Inter-α-trypsin Inhibitor Research Articles

ITIH4 Serum Concentration Increases during Acute-Phase Processes in Human Patients and Is Up-Regulated by Interleukin-6 in Hepatocarcinoma HepG2 Cells

The serum concentration of the inter-α trypsin inhibitor heavy chain 4 protein (ITIH4) increases (from 1.4–3 times) in male patients suffering of different acute-phase processes (myocardial infarction, unstable angina or programmed surgery). The concentration of C-reactive protein (CRP) in these samples ranged from 15 μg/ml to 133 μg/ml. Using the hepatocarcinoma HepG2 cell line we have observed up-regulation of ITIH4 mRNA expression upon dose-response treatments with interleukin-6 (IL-6). This effect correlates with the increase of radiolabeled ITIH4 in the cellular media of 35S-labeled HepG2 cells treated with the cytokine. A similar effect was observed for haptoglobin mRNA, used as a control for acute-phase protein expression. IL-1β, although up-regulating the expression of α1-acid glycoprotein in these cells, did not induce any effect in the expression of ITIH4. No changes were observed after TNF-α treatments. The results presented here indicate that ITIH4 is a type II acute-phase protein in humans.

Isolation and characterization of proteoglycans from human follicular fluid

Two proteoglycans differing in size and composition were isolated from human follicular fluid. The larger one of high density had a molecular mass of 3.0×106 Da, as determined by laser light-scattering, and was substituted with 15-20 chondroitin sulphate (CS) chains (Mr 60000-65000). Half of the CS disaccharides were 6-sulphated, whereas the remaining ones were non-sulphated. Digestion of the CS proteoglycan with chondroitinase ABC lyase, followed by SDS/PAGE, yielded a protein core of 600 to 700 kDa including substituted oligosaccharides, and a band of 70 kDa that was identified as the heavy-chain component of the inter-α-trypsin inhibitor (ITI). Western blotting of the CS proteoglycan showed that this had reactivity with antibodies raised against human versican. Electron microscopy (EM) of the CS proteoglycan also revealed a versican-like structure, with one globular domain at each end of a long extended segment substituted with CS side chains, as well as a structure interpreted as being the heavy chain of ITI attached to CS chains. Laser light-scattering revealed that the smaller proteoglycan had a molecular mass of 1.1×106 Da, and EM demonstrated that it had a globular-protein core structure. The core protein, which showed immunological reactivity with perlecan antibodies, was substituted with approximately seven heparan sulphate (HS) and CS chains of similar size (50-55 kDa), the CS disaccharides being mainly 6-sulphated (68%), with a small proportion being 4-sulphated. The protein core was shown to be heterogeneous, with bands occurring at 215, 330 and 400 kDa after enzymic degradation of the glycosaminoglycan chains followed by SDS/PAGE analysis. The demonstration of intact molecules and fragments obtained after stepwise degradations, as shown by gel chromatography, supported a ‘composite’ structure of this proteoglycan.

Isolation and characterization of proteoglycans from human follicular fluid

Two proteoglycans differing in size and composition were isolated from human follicular fluid. The larger one of high density had a molecular mass of 3.0x10(6) Da, as determined by laser light-scattering, and was substituted with 15-20 chondroitin sulphate (CS) chains (Mr 60000-65000). Half of the CS disaccharides were 6-sulphated, whereas the remaining ones were non-sulphated. Digestion of the CS proteoglycan with chondroitinase ABC lyase, followed by SDS/PAGE, yielded a protein core of 600 to 700 kDa including substituted oligosaccharides, and a band of 70 kDa that was identified as the heavy-chain component of the inter-alpha-trypsin inhibitor (ITI). Western blotting of the CS proteoglycan showed that this had reactivity with antibodies raised against human versican. Electron microscopy (EM) of the CS proteoglycan also revealed a versican-like structure, with one globular domain at each end of a long extended segment substituted with CS side chains, as well as a structure interpreted as being the heavy chain of ITI attached to CS chains. Laser light-scattering revealed that the smaller proteoglycan had a molecular mass of 1. 1x10(6) Da, and EM demonstrated that it had a globular-protein core structure. The core protein, which showed immunological reactivity with perlecan antibodies, was substituted with approximately seven heparan sulphate (HS) and CS chains of similar size (50-55 kDa), the CS disaccharides being mainly 6-sulphated (68%), with a small proportion being 4-sulphated. The protein core was shown to be heterogeneous, with bands occurring at 215, 330 and 400 kDa after enzymic degradation of the glycosaminoglycan chains followed by SDS/PAGE analysis. The demonstration of intact molecules and fragments obtained after stepwise degradations, as shown by gel chromatography, supported a 'composite' structure of this proteoglycan.

Urinary trypsin inhibitor suppresses vascular smooth muscle contraction by inhibition of Ca 2+ influx

Urinary trypsin inhibitor (UTI) and its precursor form inter- α trypsin inhibitor (ITI) are present in plasma. To determine the action of UTI on blood vessels, we performed isometric vascular muscle contraction tests, microcirculation studies and measurement of cytosolic free Ca 2+ in vascular smooth muscle cells. An isometric vascular muscle contraction test showed that the contractions stimulated by endothelin-1 or norepinephrine were suppressed in the presence of UTI, and that the contractions were not inhibited in the presence of ITI. The microcirculation study showed that the contraction of mesenteric arterioles of WKY rats induced by norepinephrine were inhibited by treatment of UTI, and that they did not alter by treatment of ITI. Pre-incubation of UTI, but not ITI, with vascular smooth muscle cells inhibited the increase of cytosolic free Ca 2+ induced by endothelin-1 or norepinephrine. Cell-binding study by biotinylated UTI showed that vascular smooth muscle cells have specific binding site for UTI, but not for ITI. We propose that circulating UTI converted from ITI has a regulatory effect on local vascular tone by regulation of Ca 2+ influx into smooth muscle cells.

Inter-alpha-trypsin inhibitor proteoglycan family--a group of proteins binding and stabilizing the extracellular matrix.

Extracellular matrix (ECM) is composed of several macromolecules associated in a complex network. This structure allows cells to adhere, migrate and interact. Hyaluronic acid (HA) is a glycosaminoglycan (GAG) and a major representative of ECM. HA-binding proteins such as CD44, aggrecan, and versican, have been implicated in structuring the ECM by stabilizing large macromolecular aggregates. They also play an important role in tumor metastasis and cell motility. Recently, further HA-binding proteins were identified: the inter-alpha-trypsin inhibitor(ITI)-related proteins. ITI is a glycoprotein composed of three polypeptides: two heavy chains (HC1 and HC2) and one light chain (bikunin). Bikunin confers the protease-inhibitor function. The heavy chains' function was unknown. Recent studies have shown that HC1 and HC2 are linked in vivo and in vitro to hyaluronic acid. This linkage greatly improves extracellular matrix stability. It also demonstrates that ITI-related proteins might be considered as HA-binding proteins (HABP). The ITI related proteins are composed of four polypeptides (HC1, HC2, HC3 and the bikunin) encoded by four genes H1, H2, H3 and L. Unlike the majority of plasma protein a non-disulfide covalent linkage exists between heavy chains and bikunin. This review presents the recent progress concerning the interactions between ITI and ECM showing that ITI-related proteins are HABP members. We will focus on the heavy chain linkage with HA, which represents the demonstration of covalent binding between proteins and HA.

Development of an enzyme-linked immunosorbent assay for human plasma inter-α-trypsin inhibitor (ITI) using specific antibodies against each of the H 1 and H 2 heavy chains

Inter-α-trypsin inhibitor (ITI) is a serine-proteinase inhibitor of human plasma enzymes. ITI is composed of three polypeptide chains covalently linked: bikunin, responsible for the antiprotease activity and two heavy chains H 1 and H 2. Human plasma also contains other components immunologically related to ITI such as pre-α-trypsin inhibitor (paI), inter-α-like inhibitor (IαLI) and free bikunin. The ELISA procedure we propose exclusively measures native ITI within the range 12.5–200 μg/l. The intra- and interassay coefficients of variation were less than 5.6% and 8.7%, respectively. When ITI was added to plasma samples, full recovery was obtained. EDTA-plasma from 30 healthy individuals revealed a mean level of 241.5 mg/l (range 145.5–506). The high specificity, sensitivity, reproducibility and accuracy of the present assay should facilitate the specific measurement of native ITI in blood and thus might represent a useful tool for further physiopathological studies.

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

Partial cDNAs coding for each of the three human inter-α-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 H1-related HC/bikunin component. The relative levels of ITI family members was further studied in baboon and foetal calf sera.

Gene expression and protein distribution of inter-α-trypsin inhibitor in three human hepatoma cell lines.

In standard culture conditions, three human hepatoma cell lines, Hep3B, PLC/PRF/5 and HepG2, were characterised by a predominant transcription of only two (H2 and L) among the four genes involved in the synthesis of inter-α-trypsin inhibitor (ITI)-related proteins. Pulse-chase experiments followed by immunoprecipitation with specific anti-L and anti-H ITI antisera showed that the proteins synthesised displayed a restricted L and/or H2 antigenic reactivity. Furthermore, while Hep3B and PLC/PRF/5 lines only synthesised ITI precursors (mainly the L-form), HepG2 cells were able to secrete an ITI-like protein. Immunocytochemical analyses substantiated these results with uneven distribution of heavy and light-chain polypeptide reactivity among the cells. The use of hepatoma cell models for the study of protein synthesis and assembly must therefore be considered cautiously.

Simple detection of the inter-α-trypsin-inhibitor (ITI) polymorphism by isoelectric focusing with direct immunofixation

The inter-α-trypsin inhibitor (ITI) polymorphism was analysed in a German population, using polyacrylamide gel isoelectric focusing with subsequent direct immunofixation with monospecific ITI antisera. Gene frequencies of ITI*1, ITI*2, ITI*3, and ITI*4 were calculated to be 0.6150, 0.3753, 0.0078 and 0.0019, respectively. In our study the allele ITI*4 is described for the first time in a German population.

Kunitz-Type Trypsin Inhibitor Prevents LPS-Induced Increase of Cytosolic Free Ca 2+ in Human Neutrophils and HUVEC Cells

The protease inhibitor part of inter-α trypsin inhibitor is identical to urinary trypsin inhibitor (UTI). Preincubation of neutrophils and HUVEC cells with UTI inhibited increase of cytosolic free Ca 2+ induced by LPS. Increase of cytosolic free Ca 2+ induced by LPS in the presence of EGTA was also inhibited by UTI. In contrast, UTI did not inhibit increase of cytosolic free Ca 2+ in cells stimulated by Ca 2+ ionophore with or without EGTA. The effects of nine synthetic peptides of UTI on the concentration of cytosolic free Ca 2+ in the neutrophils induced by LPS were examined. Preincubation with a peptide of UTI domain 2, NLPIVRGPCRAFIQL (83-97), was completely inhibited by the increase of cytosolic free Ca 2+ in neutrophils. This region is identical to the trypsin inhibitor site of UTI. We propose that a function of UTI other than as a protease inhibitor is in regulation of intracellular Ca 2+ and that this is due to its trypsin inhibitor region.

CDNA and deduced amino acid sequence of human PK-120, a plasma kallikrein-sensitive glycoprotein

PK-120 is a substrate for plasma kallikrein (PK), recently purified from human plasma. Here we have established the cDNA sequence for human PK-120 mRNA. The deduced amino sequence of PK-120 revealed that it consists of 902 amino acid residues with a calculated mass of 116,423 Da. The putative cleavage sites by PK have been proposed, suggesting that PK-120 may be a precursor of a bioactive peptide. Most interestingly, PK-120 showed significant sequence identities to heavy chains (HCs) of the inter-α-trypsin inhibitor (ITI) superfamily.

The polymorphism of the plasma inter-alpha-trypsin inhibitor (ITI) and its relationship to the heavy chain H1 subunit gene (ITIH1) at 3p211-212.

We investigated the ITI protein polymorphism in linkage analysis, using DraI and SstI as restriction fragment length polymorphism (RFLP) markers for the ITIH1 gene. Isoelectric focusing (IEF) classification from 76 individual plasma samples and RFLP analysis from the corresponding DNA preparations disclosed linkage disequilibrium between the phenotypic IEF patterns of the two common ITI alleles, ITI*1 and ITI*2, and the diallelic DNA polymorphisms of two ITIH1 RFLPs, represented by DraI 4.0 kb and DraI 2.4 + 1.6 kb, and by SstI 6.7 kb and SstI 6.0 + 0.7 kb, for the ITI 1 and ITI 2 IEF phenotypes, respectively, and by DraI 4.0/2.4 + 1.6 kb and SstI 6.7/6.0 + 0.7 kb for the heterozygous ITI 1-2 IEF phenotype. Linked segregation between either of the RFLPs and the polymorphic ITI plasma protein locus has been established in nine informative family pedigrees. The less frequent allele in Europeans, ITI*3, is not represented by a further allelic restriction fragment in either RFLP. The significant linkage disequilibrium observed in this genetic study indicates that the ITI locus, with the alleles ITI*1 and ITI*2, must be close to, or reside within, the ITIH1 gene. The diallelic ITI protein polymorphism therefore provides an informative phenotypic marker system for chromosome 3p211-212.

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

Inter-α-trypsin inhibitor (ITI) is a serine protease inhibitor found in human plasma. Its antiprotease activity is due to bikunin which is effective in various types of experimental shock and pancreatitis. Therefore ITI, which releases bikunin by proteolytic cleavage, could be of therapeutic interest. A method for the large- scale isolation of ITI from human plasma is described. ITI was purified from the prothrombin complex concentrate (PCC) by diethylaminoethyl-Sepharose fast- flow chromatography followed by a chromatographic step on immobilized heparin designed to remove C4, factor X and protein C. With this procedure, which was performed under mild conditions, a homogeneous preparation of native ITI was obtained, as demonstrated by electrophoretic and chromatographic analyses. ITI maintained its biological activity, as exhibited by its specific antitryptic activity of 420±65 IU/g. In order to decrease or eliminate the risk of transmission of viral disease due to lipid-enveloped viruses, the process incorporated a solvent- detergent treatment. Animal studies on the final product revealed no adverse side-effects in terms of toxicity, thrombogenicity or hypotension. This preparation appears suitable for therapeutic evaluation in animal experimental models.

Population studies on the inter-α-trypsin inhibitor (ITI) polymorphism

Serum samples from four European populations — Germans from Jena, Germans from Hannover, Italians from Udine, and Slovakians from Bratislava — were tested for the inter-α-trypsin inhibitor (ITI) polymorphism. The results are compared with those reported for other European, Asiatic and African populations. Reviewing all the hitherto published ITI allele frequencies one can see that the variability among European Populations seems to be rather small, whereas Asiatic and African populations are striking by quite different distribution patterns.

Human inter-α-trypsin inhibitor: Full-length cDNA sequence of the heavy chain H1

Inter-α-trypsin inhibitor (ITI), called inter-α-inhibitor, is a 220 kDa serine proteinase inhibitor found in human serum. It is composed of at least three distinct polypeptide chains. These chains, named H1, H2 and L, are an independently synthesized and proteolytically processed precursor protein. Only the complete structure of H2 and L has been established so far. We used a PCR-based cloning approach and a cDNA screening library to isolate the full-length cDNA H1. The amino acid sequences of the two heavy chains deduced from the cDNA are highly similar (40% identity). Nevertheless, the structure of the signal peptide and propeptide in the N-terminal region is different in these two chains. A complex posttranslational cleavage at both ends of H1 and H2 may be proposed prior to assembly of the ITI chains.

Demonstration of an active component of inter-α-trypsin inhibitor in the brains of Alzheimer type dementia

The putative precursor of A4 amyloid protein associated with Alzheimer's disease is known to have a domain with an amino acid sequence characteristic of a Kunitz-type serine protease inhibitor. Human serum inter-α-trypsin inhibitor (ITI) is the most similar inhibitor. We screened brain tissues with senile dementia of the Alzheimer type in an attempt to detect ITI immunoreactivity employing immunohistochemical methods. For this purpose, we used the antibody raised against acid-stable proteinase inhibitor (ASPI) which is an active component of ITI. ASPI immunoreactivity was found to be localized in diffuse type senile plaques, the perivascular area and subpial layer. Reactive astrocytes with intense ASPI immunoreactivity were present in the pyramidal layer of the parahippocampus, where loss of neurons was observed. These findings suggest that ITI may be related to the pathogenesis of Alzheimer type dementia.

Plasma proteins immunologically related to inter-α-trypsin inhibitor

SDS-polyacrylamide gel electrophoresis and immunoblot were applied to analysis of plasma proteins immunologically related to inter-α-trypsin inhibitor (ITI). In this system, anti-ITI sera were able to identify ITI and other components with an M r near 120 kDa which would be degradation products of ITI by limited proteolysis. An anti-UTI (urinary trypsin-inhibitor) serum could detect, beside these derivatives, two minor components ( M r values near 90 and 60 kDa). Analysis of perchloric acid supernatants of plasma samples, using the same technic, induced visualization of a new component, similar to urinary trypsin inhibitor which could not be detected by direct analysis. This one was also characterized in a higher content in pathological samples (renal failure and infectious diseases).

Human inter-α trypsin inhibitor and immunologically related inhibitors investigated by quantitative immunoelectrophoresis. I. Method and reference material

The concentration in serum of inter-α trypsin inhibitor (IαI) and its immunologically related inhibitor, the prealbumin-like proteinase inhibitor (pA-PI), was investigated by quantitative crossed immunoelectrophoresis (CIE) in 33 healthy persons. The corresponding urinary excretion over a 4 h period of the immunologically related urinary proteinase inhibitor (UPI) was measured by rocket immunoelectrophoresis. The concentration of pA-PI increased significantly ( p < 0.001) with age whereas that of IαI and the urinary excretion of UPI were independent of age. There was no significantly difference in the concentration of the inhibitors in serum between the sexes, but females were found to have a lower urinary excretion of UPI than males ( p < 0.05). Furthermore we found no significant variation in the concentration of the inhibitors during the course of the day or from day to day over a 5 day period. Results obtained by rocket immunoelectrophoresis correlated well with enzymatically measured trypsin inhibitory activity.

Human Inter-alpha-Trypsin-Inhibitor: Characterization and partial nucleotide sequencing of a light chain-encoding cDNA

A synthetic Inter-α-Trypsin-Inhibitor (ITI)-specific oligonucleotide probe was used to isolate a clone from a human liver cDNA library. The amino-acid sequence deduced from partial nucleotide sequencing of the corresponding cDNA insert perfectly matched a known ITI sequence, apart from an as yet unreported C-terminal dipeptide. Hybridization on Northern blots evidenced that this cDNA insert originated from an ITI light chain-encoding mRNA whose size was estimated to be 1 300 bases.

Inter-α-trypsin inhibitor (ITI): Use of new antisera for qualitative studies and discrete quantitation of ITI and its derivatives

Inter-α-trypsin inhibitor (ITI) is a human protease inhibitor characterized by its coexistence with several physiological derivatives displaying immunological cross-reactivity when analyzed in plasma with usual anti-ITI antisera. Taking advantage of the presence in urine of a particular ITI derivative, antisera with restricted specificity for either ITI or its derivatives could be prepared. Some applications of these new reagents are given, including qualitative studies and discrete quantitation—by electroimmunoassay—of ITI and ITI derivatives. The procedures herein described should prove useful for qualitative and quantitative analysis to investigators dealing with mixtures of antigenically related proteins.