Preparation and Properties of a Therapeutic Inter-Alpha-Trypsin Inhibitor Concentrate from Human Plasma

Michalski, Catherine; Piva, Frank; Balduyck, Malika; Mizon, Charlotte; Burnouf, Thierry; Huart, J. J.; Mizon, J

doi:10.1159/000462634

Cited by 13 publications

(19 citation statements)

References 16 publications

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“…We cannot exclude the possibility that the bikunin-like peptides observed in CaOx crystals by Atmani et al [16] and ourselves may be breakdown products of one of the numerous combinations of the various chains of IαI, caused by the crystallization process itself, which unlike crystal formation in vivo was induced by extremely high concentrations of oxalate. These peptides were obvious in the gels and blots of the hydrolysed IαI used as a standard, which confirms previous reports that alkaline hydrolysis causes significant fragmentation of the IαI molecule [22,23]. A large and confusing array of protein bands, all of which reacted with the IαI antibody, were seen after alkaline treatment of intact IαI.…”

Section: Discussionsupporting

confidence: 88%

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Inter-α-inhibitor in calcium stones

DAWSON¹,

Grover²,

KANELLOS³

et al. 1998

Clinical Science

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1. A broad spectrum of proteins has been detected within calcium stones. A newcomer to the field of urolithiasis is the blood protein inter-alpha-inhibitor. Inter-alpha-inhibitor comprises three protein chains linked by chondroitin sulphate: two heavy chains, H1 (65 kDa) and H2 (70 kDa) and a light chain (approx. 30 kDa) most commonly known as bikunin. The physiological function of the two heavy chains is unknown; nor has their presence been reported in urine. However, bikunin has been implicated in various renal diseases, including urolithiasis. 2. This study was undertaken to determine which chains of inter-alpha-inhibitor are actually present in calcium kidney stones. Organic extracts were obtained from 10 calcium stones and analysed by SDS/PAGE and Western blotting. The H1 and H2 chains of inter-alpha-inhibitor were detected in 9 of the 10 stones, but only one stone contained a protein with a molecular mass close to that of bikunin (30-35 kDa). 3. These results demonstrate for the first time that H1 and H2 are present in stones and show that the bikunin chain of inter-alpha-inhibitor may not be the only part of the molecule implicated in stone formation.

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Section: Discussionsupporting

confidence: 88%

“…Alkaline hydrolysis of intact IαI was based upon the methods of Malki et al [22] and Michalski et al [23]. Briefly, IαI (1.75 mg\ml) in a citric acid\phosphate buffer (see above) was adjusted to a pH of approximately 13.0 with NaOH and stirred for 10 min at room temperature.…”

Section: Hydrolysis Of Iαimentioning

confidence: 99%

Inter-α-inhibitor in calcium stones

DAWSON¹,

Grover²,

KANELLOS³

et al. 1998

Clinical Science

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“…I␣I, purified from human serum (29), and bikunin, prepared by NH 2 OH dissociation of purified I␣I followed by ion-exchange chromatography (30), were kindly supplied by Dr. Jacques Mizon (Université de Lille II, France). Purity of the isolated proteins was assessed by SDS-PAGE followed by silver staining.…”

Section: Methodsmentioning

confidence: 99%

PTX3 Interacts with Inter-α-trypsin Inhibitor

Scarchilli

Camaioni

Bottazzi

et al. 2007

Journal of Biological Chemistry

137

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Pentraxin 3 (PTX3) and heavy chains (HCs) of inter-␣-trypsin inhibitor (I␣I) are essential for hyaluronan (HA) organization within the extracellular matrix of the cumulus oophorus, which is critical for in vivo oocyte fertilization and female fertility. In this study, we examined the possibility that these molecules interact and cooperate in this function. We show that HCs and PTX3 colocalize in the cumulus matrix and coimmunoprecipitate from cumulus matrix extracts. Coimmunoprecipitation experiments and solid-phase binding assays performed with purified human I␣I and recombinant PTX3 demonstrate that their interaction is direct and not mediated by other matrix components. PTX3 does not bind to I␣I subcomponent bikunin and, accordingly, bikunin does not compete for the binding of PTX3 to I␣I, indicating that PTX3 interacts with I␣I subcomponent HC only. Recombinant PTX3-specific N-terminal region, but not the PTX3-pentraxin C-terminal domain, showed the same ability as full-length protein to bind to HCs and to enable HA organization and matrix formation by Ptx3 ؊/؊ cumulus cell oocyte complexes cultured in vitro. Furthermore, a monoclonal antibody raised against PTX3 N terminus, which inhibits PTX3/I␣I interaction, also prevents recombinant fulllength PTX3 from restoring a normal phenotype to in vitrocultured Ptx3 ؊/؊ cumuli. These results indicate that PTX3 directly interacts with HCs of I␣I and that such interaction is essential for organizing HA in the viscoelastic matrix of cumulus oophorus, highlighting a direct functional link between the two molecules.

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“…UAP was isolated from healthy male human urine using three chromatographic steps on DEAE-Sephacel, Sephacryl S-300 and Mono Q HR 5/5 column as earlier described [3]. IaI was isolated from male human plasma according to the procedure described by Michalski et al [29] using DEAE-Sephadex, then DEAE-Sepharose fast flow chromatography followed by a chromatographic step on immobilized heparin. Urinary bikunin was prepared from male human urine following the procedure described by Balduyck et al [30] using DEAE-Sephacel, gel-filtration on Sephacryl S-200, DEAE-Trisacryl chromatography, affinity chromatography on ConA-Sepharose and finally gel filtration on Sephacryl S-200.…”

Section: Methodsmentioning

confidence: 99%

Identification of Uronic‐Acid‐Rich Protein as Urinary Bikunin, the Light Chain of Inter‐α‐Inhibitor

Atmani

Mizon

Khan

1996

European Journal of Biochemistry

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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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Preparation and Properties of a Therapeutic Inter-Alpha-Trypsin Inhibitor Concentrate from Human Plasma

Cited by 13 publications

References 16 publications

Inter-α-inhibitor in calcium stones

Inter-α-inhibitor in calcium stones

PTX3 Interacts with Inter-α-trypsin Inhibitor

Identification of Uronic‐Acid‐Rich Protein as Urinary Bikunin, the Light Chain of Inter‐α‐Inhibitor

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