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• W3010056472 abstract "Inter-α-inhibitor is a proteoglycan essential for mammalian reproduction and also plays a less well-characterized role in inflammation. It comprises two homologous “heavy chains” (HC1 and HC2) covalently attached to chondroitin sulfate on the bikunin core protein. Before ovulation, HCs are transferred onto the polysaccharide hyaluronan (HA) to form covalent HC·HA complexes, thereby stabilizing an extracellular matrix around the oocyte required for fertilization. Additionally, such complexes form during inflammatory processes and mediate leukocyte adhesion in the synovial fluids of arthritis patients and protect against sepsis. Here using X-ray crystallography, we show that human HC1 has a structure similar to integrin β-chains, with a von Willebrand factor A domain containing a functional metal ion-dependent adhesion site (MIDAS) and an associated hybrid domain. A comparison of the WT protein and a variant with an impaired MIDAS (but otherwise structurally identical) by small-angle X-ray scattering and analytical ultracentrifugation revealed that HC1 self-associates in a cation-dependent manner, providing a mechanism for HC·HA cross-linking and matrix stabilization. Surprisingly, unlike integrins, HC1 interacted with RGD-containing ligands, such as fibronectin, vitronectin, and the latency-associated peptides of transforming growth factor β, in a MIDAS/cation-independent manner. However, HC1 utilizes its MIDAS motif to bind to and inhibit the cleavage of complement C3, and small-angle X-ray scattering–based modeling indicates that this occurs through the inhibition of the alternative pathway C3 convertase. These findings provide detailed structural and functional insights into HC1 as a regulator of innate immunity and further elucidate the role of HC·HA complexes in inflammation and ovulation. Inter-α-inhibitor is a proteoglycan essential for mammalian reproduction and also plays a less well-characterized role in inflammation. It comprises two homologous “heavy chains” (HC1 and HC2) covalently attached to chondroitin sulfate on the bikunin core protein. Before ovulation, HCs are transferred onto the polysaccharide hyaluronan (HA) to form covalent HC·HA complexes, thereby stabilizing an extracellular matrix around the oocyte required for fertilization. Additionally, such complexes form during inflammatory processes and mediate leukocyte adhesion in the synovial fluids of arthritis patients and protect against sepsis. Here using X-ray crystallography, we show that human HC1 has a structure similar to integrin β-chains, with a von Willebrand factor A domain containing a functional metal ion-dependent adhesion site (MIDAS) and an associated hybrid domain. A comparison of the WT protein and a variant with an impaired MIDAS (but otherwise structurally identical) by small-angle X-ray scattering and analytical ultracentrifugation revealed that HC1 self-associates in a cation-dependent manner, providing a mechanism for HC·HA cross-linking and matrix stabilization. Surprisingly, unlike integrins, HC1 interacted with RGD-containing ligands, such as fibronectin, vitronectin, and the latency-associated peptides of transforming growth factor β, in a MIDAS/cation-independent manner. However, HC1 utilizes its MIDAS motif to bind to and inhibit the cleavage of complement C3, and small-angle X-ray scattering–based modeling indicates that this occurs through the inhibition of the alternative pathway C3 convertase. These findings provide detailed structural and functional insights into HC1 as a regulator of innate immunity and further elucidate the role of HC·HA complexes in inflammation and ovulation. Inter-α-inhibitor (IαI) 3The abbreviations used are: IαIinter-α-inhibitorAUCanalytical ultracentrifugationCMG2capillary morphogenesis protein-2COCcumulus–oocyte complexCSchondroitin sulfateFBcomplement factor BHAhyaluronanHCheavy chainITGBintegrin β-chainLAPlatency associated peptideLLClarge latent complexLTBPlatent TGFβ-binding proteinMIDASmetal ion-dependent adhesion sitePαIpre-α-inhibitorrHC1recombinant HC1SAXSsmall-angle X-ray scatteringSLCsmall latent complexTEM8tumor endothelial marker-8TGFβtransforming growth factor βTSG-6tumor necrosis factor–stimulated gene 6vWFAvon Willebrand factor ARMSDroot-mean-square deviationPDBProtein Data BankSPRsurface plasmon resonanceFHfactor HNT1N-terminal regionCTC-terminal regionEGFepidermal growth factor. is a plasma proteoglycan composed of two homologous “heavy chains” (HC1 and HC2) covalently attached to chondroitin sulfate (CS) on the bikunin core protein (1Enghild J.J. Thøgersen I.B. Pizzo S.V. Salvesen G. Analysis of inter-α-trypsin inhibitor and a novel trypsin inhibitor, pre-α-trypsin inhibitor, from human plasma: polypeptide chain stoichiometry and assembly by glycan.J. Biol. Chem. 1989; 264 (2476436): 15975-15981Abstract Full Text PDF PubMed Google Scholar; see Fig. S1A). IαI plays a critical role in mammalian reproductive biology such that female mice with the bikunin gene deleted, and consequently lacking IαI as well as the related pre-α-inhibitor (PαI), are infertile (2Zhuo L. Yoneda M. Zhao M. Yingsung W. Yoshida N. Kitagawa Y. Kawamura K. Suzuki T. Kimata K. Defect in SHAP-hyaluronan complex causes severe female infertility: a study by inactivation of the bikunin gene in mice.J. Biol. Chem. 2001; 276 (11145954): 7693-769610.1074/jbc.C000899200Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 3Sato H. Kajikawa S. Kuroda S. Horisawa Y. Nakamura N. Kaga N. Kakinuma C. Kato K. Morishita H. Niwa H. Miyazaki J. Impaired fertility in female mice lacking urinary trypsin inhibitor.Biochem. Biophys. Res. Commun. 2001; 281 (11243855): 1154-116010.1006/bbrc.2001.4475Crossref PubMed Scopus (88) Google Scholar). This is due to the impaired formation of the cumulus extracellular matrix that normally drives the expansion of the cumulus–oocyte complex (COC). This elastic matrix (4Chen X. Bonfiglio R. Banerji S. Jackson D.G. Salustri A. Richter R.P. Micromechanical analysis of the hyaluronan-rich matrix surrounding the oocyte reveals a uniquely soft and elastic composition.Biophys. J. 2016; 110 (27332136): 2779-278910.1016/j.bpj.2016.03.023Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) protects the oocyte during the expulsion of the COC from the ovarian follicle and also provides a large surface area facilitating sperm capture in vivo (5Russell D.L. Salustri A. Extracellular matrix of the cumulus–oocyte complex.Semin. Reprod. Med. 2006; 24 (16944419): 217-22710.1055/s-2006-948551Crossref PubMed Scopus (130) Google Scholar, 6Nagyova E. Organization of the expanded cumulus–extracellular matrix in preovulatory follicles: a role for inter-α-trypsin inhibitor.Endocr. Regul. 2015; 49 (25687679): 37-4510.4149/endo_2015_01_37Crossref PubMed Scopus (12) Google Scholar). The cumulus matrix is rich in the nonsulfated glycosaminoglycan hyaluronan (HA), where this high-molecular-weight polysaccharide becomes modified by the covalent attachment of HC1 and HC2 from IαI and HC3 from PαI (7Mukhopadhyay D. Hascall V.C. Day A.J. Salustri A. Fülöp C. Two distinct populations of tumor necrosis factor-stimulated gene-6 protein in the extracellular matrix of expanded mouse cumulus cell–oocyte complexes.Arch. Biochem. Biophys. 2001; 394 (11594731): 173-18110.1006/abbi.2001.2552Crossref PubMed Scopus (106) Google Scholar). TSG-6 plays a catalytic role in transferring HCs from the CS chains of IαI and PαI onto HA to form HC·HA complexes (8Rugg M.S. Willis A.C. Mukhopadhyay D. Hascall V.C. Fries E. Fülöp C. Milner C.M. Day A.J. Characterization of complexes formed between TSG-6 and inter-α-inhibitor that act as intermediates in the covalent transfer of heavy chains onto hyaluronan.J. Biol. Chem. 2005; 280 (15840581): 25674-2568610.1074/jbc.M501332200Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 9Day A.J. Milner C.M. TSG-6: A multifunctional protein with anti-inflammatory and tissue-protective properties.Matrix Biol. 2019; 78-79: 60-83Crossref PubMed Scopus (146) Google Scholar), where this is essential for female fertility (10Briggs D.C. Birchenough H.L. Ali T. Rugg M.S. Waltho J.P. Ievoli E. Jowitt T.A. Enghild J.J. Richter R.P. Salustri A. Milner C.M. Day A.J. Metal ion-dependent heavy chain transfer activity of TSG-6 mediates assembly of the cumulus-oocyte matrix.J. Biol. Chem. 2015; 290 (26468290): 28708-2872310.1074/jbc.M115.669838Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 11Fülöp C. Szántó S. Mukhopadhyay D. Bárdos T. Kamath R.V. Rugg M.S. Day A.J. Salustri A. Hascall V.C. Glant T.T. Mikecz K. Impaired cumulus mucification and female sterility in tumor necrosis factor-induced protein-6 deficient mice.Development. 2003; 130 (12668637): 2253-226110.1242/dev.00422Crossref PubMed Scopus (328) Google Scholar, 12Ochsner S.A. Day A.J. Rugg M.S. Breyer R.M. Gomer R.H. Richards J.S. Disrupted function of tumor necrosis factor-α-stimulated gene 6 blocks cumulus cell-oocyte complex expansion.Endocrinology. 2003; 144 (12959984): 4376-438410.1210/en.2003-0487Crossref PubMed Scopus (128) Google Scholar, 13Salustri 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. et al.PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization.Development. 2004; 131 (14998931): 1577-158610.1242/dev.01056Crossref PubMed Scopus (363) Google Scholar). As well as being expressed by cumulus cells during ovulation, TSG-6 is also produced in the context of inflammation, where it mediates the formation of HC·HAs (14Ni K. Gill A. Cao D. Koike K. Schweitzer K.S. Garantziotis S. Petrache I. Intravascular heavy chain-modification of hyaluronan during endotoxic shock.Biochem. Biophys. Rep. 2019; 17 (30623115): 114-12110.1016/j.bbrep.2018.12.007PubMed Google Scholar), e.g. when IαI/PαI leaks into tissues from the blood circulation (reviewed in Ref. 9Day A.J. Milner C.M. TSG-6: A multifunctional protein with anti-inflammatory and tissue-protective properties.Matrix Biol. 2019; 78-79: 60-83Crossref PubMed Scopus (146) Google Scholar). inter-α-inhibitor analytical ultracentrifugation capillary morphogenesis protein-2 cumulus–oocyte complex chondroitin sulfate complement factor B hyaluronan heavy chain integrin β-chain latency associated peptide large latent complex latent TGFβ-binding protein metal ion-dependent adhesion site pre-α-inhibitor recombinant HC1 small-angle X-ray scattering small latent complex tumor endothelial marker-8 transforming growth factor β tumor necrosis factor–stimulated gene 6 von Willebrand factor A root-mean-square deviation Protein Data Bank surface plasmon resonance factor H N-terminal region C-terminal region epidermal growth factor. In IαI, HC1, and HC2 (the protein products of the ITIH1 and ITIH2 genes) are covalently bound via ester bonds linking their C termini to GalNAc sugars within the CS chain (15Enghild J.J. Salvesen G. Thøgersen I.B. Valnickova Z. Pizzo S.V. Hefta S.A. Presence of the protein–glycosaminoglycan–protein covalent cross-link in the inter-α-inhibitor–related proteinase inhibitor heavy chain 2/bikunin.J. Biol. Chem. 1993; 268 (7682553): 8711-8716Abstract Full Text PDF PubMed Google Scholar, 16Morelle W. Capon C. Balduyck M. Sautiere P. Kouach M. Michalski C. Fournet B. Mizon J. Chondroitin sulphate covalently cross-links the three polypeptide chains of inter-α-trypsin inhibitor.Eur. J. Biochem. 1994; 221 (7513643): 881-88810.1111/j.1432-1033.1994.tb18803.xCrossref PubMed Scopus (68) Google Scholar). The two HCs are attached to sugars one or two disaccharides apart, with HC2 positioned closer to bikunin than HC1 (17Enghild J.J. Thøgersen I.B. Cheng F. Fransson L.A. Roepstorff P. Rahbek-Nielsen H. Organization of the inter-α-inhibitor heavy chains on the chondroitin sulfate originating from Ser10 of bikunin: posttranslational modification of IαI-derived bikunin.Biochemistry. 1999; 38 (10512637): 11804-1181310.1021/bi9908540Crossref PubMed Scopus (63) Google Scholar, 18Ly M. Leach 3rd, F.E. Laremore T.N. Toida T. Amster I.J. Linhardt R.J. Linhardt R.J. The proteoglycan bikunin has a defined sequence.Nat. Chem. Biol. 2011; 7 (21983600): 827-83310.1038/nchembio.673Crossref PubMed Scopus (148) Google Scholar). HC1 and HC2 are ∼80 kDa in size and share ∼39% sequence identity. They are synthesized with C-terminal pro-domains (of 239 and 244 amino acid residues, respectively) that are removed when the HCs are covalently attached to the bikunin CS chain (19Kaczmarczyk A. Thuveson M. Fries E. Intracellular coupling of the heavy chain of pre-α-inhibitor to chondroitin sulfate.J. Biol. Chem. 2002; 277 (11827976): 13578-1358210.1074/jbc.M200288200Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 20Zhuo L. Hascall V.C. Kimata K. Inter-α-trypsin inhibitor, a covalent protein–glycosaminoglycan–protein complex.J. Biol. Chem. 2004; 279 (15151994): 38079-3808210.1074/jbc.R300039200Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). HC3 (ITIH3; 54% identical to HC1) can also link to the bikunin CS proteoglycan (Fig. S1A) to form PαI (1Enghild J.J. Thøgersen I.B. Pizzo S.V. Salvesen G. Analysis of inter-α-trypsin inhibitor and a novel trypsin inhibitor, pre-α-trypsin inhibitor, from human plasma: polypeptide chain stoichiometry and assembly by glycan.J. Biol. Chem. 1989; 264 (2476436): 15975-15981Abstract Full Text PDF PubMed Google Scholar, 21Enghild J.J. Salvesen G. Hefta S.A. Thøgersen I.B. Rutherfurd S. Pizzo S. V Chondroitin 4-sulfate covalently cross-links the chains of the human blood protein pre-α-inhibitor.J. Biol. Chem. 1991; 266 (1898736): 747-751Abstract Full Text PDF PubMed Google Scholar), and there is evidence that the related HC5, and likely HC6, can also become attached to CS in this way (9Day A.J. Milner C.M. TSG-6: A multifunctional protein with anti-inflammatory and tissue-protective properties.Matrix Biol. 2019; 78-79: 60-83Crossref PubMed Scopus (146) Google Scholar, 22Martin J. Midgley A. Meran S. Woods E. Bowen T. Phillips A.O. Steadman R. Tumor Necrosis Factor-stimulated Gene 6 (TSG-6)–mediated interactions with the inter-α-inhibitor heavy chain 5 facilitate tumor growth factor β1 (TGFβ1)–dependent fibroblast to myofibroblast differentiation.J. Biol. Chem. 2016; 291 (27143355): 13789-1380110.1074/jbc.M115.670521Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). All HCs are predicted to contain a single von Willebrand factor type A (vWFA) domain; however, there are no structural data yet available for heavy chains. The covalent attachment of HCs to HA changes the physical properties of this ubiquitous glycosaminoglycan. For example in synovial fluid from rheumatoid arthritis patients, where on average three to five HCs are attached to an HA chain of ∼2 MDa, the polysaccharide is more aggregated compared with unmodified HA (23Yingsung W. Zhuo L. Morgelin M. Yoneda M. Kida D. Watanabe H. Ishiguro N. Iwata H. Kimata K. Molecular heterogeneity of the SHAP–hyaluronan complex: Isolation and characterization of the complex in synovial fluid from patients with rheumatoid arthritis.J. Biol. Chem. 2003; 278 (12799384): 32710-3271810.1074/jbc.M303658200Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar); this has been attributed to cross-linking of HC·HA complexes via interactions between HCs based on their apparent associations visualized by EM. Given that HC1, HC2, and HC3 can all be transferred onto HA during arthritis (24Zhao M. Yoneda M. Ohashi Y. Kurono S. Iwata H. Ohnuki Y. Kimata K. Evidence for the covalent binding of SHAP, heavy chains of inter-α-trypsin inhibitor, to hyaluronan.J. Biol. Chem. 1995; 270 (7592891): 26657-2666310.1074/jbc.270.44.26657Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), such cross-linking could potentially be mediated by homotypic and/or heterotypic HC–HC interactions; however, currently there are no biophysical data to support this. Irrespective of the mechanism, the formation of HC·HA in arthritic joints enhances the binding of HA to its major cell surface receptor, CD44, on leukocytes (25Zhuo L. Kanamori A. Kannagi R. Itano N. Wu J. Hamaguchi M. Ishiguro N. Kimata K. SHAP potentiates the CD44-mediated leukocyte adhesion to the hyaluronan substratum.J. Biol. Chem. 2006; 281 (16702221): 20303-2031410.1074/jbc.M506703200Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). However, it is unknown whether this, or indeed the altered hydrodynamic properties of the modified HA (26Baranova N.S. Inforzato A. Briggs D.C. Tilakaratna V. Enghild J.J. Thakar D. Milner C.M. Day A.J. Richter R.P. Incorporation of pentraxin 3 into hyaluronan matrices is tightly regulated and promotes matrix cross-linking.J. Biol. Chem. 2014; 289 (25190808): 30481-3049810.1074/jbc.M114.568154Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), are part of a protective process or contribute to arthritis pathology (9Day A.J. Milner C.M. TSG-6: A multifunctional protein with anti-inflammatory and tissue-protective properties.Matrix Biol. 2019; 78-79: 60-83Crossref PubMed Scopus (146) Google Scholar). In this regard, HC·HA complexes from human amniotic membrane, which are reported to contain only HC1 (27Zhang S. He H. Day A.J. Tseng S.C. Constitutive expression of inter-α-inhibitor (IαI) family proteins and tumor necrosis factor-stimulated gene-6 (TSG-6) by human amniotic membrane epithelial and stromal cells supporting formation of the heavy chain-hyaluronan (HC-HA) complex.J. Biol. Chem. 2012; 287 (22351758): 12433-1244410.1074/jbc.M112.342873Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), are potently anti-fibrotic and anti-inflammatory (9Day A.J. Milner C.M. TSG-6: A multifunctional protein with anti-inflammatory and tissue-protective properties.Matrix Biol. 2019; 78-79: 60-83Crossref PubMed Scopus (146) Google Scholar, 28Ogawa Y. He H. Mukai S. Imada T. Nakamura S. Su C.-W. Mahabole M. Tseng S.C. Tsubota K. Heavy chain-hyaluronan/pentraxin 3 from amniotic membrane suppresses inflammation and scarring in murine lacrimal gland and conjunctiva of chronic graft-versus-host disease.Sci. Rep. 2017; 7 (28165063): 4219510.1038/srep42195Crossref PubMed Scopus (24) Google Scholar); HC·HAs also protect against endotoxic shock and sepsis (14Ni K. Gill A. Cao D. Koike K. Schweitzer K.S. Garantziotis S. Petrache I. Intravascular heavy chain-modification of hyaluronan during endotoxic shock.Biochem. Biophys. Rep. 2019; 17 (30623115): 114-12110.1016/j.bbrep.2018.12.007PubMed Google Scholar, 29Stober V.P. Lim Y.-P. Opal S. Zhuo L. Kimata K. Garantziotis S. Inter-α-inhibitor ameliorates endothelial inflammation in sepsis.Lung. 2019; 197 (31028466): 361-36910.1007/s00408-019-00228-1Crossref PubMed Scopus (19) Google Scholar, 30Htwe S.S. Wake H. Liu K. Teshigawara K. Stonestreet B.S. Lim Y.-P. Nishibori M. Inter-α inhibitor proteins maintain neutrophils in a resting state by regulating shape and reducing ROS production.Blood Adv. 2018; 2 (30093530): 1923-193410.1182/bloodadvances.2018018986Crossref PubMed Scopus (17) Google Scholar). However, the role of HCs (including HC1) has not been determined in these processes. IαI has been implicated as a regulator of innate immunity having been shown to be an inhibitor of the complement system, affecting the alternative, classical, and lectin activation pathways (31Adair J.E. Stober V. Sobhany M. Zhuo L. Roberts J.D. Negishi M. Kimata K. Garantziotis S. Inter-α-trypsin inhibitor promotes bronchial epithelial repair after injury through vitronectin binding.J. Biol. Chem. 2009; 284 (19395377): 16922-1693010.1074/jbc.M808560200Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 32Garantziotis S. Hollingsworth J.W. Ghanayem R.B. Timberlake S. Zhuo L. Kimata K. Schwartz D.A. Inter-α-trypsin inhibitor attenuates complement activation and complement-induced lung injury.J. Immunol. 2007; 179 (17785858): 4187-419210.4049/jimmunol.179.6.4187Crossref PubMed Scopus (61) Google Scholar, 33Okroj M. Holmquist E. Sjölander J. Corrales L. Saxne T. Wisniewski H.-G. Blom A.M. Heavy chains of inter α inhibitor (IαI) inhibit the human complement system at early stages of the cascade.J. Biol. Chem. 2012; 287 (22528482): 20100-2011010.1074/jbc.M111.324913Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The inhibition of the alternative and classical pathways of complement is thought to be dependent on HCs rather than bikunin (32Garantziotis S. Hollingsworth J.W. Ghanayem R.B. Timberlake S. Zhuo L. Kimata K. Schwartz D.A. Inter-α-trypsin inhibitor attenuates complement activation and complement-induced lung injury.J. Immunol. 2007; 179 (17785858): 4187-419210.4049/jimmunol.179.6.4187Crossref PubMed Scopus (61) Google Scholar, 33Okroj M. Holmquist E. Sjölander J. Corrales L. Saxne T. Wisniewski H.-G. Blom A.M. Heavy chains of inter α inhibitor (IαI) inhibit the human complement system at early stages of the cascade.J. Biol. Chem. 2012; 287 (22528482): 20100-2011010.1074/jbc.M111.324913Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar); however, most of the available data were generated using IαI, and the HC-mediated mechanisms have not been determined. In the case of the alternative pathway, IαI was found to inhibit the factor D–mediated cleavage of factor B (FB) to Bb, which occurs during the formation of the C3 convertase (C3bBb). IαI has been found to bind to vitronectin (31Adair J.E. Stober V. Sobhany M. Zhuo L. Roberts J.D. Negishi M. Kimata K. Garantziotis S. Inter-α-trypsin inhibitor promotes bronchial epithelial repair after injury through vitronectin binding.J. Biol. Chem. 2009; 284 (19395377): 16922-1693010.1074/jbc.M808560200Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), a multifunctional plasma and matrix protein that, as a well as being a regulator of complement system terminal pathway, also mediates binding to αV integrins (34Preissner K.T. Reuning U. Vitronectin in vascular context: facets of a multitalented matricellular protein.Semin. Thromb. Hemost. 2011; 37 (21805447): 408-42410.1055/s-0031-1276590Crossref PubMed Scopus (93) Google Scholar). Vitronectin's integrin-binding activity has an important role in epithelial repair in the context of lung homeostasis, and the adhesion and migration of epithelial cells was promoted by its interaction with IαI (31Adair J.E. Stober V. Sobhany M. Zhuo L. Roberts J.D. Negishi M. Kimata K. Garantziotis S. Inter-α-trypsin inhibitor promotes bronchial epithelial repair after injury through vitronectin binding.J. Biol. Chem. 2009; 284 (19395377): 16922-1693010.1074/jbc.M808560200Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar); moreover, IαI-deficient mice had impaired recovery in experimental lung injury. The association between IαI and vitronectin is reported to be of high affinity and inhibited by RGD peptides, implicating IαI's vWFA domain in the interaction. To explore and better explain the functions of HCs, we undertook structural and biophysical characterization of the prototypical heavy chain, HC1. Here we present the crystal structure of HC1 and reveal that HC1 can form metal ion-dependent homodimers, which require a functional metal ion-dependent adhesion site (MIDAS) motif within its vWFA domain. We also show that the MIDAS is important in HC1-mediated inhibition of the alternative pathway C3 convertase via its interaction with C3 and demonstrate that HC1 can interact with vitronectin and other novel ligands (e.g. fibronectin and small latent complexes of transforming growth factor β (TGFβ)) in a noncanonical MIDAS-independent manner. Crystal structures were obtained for the WT recombinant HC1 (rHC1), encompassing the entire 638-residue mature protein sequence, and for the corresponding D298A single-site mutant, at 2.34 and 2.20 Å resolution, respectively (Table 1). This revealed that heavy chains are composed of three distinct domains (Fig. 1A and Fig. S1B); these domains include a vWFA domain (residues 288–477), which is inserted into a loop in an integrin-like hybrid domain (termed here HC–Hybrid1) composed of residues 266–287 and 478–543. These two domains sit atop a large, novel, 16-stranded β-sandwich, composed of residues 45–265 and 601–652, along with 3 α-helices (residues 544–600), which together we call the HC–Hybrid2 domain (Fig. 1A).Table 1Data collection and refinement statistics for rHC1WTD298AProtein Data Bank code6FPY6FPZWavelength0.92 Å0.92 ÅResolution rangeaStatistics for the highest-resolution shell are shown in parentheses.71–2.3 (2.4–2.3)56.8–2.2 (2.3–2.2)Space groupP 42P 42Unit cell158.8, 158.8, 65.4, 90, 90, 90159.7, 159.7, 65.79, 90, 90, 90Total reflections455,794 (45,882)312,045 (26,776)Unique reflections69,109 (6862)83,837 (8233)Multiplicity6.6 (6.7)3.7 (3.3)Completeness (%)91.94 (87.58)94.27 (88.13)Mean I/σI8.02 (1.88)10.93 (2.05)Wilson B-factor37.4634.70Rmerge0.1432 (0.8002)0.06619 (0.4731)Rmeas0.1554 (0.8681)0.07734 (0.5596)Rpim0.05991 (0.3337)0.03928 (0.2917)CC½0.994 (0.523)0.995 (0.478)CC*0.998 (0.829)0.999 (0.804)Reflections used in refinement63,610 (6027)79,667 (7405)Reflections used for Rfree3194 (306)3983 (347)Rwork0.2317 (0.3810)0.2174 (0.3701)Rfree0.2605 (0.4195)0.2475 (0.3641)CCwork0.953 (0.726)0.954 (0.730)CCfree0.946 (0.696)0.948 (0.768)Number of non-hydrogen atoms970210,106Macromolecules93199335Ligands3858Solvent345713Protein residues12011196RMSDBonds0.0040.003Angles1.030.92Ramachandran (%)Favored97.6598.15Allowed2.271.85Outliers0.080.00Rotamer outliers (%)1.201.00Clashscore1.781.71Average B-factor49.6745.75Macromolecules49.9245.76Ligands62.8271.64Solvent41.5543.54Number of TLS groups86a Statistics for the highest-resolution shell are shown in parentheses. Open table in a new tab The construct-derived His6 tag and residues 35–44, 631–638, and 653–672 of HC1, which were clearly present in the protein preparation as determined by MS, were not visible in the electron density and are therefore assumed to be unstructured or highly conformationally labile. This includes the native C terminus of HC1, which is covalently attached to CS in IαI and to HA in the context of HC·HA complexes. These missing residues were modeled using small angle X-ray scattering (SAXS) data for (monomeric) D298A as a restraint target (Fig. 1, C and D); as can be seen, the AllosMod model fits better than the crystal structure alone to the experimental SAXS curve, with χ values of 1.56 and 2.68, respectively. Despite low sequence identities (17 and 15%, respectively), the HC1 vWFA domain is structurally most similar to the vWFA domains from capillary morphogenesis protein 2 (CMG2 (35Lacy D.B. Wigelsworth D.J. Scobie H.M. Young J.A. Collier R.J. Crystal structure of the von Willebrand factor A domain of human capillary morphogenesis protein 2: an anthrax toxin receptor.Proc. Natl. Acad. Sci. U.S.A. 2004; 101 (15079089): 6367-637210.1073/pnas.0401506101Crossref PubMed Scopus (102) Google Scholar)) and tumor endothelial marker-8 (TEM8 (36Fu S. Tong X. Cai C. Zhao Y. Wu Y. Li Y. Xu J. Zhang X.C. Xu L. Chen W. Rao Z. The structure of tumor endothelial marker 8 (TEM8) extracellular domain and implications for its receptor function for recognizing anthrax toxin.PLoS One. 2010; 5 (20585457): e1120310.1371/journal.pone.0011203Crossref PubMed Scopus (28) Google Scholar)), with PDBeFold Q scores of 0.56 and 0.52, respectively. These are both transmembrane proteins that serve as functional receptors for the anthrax toxin (37Liu S. Zhang Y. Hoover B. Leppla S. The receptors that mediate the direct lethality of anthrax toxin.Toxins (Basel). 2012; 5 (23271637): 1-810.3390/toxins5010001Crossref PubMed Scopus (34) Google Scholar). HC1 also shows significant structural similarity to the vWFA domains of various integrin I domains, with the highest Q score (0.50) for integrin αM (also known as CD11b or as complement receptor type 3 (CR3)), and the vWFA domain of complement FB (Q score 0.37); FB and integrin αM are C3-binding proteins, with roles in complement activation/amplification and complement-mediated phagocytosis, respectively (38Ricklin D. Reis E.S. Lambris J.D. Complement in disease: a defence system turning offensive.Nat. Rev. Nephrol. 2016; 12 (27211870): 383-40110.1038/nrneph.2016.70Crossref PubMed Scopus (326) Google Scholar). From the structure of WT rHC1, it is apparent that its vWFA domain contains a MIDAS motif (Fig. 1B), where residues Asp298, Ser300, Ser302, and Asp403 chelate a magnesium ion, the identity of which can be inferred from the trigonal bipyramid coordination geometry, bond distances, and refined atomic displacement parameters. The D298A mutant has no bound Mg2+ ion but is otherwise essentially identical to the WT structure, with a RMSD between the two most similar chains of 0.24 Å over 598 C-α atoms. The HC–Hybrid1 domain of HC1 is composed of two four-stranded β-sheets, where two of the β-strands are formed from amino acid residues before the vWFA domain and the remaining six β-strands are from sequence after it; these regions (residues 266–287 and 478–543, respectively) are connected by a disulfide bond between Cys268 and Cys540 (39Olsen E.H. Rahbek-Nielsen H. Thogersen I.B. Roepstorff P. Enghild J.J. Posttranslational modifications of human inter-α-inhibitor: identification of glycans and disulfide bridges in heavy chains 1 and 2.Biochemistry. 1998; 37 (9425062): 408-41610.1021/bi971137dCrossref PubMed Scopus (18) Google Scholar). This arrangement of the HC–Hybrid1 and vWFA domains is highly reminiscent of integrin β-chains, as illustrated in Fig. 2 for a comparison of rHC1 with ITGB3. Here the topologies of the β-strands in the hybrid domains are similar (Fig. 2A), as are the relativ" @default.
• W3010056472 created "2020-03-13" @default.
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• W3010056472 date "2020-04-01" @default.
• W3010056472 modified "2023-10-10" @default.
• W3010056472 title "Inter-α-inhibitor heavy chain-1 has an integrin-like 3D structure mediating immune regulatory activities and matrix stabilization during ovulation" @default.
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