Glycosaminoglycan-bound and free inter-alpha-trypsin inhibitor components of follicular fluid.

The proteinase inhibitor inter-alpha trypsin inhibitor (ITI) is a blood-derived protein necessary for normal female fertility. Absence of ITI leads to ovulation of naked oocytes that cannot fertilise. ITI consists of two heavy chains (ITI-HC) and bikunin linked by a chrondroitin sulphate. By binding to hyaluronate, ITI-HC stabilises the extracellular matrix, but ITI-HC also binds to proteoglycans in follicular fluid. In vivo concentrations of ITI components in preovulatory follicular fluid, free as well as bound to hyaluronate or proteoglycan, are unknown. In order to quantify these components, 58 follicular fluids and 13 blood samples were collected in connection with in vitro fertilisation and embryo transfer treatment of 13 women. Quantitation of glycosaminoglycan-bound ITI-HC was performed after separation from free ITI in agarose gel. ITI components were determined by immunoelectrophoresis and hyaluronate by an ELISA method. The follicular fluid concentration of ITI was on average 70% of that in plasma and the concentration of hyaluronate remained low despite follicular production, suggesting that the production of hyaluronate is the rate-limiting step in the formation of the extracellular matrix of the oocyte-cumulus complex. In follicular fluid, the concentration of free ITI-HC was higher than that of glycosaminoglycan-bound ITI-HC. Addition of exogeneous hyaluronate doubled the amount of hyaluronate-bound ITI-HC, further supporting the notion that ITI in follicular fluid is not rate-limiting for cumulus expansion in vivo.     

Immunohistochemical distribution of inter-alpha-trypsin inhibitor chains in normal and malignant human lung tissue.

The inter-alpha-trypsin inhibitor (ITI) family is a group of plasma proteins built up from heavy (HC1, HC2, HC3) and light (bikunin) chains synthesized in the liver. In this study we determined the distribution of ITI constitutive chains in normal and cancerous lung tissues using polyclonal antibodies. In normal lung tissue, H2, H3, and bikunin chains were found in polymorphonuclear cells, whereas H1 and bikunin proteins were found in mast cells. Bikunin was further observed in bronchoepithelial mucous cells. In lung carcinoma, similar findings were obtained on infiltrating polymorphonuclear and mast cells surrounding the tumor islets. Highly differentiated cancerous cells displayed strong intracytoplasmic staining with H1 and bikunin antiserum in both adenocarcinoma and squamous cell carcinoma. Moreover, weak but frequent H2 expression was observed in adenocarcinoma cells, whereas no H3-related protein could be detected in cancer cells. Local lung ITI expression was confirmed by RT-PCR. Although the respective role of inflammatory and tumor cells in ITI chain synthesis cannot be presently clarified, these results show that heavy chains as well as bikunin are involved in malignant transformation of lung tissue.(J Histochem Cytochem 47:1625-1632, 1999)     

Biosynthesis of bikunin (urinary trypsin inhibitor) in rat hepatocytes.

One of the major sulfated proteins secreted by rat hepatocytes contains a low-sulfated chondroitin sulfate chain and its apparent molecular mass upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis shifts from 40 to 28 kDa upon chondroitinase ABC treatment (E. M. Sj?berg and E. Fries, 1990, Biochem. J. 272, 113-118). These properties suggest that this protein is the rat homologue of the major trypsin inhibitor of human urine which was recently named bikunin. In serum, bikunin occurs mainly as a subunit of the pre-alpha-inhibitor and the inter-alpha-inhibitor; in these proteins it is covalently linked to the other polypeptides through its chondroitin sulfate chain. Bikunin has been shown to be synthesized by liver cells as a 42-kDa precursor, in which it is linked to alpha 1-microglobulin by two basic amino acids. We have isolated bikunin from rat urine and prepared antibodies against it. In rat hepatocytes pulse-labeled with [35S]methionine, these antibodies precipitated a labeled protein of 42 kDa. Upon chase, three different labeled proteins were recognized by the antibodies in the medium: one protein of 40 kDa (free bikunin), one of 125 kDa (presumably pre-alpha-inhibitor), and one greater than 240 kDa (possibly a protein related to the inter-alpha-inhibitor). Pulse-chase experiments with [35S]sulfate showed that these proteins occurred intracellularly as precursors containing alpha 1-microglobulin. These results demonstrate that the completion of the chondroitin sulfate chain and its coupling to other polypeptide chains occur before the cleavage of the alpha 1-microglobulin/bikunin precursor.     

Proteomic characterization of inter-alpha inhibitor proteins from human plasma

Inter-alpha inhibitor proteins (IaIp) are a family of structurally related serine protease inhibitors found in relatively high concentrations in human plasma. Recent studies have implicated a role for IaIp in sepsis, and have demonstrated their potential as biomarkers in sepsis and cancer. For characterization of isolated IaI proteins and contaminating proteins during the last steps of the purification process, SELDI-TOF MS and HPLC-ESI-MS/MS were used. After separation by SDS-PAGE or 2-DE, polypeptide bands of 80, 125 and 250 kDa were excised from gels and digested by trypsin. The tryptic peptides were analyzed by both MS methods. The main contamination during the purification process, a band of 80 kDa, contains mainly IaIp heavy chain (HC) H3. HC H1 and H2 were also found in this band. In addition, some vitamin K-dependent clotting factors and inhibitors and other plasma proteins were identified. The 125-kDa band, representing the pre-alpha inhibitor, was found to contain both bikunin and HC H3. The presence of other HC H1, H2 and the recently described HC H4 was also detected by SELDI-TOF MS. The presence of HC H1, H2, and H3 in the 125-kDa band was confirmed by ESI-MS/MS, but not the presence of the H4. Three polypeptides, H1 and H2 together with bikunin, were identified in the 250-kDa band, representing the ITI, by both MS techniques. Once again, the presence of H4 was detected in this band only by SELDI-TOF MS, but the number of corresponding peptides was still not sufficient for final identification of this polypeptide. The importance of the application of proteomic methods for the proper evaluation of therapeutic drugs based on human plasma is discussed.     

Crossed immunoelectrophoresis does not allow accurate determination of inter-alpha-trypsin inhibitor and its derivatives in plasma.

By crossed-immunoelectrophoresis (CIE) of plasma, using anti-inter-alpha-trypsin inhibitor (ITI) immunoglobulins, beside native ITI, related components are visualized as an heterogeneous peak migrating farther than ITI. The area corresponding to this peak is largely increased in case of inflammatory disease. So, quantitative CIE has been previously proposed for discrete evaluation of ITI and its derivatives. We herein present evidence that CIE does not allow a clear separation of ITI from its derivatives: in this system, they are to some extent coprecipitated. Therefore the resulting overestimation of ITI may explain the previously reported absence of inverse relation between relative contents of ITI and derivatives in case of inflammatory disorders. Our data confirm the view that ITI acts as a precursor of smaller immunologically related inhibitors.     

Homologous chromosomal locations of the four genes for inter-alpha-inhibitor and pre-alpha-inhibitor family in human and mouse: assignment of the ancestral gene for the lipocalin superfamily.

The inter-alpha-inhibitor (I alpha I) and pre-alpha-inhibitor (P alpha I) family is composed of three plasma protease inhibitors, I alpha I, P alpha I, and bikunin, whose chains are encoded by a set of three evolutionarily related heavy (H) chain genes designated H1, H2, and H3 and a fourth gene, the so-called alpha 1-microglobulin/bikunin precursor (AMBP) gene. The latter codes for a precursor that splits into: (i) alpha 1-microglobulin, which belongs to the lipocalin superfamily; and (ii) bikunin, which is made up of two tandemly arranged protease inhibitor domains and belongs to the superfamily of Kunitz-type protease inhibitors. The bikunin chain is found in I alpha I and P alpha I molecules and it is also present as a free molecule in plasma. In human, the AMBP and H2 genes have been mapped to 9q32-q34 and 10p14-p15, respectively, while the H1 and H3 genes are tandemly located at 3p21.1-p21.2. In situ hybridization mappings indicate that the mouse AMBP gene (Intin-4) is located at 4C1----C4, and the H1 (Intin-1) and H3 (Intin-3) genes are colocated at 14A2----C1. In interspecific backcrosses (C57BL/6Pas x Mus spretus) a TaqI restriction variant in (and/or near) the H2 (Intin-2) gene identified a linkage of this gene with other polymorphic loci, which assigns Intin-2 to the centromeric area of chromosome 2. All such assignments are in conserved chromosomal regions between human and mouse. Therefore the genetic events that gave rise to the four I alpha I family genes took place prior to the divergence between human and mouse.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heavy chains of inter alpha inhibitor (IαI) inhibit the human complement system at early stages of the cascade

Inter alpha inhibitor (IαI) is an abundant serum protein consisting of three polypeptides: two heavy chains (HC1 and HC2) and bikunin, a broad-specificity Kunitz-type proteinase inhibitor. The complex is covalently held together by chondroitin sulfate but during inflammation IαI may interact with TNF-stimulated gene 6 protein (TSG-6), which supports transesterification of heavy chains to hyaluronan. Recently, IαI was shown to inhibit mouse complement in vivo and to protect from complement-mediated lung injury but the mechanism of such activity was not elucidated. Using human serum depleted from IαI, we found that IαI is not an essential human complement inhibitor as was reported for mice and that such serum has unaltered hemolytic activity. However, purified human IαI inhibited classical, lectin and alternative complement pathways in vitro when added in excess to human serum. The inhibitory activity was dependent on heavy chains but not bikunin and detected at the level of initiating molecules (MBL, properdin) in the lectin/alternative pathways or C4b in the classical pathway. Furthermore, IαI affected formation and assembly of the C1 complex and prevented assembly of the classical pathway C3-convertase. Presence and putative interactions with TSG-6 did not affect the ability of IαI to inhibit complement thus implicating IαI as a potentially important complement inhibitor once enriched onto hyaluronan moieties in the course of local inflammatory processes. In support of this, we found a correlation between IαI/HC-containing proteins and hemolytic activity of synovial fluid from patients suffering from rheumatoid arthritis.     

A physiological function of serum proteoglycan bikunin: the chondroitin sulfate moiety plays a central role

Bikunin is a small chondroitin sulfate proteoglycan that occurs in blood as the light chain of inter--trypsin inhibitor (ITI) family members. The relatively short chondroitin sulfate chain of bikunin shows a characteristic pattern of sulfation in both the linkage region and the chondroitin sulfate backbone. To the internal N-acetylgalactosamines in the lower sulfated portion near the non-reducing end, up to two side proteins could bind covalently via a unique ester bond to form core protein-glycosaminoglycan-side protein complexes, the ITI family. ITI molecules are synthesized in hepatocytes, and then secreted into circulation at high concentrations. In the presence of yet unidentified factors, the side proteins are transferred from chondroitin sulfate to hyaluronan by a transesterification reaction to form what has been described as the Serum-derived Hyaluronan-Associated Protein (SHAP)-hyaluronan complex. The formation of this complex is required for the stabilization of the extracellular matrix of fibroblasts, mesothelial cells, and cumuli oophori. When the gene for bikunin is inactivated, female mice exhibit severe infertility as a consequence of a defect of the side protein precursor in forming a complex with the hyaluronan in cumulus oophorus before ovulation. Therefore, the chondroitin sulfate moiety of bikunin is essential for presenting SHAP to hyaluronan, which is indispensable for ovulation and fertilization in mammals. Published in 2003.     

Expression of a functional proteinase inhibitor capable of accepting xylose: bikunin

Bikunin is a Kunitz-type proteinase inhibitor, which is cross-linked to heavy chains via a chondroitin sulfate chain, forming inter-alpha-inhibitor and related molecules. Rat bikunin was produced by baculovirus-infected insect cells. The protein could be purified with a total yield of 20 mg/liter medium. Unlike naturally occuring bikunin the recombinant protein had no galactosaminoglycan chain. Endoglycosidase digestion also suggested that the recombinant form lacked N-linked oligosaccharides. Bikunin is translated as a part of a precursor, alpha1-microglobulin/bikunin, but the functional significance of the cotranslation is unknown. Our results indicate that the proteinase inhibitory function of bikunin is not regulated by the alpha1-microglobulin-part of the alpha1-microglobulin/bikunin precursor since recombinant bikunin had the same trypsin inhibitory activity as the recombinant precursor. Both free bikunin and the precursor were also functional as a substrate in an in vitro xylosylation system. This demonstrates that the alpha1-microglobulin-part is not necessary for the first step of galactosaminoglycan assembly.     

Rat alpha 1-microglobulin: co-expression in liver with the light chain of inter-alpha-trypsin inhibitor.

A 1162 bp rat liver cDNA clone encoding the immunoregulatory plasma protein alpha 1-microglobulin was isolated and sequenced. The open reading frame encoded a 349 amino acid polyprotein, including alpha 1-microglobulin, 182 amino acids, and bikunin, the light chain of the plasma protein inter-alpha-trypsin inhibitor, 145 amino acids. The alpha 1-microglobulin/bikunin mRNA was found only in the liver when different tissues were examined. Free alpha 1-microglobulin and a polyprotein, containing both alpha 1-microglobulin and inter-alpha-trypsin inhibitor epitopes, were found in the microsomal fraction from rat liver homogenates.     

Defect in SHAP-hyaluronan complex causes severe female infertility. A study by inactivation of the bikunin gene in mice

Hyaluronan (HA) associates with proteins and proteoglycans to form the extracellular HA-rich matrices that significantly affect cellular behaviors. So far, only the heavy chains of the plasma inter-alpha-trypsin inhibitor (ITI) family, designated as SHAPs (serum-derived hyaluronan-associated proteins), have been shown to bind covalently to HA. The physiological significance of such a unique covalent complex has been unknown but is of great interest, because HA and the ITI family are abundant in tissues and in plasma, respectively, and the SHAP-HA complex is formed wherever HA meets plasma. We abolished the formation of the SHAP-HA complex in mice by targeting the gene of bikunin, the light chain of the ITI family members, which is essential for their biosynthesis. As a consequence, the cumulus oophorus, an investing structure unique to the oocyte of higher mammals, had a defect in forming the extracellular HA-rich matrix during expansion. The ovulated oocytes were completely devoid of matrix and were unfertilized, leading to severe female infertility. Intraperitoneal administration of ITI, accompanied by the formation of the SHAP-HA complex, fully rescued the defects. We conclude that the SHAP-HA complex is a major component of the HA-rich matrix of the cumulus oophorus and is essential for fertilization in vivo.     

Inter-alpha-inhibitor, hyaluronan and inflammation

Inter-alpha-inhibitor is an abundant plasma protein whose physiological function is only now beginning to be revealed. It consists of three polypeptides: two heavy chains and one light chain called bikunin. Bikunin, which has antiproteolytic activity, carries a chondroitin sulphate chain to which the heavy chains are covalently linked. The heavy chains can be transferred from inter-alpha-inhibitor to hyaluronan molecules and become covalently linked. This reaction seems to be mediated by TSG-6, a protein secreted by various cells upon stimulation by inflammatory cytokines. Inter-alpha-inhibitor has been shown to be required for the stabilization of the cumulus cell-oocyte complex during the expansion that occurs prior to ovulation. Hyaluronan-linked heavy chains in the extracellular matrix of this cellular complex have recently been shown to be tightly bound to TSG-6. Since TSG-6 binds to hyaluronan, its complex with heavy chains could stabilize the extracellular matrix by cross-linking hyaluronan molecules. Heavy chains linked to hyaluronan molecules have also been found in inflamed tissues. The physiological role of these complexes is not known but there are indications that they might protect hyaluronan against fragmentation by reactive oxygen species. TSG-6 also binds to bikunin thereby enhancing its antiplasmin activity. Taken together, these results suggest that inter-alpha-inhibitor is an anti-inflammatory agent which is activated by TSG-6.     

Intracellular coupling of the heavy chain of pre-alpha-inhibitor to chondroitin sulfate

Pre-alpha-inhibitor is a serum protein consisting of two polypeptides, the heavy chain and bikunin, covalently linked through an ester bond between the chondroitin sulfate chain of bikunin and the alpha-carboxyl group of the carboxyl-terminal residue of the heavy chain. The heavy chain is synthesized with a carboxyl-terminal extension, which is cleaved off just before the link to bikunin is formed. Our earlier studies indicate that this extension mediates the cleavage, and we have now found that a short segment on the amino-terminal side of the cleavage site is also required for the reaction. Furthermore, we previously showed that coexpression of the heavy chain precursor and bikunin in COS-1 cells leads to linkage, and we have now used this system to identify a His residue in the carboxyl-terminal extension that is specifically required for the intracellular coupling of the two proteins. In addition, we have shown that another chondroitin sulfate-containing protein, decorin, will also form a complex with the heavy chain, as will free chondroitin sulfate chains. These results suggest that in vivo there might be other, as yet unknown, chondroitin sulfate-containing polypeptides linked to the heavy chain.     

Cloning and expression of two human p70 S6 kinase polypeptides differing only at their amino termini.

Two classes of human cDNA encoding the insulin/mitogen-activated p70 S6 kinase have been isolated; the two classes differ only in the 5' region, such that the longer polypeptide (p70 S6 kinase alpha I; calculated Mr 58,946) consists of 525 amino acids, of which the last 502 residues are identical in sequence to the entire polypeptides encoded by the second cDNA (p70 S6 kinase alpha II; calculated Mr 56,153). Both p70 S6 kinase polypeptides predicted by these cDNAs are present in p70 S6 kinase purified from rat liver, and each is thus expressed in vivo. Moreover, both polypeptides are expressed from a single mRNA transcribed from the (longer) p70 S6 kinase alpha I cDNA through the utilization of different translational start sites. Although the two p70 S6 kinase polypeptides differ by only 23 amino acid residues, the slightly longer alpha I polypeptide exhibits anomalously slow mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), migrating at an apparent Mr of 90,000 probably because of the presence of six consecutive Arg residues immediately following the initiator methionine. Transient expression of p70 alpha I and alpha II S6 kinase cDNA in COS cells results in a 2.5- to 4-fold increase in overall S6 kinase activity. Upon immunoblotting, the recombinant p70 polypeptides appear as a closely spaced ladder of four to five bands between 65 and 70 kDa (alpha II) and 85 and 90 kDa (alpha I). Transfection with the alpha II cDNA yields only the smaller set of bands, while transfection with the alpha I cDNA generates both sets of bands. Mutation of Met-24 in the alpha I cDNA to Leu or Thr suppresses synthesis of the alpha II polypeptides. Only the p70 alpha I and alpha II polypeptides of slowest mobility on SDS-PAGE comigrate with the 70- and 90-kDa proteins observed in purified rat liver S6 kinase. Moreover, it is the recombinant p70 polypeptides of slowest mobility that coelute with S6 kinase activity on anion-exchange chromatography. The slower mobility and higher enzymatic activity of these p70 proteins is due to Ser/Thr phosphorylation, inasmuch as treatment with phosphatase 2A inactivates kinase activity and increases the mobility of the bands on SDS-PAGE in an okadaic acid-sensitive manner. Thus, the recombinant p70 S6 kinase undergoes multiple phosphorylation and partial activation in COS cells. Acquisition of S6 protein kinase catalytic function, however, is apparently restricted to the most extensively phosphorylated recombinant polypeptides.     

Plasma proteins immunologically related to inter-alpha-trypsin inhibitor

SDS-polyacrylamide gel electrophoresis and immunoblot were applied to analysis of plasma proteins immunologically related to inter-alpha-trypsin inhibitor (ITI). In this system, anti-ITI sera were able to identify ITI and other components with an Mr 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 (Mr 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).     

Intracellular proteolytic processing of the heavy chain of rat pre-alpha-inhibitor. The COOH-terminal propeptide is required for coupling to bikunin

Pre-alpha-inhibitor is a serum protein consisting of two polypeptides named bikunin and heavy chain 3 (H3). Both polypeptides are synthesized in hepatocytes and while passing through the Golgi complex, bikunin, which carries a chondroitin sulfate chain, becomes covalently linked to the COOH-terminal amino acid residue of H3 via its polysaccharide. Immediately prior to this reaction, a COOH-terminal propeptide of 33 kDa is cleaved off from the heavy chain. Using COS-1 cells transfected with rat H3, we found that in the absence of bikunin, the cleaved propeptide remained bound to the heavy chain and that H3 lacking the propeptide sequence did not become linked to coexpressed bikunin. Sequencing of H3 secreted from COS-1 cells showed that part of the molecules had a 12-amino acid residue long NH2-terminal propeptide. Cleavage of this propeptide, which occurred in the endoplasmic reticulum, was found to require basic amino acid residues at P1, P2, and P6 suggesting that it is mediated by a Golgi enzyme in transit. Deletion of the NH2-terminal propeptide or blocking of its release affected neither transport nor coupling of the heavy chain to bikunin.     

cDNA cloning of human inter-alpha-trypsin inhibitor discloses three different proteins

Inter-alpha-trypsin inhibitor (ITI) is a serum protein of unknown function. Part of the molecule (formerly called HI30) is closely related to a tumor-derived protein acting as a growth factor for endothelial cells. We screened a human liver cDNA expression library with antibodies raised against human ITI and isolated several clones which could be divided into three groups according to their DNA sequences. The cDNA of the first group codes for a protein composed of alpha 1-microglobulin (alpha 1M) and urinary trypsin inhibitor (UTI) and is identical to that encoded by a clone originally found by screening a human liver cDNA library with oligonucleotides derived from amino-acid sequences of the two Kunitz-type domains of UTI. The proteins derived from the cDNA of the second and the third group of clones are distantly related to each other, but unrelated to the protein derived from group 1 clones. Partial amino-acid sequencing of ITI isolated from serum allowed the verification of large parts of the cDNA-derived amino-acid sequences. The results favour the view that ITI is not a single chain protein, but rather a very tight complex of several components or a mixture of such complexes.     

Evolutionary conservation of heavy chain protein transfer between glycosaminoglycans

The bikunin proteins are composed of heavy chains (HCs) covalently linked to a chondroitin sulfate chain originating from Ser-10 of bikunin. Tumor necrosis factor stimulated gene-6 protein (TSG-6)/heavy chain 2 (HC2) cleaves this unique cross-link and transfers the HCs to hyaluronan and other glycosaminoglycans via a covalent HC•TSG-6 intermediate. In the present study, we have investigated if this reaction is evolutionary conserved based on the hypothesis that it is of fundamental importance. The results revealed that plasma/serum samples from mammal, bird, and reptile were able to form TSG-6 complexes suggesting the presence of proteins with the same function as the human bikunin proteins. To substantiate this, the complex forming protein from Gallus gallus (Gg) plasma was purified and identified as a Gg homolog of human HC2•bikunin. In addition, Gg pre-α-inhibitor and smaller amount of high molecular weight forms composed of bikunin and two HCs were purified. Like the human bikunin proteins, the purified Gg proteins were all stabilized by a protein–glycosaminoglycan–protein cross-link, i.e. the HCs were covalently attached to a chondroitin sulfate originating from bikunin. Furthermore, the complex formed between Gg HC2•bikunin and human TSG-6 appeared to be identical to that of the human proteins. Akin to human, Gg HC2 was further transferred to hyaluronan when present, and when incubated in vitro, Gg pre-α-inhibitor and TSG-6, failed to form the intermediate covalent complex, essential for HC transfer. Significantly, Gg HC2, analogous to human HC2, promoted complex formation between human HC3 and human TSG-6, substantiating the evolutionary conservation of these interactions. The present study demonstrates that the unique interactions between bikunin proteins, glycosaminoglycans, and TSG-6 are evolutionary conserved, emphasizing the physiological importance of the TSG-6/HC2-mediated HC-transfer reaction. In addition, the data show that the evolution of HC transfer is likely to predate the role of HC·HA complexes in female fertility and thus has evolved in the context of inflammation rather than fertility.