Intracellular proteolytic processing of the heavy chain of rat pre-alpha-inhibitor. The COOH-terminal propeptide is required for coupling to bikunin.
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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. (+info)
Reduction of sodium deoxycholic acid-induced scratching behaviour by bradykinin B2 receptor antagonists.
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1. Subcutaneous injection of sodium deoxycholic acid into the anterior of the back of male ddY mice elicited dose-dependent scratching of the injected site with the forepaws and hindpaws. 2. Up to 100 microg of sodium deoxycholic acid induced no significant increase in vascular permeability at the injection site as assessed by a dye leakage method. 3. Bradykinin (BK) B2 receptor antagonists, FR173657 and Hoe140, significantly decreased the frequency of scratching induced by sodium deoxycholic acid. 4. Treatment with aprotinin to inhibit tissue kallikrein reduced the scratching behaviour induced by sodium deoxycholic acid, whereas treatment with soybean trypsin inhibitor to inhibit plasma kallikrein did not. 5. Although injection of kininase II inhibitor, lisinopril together with sodium deoxycholic acid did not alter the scratching behaviour, phosphoramidon, a neutral endopeptidase inhibitor, significantly increased the frequency of scratching. 6. Homogenates of the skin excised from the backs of mice were subjected to gel-filtration column chromatography followed by an assay of kinin release by trypsin from each fraction separated. Less kinin release from the fractions containing kininogen of low molecular weight was observed in the skin injected with sodium deoxycholic acid than in normal skin. 7. The frequency of scratching after the injection of sodium deoxycholic acid in plasma kininogen-deficient Brown Norway Katholiek rats was significantly lower than that in normal rats of the same strain, Brown Norway Kitasato rats. 8. These results indicate that BK released from low-molecular-weight kininogen by tissue kallikrein, but not from high-molecular-weight kininogen by plasma kallikrein, may be involved in the scratching behaviour induced by the injection of sodium deoxycholic acid in the rodent. (+info)
Angiostatin formation involves disulfide bond reduction and proteolysis in kringle 5 of plasmin.
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Plasmin is processed in the conditioned medium of HT1080 fibrosarcoma cells producing fragments with the domain structures of the angiogenesis inhibitor, angiostatin, and microplasmin. Angiostatin consists of kringle domains 1-4 and part of kringle 5, while microplasmin consists of the remainder of kringle 5 and the serine proteinase domain. Our findings indicate that formation of angiostatin/microplasmin involves reduction of plasmin by a plasmin reductase followed by proteolysis of the reduced enzyme. We present evidence that the Cys461-Cys540 and Cys511-Cys535 disulfide bonds in kringle 5 of plasmin were reduced by plasmin reductase. Plasmin reductase activity was secreted by HT1080 and Chinese hamster ovary cells and the human mammary carcinoma cell lines MCF-7, MDA231, and BT20 but not by the monocyte/macrophage cell line THP-1. Neither primary foreskin fibroblasts, blood monocyte/macrophages, nor macrovascular or microvascular endothelial cells secreted detectable plasmin reductase. In contrast, cultured bovine and rat vascular smooth muscle cells secreted small but reproducible levels of plasmin reductase. Reduction of the kringle 5 disulfide bonds triggered cleavage at either Arg529-Lys530 or two other positions C-terminal of Cys461 in kringle 5 by a serine proteinase. Plasmin autoproteolysis could account for the cleavage, although another proteinase was mostly responsible in HT1080 conditioned medium. Three serine proteinases with apparent Mr of 70, 50, and 39 were purified from HT1080 conditioned medium, one or more of which could contribute to proteolysis of reduced plasmin. (+info)
Temporal changes in mRNA expression for bikunin in the kidneys of rats during calcium oxalate nephrolithiasis.
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Inter-alpha-inhibitor and other bikunin-containing proteins are synthesized in relatively large quantities by the liver. These proteins function as Kunitz-type serine protease inhibitors and appear capable of inhibiting calcium oxalate (CaOx) crystallization in vitro. Preliminary studies have shown that renal tubular epithelial cells synthesize bikunin in response to CaOx challenge. To examine this response in vivo, a sensitive reverse transcription-quantitative competitive template-PCR was developed to detect and quantify poly(A)+ -tailed bikunin mRNA expression in kidney tissue from normal rats and rats developing CaOx nephrolithiasis after challenge with ethylene glycol. Bikunin mRNA expression in rat liver tissue was assessed as a positive control. The expression of bikunin mRNA in liver did not differ significantly between normal control rats and experimental rats with induced hyperoxaluria and renal CaOx crystallization. In contrast, there were significant temporal increases in the levels of bikunin mRNA expression in rat kidneys during CaOx nephrolithiasis after challenge with ethylene glycol. Urinary excretion of bikunin-containing proteins seemed to increase concomitantly. These findings indicate an association between the induction of hyperoxaluria/CaOx nephrolithiasis and the expression of the bikunin gene in rat kidneys. (+info)
Factor XII Tenri, a novel cross-reacting material negative factor XII deficiency, occurs through a proteasome-mediated degradation.
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A homozygous cross-reacting material negative factor XII-deficient patient with 3% antigen and activity levels of factor XII was screened for the identification of a mutation at the genomic level. Low-ionic strength single-stranded conformation polymorphism (SSCP) analysis and sequence analysis showed that the proband's gene for factor XII had an A-->G substitution at nucleotide position 7832 in exon 3, resulting in a Tyr34 to Cys substitution in the NH2-terminal type II domain of factor XII. We designated this mutation as factor XII Tenri. Mutagenic polymerase chain reaction (PCR), followed by KpnI digestion, showed a homozygous mutation in the proband's gene and heterozygous mutations in his parents and sister. Immunoprecipitation and Western blot analyses of plasma samples from the factor XII Tenri family indicated that the proband had a trace amount of variant factor XII with an apparent molecular mass of 115 kD, which was converted to the normal 80-kD form after reduction, suggesting that factor XII Tenri was secreted as a disulfide-linked heterodimer with a approximately 35-kD protein, which we identified as alpha1-microglobulin by immunoblotting. Pulse-chase experiments using baby hamster kidney (BHK) cells showed that Tenri-type factor XII was extensively degraded intracellularly, but the addition of cystine resulted in increased secretion of the mutant. Using membrane-permeable inhibitors, we observed that the degradation occurred in the pre-Golgi, nonlysosomal compartment and a proteasome appeared to play a major role in this process. On the basis of these in vitro results, we speculate that the majority of the factor XII Tenri is degraded intracellularly through a quality control mechanism in the endoplasmic reticulum (ER), and a small amount of factor XII Tenri that formed a disulfide-linked heterodimer with alpha1-microglobulin is secreted into the blood stream. (+info)
Analysis of renal function in onchocerciasis patients before and after therapy.
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The occurrence of renal abnormalities was investigated in patients with onchocerciasis in comparison to individuals without onchocerciasis in Guinea. Serum creatinine levels, excretion of urinary marker proteins, and kidney size by ultrasound were determined. A high prevalence of glomerular as well as tubular dysfunctions was observed; however, no association with onchocerciasis could be detected. We also hypothesized that patients with hyperreactive onchocerciasis might be prone to develop immune-mediated glomerular disorders; however, this could not be verified. Following treatment with ivermectin, a slight but significant increase in the excretion of urinary albumin and alpha1-microglobulin was seen five days after treatment in all treated patients, whereas levels of proteinuria were significantly higher five days after treatment only in patients with high microfilarial densities. Our results indicate that ivermectin can cause glomerular and tubular disturbances in patients with onchocerciasis; however, these are minor and do not seem to be clinically relevant. (+info)
Bikunin, a serine protease inhibitor, is present on the cell boundary of epidermis.
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Bikunin, which is an inhibitor of serine proteases, is widely distributed in human tissues, including liver, kidney, and mucous membranes of the stomach and colon. The aim of this study was to clarify whether bikunin is expressed in human epidermis and its appendages. Immunoblot analysis using a specific polyclonal antibody to bikunin revealed that a single 43 kDa protein is present in the cell lysate from the human keratinocyte cell line HaCaT. Immunohistochemically, dotted reaction products stained with anti-bikunin antibody were localized on the cell boundary in both basal and spinous cell layers, except on the cell boundary of the basal cells facing the basal membrane. There were no reaction products in the granular-horny cell layers. Reaction products stained with anti-bikunin antibody were also observed on the hair bulb cells and eccrine sweat gland cells, but not on apocrine sweat glands. Also, reaction products were observed on the luminal surface of the renal proximal tubules and in the cytoplasm of these cells. In immunoelectron microscopy, gold particles were observed on the cell membranes close to the desmosomal structures. Reverse transcription-polymerase chain reaction and northern blot analyses showed that mRNA specific for bikunin was expressed in HaCaT cells and human epidermal keratinocytes obtained from suction blisters, and was contained in a commercially available human keratinocyte cDNA preparation. These findings indicate that bikunin is expressed in keratinocytes and may play an important part in regulating keratinocytes in either mitosis or inflammation. (+info)
Generation of catalytically active granzyme K from Escherichia coli inclusion bodies and identification of efficient granzyme K inhibitors in human plasma.
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Granzymes are granule-stored lymphocyte serine proteases that are implicated in T- and natural killer cell-mediated cytotoxic defense reactions after target cell recognition. A fifth human granzyme (granzyme 3, lymphocyte tryptase-2), renamed as granzyme K (gene name GZMK), has recently been cloned from lymphocyte tissue. For its further characterization we successfully generated catalytically active enzyme in milligram quantities per liter of Escherichia coli culture. The natural proform of granzyme K with the amino-terminal propeptide Met-Glu was expressed as inclusion bodies and converted to its active enzyme by cathepsin C after refolding of precursor molecules. Recombinant granzyme K cleaves synthetic thiobenzyl ester substrates after Lys and Arg with k(cat)/K(m) values of 3.7 x 10(4) and 4.4 x 10(4) M(-1) s(-1), respectively. Granzyme K activity was shown to be inhibited by the synthetic compounds Phe-Pro-Arg-chloromethyl ketone, phenylmethylsulfonyl fluoride, PefablocSC, and benzamidine, by the Kunitz-type inhibitor aprotinin and by human blood plasma. The plasma-derived inter-alpha-trypsin inhibitor complex, its bikunin subunit, and the second carboxyl-terminal Kunitz-type domain of bikunin were identified as genuine physiologic inhibitors with K(i) values of 64, 50, and 22 nM, respectively. Inter-alpha-trypsin inhibitor and free bikunin have the potential to neutralize extracellular granzyme K activity after T cell degranulation and may thus control unspecific damage of bystander cells at sites of inflammatory reactions. (+info)