa Z. Rugg M.S. Marsden B.D. Cordell M.R. Mahoney D.J. Jenkins S.C. Dowling B. Fries E. Milner C.M. Loughlin J. Day A.J. J. Biol. Chem. 2002; 277: 15354-15362Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). To vary the pH of the binding reaction, after the initial washes with standard assay buffer (SAB; 100 mm NaCl, 50 mm sodium acetate, 0.05% (v/v) Tween 20, pH 6.0), individual wells were rewashed once with buffers of specific pH (containing 100 mm NaCl, 50 mm Na-HEPES, 0.05% (v/v) Tween 20) and dried, then biotinylated HA (27Mahoney D.J. Blundell C.D. Day A.J. J. Biol. Chem. 2001; 276: 22764-22771Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) in the appropriate pH buffer was added to each well (12.5 ng/well), and the mixture was incubated at room temperature for 4 h. Wells were then rewashed in SAB, and the assay was continued as described previously (27Mahoney D.J. Blundell C.D. Day A.J. J. Biol. Chem. 2001; 276: 22764-22771Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). General NMR Methodology—Samples for NMR analysis comprised 15N- or 15N,13C-labeled WT Link_TSG6 with or without oligosaccharide (in a 1:1 molar ratio) in 600 μl of 10% (v/v) D2O, 0.02% (w/v) NaN3. All NMR experiments were performed at 25 °C on spectrometers at the Oxford Centre for Molecular Sciences (University of Oxford, UK) at a proton resonance frequency of 500 MHz (unless otherwise stated) following the methods detailed previously (26Kahmann J.D. O'Brien R. Werner J.M. Heinegard D. Ladbury J.E. Campbell I.D. Day A.J. Structure (Lond.). 2000; 8: 763-774Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 28Blundell C.D. Mahoney D.J. Almond A. DeAngelis P.L. Kahmann J.D. Teriete P. Pickford A.R. Campbell I.D. Day A.J. J. Biol. Chem. 2003; 278: 49261-49270Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 29Blundell C.D. Almond A. Mahoney D.J. DeAngelis P.L. Campbell I.D. Day A.J. J. Biol. Chem. 2005; 280: 18189-18201Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Data were processed using FELIX 2.3 and referenced and analyzed with XEasy. NMR Analysis of Free and H" @default.
W1999080471 created "2016-06-24" @default.
W1999080471 creator A5006588214 @default.
W1999080471 creator A5018635238 @default.
W1999080471 creator A5019105439 @default.
W1999080471 creator A5030688126 @default.
W1999080471 creator A5062219892 @default.
W1999080471 creator A5062909663 @default.
W1999080471 creator A5070614775 @default.
W1999080471 creator A5079876267 @default.
W1999080471 creator A5086306406 @default.
W1999080471 date "2007-04-01" @default.
W1999080471 modified "2023-09-29" @default.
W1999080471 title "Determining the Molecular Basis for the pH-dependent Interaction between the Link Module of Human TSG-6 and Hyaluronan" @default.
W1999080471 cites W1585907324 @default.
W1999080471 cites W1652278102 @default.
W1999080471 cites W1893337122 @default.
W1999080471 cites W1971859070 @default.
W1999080471 cites W1974527403 @default.
W1999080471 cites W1977782939 @default.
W1999080471 cites W1983656706 @default.
W1999080471 cites W1989241075 @default.
W1999080471 cites W1991333792 @default.
W1999080471 cites W1992083598 @default.
W1999080471 cites W1996921732 @default.
W1999080471 cites W1998389103 @default.
W1999080471 cites W2011291067 @default.
W1999080471 cites W2017606019 @default.
W1999080471 cites W2023208535 @default.
W1999080471 cites W2026831675 @default.
W1999080471 cites W2039395012 @default.
W1999080471 cites W2053405152 @default.
W1999080471 cites W2059217857 @default.
W1999080471 cites W2060665961 @default.
W1999080471 cites W2069649792 @default.
W1999080471 cites W2071479693 @default.
W1999080471 cites W2079586552 @default.
W1999080471 cites W2086889757 @default.
W1999080471 cites W2088128469 @default.
W1999080471 cites W2089753996 @default.
W1999080471 cites W2091120027 @default.
W1999080471 cites W2092704138 @default.
W1999080471 cites W2093681883 @default.
W1999080471 cites W2110434905 @default.
W1999080471 cites W2115525854 @default.
W1999080471 cites W2118875207 @default.
W1999080471 cites W2123392882 @default.
W1999080471 cites W2126390088 @default.
W1999080471 cites W2129082014 @default.
W1999080471 cites W2129520501 @default.
W1999080471 cites W2136398593 @default.
W1999080471 cites W2138190971 @default.
W1999080471 cites W2138948612 @default.
W1999080471 cites W2139519365 @default.
W1999080471 cites W2142159393 @default.
W1999080471 cites W2146505397 @default.
W1999080471 cites W2147020424 @default.
W1999080471 cites W2147716262 @default.
W1999080471 cites W2153729118 @default.
W1999080471 cites W2161620948 @default.
W1999080471 cites W2163279804 @default.
W1999080471 cites W2170726898 @default.
W1999080471 cites W2170982648 @default.
W1999080471 doi "https://doi.org/10.1074/jbc.m611713200" @default.
W1999080471 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17307731" @default.
W1999080471 hasPublicationYear "2007" @default.
W1999080471 type Work @default.
W1999080471 sameAs 1999080471 @default.
W1999080471 citedByCount "32" @default.
W1999080471 countsByYear W19990804712012 @default.
W1999080471 countsByYear W19990804712013 @default.
W1999080471 countsByYear W19990804712014 @default.
W1999080471 countsByYear W19990804712015 @default.
W1999080471 countsByYear W19990804712016 @default.
W1999080471 countsByYear W19990804712019 @default.
W1999080471 countsByYear W19990804712021 @default.
W1999080471 countsByYear W19990804712022 @default.
W1999080471 countsByYear W19990804712023 @default.
W1999080471 crossrefType "journal-article" @default.
W1999080471 hasAuthorship W1999080471A5006588214 @default.
W1999080471 hasAuthorship W1999080471A5018635238 @default.
W1999080471 hasAuthorship W1999080471A5019105439 @default.
W1999080471 hasAuthorship W1999080471A5030688126 @default.
W1999080471 hasAuthorship W1999080471A5062219892 @default.
W1999080471 hasAuthorship W1999080471A5062909663 @default.
W1999080471 hasAuthorship W1999080471A5070614775 @default.
W1999080471 hasAuthorship W1999080471A5079876267 @default.
W1999080471 hasAuthorship W1999080471A5086306406 @default.
W1999080471 hasBestOaLocation W19990804711 @default.
W1999080471 hasConcept C12426560 @default.
W1999080471 hasConcept C12554922 @default.
W1999080471 hasConcept C185592680 @default.
W1999080471 hasConcept C2524010 @default.
W1999080471 hasConcept C2778753846 @default.
W1999080471 hasConcept C31258907 @default.
W1999080471 hasConcept C33923547 @default.
W1999080471 hasConcept C41008148 @default.
W1999080471 hasConcept C55493867 @default.