Inter-α-trypsin Inhibitor, a Covalent Protein-Glycosaminoglycan-Protein Complex

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. 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