The Heavy Chains of Human Plasma Inter-α-Trypsin Inhibitor: Their Isolation, Their Identification by Electrophoresis and Partial Sequencing. Differential Reactivity with Concanavalin A

Malki, Nouzha; Balduyck, Malika; Mäes, Pierrette; Capon, Calliope; Mizon, Charlotte; Han, Kia Ki; Tartar, André; Fournet, Bernard; Mizon, J

doi:10.1515/bchm3.1992.373.2.1009

Cited by 40 publications

(39 citation statements)

References 17 publications

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“…The specificity of the anti-MBP-H antisera was investigated by: (1) immunoprecipitation of the translation products of human liver mRNAs obtained in mRNA-dependent rabbit reticulocyte lysates in the presence of [35S]methionine as previously described [4]; (2) analysis by Western blotting of serum proteins submitted to chondroitin AC lyase digestion according to [23], except that 4 mU of enzyme was used.…”

Section: Characterization Of Anti-mbp-h Antiseramentioning

confidence: 99%

“…Serum submitted to limited digestion by chondroitin AC lyase, a procedure known to give rise to free ITI heavy chains [23], was analysed by SDS-PAGE and Western blotting with anti-ITl antisera. As shown in Fig.…”

Section: Obtaining Of H-specific Antisera From Bacterial Expression Omentioning

confidence: 99%

“…Contrasting results were obtained in foetal calf serum: besides a slightly higher molecular mass, ITI only displayed L, H2 and H3 reactivities and the 120-kDa protein group was restricted to HC2/bikunin. Despite the low reactivity of our antisera towards bovine proteins, these results are in total agreement with data obtained by protein-sequencing [29] where ITI (236 kDa) was composed of H2 and H3 heavy chains linked to bikunin and a 126-kDa form involved one H2 heavy chain + bikunin; the latter complex would correspond to ESF, the extracellular matrix stabilizing factor previously reported [11. In the originally described composition of ITI [13] as well as in a further study [23], chemical treatment and/or enzymatic digestion of ITI were used before the identification of its components by N-terminal-sequencing; the number and nature of chains involved in the native molecule could, therefore, not be accurately determined. Indeed, several elements challenge the commonly accepted composition of ITI, viz.…”

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Involvement of the three inter‐α‐trypsin inhibitor (ITI) heavy chains in each member of the serum ITI family

et al. 1995

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Partial cDNAs coding for each of the three human inter-a-trypsin inhibitor (ITI) heavy chains were expressed in a bacterial plasmid system and rabbits were immunised with the fusion peptides obtained. Despite the strong sequence homology of these chains, the antisera turned out to be highly specific in the analysis of corresponding mRNA translation products or partially digested serum ITI. Besides classical serum ITI members, their use in Western blotting made it possible to evidence an H3-related ITI form and a low-amount HI-related HC/bikunin component. The relative levels of ITI family members was further studied in baboon and foetal calf sera.

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Section: Characterization Of Anti-mbp-h Antiseramentioning

confidence: 99%

Section: Obtaining Of H-specific Antisera From Bacterial Expression Omentioning

confidence: 99%

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Involvement of the three inter‐α‐trypsin inhibitor (ITI) heavy chains in each member of the serum ITI family

et al. 1995

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“…More recently, Malki et al [31] have described a new Nterminal sequence SATGR (Fig. 4A, amino acids -5 to -1) for H1.…”

Section: Two Mrnas For It1 Heavy Chain H1mentioning

confidence: 99%

Isolation and characterization of the human inter‐α‐trypsin inhibitor heavy‐chain H1 gene

Bost

Bourguignon

Martín

et al. 1993

European Journal of Biochemistry

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The human inter-a-trypsin inhibitor heavy chain HI (IT1 heavy chain H1) gene was isolated from two overlapping clones. It spans 14 kbp and is composed of 22 exons from 15 bp to 281 bp in size and has consensus splice sites. Intron sizes range from 80 bp to 2000 bp. It codes for the precursor of HCI that is part of the serum IT1 form of 220 kDa. Two major transcriptional initiation sites were identified in the 5'-flanking region, which contained putative promoter elements, but no typical TATA and CAAT boxes. mRNA for the IT1 heavy chain H1 was found only in liver. The tissue-specific transcription of the gene might be due to the presence of binding sites for the hepatocyte nuclear factor HNF-5 and to the octameric motifs. A previous overlapping of cDNA clones indicated the absence of 29 bp in one of these clones. The present study shows that the 29 bp is located within the gene at the end of exon 21. A reverse-transcriptase polymerase chain reaction mapping analysis of liver mRNA identified the two types of the mRNA for IT1 heavy chain H1. Accordingly, the data demonstrate that there is alternative splicing of at least one exon of the IT1 heavy chain H1 gene.The human inter-a-trypsin inhibitor (ITI) heavy-chain H1 and H3 genes are located exclusively in the p21.1-21.2 region of chromosome 3 [l]. The IT1 family members are plasma serine proteinase inhibitors in mammals (reviewed in [2]). We have described the four chains that are implicated in this family, i.e. three heavy chains (HI, H2, H3) and one light chain (L) [l]. The four genes of the IT1 family are mapped to three differents chromosomes 111. Actually, the IT1 family is composed of at least four members: the IT1 itself, a 220-kDa glycoprotein made of H1, H2 and bikunin chains; the pre-a-trypsin inhibitor (PaI), composed of H3 and bikunin chains; an uncomplexed bikunin molecule, and a 130-kDa trypsin inhibitor molecule made of H2 and bikunin chains [3]. The chains are linked by a glycosaminoglycan chain [3-51. Only the structure of the human light-chain gene has been established so far. Separate exons of the L precursor code for two proteins, a,-microglobulin and bikunin, that have no typical functional feature [6, 71. The ex-

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“…The three chains of IaI are held together in a unique complex b! , way of a carbohydrate chain [13,241. This chain was subsequently demonstrated to be a glycosaminoglycan, which was sensitive to chondroitinase hydrolysis [ 16,25,261.…”

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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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The Heavy Chains of Human Plasma Inter-α-Trypsin Inhibitor: Their Isolation, Their Identification by Electrophoresis and Partial Sequencing. Differential Reactivity with Concanavalin A

Cited by 40 publications

References 17 publications

Involvement of the three inter‐α‐trypsin inhibitor (ITI) heavy chains in each member of the serum ITI family

Involvement of the three inter‐α‐trypsin inhibitor (ITI) heavy chains in each member of the serum ITI family

Isolation and characterization of the human inter‐α‐trypsin inhibitor heavy‐chain H1 gene

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

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