Structural Characterization of Inter-α-inhibitor

Abstract

Inter-α-inhibitor (IαI) is a 180-kDa serum protein consisting of three polypeptides. Two of these, the heavy chains 1 and 2 (H1 and H2), are of 75–80 kDa and have similar amino acid sequences. The third polypeptide, bikunin, has a molecular mass of 25 kDa and contains a 7-kDa chondroitin sulfate chain that is covalently linked to the C-terminal amino acid residues of H1 and H2. IαI has been shown to be required for the formation of the hyaluronan-containing extracellular matrix of certain cell types. How IαI exerts this function is not known, but it appears that upon interaction with cells, the heavy chains are released and become covalently linked to hyaluronan. Our results indicate that IαI and its heavy chains are extended molecules; thus, upon electron microscopy, IαI appeared to consist of two globular domains connected by a thin structure 31-nm long and the isolated heavy chains of a globular domain and a “tail” about 15-nm long. Analysis of the heavy chains by partial proteolysis showed that the C-terminal halves are particularly sensitive to hydrolysis indicating that they are loosely folded. Furthermore, electron microscopy showed that partially degraded heavy chains lacked the extended regions. Taken together, these results suggest that the N-terminal half of the heavy chains forms a globular domain, whereas the other half has an extended and loosely folded structure. Inter-α-inhibitor (IαI) is a 180-kDa serum protein consisting of three polypeptides. Two of these, the heavy chains 1 and 2 (H1 and H2), are of 75–80 kDa and have similar amino acid sequences. The third polypeptide, bikunin, has a molecular mass of 25 kDa and contains a 7-kDa chondroitin sulfate chain that is covalently linked to the C-terminal amino acid residues of H1 and H2. IαI has been shown to be required for the formation of the hyaluronan-containing extracellular matrix of certain cell types. How IαI exerts this function is not known, but it appears that upon interaction with cells, the heavy chains are released and become covalently linked to hyaluronan. Our results indicate that IαI and its heavy chains are extended molecules; thus, upon electron microscopy, IαI appeared to consist of two globular domains connected by a thin structure 31-nm long and the isolated heavy chains of a globular domain and a “tail” about 15-nm long. Analysis of the heavy chains by partial proteolysis showed that the C-terminal halves are particularly sensitive to hydrolysis indicating that they are loosely folded. Furthermore, electron microscopy showed that partially degraded heavy chains lacked the extended regions. Taken together, these results suggest that the N-terminal half of the heavy chains forms a globular domain, whereas the other half has an extended and loosely folded structure. inter-α-inhibitor heavy chain 1 and 2, respectively polyacrylamide gel electrophoresis phosphate-buffered saline N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]glycine. Inter-α-inhibitor (IαI)1 is a plasma protein synthesized by hepatocytes. It consists of bikunin, which has a molecular mass of about 25 kDa (1Olsen E.H.N. Rahbek-Nielsen H. Thogersen I.B. Roepstorff P. Enghild J.J. Biochemistry. 1998; 37: 408-416Crossref PubMed Scopus (18) Google Scholar, 2Lindström K.E. Blom A. Johnsson E. Haraldsson B. Fries E. Kidney Int. 1997; 51: 1053-1058Abstract Full Text PDF PubMed Scopus (41) Google Scholar), and two other polypeptides of 75–80 kDa, the heavy chains 1 and 2 (H1 and H2) (3Salier J.-P. Rouet P. Raguenez G. Daveau M. Biochem. J. 1996; 315: 1-9Crossref PubMed Scopus (234) Google Scholar). Bikunin carries a 7-kDa chondroitin sulfate chain (4Enghild J.J. Salvesen G. Hefta S.A. Thogersen I.B. Rutherfurd S. Pizzo S.V. J. Biol. Chem. 1991; 266: 747-751Abstract Full Text PDF PubMed Google Scholar, 5Toyoda H. Kobayashi S. Sakamoto S. Toida T. Imanari T. Biol. Pharm. Bull. 1993; 16: 945-947Crossref PubMed Scopus (40) Google Scholar), which is linked to the α-carbons of the C-terminal amino acid residues of H1 and H2 through two ester bonds (6Morelle W. Capon C. Balduyck M. Sautiere P. Kouach M. Michalski C. Fournet B. Mizon J. Eur. J. Biochem. 1994; 221: 881-888Crossref PubMed Scopus (68) Google Scholar). Pulse-chase experiments with isolated hepatocytes have shown that the coupling of bikunin to the heavy chains occurs in the Golgi complex immediately after the chondroitin sulfate chain has been completed (7Sjöberg E.M. Fries E. Arch. Biochem. Biophys. 1992; 295: 217-222Crossref PubMed Scopus (43) Google Scholar, 8Thogersen I.B. Enghild J.J. J. Biol. Chem. 1995; 270: 18700-18709Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Bikunin, which occurs in plasma also in free form (9Hochstrasser K. Niebel J. Feuth H. Lempart K. Klin. Wochenschr. 1977; 55: 337-342Crossref PubMed Scopus (23) Google Scholar), is a proteinase inhibitor with a wide specificity range but with relatively low affinity to the proteolytic enzymes tested so far (10Potempa J. Kwon K. Chawla R. Travis J. J. Biol. Chem. 1989; 264: 15109-15114Abstract Full Text PDF PubMed Google Scholar). Although IαI was isolated more than 30 years ago (11Steinbuch M. Loeb J. Nature. 1961; 192: 1196Crossref PubMed Scopus (32) Google Scholar), its physiological role is still unclear (for a review see Ref. 3Salier J.-P. Rouet P. Raguenez G. Daveau M. Biochem. J. 1996; 315: 1-9Crossref PubMed Scopus (234) Google Scholar). Recent findings indicate that this protein is required for the maturation of oocytes; in vitro experiments have shown that IαI stabilizes the hyaluronan-containing extracellular matrix that is formed by the cells surrounding the oocytes (12Chen L. Mao S.J. Larsen W.J. J. Biol. Chem. 1992; 267: 12380-12386Abstract Full Text PDF PubMed Google Scholar). The observation that the capillaries near the follicles become leaky to plasma proteins during ovulation supports the idea that the observed effect occursin vivo (13Chen L. Mao S.J. McLean L.R. Powers R.W. Larsen W.J. J. Biol. Chem. 1994; 269: 28282-28287Abstract Full Text PDF PubMed Google Scholar). IαI has also been shown to be required for the formation of the hyaluronan-containing structure that surrounds fibroblasts and mesothelial cells (14Blom A. Pertoft H. Fries E. J. Biol. Chem. 1995; 270: 9698-9701Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The physiological role of these coats is unclear, but it has been suggested that they may protect against compression (15Lee G.M. Johnstone B. Jacobson K. Caterson B. J. Cell Biol. 1993; 123: 1899-1907Crossref PubMed Scopus (124) Google Scholar), promote cell migration (16Banerjee S.D. Toole B.P. J. Cell Biol. 1992; 119: 643-652Crossref PubMed Scopus (48) Google Scholar), and help to keep cells separated (15Lee G.M. Johnstone B. Jacobson K. Caterson B. J. Cell Biol. 1993; 123: 1899-1907Crossref PubMed Scopus (124) Google Scholar). It is not clear in what way IαI stabilizes this extracellular structure, but it appears that when the bikunin-containing proteins interact with cells, the bikunin moiety is displaced by hyaluronan molecules that become covalently linked to the heavy chains (17Huang L. Yoneda M. Kimata K. J. Biol. Chem. 1993; 268: 26725-26730Abstract Full Text PDF PubMed Google Scholar, 18Chen L. Zhang H. Powers R.W. Russell P.T. Larsen W.J. J. Biol. Chem. 1996; 271: 19409-19414Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Recent observations have indicated that IαI may also play a role in inflammation. Thus hyaluronan isolated from the synovial fluid of patients with rheumatoid arthritis has been found to contain covalently linked heavy chains (19Jessen T.E. Odum L. Johnsen A.H. Biol. Chem. Hoppe-Seyler. 1994; 375: 521-526Crossref PubMed Scopus (46) Google Scholar, 20Zhao M. Yoneda M. Okashi Y. Kurono S. Iwata H. Ohnuki Y. Kimata K. J. Biol. Chem. 1995; 270: 26657-26663Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Furthermore, upon inflammation, fibroblasts and monocytes secrete a protein named TSG-6, which reacts with IαI by displacing H1 and forming a covalent link with the chondroitin sulfate of bikunin (21Wisniewski H.G. Burgess W.H. Oppenheim J.D. Vilcek J. Biochemistry. 1994; 33: 7423-7429Crossref PubMed Scopus (99) Google Scholar). Upon this reaction, the ability of bikunin to inhibit plasmin is enhanced, and the formed complex appears to have a strong anti-inflammatory capacity (22Wisniewski H. Hua J.-C. Poppers D.M. Naime D. Vilcek J. Cronstein B.N. J. Immunol. 1996; 156: 1609-1615PubMed Google Scholar). The molecular details of the interactions of IαI and TSG-6 are still unknown. For further functional studies of IαI, detailed structural information will be helpful. The amino acid sequences of the heavy chains and bikunin are known, as are the structures of their glycans and disulfide arrangements (1Olsen E.H.N. Rahbek-Nielsen H. Thogersen I.B. Roepstorff P. Enghild J.J. Biochemistry. 1998; 37: 408-416Crossref PubMed Scopus (18) Google Scholar, 23Wachter E. Hochstrasser K. Hoppe-Seyler's Z. Physiol. Chem. 1981; 362: 1351-1355Crossref PubMed Scopus (93) Google Scholar). In this study, we have investigated the conformation of IαI and its isolated heavy chains with various techniques. Chymotrypsin and elastase from pig pancreas were purchased from Boehringer-Mannheim. Thermolysin (type X) was from Sigma, hyaluronidase from ovine testes from Calbiochem, and chondroitinase AC II Arthro from Seikagaku Corp., Tokyo, Japan. For the isolation of IαI, a side fraction from the commercial production of factor IX was used (kindly provided by I.-M. Lööf; Pharmacia-Upjohn, Stockholm, Sweden). After dialysis against phosphate-buffered saline (PBS) and removal of insoluble protein, this material was subjected to gel filtration on Sepharose S-400, which yielded pure IαI. For the release of the heavy chains, 2m NaOH was added to a solution of IαI (1 mg/ml in PBS) to give a final concentration of 0.05 m (24Enghild J.J. Thogersen I.B. Pizzo S.V. Salvesen G. J. Biol. Chem. 1989; 264: 15975-15981Abstract Full Text PDF PubMed Google Scholar). After 15 min at room temperature, Tris-HCl, pH 8.0, was added to yield a final concentration of 0.25 m. Alternatively, one volume of IαI (1 mg/ml in PBS) was mixed with two volumes of 0.4 m sodium acetate, pH 6.0, followed by the addition of chondroitinase and phenylmethanesulfonyl fluoride to yield the final concentrations 20 milliunits/ml and 0.2 mm, respectively (25Balduyck M. Laroui S. Mizon C. Mizon J. Biol. Chem. Hoppe-Seyler. 1989; 370: 329-336Crossref PubMed Scopus (56) Google Scholar). The mixture was incubated for 20 h at 37 °C. The sample was then dialyzed against 20 mm sodium phosphate, pH 7.6, and applied to an anion exchange gel (MonoQ HR 5/5; Pharmacia Biotech) equilibrated with the same buffer. The proteins were eluted at 4 °C at a linear flow rate of 20 cm × h−1 with 100 ml of a gradient from 0 to 0.7 m NaCl in 20 mm sodium phosphate, pH 7.6 (26Balduyck M. Piva F. Mizon C. Maes P. Malki N. Gressier B. Michalski C. Mizon J. Biol. Chem. Hoppe-Seyler. 1993; 374: 895-901Crossref PubMed Scopus (18) Google Scholar). The fractions were analyzed by SDS-PAGE followed by staining with Coomassie Brilliant Blue. Unless specified otherwise, protein concentrations were determined by UV measurements. The absorbance coefficients for the protein moieties of IαI, H1, and H2 were calculated from the amino acid compositions (Lasergene, DNASTAR Inc.). The published carbohydrate contents were then used for the calculation of the corresponding values for the whole proteins: 0.60, 0.47, and 0.72 mg−1 ml cm−1, respectively. The protein solutions were stored at −20 °C until they were used for experiments. The proteins were applied to a Superdex 200 gel (3.2/30; Pharmacia Biotech) equilibrated with PBS at a flow rate of 50 μl/min; the absorbances at 280, 254, and 214 nm of the eluate were continuously measured. Thymidine and blue dextran were used as a markers forV t and V 0, respectively. Calibration curves for Stokes radius and molecular mass were obtained with thyroglobulin (8.5 nm, 668 kDa), ferritin (6.1 nm, 440 kDa), catalase (5.2 nm, 232 kDa), aldolase (4.8 nm, 158 kDa), and bovine serum albumin (3.55 nm, 67 kDa), all were obtained from Pharmacia Biotech. After thawing the protein samples, proteinase inhibitors were added: 0.1 m 6-aminohexanoic acid, 5 mm N-ethylmalemide, 5 mm benzamidine hydrochloride, and 0.5 mm phenylmethanesulfonyl fluoride. The solutions were then dialyzed at 4 °C overnight against 0.2 mNH4HCO3, pH 7.9. Glycerol and were as J. H. Scholar, J. Biol. PubMed Scopus Google Scholar, D.M. D. J. Biol. PubMed Scopus Google Scholar, D. J. PubMed Scopus Google Scholar). and were added to the final concentrations of protein and 5 and respectively. The was by the addition of The were in and at with through electron in a The were observed in a electron at of the from the electron was as J. H. Scholar). For the removal of bikunin from IαI, the of Enghild (24Enghild J.J. Thogersen I.B. Pizzo S.V. Salvesen G. J. Biol. Chem. 1989; 264: 15975-15981Abstract Full Text PDF PubMed Google was a sample mg/ml IαI and hyaluronidase in PBS was incubated for h at 37 °C. H2 was by elastase as by Balduyck (26Balduyck M. Piva F. Mizon C. Maes P. Malki N. Gressier B. Michalski C. Mizon J. Biol. Chem. Hoppe-Seyler. 1993; 374: 895-901Crossref PubMed Scopus (18) Google an to protein of with the for of the isolated heavy of H1 was as with an to protein of of the protein showed with the molecular 2 of a solution with concentrations of was added to of a solution heavy chain in 100 mm Tris-HCl, pH 50 mm and 1 mm to give various of to heavy After min of at 37 the was by the addition of phenylmethanesulfonyl fluoride to a final concentration of 2 The was as for the was 100 mm Tris-HCl, pH with 50 mm NaCl and 20 mm The was as for the was 100 mm Tris-HCl, pH and 50 mm The was as for elastase but was by the addition of to a final concentration of After the proteins were separated by electrophoresis in polyacrylamide in H. Biochem. PubMed Scopus Google Scholar). The proteins were by electrophoresis a The was and the and subjected to N-terminal in a amino acid mg/ml protein were dialyzed against 25 mm sodium and the protein concentrations were determined by amino acid were obtained at room on a with a 0.1 cm were from to nm in 0.5 nm at a rate of 50 for were and The of structural were with the of a of proteins with structure P. Biochem. PubMed Scopus Google Scholar, Biochemistry. 1981; PubMed Scopus Google Scholar). Analysis of IαI, H1, and H2 by gel filtration showed that more than of the applied material eluted as one on their amino acid sequences and carbohydrate the molecular of the of IαI and are (1Olsen E.H.N. Rahbek-Nielsen H. Thogersen I.B. Roepstorff P. Enghild J.J. Biochemistry. 1998; 37: 408-416Crossref PubMed Scopus (18) Google Scholar), and 25 kDa (1Olsen E.H.N. Rahbek-Nielsen H. Thogersen I.B. Roepstorff P. Enghild J.J. Biochemistry. 1998; 37: 408-416Crossref PubMed Scopus (18) Google Scholar, 2Lindström K.E. Blom A. Johnsson E. Haraldsson B. Fries E. Kidney Int. 1997; 51: 1053-1058Abstract Full Text PDF PubMed Scopus (41) Google Scholar), a mass of kDa for the whole The gel filtration experiments showed that IαI, H1, and H2 globular proteins of 100 and 1 kDa 1 three indicating that they have extended The corresponding Stokes are and 0.1 nm, respectively 1 The these and of proteins of the same the are and respectively Biophys. PubMed Scopus Google Scholar). IαI molecules by and showed with a of 2 of these by a thin The the of the the was nm that the was about 31-nm long. In the of the was a globular structure the of which was at the of the of the 2 the idea that this was bikunin, IαI was subjected to hyaluronidase which has been shown to bikunin (24Enghild J.J. Thogersen I.B. Pizzo S.V. Salvesen G. J. Biol. Chem. 1989; 264: 15975-15981Abstract Full Text PDF PubMed Google Scholar). shown in 2 the molecules lacked the of the further the of the structures by electron microscopy, H2 was released from IαI by elastase (26Balduyck M. Piva F. Mizon C. Maes P. Malki N. Gressier B. Michalski C. Mizon J. Biol. Chem. Hoppe-Seyler. 1993; 374: 895-901Crossref PubMed Scopus (18) Google Scholar). 2 that this to the of one of the globular whereas the by H1 with the same 2, and a globular domain 2 with a thin the of the was 20 5 nm the about 15 proteolytic of H1 2 in removal of the whereas the of the globular domain results were obtained with H2 not The extended structure of IαI and its isolated heavy chains was also with material not of molecular structures by electron the the of the two in 100 IαI molecules the one globular domain were not in the shown are the from the of the globular to the of the in 100 H1 molecules of IαI and H1 as by electron For the of the molecules are shown as extended The the as obtained by on the of proteins are more sensitive to proteolytic than the folded A. G. C. D. M. M. Biochemistry. PubMed Scopus Google Scholar). proteolysis be used for the structural of 4 the effect of H1 with of as by SDS-PAGE followed by staining with Coomassie Brilliant Blue. Upon at a of to H1 of the molecular mass of the from to kDa, and three of of about and kDa The separated were to a and the N-terminal sequences of two of these 1 and t were The obtained sequences were the same as the N-terminal of the protein 4 that the been formed by C-terminal the of the in the t 2 and the of three of and kDa t t showed was an N-terminal and 4 and t 5 were the and N-terminal halves of t in the concentration to the of t and a of t 4 and t 5 and by its molecular mass on t 4 appears to the of t 4 this is the t 4 upon electrophoresis to be of H1 with yielded with molecular to obtained with the were the same as found for and The by H1 with elastase were similar in to obtained with the two other their N-terminal amino acid sequences were not also used partial proteolysis to the structure of H2 a of a of in the range of kDa were formed of these were analyzed by amino acid and found to have the of the and It be that this at a proteinase concentration than that required to a similar effect on the concentration of was appeared through of the C-terminal the proteinase concentrations used near the also t results were obtained with and elastase The of structures in the heavy chains was by measurements. The of the two proteins were The of structures were obtained by the to of proteins with structure P. Biochem. PubMed Scopus Google Scholar, Biochemistry. 1981; PubMed Scopus Google Scholar). were values than The in the was than for of structure. yielded the and similar compositions have been obtained for globular proteins N. Biochemistry. PubMed Scopus Google Scholar). In this study, we have found that IαI and its isolated heavy chains as extended molecules in free their Stokes as by gel filtration are than of globular proteins of the same results are by electron microscopy, which that IαI consists of two globular domains connected by a thin structure 2, the same the isolated heavy chains to consist of a globular domain of the same as in IαI and of a thin is about half that of the the globular domains in the whole and to be the C-terminal of the upon of the heavy chains with low concentrations of the C-terminal of the polypeptides are degraded 4 and 5 and the extended by electron microscopy has shown that the C-terminal of the two heavy chains in IαI are covalently linked to the chondroitin sulfate chain of bikunin (6Morelle W. Capon C. Balduyck M. Sautiere P. Kouach M. Michalski C. Fournet B. Mizon J. Eur. J. Biochem. 1994; 221: 881-888Crossref PubMed Scopus (68) Google Scholar). In the electron of IαI, a be in the of the the globular domains 2 structure appears to be bikunin, it is not in molecules subjected to hyaluronidase 2 a the bikunin moiety (24Enghild J.J. Thogersen I.B. Pizzo S.V. Salvesen G. J. Biol. Chem. 1989; 264: 15975-15981Abstract Full Text PDF PubMed Google Scholar). It is that the chondroitin sulfate chain of bikunin of the structure the globular domains in IαI. the that the of the isolated heavy chains are about half of the whole that the of the is the that the bikunin is released upon hyaluronidase be of the heavy chains linked to the chondroitin sulfate chain at relatively (24Enghild J.J. Thogersen I.B. Pizzo S.V. Salvesen G. J. Biol. Chem. 1989; 264: 15975-15981Abstract Full Text PDF PubMed Google Scholar). for results is that the release of the heavy chains from IαI was and that the are the observation that the heavy chains as upon SDS-PAGE with an molecular mass of the against this In this study, we used two for the heavy chains from to pH and chondroitinase The is the to but about we have not been to heavy chains by Our results suggest that the heavy chains of IαI have a structure. are of proteins of this For the of the matrix protein which has a molecular mass of about kDa, appears to consist of a globular domain with a of 5 nm and a thin of nm M. D. J. M. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). protein structures are with carbohydrate and it has been suggested that one of their is to the of the chain N. Biochem. Full Text PDF PubMed Scopus Google Scholar). It is to that of the carbohydrate on H1 and H2 are near the In one of the heavy chains is a disulfide the and N-terminal halves of the The of this that the extended of the is formed by the this which contains about amino acid this of the form an it have a of about 20 by electron microscopy, the of the is about 15 nm that the extended of the heavy chains contains structure. Our observation that proteolytic in this of the protein occurs at 4 and 5 is in with this Furthermore, is a of the amino acid near the of H1 and H2 J. L. F. R. Eur. J. Biochem. 1993; PubMed Scopus Google Scholar). The that the of the heavy chains half the polypeptides that are other than the that contain as by electron microscopy and gel M. Blom and E. the of the globular domains were not by partial proteolytic findings indicate that the formed from the globular domains were not It is that the structures of IαI and its isolated heavy chains were during the for electron The that this not in the H1 and whereas gel filtration showed that that H1 was extended than that this is the electron microscopy of the at more physiological The experiments with partial proteolytic in this is an of an in which it was shown that upon of IαI with of H1 and H2 were released by C-terminal (26Balduyck M. Piva F. Mizon C. Maes P. Malki N. Gressier B. Michalski C. Mizon J. Biol. Chem. Hoppe-Seyler. 1993; 374: 895-901Crossref PubMed Scopus (18) Google Scholar). results were also obtained upon of IαI with cells H. J. Y. T. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). this is of physiological to be It is still unclear IαI supports the formation of the hyaluronan-containing on various cell types. It has been suggested that the heavy chains to the cell and then become covalently linked to hyaluronan molecules through their C-terminal M. S. Kimata K. J. Biol. Chem. Full Text PDF PubMed Google Scholar). this is the it for the globular domains to be to the cell and the C-terminal to of the N. Biochem. Full Text PDF PubMed Scopus Google Scholar). for help with the gel filtration experiments and T. C. for