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)     

Bikunin and α1-microglobulin in human zona pellucida and connective tissue

The occurrence of bikunin and ·₁-microglobuli n was investigated in human ovary and Fallopian tubes. Bikunin and ·₁-microglobulin are transcribed in the liver from a common gene. Bikunin immunoreactivity was detected in the zona pellucida. A positive reaction for bikunin was also observed in connective tissue of the oviduct. In addition, mast cells showed a more intense posi tive reaction than the surrounding connective tissue. Specific displaceable ·₁-microglobulin immunoreactivity was revealed in the zona pellucida. The data suggest that bikunin and ·₁- microglobulin are trapped in the zona pellucida. This revised version was published online in November 2006 with corrections to the Cover Date.     

Sulfation of the Bikunin Chondroitin Sulfate Chain Determines Heavy Chain·Hyaluronan Complex Formation

Inter-α-trypsin inhibitor (IαI) is a complex comprising two heavy chains (HCs) that are covalently bound by an ester bond to chondroitin sulfate (CS), which itself is attached to Ser-10 of bikunin. IαI is essential for the trans-esterification of HCs onto hyaluronan (HA). This process is important for the stabilization of HA-rich matrices during ovulation and some inflammatory processes. Bikunin has been isolated previously by anion exchange chromatography with a salt gradient up to 0.5 m NaCl and found to contain unsulfated and 4-sulfated CS disaccharides. In this study, bikunin-containing fractions in plasma and urine were separated by anion exchange chromatography with a salt gradient of 0.1–1.0 m NaCl, and fractions were analyzed for their reactivity with the 4-sulfated CS linkage region antibody (2B6). The fractions that reacted with the 2B6 antibody (0.5–0.8 m NaCl) were found to predominantly contain sulfated CS disaccharides, including disulfated disaccharides, whereas the fractions that did not react with this antibody (0.1–0.5 m NaCl) contained unsulfated and 4-sulfated CS disaccharides. IαI in the 0.5–0.8 m NaCl plasma fraction was able to promote the trans-esterification of HCs to HA in the presence of TSG-6, whereas the 0.1–0.5 m NaCl fraction had a much reduced ability to transfer HC proteins to HA, suggesting that the CS containing 4-sulfated linkage region structures and disulfated disaccharides are involved in the HC transfer. Furthermore, these data highlight that the structure of the CS attached to bikunin is important for the transfer of HC onto HA and emphasize a specific role of CS chain sulfation.     

The Compact and Biologically Relevant Structure of Inter-α-inhibitor Is Maintained by the Chondroitin Sulfate Chain and Divalent Cations

Inter-α-inhibitor is a proteoglycan of unique structure. The protein consists of three subunits, heavy chain 1, heavy chain 2, and bikunin covalently joined by a chondroitin sulfate chain originating at Ser-10 of bikunin. Inter-α-inhibitor interacts with an inflammation-associated protein, tumor necrosis factor-inducible gene 6 protein, in the extracellular matrix. This interaction leads to transfer of the heavy chains from the chondroitin sulfate of inter-α-inhibitor to hyaluronan and consequently to matrix stabilization. Divalent cations and heavy chain 2 are essential co-factors in this transfer reaction. In the present study, we have investigated how divalent cations in concert with the chondroitin sulfate chain influence the structure and stability of inter-α-inhibitor. The results showed that Mg²⁺ or Mn²⁺, but not Ca²⁺, induced a conformational change in inter-α-inhibitor as evidenced by a decrease in the Stokes radius and a bikunin chondroitin sulfate-dependent increase of the thermodynamic stability. This structure was shown to be essential for the ability of inter-α-inhibitor to participate in extracellular matrix stabilization. In addition, the data revealed that bikunin was positioned adjacent to both heavy chains and that the two heavy chains also were in close proximity. The chondroitin sulfate chain interacted with all protein components and inter-α-inhibitor dissociated when it was degraded. Conventional purification protocols result in the removal of the Mg²⁺ found in plasma and because divalent cations influence the conformation and affect function it is important to consider this when characterizing the biological activity of inter-α-inhibitor.     

New human colorectal carcinoma cell lines that secrete proteinase inhibitors in vitro

Two new human cell lines, RCM-1 and CoCM-1, have been established from primary colorectal adenocarcinomas. Both cell lines were unique in that the cultures secreted trypsin inhibitors in vitro. The activities of these inhibitors were accumulated in serum-free media of both cell lines over a period of several days. Two inhibitors (PI-1 and PI-2) were isolated from serum-free conditioned medium in which RCM-1 was grown by anion-exchange and gel filtration high-performance liquid chromatography. PI-1 inhibited trypsin and chymotrypsin strongly, and pancreatic elastase weakly. Its molecular weight was about 57 kilodaltons (Kd) as determined by gel filtration chromatography. It cross-reacted with the antiserum elicited against human α₁-antitrypsin in double immunodiffusion. PI-1 corresponding to α₁ - antitrypsin was also demonstrated immunohistochemically in both cell lines. PI-2 inhibited trypsin strongly, and chymotrypsin, kallikrein and plasmin weakly. It had higher molecular weight (200–300 Kd) than that of PI-1, and did not crossreact with antisera against human α₁-antitrypsin, α₂-macroglobulin, α₁-antichymotrypsin, α₂-plasmin inhibitor, inter-α-trypsin inhibitor and urinary trypsin inhibitor. RCM-1 and CoCM-1 are the first colorectal adenocarcinoma cell lines that secrete functionally active trypsin inhibitors, including α₁-antitrypsin in vitro, and are useful for the study of tumor-cell derived proteinase inhibitors.     

Inhibition of Tryptase TL2 from Human T4+ Lymphocytes and Inhibition of HIV-1 Replication in H9 Cells by Recombinant Aprotinin and Bikunin Homologues

The serine esterase TL₂ from human T4+ lymphocytes is a binding component to HIV-1 glycoprotein gp120 and seems to play a role in the HIV-1 infection mechanism. Recombinant variants of the Kunitz-type serine proteinase inhibitor aprotinin were investigated for their ability to inhibit tryptase TL₂ and the binding of gp120 to this enzyme. Furthermore, the viral replication of HIV-1 was investigated in H9 cell cultures under the influence of recombinant aprotinin and bikunin variants. In contrast to native aprotinin, the recombinant variant [Arg¹⁵, Phe¹⁷, Glu⁵²]aprotinin with a reactive-site sequence homologous to the V3 loop of HIV-1 gp120 showed a specific inhibition of tryptase TL₂ (>80%). However, the [Leu¹⁵, Phe¹⁷, Glu⁵²]aprotinin variant with hydrophobic subsites was the most potent inhibitor of the binding of gp120 to tryptase TL₂ (68%). Our results show that the enzyme activity of purified tryptase TL₂ is inhibited not only by variants with basic amino acids, but also those with hydrophobic residues in the reactive-site region. Therefore, tryptase TL₂ is not a typical trypsin-like or chymotrypsin-like protease. Investigations on inhibition of HIV-1 replication in H9 cell cultures showed that tryptase TL₂ is involved in the mechanism of virus internalization into human lymphocytes. The [Leu¹⁵, Phe¹⁷, Glu⁵²]aprotinin showed a significant retardation of syncytium formation over a period of 5 days in a 1 M concentration. Similar investigations were performed with recombinant variants of bikunin, the light chain of human inter--trypsin inhibitor. Only the single-headed variant [Arg⁹⁴]⁸²bikunin inhibited slightly the syncytium formation over a period of 2 days in a 2.2 M concentration. Wild-type bikunin and all full-length variants showed no effect, possibly due to steric hindrance by the second domain of the double-headed inhibitor.     

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.     

Increased Immunoreactivities of Cleaved αII-Spectrin and Cleaved Caspase-3 in the Aged Dog Spinal Cord

The activation of caspase-3 is considered to be a reliable marker for apoptotic cell death, and a 120-kDa fragment of αII-spectrin is generated by caspase-3 mediated cleavage of this structural protein. In the present study, we compared cleaved αII-spectrin (120-kDa) and cleaved caspase-3-immunoreactive cells and their protein levels in the cervical (C₅–C₆) and lumbar (L₃–L₄) levels of the spinal cord in adult (1–2 year-old) and aged (10–12 year-old) dogs (German shepherds). Weak cleaved αII-spectrin and cleaved caspase-3 immunoreactivity was found in neurons of the adult group; however, their immunoreactivity was distinctively increased in the neuronal cytoplasm in the aged group compared to those in the adult group, although the distribution pattern of their neurons was similar between the adult and age group. In addition, cleaved αII-spectrin and cleaved caspase-3 levels in the aged spinal cord were markedly increased compared to those in the adult group. These findings suggest that the increases of cleaved αII-spectrin and cleaved caspase-3 immunoreactivity may be related to aging of the spinal cord in dogs.     

Inhibition of Tryptase TL2 from Human T4+ Lymphocytes and Inhibition of HIV-1 Replication in H9 Cells by Recombinant Aprotinin and Bikunin Homologues

The serine esterase TL₂ from human T4+ lymphocytes is a binding component to HIV-1 glycoprotein gp120 and seems to play a role in the HIV-1 infection mechanism. Recombinant variants of the Kunitz-type serine proteinase inhibitor aprotinin were investigated for their ability to inhibit tryptase TL₂ and the binding of gp120 to this enzyme. Furthermore, the viral replication of HIV-1 was investigated in H9 cell cultures under the influence of recombinant aprotinin and bikunin variants. In contrast to native aprotinin, the recombinant variant [Arg¹⁵, Phe¹⁷, Glu⁵²]aprotinin with a reactive-site sequence homologous to the V3 loop of HIV-1 gp120 showed a specific inhibition of tryptase TL₂ (>80%). However, the [Leu¹⁵, Phe¹⁷, Glu⁵²]aprotinin variant with hydrophobic subsites was the most potent inhibitor of the binding of gp120 to tryptase TL₂ (68%). Our results show that the enzyme activity of purified tryptase TL₂ is inhibited not only by variants with basic amino acids, but also those with hydrophobic residues in the reactive-site region. Therefore, tryptase TL₂ is not a typical trypsin-like or chymotrypsin-like protease. Investigations on inhibition of HIV-1 replication in H9 cell cultures showed that tryptase TL₂ is involved in the mechanism of virus internalization into human lymphocytes. The [Leu¹⁵, Phe¹⁷, Glu⁵²]aprotinin showed a significant retardation of syncytium formation over a period of 5 days in a 1 μM concentration. Similar investigations were performed with recombinant variants of bikunin, the light chain of human inter-α-trypsin inhibitor. Only the single-headed variant [Arg⁹⁴]⁸²bikunin inhibited slightly the syncytium formation over a period of 2 days in a 2.2 μM concentration. Wild-type bikunin and all full-length variants showed no effect, possibly due to steric hindrance by the second domain of the double-headed inhibitor.