Inter-a-trypsin inhibitor (ITI) is a tight complex of three different proteins : bikunin and two heavy chains H1 and H2. In order to demonstrate that the three chains are covalently linked by a chondroitin sulphate chain as previously proposed [Enghild, J. J., Salvesen, G., Hefta, S. A., Thogersen, I. B., Rutherford, S. and Pizzo, S. V. (1991) J. Biol. Chem. 266, IT1 was extensively digested with thermolysin and the glycosaminoglycan-containing fragment was isolated from the digest by ion-exchange chromatography. Its peptide structural determination and mass spectrometry analysis both provide evidence that the different peptide chains constituting IT1 are associated by the new cross-link described as the protein-glycosaminoglycan-protein cross-link. Various biological and physiological functions have been described for glycosaminoglycans (GAG). Enghild et al.[ 11 recently demonstrated that a chondroitin sulphate chain covalently links two different proteins: bikunin and the heavy chain H3, thus constituting pre-a-trypsin inhibitor (paI). The chondroitin sulphate chain is bound at its reducing end by the typical linkage structure Gal-Gal-Xyl to the Ser10 of bikunin whereas the C-terminal Asp of the heavy chain H3 esterifies C6 of an internal N-acetylgalactosamine inside the GAG chain. A similar protein-GAG-protein structure cross-links bikunin and the heavy chain H2 [2].In human plasma, the antiprotease activity due to bikunin is for the main part associated with inter-a-trypsin inhibitor (ITI), this being made up by bikunin associated to both the H1 and H2 heavy chains [3-61. H1, H2 and H3 exhibit numerous structural similarities [7]. Moreover IT1 and p d , which both move as a unique band in SDSPAGE under reducing conditions, may be identically dissociated in their constitutive subunits by chondroitinase digestion, trifluoromethanesulfonic acid deglycosylation or alkaline treatment

Inter-a-trypsin inhibitor (ITI) is a complex protein made up of a light chain so-called bikunin and two heavy chains (apparent Mr values 96000 and 86000 in SDS/PAGE in non-reducing conditions). By sequence analysis, we clearly identified those two components as HI and H 2 , respectively.We demonstrate that alkaline treatment (50mM NaOH during 5 min at room temperature) as well as chondroitinase digestion both lead to the dissociation of ITI. The conditions used for alkaline treatment were previously reported for cleavage of the covalent linkage between bikunin and H 3 inside pre-a-trypsin inhibitor (Enghild et al. (1991) /. Biol. Chem. 266, 747-751).Carbohydrate analysis of the two heavy chains isolated by ion-exchange chromatography suggests the presence of complex-type ./V-glycans in both H! and H 2 and that of O-glycans in H 2 . HI is eluted from Con-A Sepharose by cc-methylmannoside, in agreement with the existence of at least one biantennary glycan chain. In contrast, H 2 remains strongly bound to this support when submitted to the same conditions. Therefore this binding does not depend on carbohydrates.The capacity of H 2 to develop such interactions is discussed with regard to the unusual bindings likely to exist between the different peptide chains constituting ITI. Die schweren Ketten des Inter-a-Trypsininhibitors: Isolierung und Identifizierung durch Elektrophorese und Teilsequenzierung. Unterschiedliche Reaktivitäten gegenüber Concanavalin A.Zusammenfassung: Der Inter-a-Trypsininhibitor (ITI) ist ein komplexes Protein, das aus einer leichten Kette, dem sogenannten Bikunin besteht sowie aus zwei schweren Ketten mit den apparenten M r -Werten 96000 und 86000 (in der SDS/PAGE unter nicht reduzierenden Bedingungen). Durch Sequenzanalyse konnten wir diese beiden Komponenten klar als HI und H 2 identifizieren.Wir zeigen, daß sowohl alkalische Behandlung (50mM NaOH 5 min bei Raumtemperatur) wie Chondroitase-Verdauung zur Dissoziation von ITI führen. Die für die Alkalibehandlung verwendeten Bedingungen wurden bereits für die Spaltung der kovalenten Bindung zwischen Bikunin und H 3 im Prä-atrypsininhibitor mitgeteilt (Enghild et al. (1991) /. Biol. Chem. 266, 747-751).

Uronic-acid-rich protein (UAP) is a urinary glycoprotein that inhibits calcium oxalate crystallization in vitro. It shows a structural similarity to bikunin, a component of inter-a-inhibitor (Id) known for its inhibition of the action of many serine proteinases like trypsin and chymotrypsin. To clarify the relationship between these macromolecules, UAP, IaI, urinary bikunin, and plasma bikunin were purified and studied. Their calcium oxalate crystallization inhibitory activity was assayed before and after treatment with chondroitinase AC and pronase. Their molecular mass was determined by using SDS/PAGE before and after these treatments. Polyclonal bikunin antibody was used on Western blots for immunological identification. The partial amino acid sequence of UAP before and after chondroitinase treatment was determined. Also, the antitryptic activity of UAP was measured and compared to that of bikunin, which is responsible for the antiprotease activity of IaI. UAP exhibited a strong calcium oxalate crystallization inhibitory activity. I d and both bikunins were less inhibitory. Chondroitinase AC had no effect on inhibitory activity of these proteins even when their molecular mass changed. However, after pronase treatment, the inhibitory activity of both bikunins and UAP was completely destroyed. The antitryptic activity of UAP was found to be 0.78 U/mg which is lower than that of bikunin which is about 1.9 U/mg. On Western blotting, bikunin antibody immunoreacted with UAP and both urinary and plasma bikunins. Partial amino acid sequence confirmed the identity of UAP as urinary bikumin.Keywords: nephrolithiasis ; calcium oxalate ; inter-a-inhibitor ; uronic-acid-rich protein ; bikunin.Kidneys excrete a urine normally supersaturated with respect to many salts including calcium oxalate, the most common compound found in kidney stones [l, 21. But normal urine does not support crystallization because of the presence of many natural inhibitors. Purification, identification, and characterization of such macromolecules have been the object of extensive studies [3 -91. Among these inhibitors is uronic-acid-rich protein (UAP), a glycoprotein of 35 kDa isolated from human [3] and rat urine [ 101 which strongly inhibits calcium oxalate crystallization in vitro. The same inhibitor isolated from the urine of stone formers showed less inhibitory activity when compared to that purified from the urine of healthy subjects [ll]. This result suggests a structural abnormality of UAP obtained from stone-forming patients. Interestingly, both human and rat UAP exhibited structural similarity to bikunin, a subunit of inter-a-inhibitor ( I d ) [lo, 121. Indeed, the sequence of the first 18 amino acid residues of UAP was identical to I d and bikunin. Furthermore, on Western blotting, the immuoreaction between UAP and I d antibody was positive [lo].IaI, with a molecular mass of approximately 220 kDa, is one of the plasma protease inhibitors known formerly as inter-a-trypsin inhibitor (ITI be the active part of I d involved in the inhibition...

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