Bikunin (Bk) is a Künitz-type serine proteinase inhibitor, which occurs in human plasma, mainly as covalent complexes with one or two of the three peptide heavy chains. The leading member of this glycoprotein family is inter-α-inhibitor (IαI), which consists of two heavy chains (H1 and H2) linked to Bk. Bk carries a glycosaminoglycan (GAG) chain, which is linked by ester bonds to the heavy chains of IαI. Furthermore, Bk, IαI and related components such as pre-α-inhibitor (PαI), all together making up the IαI family, present antiinflammatory and antimetastatic effects that hinge on this GAG chain. Recently (Eur. J. Biochem. 268 (2001) 2717), we provided evidence that, during acute phase response, the GAG chain of Bk, which is a low-sulphated chondroitin-sulphate, increases in size according to the severity of the inflammatory disease. This increase affects Bk-containing proteins in circulating blood as well as Bk excreted in higher amounts in urine of these patients. In this work, we have more extensively analysed the GAG chain of Bk isolated from urine collected from a unique patient with septic shock. Using MALDI-TOF-MS and HPLC analyses of chondrodisaccharides released by enzymatic digestion, we have demonstrated that the GAG chain is clearly modified; it consists of 20 ± 5 disaccharide units vs. 14 ± 3 for reference Bk originating from healthy donors. Among them, only 3 ± 2.5 units are 4-sulphated for patient’s Bk vs. 5 ± 1.5 for reference Bk. Therefore, the non-sulphated region of the GAG chain, which is located towards its non-reducing end, where the heavy chains are positioned, is lengthened from 9 for reference Bk to 17 disaccharide units. We suggest that the biological effects of Bk-proteins may hereby be modulated during inflammatory diseases.

Bikunin (BK) is a Kunitz-type proteinase inhibitor responsible for most of the antitryptic activity of urine and so is known as the urinary trypsin inhibitor. As its excretion increases in inflammatory conditions, it is often considered to be a positive acute phase protein (APP). However, the gene for BK is downregulated in inflammation. In human plasma the major part of BK is covalently linked through a glycosaminoglycan chain to one or two homologous peptide heavy chains, thus forming high molecular weight proteinase inhibitors called pre-alpha-inhibitor (PalphaI) and inter-alpha-inhibitor (IalphaI), respectively. The C-terminal parts of these heavy chains are very sensitive to proteolysis. Neutrophil proteinases in particular are able to release from IalphaI and PalphaI BK (M, about 25,000) which retains its antitryptic activity and is quickly excreted in urine. It was therefore an early supposition that the higher urinary excretion of BK occurring during inflammatory diseases should be, at least in some respect, related to a partial proteolysis of IalphaI and PalphaI. In this study we observed that BK, determined as antitryptic activity, was clearly increased in urine from 35 patients with inflammatory diseases varying in origin and severity (76.5 +/- 75.5 IU/g vs. reference value <10 IU/g creatinine). This increase seems mainly to be associated with polymorphonuclear leukocyte activation, monitored by human leukocyte elastase (HLE) determination rather than with the acute phase response assessed by C-reactive protein (CRP) measurement. For all the patients we found that the urinary levels of BK and serum concentration of intact IalphaI correlated inversely (r=-0.36; p=0.03), in agreement with the presumed precursor-product relationship linking IalphaI and BK. We also proved that urinary BK was significantly higher, and serum IalphaI was significantly lower, in samples with plasma HLE values above the reference: 90 microg/l. Taken together, our results demonstrate that BK, the urinary excretion of which is increased in inflammatory conditions, originates, at least partly, from IalphaI and PalphaI by proteolytic cleavage. Consequently, urinary BK determination provides information on the severity of systemic proteolysis occurring in inflammation. We also demonstrated that during inflammatory diseases IalphaI and PalphaI concentrations in serum are dependent on their increased utilization as well as on the regulation of their biosynthesis.

Inter-alpha -inhibitor (I alphaI) and pre-alpha -inhibitor (P alphaI) are the main members of a set of multichain serine proteinase inhibitors. Present in human plasma, they may be involved in control of the inflammatory process. They are composed of homologous heavy chains (H1 and H2 for I alphaI; H3 for P alphaI) covalently linked by a protein-glycosaminoglycan-protein cross-link to bikunin, which is a chondroitin 4-sulfate proteoglycan. During the acute-phase response, biosynthesis of I alphaI and P alphaI is downregulated and upregulated, respectively.In this work, we provide evidence that, in inflammatory diseases, the chondroitin sulfate chain of bikunin increases in size proportionally to the severity of the inflammatory response. As a consequence, all I alphaI-related components that contain bikunin are structurally modified. Therefore, the changes in glycosylation of the acute-phase proteins are not restricted to N-linked glycans but also affect glycosaminoglycans.The implications of these findings are discussed with regard to biosynthesis and biological role, especially the anti-inflammatory effects of I alphaI-related proteinase inhibitors.

The inter-alpha-inhibitor (I alpha I) family gathers together several plasma protease inhibitors such as I alpha I and pre-alpha-inhibitor (P alpha I) that are variously assembled from a set of polypeptide chain precursors designated H1P to H3P. In addition to their protease inhibitory activity, a major physiological function of I alpha I family members is hyaluronan (HA) binding and HA-dependent stabilization of the extracellular matrix surrounding various cell types. Also, binding of HA to these molecules has been shown to be an important event in tumor cell proliferation and rheumatoid arthritis. However, how HA and I alpha I family members first recognize each other has so far remained elusive. The so-called BX7B domain found in some HA-binding proteins is an HA-binding site in which B represents a basic amino-acid residue and X represents any nonacidic residue. This domain has now been identified in the N-terminal end of H3P that is a precursor of P alpha I. A series of wild-type or mutant recombinant H3P chains produced with a mouse cDNA expressed in Escherichia coli allowed us to demonstrate that this domain binds HA in a noncovalent fashion. Furthermore, unmasking this HA-binding activity required most of H3P to be trimmed off at its C-terminal end. The latter observation was confirmed with a natural, mature H3 chain purified from human plasma. Indeed, a thermolysin-generated, N-terminal fragment of this H3 chain strongly bound HA whereas the intact H3 chain did not. Therefore, in vivo, the HA-binding activity of the mature H3 chain within P alpha I may vary with the folding and/or fragmentation of this protein.

BACKGROUND Pre-alpha-inhibitor (PalphaI) is a human plasma serine-proteinase inhibitor that is structurally related to inter-alpha-inhibitor (IalphaI). It is composed of a heavy chain named H3 covalently linked to bikunin by means of a glycosaminoglycan chain. We developed an ELISA procedure making it possible to measure PalphaI for the first time and we investigated its levels in sera from patients with inflammatory diseases. MATERIALS AND METHODS We generated rabbit anti-H3 immunoglobulins, which were used on solid phase and biotinylated antibikunin immunoglobulins to detect trapped PalphaI. RESULTS We demonstrate that PalphaI is more susceptible than IalphaI to in vitro proteolysis by stimulated neutrophils. However, the degradation products thus released as well as the other members of the IalphaI family present in serum do not affect the ELISA test. In a panel of control sera we observed PalphaI concentrations of 25.6 +/- 7.8 mg L-1 (mean +/- SD; n = 30). These values increased to 64.2 +/- 16.06 mg L-1 (mean +/- SD; n = 15) in patients with inflammatory diseases, concording with the positive acute-phase protein nature of PalphaI. However, for all these patients, the serum concentrations of PalphaI and C-reactive protein poorly correlated (r = 0.476; P = 0.076). Indeed, four patients had a relatively weaker increase in their PalphaI level than that of C-reactive protein. More often than not their plasma elastase content was then elevated. CONCLUSION During inflammatory diseases plasma PalphaI levels may be dependent on increased synthesis in combination with enhanced catabolism, perhaps implicating neutrophil or other proteinases.

Inter-α-inhibitor (IαI) is a human plasma serine proteinase inhibitor. It contains one light peptide chain called bikunin that exerts antiproteinase activity and other anti-inflammatory functions. Bikunin is covalently linked to two heavy chains that, after tissular diffusion, stabilize the extracellular matrix. Owing to its negative acute-phase reactant character and its susceptibility to proteolysis, IαI has been implicated in the pathophysiology of sepsis. Moreover, IαI has been shown to exert a protective effect on a pig model of endotoxic shock. Twenty patients admitted to the intensive care unit (ICU) for a septic syndrome were included in the present study. IαI and antithrombin III (ATIII) levels were measured on admission. Sequential measurements of IαI could be done in 4 patients. We demonstrate that IαI levels are significantly decreased in plasma samples collected on admission from patients with sepsis (59 ± 32 mg/L vs 241 ± 70 mg/L; P <.0001). This decrease was greater in severe sepsis and septic shock than in sepsis. Death was not predictable from initial IαI levels. In 2 patients with a favorable course, IαI values regularly increased during the ICU stay. By sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by immunoblot analysis and microsequencing, we characterized IαI-related components in plasma from several patients; they obviously arise from IαI through proteolytic cleavage. Thus, systemic proteolysis and decreased biosynthesis both contribute to the fall in the plasma level of IαI. Because IαI is very sensitive to proteolysis by polymorphonuclear granulocytes (PMNs) that are stimulated during sepsis, we suggest that IαI plasma level would be a useful marker for neutrophil proteinase activity. ATIII, as well as IαI, is considered a negative acute phase protein. Because in vitro ATIII is less susceptible than IαI to proteolysis by PMNs and because their relative levels weakly correlated, we suggest that an unspecific systemic proteolysis is not significantly involved in the ATIII deficiency occurring in sepsis. (J Lab Clin Med 2000;135:188-98)

Human inter-alpha-inhibitor (IalphaI) has been shown to exert a beneficial therapeutic effect in a porcine model of endotoxin shock. It is therefore useful to have a better understanding of IalphaI metabolism during severe inflammatory syndromes. Experimental bacterial pneumonia was induced in pigs. The acute phase response was highlighted by an increase in pig major acute phase protein (pig-MAP) and haptoglobin concentrations in plasma collected daily over 4 days. In the same samples, the IalphaI levels remained unchanged. Moreover, crossed-immunoelectrophoretic and immunoblot analyses did not show any qualitative modification of IalphaI throughout the experiment. IalphaI has been reported to be a negative acute phase protein in both humans and rats. Here we demonstrated that IalphaI behavior clearly differs in humans and pigs and is definitively species specific.

Abstract Urinary trans,trans-muconic acid is a sensitive biomarker for low level benzene exposure. The method described by Ducos et al. (Int Arch Occup Environ Health 1990; 62:529–34) is commonly used for its determination. In this study, we demonstrate that N-benzoyl, L-glutamic acid added to urine samples is a suitable internal standard to control trans,trans-muconic acid recovery after solid phase extraction of urine and to compensate for variations which might occur during high-performance liquid chromatography analysis.

Human inter-alpha-inhibitor (IalphaI) is a plasma serine-proteinase inhibitor. It consists of three polypeptide chains covalently linked by a glycosaminoglycan chain: a light chain named bikunin carrying the anti-proteinase activity and two heavy chains, H1 and H2, which exhibit specific properties, e.g. they interact with hyaluronan thus stabilizing the extracellular matrix. In this study, using matrix-assisted laser desorption ionization-time-of-flight MS and amino acid sequencing of tryptic peptides, we provide a detailed analysis of the glycosylation pattern of both heavy chains. H1 carries two complex-type N-glycans of predominantly biantennary structure linked to asparagine residues at positions 256 and 559 respectively. In contrast, the oligosaccharides attached to H2 are a complex-type N-glycan in the N-terminal region of the protein (Asn64) and three to four type-1 core-structure O-glycans mono- or di-sialylated, clustered in the C-terminal region. We propose that these O-glycans might function as a recognition signal for the H2 heavy chain. The biological implications of this hypothesis, notably for the biosynthetic pathway of IalphaI, are discussed.

Human inter-alpha-inhibitor (IalphaI) is a plasma serine-proteinase inhibitor. It consists of three polypeptide chains covalently linked by a glycosaminoglycan: a light one named bikunin, carrying the antiproteinase activity and two heavy chains H1 and H2. The amino acid sequences of these heavy chains are highly similar; however when IalphaI is digested by neutrophil proteinases, their proteolytic susceptibility strongly differs [Balduyck, M., Piva, F., Mizon, C., Maes, P., Malki, N., Gressier, B., Michalski, C. & Mizon, J. (1993) Human leucocyte elastase (HLE) preferentially cleaves the heavy chain H2 of inter-alpha-trypsin inhibitor (ITI), Biol. Chem. Hoppe-Seyler 374, 895-901]. We mapped the disulphide topology of the IalphaI heavy chains in order to investigate whether or not disulphide bonds might be responsible for their differential susceptibility to proteolysis. Using amino acid sequencing and mass spectrometry analysis, we demonstrate that the H1 heavy chain contains one free thiol group and two disulphide bridges of which one links two largely spaced cysteine residues (Cys239 and Cys511). Thus H1 is clearly different from H2 which contains two disulphide bonds between closely located cysteine residues. However, using immunoprint analysis, we show that, when IalphaI is subjected to a limited digestion by Staphylococcus aureus V-8 proteinase, the two polypeptide chains are similarly susceptible to proteolysis. This enzyme preferentially cleaves the IalphaI heavy chains from their N-terminal extremity. These results are consistent with the circular dichroism (CD) analysis, suggesting that the conformation of the polypeptide backbone of H1 is not very different from that of H2, with calculated alpha-helicities of 24% and 28%, respectively. The CD measurements reveal that the aromatic amino acids of H1 and H2 are in a different asymmetrical environment. Inside the IalphaI molecule, the heavy chains are linked to the glycosaminoglycan chain via their C-terminal aspartic acid residue. Thus we suggest that the affinity of cationic neutrophil proteinases for the anionic glycosaminoglycan is responsible for the cleavage of the heavy chains (mainly H2) near their C-terminal end and the high susceptibility of IalphaI to these proteinases.

We investigated the effects of human inter-alpha-inhibitor (I alpha I) on hemodynamics, oxygenation, and coagulation parameters in a porcine model of endotoxic shock. Four groups of six animals were studied: (1) control, (2) I alpha I group receiving 30 mg/kg I alpha I over 30 min, (3) LPS group receiving 5 micrograms.kg/min Escherichia coli endotoxin over 30 min, and (4) LPS + I alpha I group receiving 30 min after endotoxin 30 mg/kg/30 min I alpha I. We measured hemodynamic and oxygenation parameters, usual coagulation markers and plasma levels of thrombin-antithrombin complexes, antithrombin III activity, plasminogen activator tissue type, plasminogen activator inhibitor type 1, von Willebrand factor, tumor necrosis factor-alpha, and I alpha I at baseline and at 30, 60, 90, 120, 180, 240, and 300 min. In the I alpha I group, plasma I alpha I levels reached 447 +/- 23 mg/L just after injection and 287 +/- 39 mg/L at 300 min. I alpha I half-life was 7.3 +/- 1.9 h. In the IPS + I alpha I group, I alpha I plasma levels decreased more rapidly, reaching 260 mg/L at 300 min. Compared with the LPS group, administration of I alpha I normalized the mean arterial pressure and cardiac index, improved the LPS-induced pulmonary hypertension, and resulted in the blunted increase in blood lactate and oxygen extraction ratio. A significant decrease in thrombin-antithrombin complexes and plasminogen activator inhibitor type 1 levels were observed. There was no significant difference in plasma tumor necrosis factor-alpha levels. We concluded that in this hypodynamic model of endotoxin shock, I alpha I administration resulted in a marked improvement in the hemodynamic, oxygenation, and coagulation parameters.

With the view of investigating the metabolism of inter-alpha-inhibitor, a plasma serine-proteinase inhibitor, in an animal model of inflammatory syndrome, we isolated inter-alpha-inhibitor from pig plasma. A high yield was obtained (140 mg/liter) with a two-step procedure: anion-exchange chromatography followed by affinity chromatography on heparin-Sepharose. In contrast to bovine inter-alpha-inhibitor was highly similar to human inter-alpha-inhibitor: its heavy chains are homologous to the human H1 and H2 heavy chains, as shown by chromatographic and electrophoretic properties, cross-immunoreactivity and N-terminal sequencing. Pig may therefore represent a good animal model to study inter-alpha-inhibitor metabolism and elucidate its physiological role.

Digestive bacterial microflora play a major rolein the pathogenesis of Crohn's disease (CD). Bacterialenzyme activities, especially β-D-galactosidase,are decreased in fecal extracts from CD patients. We hypothesized that an alteration of thecolonic flora might be responsible for this decrease.Indeed, we demonstrate that β-D-galactosidaseproduction in supernates of anaerobic cultures wassignificantly (P < 0.01) reduced in feces from patientswith active Crohn's disease (N = 7), when compared tohealthy controls (N = 8). Therefore using X-gal andselective media, we enumerated bacteria able to releaseβ-D-galactosidase in feces from patients with active (N = 16) orquiescent disease (N = 5) and healthy controls (N = 14).Bifidobacteria numbers were significantly reduced inpatients (P < 0.01 for active; P < 0.02 forquiescent disease) whereas Bacteroides and Lactobacilli countsremained unchanged. β-D-Galactosidase activity andBifidobacteria counts were significantly correlated (P< 0.03). Bifidobacteria are regarded as beneficial for the host. The reduction in Bifidobacteriais responsible for decreased β-D-galactosidaseactivity. Thus oral administration of prebiotics thatpromote their growth might have potential therapeutic interest.

The unrestricted activity of leukocyte proteinases is thought to contribute to the degradation of plasma proteins and thus amplify the coagulation disorders occurring in septic shock. Inter-alpha-inhibitor (I alpha I) is a plasma protein particularly susceptible to their action. Therefore we investigated its behavior in a porcine model of endotoxin shock which reproduces the coagulation changes observed in human sepsis. We did not detect any qualitative or quantitative modification of porcine I alpha I in plasmas collected from pigs after endotoxin infusion. To explain these data, I alpha I was incubated with polymorphonuclear neutrophils (PMN) stimulated by FMLP in the presence of cytochalasin B. We found that, unlike human PMN, porcine cells were unable to proteolyze I alpha I. Moreover, in the incubation medium of pig PMN, triggered either by FMLP or PMA, no measurable elastase activity was evidenced. Therefore, we urge to better take into account species differences in functional responses of PMN, to explain the experimental results obtained in animal models of septic shock.

Pre-α-inhibitor (PαI) is a serine proteinase inhibitor from human plasma. It comprises bikunin (BK) responsible for antiprotease activity, covalently linked to a heavy chain H3. Here we describe its isolation from a side fraction of an industrial preparation of plasma clotting factors. By using a highly specific polyclonal antiserum prepared from rabbit immunized with a H3P polypeptide obtained in a bacterial expression system, we were able to identify the fractions containing PαI. Then, taking advantage of the differential affinity of the members of the inter-α-inhibitor family (IαI) for heparin-Sepharose and blue-Sepharose, we isolated PαI. Its specific antitryptic activity was 580 IU/g, higher than that of IαI: 420 IU/g. Its Mr, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with or without prior reduction, was 130 000. Its peptide chains were identified by N-terminal sequencing. The H3 heavy chain was isolated from PαI by alkaline dissociation and anion-exchange chromatography. Its electrophoretic mobility was compared to that of the H1 and H2 heavy chains of IαI. In reducing conditions, it was quite similar to that of H2 (Mr 85 000) but clearly different from that of H1 (Mr 78 000). Thus, the so-determined apparent Mr of H3 was overestimated since its molecular mass determined by MALDI-TOF was 74 100. This result agrees with the proposed structure for H3. Indeed, by carbohydrate analysis and PNGase F digestion, we demonstrate that the two potential N-glycosylation sites present in the core-protein (theoretical mass: 69 454) are really occupied by two N-glycans, probably of biantennary type.

Human neutrophil proteinases have been implicated in the pathogenesis of a wide variety of inflammatory diseases. The degradation of plasma proteins such as coagulation and fibrinolysis factors has been attributed to the excessive release of elastase in septicemia and in other conditions in which heightened proteolysis occurs. Inter-α-inhibitor (IαI) is particularly sensitive to cleavage by leukocyte proteinases. For this reason, the determination of IαI has been proposed as a method for evaluating plasma protein proteolysis by neutrophil enzymes. In this article we provide evidence that intact residual IαI can be accurately quantified by enzyme-linked immunosorbent assay (ELISA) determination without interference from fragments released from IαI by incubation with triggered neutrophils. We demonstrate that under these conditions IαI was quickly and steadily proteolyzed in a cell dose-dependent manner. α-1 proteinase inhibitor (α1 PI) partially protected IαI; however, the proteolysis persisted when IαI was incubated with stimulated neutrophils in the presence of a large relative excess of α1 PI over the amount of elastase theoretically present in cells. For the same amount of α1 PI, serum provided a better protection than α1 PI alone but did not completely inhibit the IαI degradation. Therefore, ELISA determination of IαI might be useful for monitoring the in vivo activity of neutrophil proteinases in systemic proteolytic states.

Bacterial glycosidase activities, specially β-galactosidase (β-gal), are reduced in faeces from patients with Crohn's disease (CD). In order to test the hypothesis that an alteration of the colonic flora might be responsible for decreased faecal β-gal activity in CD patients, we developed a new methodology designed to differentiate and enumerate anaerobic bacteria producing β-gal. We used an enriched Columbia agar medium modified by addition of 3-bromo-4-chloro-5-indolyl-β-d-galactopyranoside (X-gal) and tested it on different faecal bacterial strains. The presence of blue diffuse zones with various sizes surrounding bacterial colonies clearly differentiated Bifidobacterium, Ruminococcus, Bacteroides and one of five strains of Lactobacillus tested from other β-gal positive bacteria. When the X-gal medium was applied to the analysis of normal human faecal flora, 57%±29 (mean±S.D.) of the colonies growing on this medium were surrounded by blue haloes. Among them, all colonies, characterized by a large blue diffuse ring, were identified as bifidobacteria.

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-alpha-inhibitor (I alpha I) known for its inhibition of the action of many serine proteinases like trypsin and chymotrypsin. To clarify the relationship between these macromolecules, UAP, I alpha I, 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 I alpha I. UAP exhibited a strong calcium oxalate crystallization inhibitory activity. I alpha I 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 bikunin.