Inter-α-trypsin Inhibitor, a Covalent Protein-Glycosaminoglycan-Protein Complex (2004)

The inter-α-trypsin inhibitor (IαI) 1The abbreviations used are: IαI, inter-α-trypsin inhibitor; UTI, urinary trypsin inhibitor; PαI, pre-α inhibitor; HC, heavy chain of IαI family molecules; PGP, protein-glycosaminoglycan-protein covalent complex; SHAP, serum-derived hyaluronan-associated protein; HA, hyaluronan; CS, chondroitin sulfate; TSG6 or TNFIP6, tumor necrosis factor-stimulated gene 6. family, a typical and classical example for protein-glycosaminoglycan-protein (PGP) complexes, occurs constitutively in plasma at relatively high concentrations and is a result of alternate combinations of three kinds of heavy chains with a common light chain, the bikunin proteoglycan. The family is characterized by the unique covalent linkage between the heavy chains and the chondroitin sulfate chain of bikunin. The early studies on the IαI family molecules have largely focused on the light chain (bikunin) that is fully responsible for their protease inhibitory activity. Since the mid-1980s, the structures of the family molecules have been clarified, and this led to the discovery of their complex interaction with another glycosaminoglycan, hyaluronan (HA), generating the serum-derived hyaluronan-associated protein (SHAP)-HA complex. Research from various directions has clarified many aspects of the biological function of the PGP complexes and provides a new vista on their interesting structure-function relationships, especially on their roles in inflammation. The IαI family was first identified as a trypsin inhibitor activity in nephropathic and pneumonopathic urine in 1909 and in normal urine in 1910. One member, the urinary trypsin inhibitor (UTI), was purified much later in the 1950s (1Shulman N.R. J. Biol. Chem. 1955; 213: 655-671Abstract Full Text PDF PubMed Google Scholar, 2Astrup T. Alkjoer K. Soardi F. Scand. J. Clin. Lab. Invest. 1959; 11: 181Crossref Scopus (17) Google Scholar) when it was independently isolated by researchers working on mucopolysaccharides, leukemia, and urolithiasis and was given many names: acid-stable protease inhibitor, prealbumin-like protease inhibitor, urinastatin, HI30, mingin, EDC1, uronic acid-rich protein, and nephrocalin (3Anderson A.J. Maclagan N.F. Biochem. J. 1955; 59: 638-644Crossref PubMed Scopus (19) Google Scholar, 4Chawla R.K. Wadsworth A.D. Rudman D. J. Immunol. 1978; 121: 1636-1639PubMed Google Scholar, 5Atmani F. Mizon J. Khan S.R. Eur. J. Biochem. 1996; 236: 984-990Crossref PubMed Scopus (52) Google Scholar). Based on the presence of two tandem Kunitz-type protease inhibitory domains, a structure-based name, bikunin, was suggested in 1990 to avoid confusion (6Gebhard W. Hochstrasser K. Fritz H. Enghild J.J. Pizzo S.V. Salvesen G. Biol. Chem. Hoppe Seyler. 1990; 371: 13-22PubMed Google Scholar). It is now known that bikunin (Mr ∼ 40,000) is a proteoglycan with a chondroitin sulfate (CS) chain and is the predominant protease inhibitor in urine. Bikunin is acid- and heat-stable and has an acidic pI between 2 and 3 largely due to the presence of sialic acid and CS. There were continuous efforts to find a serum cognate based on a hypothesis that UTI has a serum origin. In the 1970s, this led to the identification of serum IαI (7Proksch G.J. Lane J. Nordschow C.D. Clin. Biochem. 1973; 6: 200-206Crossref PubMed Scopus (27) Google Scholar), a macromolecule previously purified in the 1960s (8Heide K. Heimburger N. Haupt H. Clin. Chim. Acta. 1965; 11: 82Crossref Scopus (55) Google Scholar) (Fig. 1). Conclusive evidence for the identity came from the comparison of amino acid sequences in the 1980s (9Wachter E. Hochstrasser K. Hoppe Seyler's Z. Physiol. Chem. 1981; 362: 1351-1355Crossref PubMed Scopus (93) Google Scholar). Smaller components with inhibitor activity were released when IαI was treated in vitro in acidic conditions or with proteases, such as plasmin, trypsin, and elastase (10Dietl T. Dobrinski W. Hochstrasser K. Hoppe Seyler's Z. Physiol. Chem. 1979; 360: 1313-1318Crossref PubMed Scopus (54) Google Scholar, 11Pratt C.W. Pizzo S.V. Biochemistry. 1987; 26: 2855-2863Crossref PubMed Scopus (22) Google Scholar), or when incubated with inflammatory cells, in particular neutrophils, or cancer cells (12Pratt C.W. Swaim M.W. Pizzo S.V. J. Leukocyte Biol. 1989; 45: 1-9Crossref PubMed Scopus (30) Google Scholar, 13Kobayashi H. Gotoh J. Hirashima Y. Terao T. J. Biol. Chem. 1996; 271: 11362-11367Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Cognate molecules with lower molecular weights were also found in serum, pre-α inhibitor (PαI), and inter-α-like trypsin inhibitor (14Enghild J.J. Thogersen I.B. Pizzo S.V. Salvesen G. J. Biol. Chem. 1989; 264: 15975-15981Abstract Full Text PDF PubMed Google Scholar). IαI was thought to be a single polypeptide chain until molecular biology techniques became available. Cell-free translation of liver mRNAs (15Bourguignon J. Vercaigne D. Sesboue R. Martin J.P. Salier J.P. FEBS Lett. 1983; 162: 379-383Crossref PubMed Scopus (30) Google Scholar) and subsequent cloning (16Diarra-Mehrpour M. Bourguignon J. Sesboue R. Mattei M.G. Passage E. Salier J.P. Martin J.P. Eur. J. Biochem. 1989; 179: 147-154Crossref PubMed Scopus (73) Google Scholar) indicated that IαI contains "heavy" and "light" polypeptides encoded by distinct genes. Reviews in 1990 summarized the multipeptide chain structure and defined the IαI family (6Gebhard W. Hochstrasser K. Fritz H. Enghild J.J. Pizzo S.V. Salvesen G. Biol. Chem. Hoppe Seyler. 1990; 371: 13-22PubMed Google Scholar, 17Salier J.P. Trends Biochem. Sci. 1990; 15: 435-439Abstract Full Text PDF PubMed Scopus (119) Google Scholar). IαI family molecules are encoded by at least five genes, ITIH1–ITIH4 for the four heavy chains (HCs) and AMBP for the light chain (the core protein of bikunin). Interestingly, the AMBP gene encodes both the core protein and a functionally unrelated serum protein, α1-microglobulin, as a precursor fusion protein (18Kaumeyer J.F. Polazzi J.O. Kotick M.P. Nucleic Acids Res. 1986; 14: 7839-7850Crossref PubMed Scopus (190) Google Scholar). cDNAs of the four HCs show high sequence homology except in the C-terminal one-third of HC4 (19Chan P. Risler J.L. Raguenez G. Salier J.P. Biochem. J. 1995; 306: 505-512Crossref PubMed Scopus (51) Google Scholar). The multipeptide structure of IαI was masked by its unusual PGP structure in which the polypeptide subunits are covalently linked together via a CS chain (20Enghild 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). In 1981, the UTI carbohydrates were identified as: (a) an O-linked, GalNAc-rich chain attached to serine at position 10 and (b) an N-linked oligosaccharide at position 45 (21Hochstrasser K. Schonberger O.L. Rossmanith I. Wachter E. Hoppe Seyler's Z. Physiol. Chem. 1981; 362: 1357-1362Crossref PubMed Scopus (77) Google Scholar). Chondroitinase and testicular hyaluronidase digestion of IαI and PαI identified the O-glycan as a low sulfated CS chain (22Balduyck M. Mizon C. Loutfi H. Richet C. Roussel P. Mizon J. Eur. J. Biochem. 1986; 158: 417-422Crossref PubMed Scopus (44) Google Scholar, 23Ochiai H. Toyoda H. Onodera M. Shinbo A. Shinomiya K. Imanari T. Chem. Pharm. Bull. (Tokyo). 1988; 36: 3726-3727Crossref PubMed Scopus (9) Google Scholar) and separated the HCs and bikunin (14Enghild J.J. Thogersen I.B. Pizzo S.V. Salvesen G. J. Biol. Chem. 1989; 264: 15975-15981Abstract Full Text PDF PubMed Google Scholar, 24Jessen T.E. Faarvang K.L. Ploug M. FEBS Lett. 1988; 230: 195-200Crossref PubMed Scopus (76) Google Scholar). The CS linkage to bikunin is a typical O-xylosylserine (22Balduyck M. Mizon C. Loutfi H. Richet C. Roussel P. Mizon J. Eur. J. Biochem. 1986; 158: 417-422Crossref PubMed Scopus (44) Google Scholar). Analyses also identified the novel ester bond that links the α-carboxyl of C-terminal aspartates of the HCs with C-6 hydroxyl groups of internal GalNAcs in the CS chain (20Enghild 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, 25Enghild J.J. Salvesen G. Thogersen I.B. Valnickova Z. Pizzo S.V. Hefta S.A. J. Biol. Chem. 1993; 268: 8711-8716Abstract Full Text PDF PubMed Google Scholar) (Fig. 1). The amino acid sequence at the CS attachment site in bikunin, Glu-Gly-Ser-Gly, is well conserved among all species examined. The CS chain is relatively short (Mr ∼ 8000), with 12–18 disaccharide repeats (GlcUAβ1,3-GalNAcβ1,4-) and a conventional linkage region (GlcUAβ1–3Galβ1–3Galβ1–4Xylβ1)-O-Ser (26Toyoda H. Kobayashi S. Sakamoto S. Toida T. Imanari T. Biol. Pharm. Bull. 1993; 16: 945-947Crossref PubMed Scopus (40) Google Scholar, 27Enghild J.J. Thogersen I.B. Cheng F. Fransson L.A. Roepstorff P. Rahbek-Nielsen H. Biochemistry. 1999; 38: 11804-11813Crossref PubMed Scopus (63) Google Scholar). About 30% of the GalNAc, usually those near the linkage region, are sulfated at C-4 hydroxyl groups (27Enghild J.J. Thogersen I.B. Cheng F. Fransson L.A. Roepstorff P. Rahbek-Nielsen H. Biochemistry. 1999; 38: 11804-11813Crossref PubMed Scopus (63) Google Scholar). CS chains synthesized during inflammations are shorter with decreased sulfation (28Capon C. Mizon C. Lemoine J. Rodie-Talbere P. Mizon J. Biochimie (Paris). 2003; 85: 101-107Crossref PubMed Scopus (28) Google Scholar). In the electron microscope, bikunin appears as a small sphere (diameter of ∼2 nm), whereas HCs have an N-terminal globule (diameter of ∼11 nm) with a thin tail (length of ∼15.5 nm) attached to the bikunin sphere (29Blom A.M. Morgelin M. Oyen M. Jarvet J. Fries E. J. Biol. Chem. 1999; 274: 298-304Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The two HCs of human IαI are located close to each other in the less sulfated region of the CS chain (27Enghild J.J. Thogersen I.B. Cheng F. Fransson L.A. Roepstorff P. Rahbek-Nielsen H. Biochemistry. 1999; 38: 11804-11813Crossref PubMed Scopus (63) Google Scholar). The HCs on IαI vary from species to species. Typically, human IαI contains HC1 and HC2 (14Enghild J.J. Thogersen I.B. Pizzo S.V. Salvesen G. J. Biol. Chem. 1989; 264: 15975-15981Abstract Full Text PDF PubMed Google Scholar), bovine IαI contains HC2 and HC3 (30Castillo G.M. Templeton D.M. FEBS Lett. 1993; 318: 292-296Crossref PubMed Scopus (49) Google Scholar), and rodent IαI contains both types (31Yamamoto T. Yamamoto K. Sinohara H. J. Biochem. (Tokyo). 1996; 120: 145-152Crossref PubMed Scopus (12) Google Scholar). On the other hand, the single HC in PαI is HC3 in all species examined except for bovine PαI, which contains HC2 (30Castillo G.M. Templeton D.M. FEBS Lett. 1993; 318: 292-296Crossref PubMed Scopus (49) Google Scholar). Low levels of HC2/bikunin (inter-α-like trypsin inhibitor) and HC1/bikunin are found in human serum, which seem to be degraded forms of IαI (14Enghild J.J. Thogersen I.B. Pizzo S.V. Salvesen G. J. Biol. Chem. 1989; 264: 15975-15981Abstract Full Text PDF PubMed Google Scholar, 25Enghild J.J. Salvesen G. Thogersen I.B. Valnickova Z. Pizzo S.V. Hefta S.A. J. Biol. Chem. 1993; 268: 8711-8716Abstract Full Text PDF PubMed Google Scholar). Currently, neither the reason nor the mechanism for the selectivity of HCs is known. Although IαI family genes are expressed in various tissues (32Mizushima S. Nii A. Kato K. Uemura A. Biol. Pharm. Bull. 1998; 21: 167-169Crossref PubMed Scopus (28) Google Scholar), the circulating IαI family molecules are produced principally by the liver. Interestingly, bikunin and HC cDNA cotransfection shows that cells other than hepatocytes, COS cells, also can couple a HC with the bikunin proteoglycan (33Blom A.M. Thuveson M. Fries E. Biochem. J. 1997; 328: 185-191Crossref PubMed Scopus (14) Google Scholar). Bikunin is first synthesized in fusion with α1-microglobulin (34Lindqvist A. Bratt T. Altieri M. Kastern W. Akerstrom B. Biochim. Biophys. Acta. 1992; 1130: 63-67Crossref PubMed Scopus (74) Google Scholar), and the cleavage occurs in the trans-Golgi after adding the CS and HCs (35Bratt T. Olsson H. Sjoberg E.M. Jergil B. Akerstrom B. Biochim. Biophys. Acta. 1993; 1157: 147-154Crossref PubMed Scopus (64) Google Scholar). The connecting tetrapeptide (Arg-X1-X2-Arg) conforms to a consensus sequence recognized by the intracellular endoprotease family that includes furin. However, furin does not seem to be responsible for the cleavage (36Brat T. Cedervall T. Akerstrom B. FEBS Lett. 1994; 354: 57-61Crossref PubMed Scopus (23) Google Scholar). The HCs undergo several proteolytic steps during biosynthesis. The signal and following N-terminal propeptides (19Chan P. Risler J.L. Raguenez G. Salier J.P. Biochem. J. 1995; 306: 505-512Crossref PubMed Scopus (51) Google Scholar) are removed in the endoplasmic reticulum, probably by a Golgi enzyme in transit (37Thuveson M. Fries E. J. Biol. Chem. 1999; 274: 6741-6746Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 38Thogersen I.B. Enghild J.J. J. Biol. Chem. 1995; 270: 18700-18709Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The large C-terminal extension (240–280 amino acid residues beginning with the conserved sequence Pro-His-Phe-Ile-Ile) is released when the remainder of the HC is coupled to the CS (Fig. 2) (19Chan P. Risler J.L. Raguenez G. Salier J.P. Biochem. J. 1995; 306: 505-512Crossref PubMed Scopus (51) Google Scholar). Therefore, the assembly of a HC involves two likely coordinated steps: a cleavage of the Asp-Pro bond and then the formation of the ester bond (38Thogersen I.B. Enghild J.J. J. Biol. Chem. 1995; 270: 18700-18709Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The C-terminal extension seems to be required for the formation of the ester linkage (37Thuveson M. Fries E. J. Biol. Chem. 1999; 274: 6741-6746Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). No protease with specificity toward the conserved sequence around the cleavage site has been described. As their original names suggest, the family molecules were studied extensively as protease inhibitors. Bikunin exhibits a weak inhibitory activity against proteases including trypsin, chymotrypsin, neutrophil elastase, and plasmin (39Potempa J. Kwon K. Chawla R. Travis J. J. Biol. Chem. 1989; 264: 15109-15114Abstract Full Text PDF PubMed Google Scholar). However, none has been defined as a physiological target of bikunin, which questions whether protease inhibition is a major function of IαI family molecules. Although circulating levels are high (150–500 μg/ml), they account for only ∼5% of the total protease inhibitory activity of the plasma, and the Ki values of bikunin are not low enough to be effective at physiological concentrations (39Potempa J. Kwon K. Chawla R. Travis J. J. Biol. Chem. 1989; 264: 15109-15114Abstract Full Text PDF PubMed Google Scholar). Proteases that bikunin inhibits are often more efficiently inhibited by their respective physiological inhibitors (40Odum L. Dan. Med. Bull. 1991; 38: 68-77PubMed Google Scholar). Accordingly, on the basis of clearance of injected bikunin-protease complexes, it was proposed that the IαI family functions as a shuttle that traps proteases and then transfers them to physiological inhibitors (41Pratt C.W. Pizzo S.V. Arch. Biochem. Biophys. 1986; 248: 587-596Crossref PubMed Scopus (18) Google Scholar). However, bikunin inhibits plasmin on the surfaces of cancer cells more efficiently than plasmin in solution, in contrast to α2-macroglobulin and α1-inhibitor, which may relate to the antimetastatic effect of bikunin (42Kobayashi H. Shinohara H. Takeuchi K. Itoh M. Fujie M. Saitoh M. Terao T. Cancer Res. 1994; 54: 844-849PubMed Google Scholar). In addition, bikunin may also inhibit granzyme K, the lymphocyte granule-stored serine protease that is implicated in T-cell- and natural killer cell-mediated cytotoxic defense after recognition of target cells (43Wilharm E. Parry M.A. Friebel R. Tschesche H. Matschiner G. Sommerhoff C.P. Jenne D.E. J. Biol. Chem. 1999; 274: 27331-27337Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Trypstatin, an inhibitor of chymase secreted by peritoneal mast cells, was suggested to be a processed fragment of bikunin that the cells had taken up from the circulation (44Itoh H. Ide H. Ishikawa N. Nawa Y. J. Biol. Chem. 1994; 269: 3818-3822Abstract Full Text PDF PubMed Google Scholar). Other diverse activities, particularly pharmaceutical effects, of bikunin have been described, such as mitogenic activity, inhibitory activity toward hyaluronidase, prevention from premature delivery, ischemia stress and renal failure, and suppression of pancreatitis, colitis, and arthritis, for which various mechanisms have been proposed (for review, see Ref. E. A.M. J. Biochem. Biol. PubMed Scopus Google Scholar). The studies focused on the bikunin of IαI family molecules. However, more than of circulating bikunin whereas urinary bikunin is of HCs A. M. M. J. Fries E. Biol. Chem. Hoppe Seyler. 1994; PubMed Scopus (17) Google Scholar). bikunin has a much shorter in circulation in than that of complexes in L. M. M. W. N. Y. K. T. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. H. M. E. H. 1989; PubMed Scopus Google Scholar). is by studies of after its discovery in the IαI was found to have an with D. J. J. Clin. Invest. PubMed Scopus Google Scholar, J. D. G. 1965; Google Scholar). is the major in that is responsible for its and isolated from of with or more protein than from normal with identified the protein as IαI then thought to be a single protein J. D. G. 1965; Google Scholar). The had such as and in the at an of the complex J. D. G. 1965; Google Scholar). proposed that IαI the from serum and became to HA, its J. D. G. 1965; Google Scholar, A. J. 14: PubMed Scopus Google Scholar). were except in a in that the of IαI with and that the complex was more to by N. P. 1988; PubMed Scopus Google Scholar). In a protein of was found with in the of M. S. K. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). The protein was not and was from the serum to the It was and was identified as the HCs of the IαI family molecules L. M. K. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). The is used for HCs in The complexes were then isolated from and from T.E. L. Biol. Chem. Hoppe Seyler. 1994; PubMed Scopus (46) Google Scholar). that are to by an ester bond between groups of the aspartates at their and the C-6 hydroxyl groups of internal in HA, to the original HC ester bond to CS in IαI M. M. Y. S. H. Y. K. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) (Fig. 1). several are linked to the complex is a PGP covalent complex. The linkage structures in the complex and IαI that the complex is by a of HCs from CS to HA, by the of bikunin proteoglycan (Fig. complex when and IαI were incubated with serum at M. S. K. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, M. M. Y. S. H. Y. K. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). No HC was at low or when were the presence of an as M. S. K. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, L. M. K. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). HC activity has also been in and in by cells, cells, or cells L. H. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, L. T.E. PubMed Scopus (19) Google Scholar). C. and K. tumor necrosis also has been identified as a it with both its J. J. Biol. 1992; PubMed Scopus Google Scholar) and IαI J. Biochemistry. 1994; PubMed Scopus Google Scholar). the as to the complex and to the of the during with L. M. M. W. N. Y. K. T. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. S. D. T. A.J. A. K. 2003; PubMed Scopus Google Scholar). Interestingly, TSG6 forms an complex with IαI, in which TSG6 appears to a HC on the CS J. Biochemistry. 1994; PubMed Scopus Google Scholar). complex was in and D.M. D. J. J. Immunol. 1996; Google Scholar). Although it was suggested that TSG6 as an than as the activity T.E. L. 2003; PubMed Scopus Google Scholar), has that TSG6 can HC from IαI to a complex in that can in a complex when or are D. A. A.J. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (54) Google Scholar). a proteoglycan with two tandem the formation of the complex in M. M. L. H. Y. L. S. H. T. Z. K. G. in Scholar). In the the is by cells in a complex. an the to serum, IαI, and the cells to and it an a in complex complexes in vitro when incubated in the presence of and In the of serum, is synthesized not a The serum was identified as IαI, and it was suggested that the mechanism a interaction between IαI and L. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). However, the interaction was weak at physiological its to the isolated from bikunin, that a complex is T.E. L. Biol. Chem. Hoppe Seyler. 1994; PubMed Scopus (46) Google Scholar, J. Biochemistry. 1994; PubMed Scopus Google Scholar, D. A. A.J. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (54) Google Scholar). from the contains they are not to the complex L. M. M. W. N. Y. K. T. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). are in with and subsequent was by of purified IαI not by bikunin L. M. M. W. N. Y. K. T. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of bikunin proteoglycan with serum from also to couple it with the with the that the assembly of IαI is in the Golgi of (35Bratt T. Olsson H. Sjoberg E.M. Jergil B. Akerstrom B. Biochim. Biophys. Acta. 1993; 1157: 147-154Crossref PubMed Scopus (64) Google Scholar). a novel for the function of bikunin to a CS chain to an ester bond with HCs for their subsequent to TSG6 is also in cells and in cells in the C. Y. A. 1997; PubMed Scopus Google Scholar, D. A.J. A. C. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). hyaluronidase digestion of the identified covalent complexes as well as complexes in the whereas bikunin was D. A.J. A. C. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). hyaluronidase digestion of from the a of complexes C. S. D. T. A.J. A. K. 2003; PubMed Scopus Google Scholar). TSG6 is for the of HCs from IαI in the The complex provides a mechanism for the by between the by the in and the covalent of its IαI family molecules also on human on human cells A. H. Fries E. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar), and on cells M. M. L. L. H. Y. S. H. K. J.F. Scholar), likely and IαI in tissues and the various such as and and during physiological such as is with bikunin levels in urine Scand. J. Clin. Lab. Invest. 1965; PubMed Scopus Google Scholar). Therefore, the function of the IαI and is of a a to the it is a major of the is the likely site for the formation of the complex. the complex was the at a than that in the circulation D. M. S. T. Y. T. N. H. K. J. 1999; 26: Google Scholar, W. L. Morgelin M. M. T. H. N. H. K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The complex from is in chain and in the molecular a chain of ∼2 on W. L. Morgelin M. M. T. H. N. H. K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus (93) Google Scholar). of HA, such as at low and D. J. J. Clin. Invest. PubMed Scopus Google Scholar, J. D. G. 1965; Google Scholar), and the to a W. L. Morgelin M. M. T. H. N. H. K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus (93) Google Scholar) may relate to the presence of and those that the complex has a in the inflammatory by of and are the major inflammatory of the and are often to as inflammatory The major an in and a of the cells of the The of inflammatory is including The interaction between cells and is also in the and of this in vitro that of human cells or with the acid the of and the formation of structures J. S.A. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) (Fig. The structures were in the presence of serum, and the complex was found in the structure J. S.A. J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). that cells or cells to the via a mechanism in a on their Therefore, a the may be required for the to and to of the is that is degraded at and by to the a large is and However, a is also to the circulation and by liver The of is in the of whereas the in circulation is at only in 1987; PubMed Scopus Google Scholar). However, decreased of serum the is used to and to in of and liver The and of and Scholar). for the of the complex in serum and other D. M. S. T. Y. T. N. H. K. J. 1999; 26: Google Scholar), has a between the levels of and in with serum D. M. S. T. Y. T. N. H. K. J. 1999; 26: Google Scholar, W. L. Morgelin M. M. T. H. N. H. K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus (93) Google Scholar) C. and K. and provides evidence for the formation of the complex in a studies on IαI family molecules focused on bikunin, the protease inhibitor, with the that the HCs only the function of bikunin. the complex was identified in a of IαI family molecules was and its biological was with in studies a novel for the of each of IαI, the HCs are to the physiological function of IαI to HA, and their to bikunin is for the It is now to find other with the HCs in to HA, and D. A. A.J. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (54) Google Scholar, M. M. L. L. H. Y. S. H. K. J.F. Scholar). of in the HC such as those suggested by sequence homology (19Chan P. Risler J.L. Raguenez G. Salier J.P. Biochem. J. 1995; 306: 505-512Crossref PubMed Scopus (51) Google Scholar), also be the mechanisms for the bond in IαI in the Golgi of and for the to the to be defined in both normal such as and in such as and inflammatory are much to have been in this