Molecular characterization, expression and functional analysis of two Kazal-type serine protease inhibitors from Venerupis philippinarum.

Kazal-type serine protease inhibitors (KSPIs) act as negative regulators in immune signaling pathway by controlling the extent of serine protease (SP) activities. In this study, the full-length cDNA of two KSPIs (designed as VpKSPI-1 and VpKSPI-2) were identified from Venerupis philippinarum by rapid amplification of cDNA ends (RACE) approaches. The open reading frame (ORF) of VpKSPI-1 and VpKSPI-2 was of 552 bp and 402 bp, encoding a polypeptide of 183 and 133 amino acids, respectively. The transcripts of VpKSPI-1 and VpKSPI-2 were ubiquitously expressed in all tissues tested with the highest expression level in hepatopancreas. After Vibrio anguillarum challenge, the relative mRNA expression of VpKSPI-1 and VpKSPI-2 in hepatopancreas was both up-regulated within 96 h. The recombinant VpKSPI-1 (rVpKSPI-1) displayed weak activities towards chymotrypsin, moderate inhibitory activity to trypsin, while rVpKSPI-2 showed significant inhibitory activities against chymotrypsin and trypsin. When the molar ratio of rVpKSPI-2 to chymotrypsin and trypsin reached 1:4 and 1:2, the protease activities could be almost entirely inhibited. All these results suggested that both VpKSPI-1 and VpKSPI-2 perhaps play a vital role in the innate immunity of V. philippinarum.

Purification, characterization and immunoreactivity of ß'-component, a major allergen from the roe of large yellow croaker (Pseudosciaena crocea).

Fish roe, a nutritious food, is favored by consumers, but has also been confirmed to be allergenic in salmonid fish. However, little information is available in other fish species. To determine the allergen in the roe of large yellow croaker (Pseudosciaena crocea), crude extracts were incubated with sera of allergic patients. The major allergen was purified by column chromatography methods, revealing a single band with 16 kDa and was confirmed as ß'-component (ß'-c) by mass spectrometry. The results of physicochemical characterization showed that ß'-c was a glycoprotein and was relatively stable following thermal or acid/alkali treatment. Furthermore, ß'-c was easily degraded by pepsin, but was resistant to trypsin and α-chymotrypsin. After treatment with different processing methods, including Maillard reaction (MR), ultraviolet radiation (UVR), ultrasound-heat (UH), and retorting (RT), the IgG-binding activity of ß'-c decreased obviously by MR, but decreased slightly by UVR and UH. Cross-immunoreactivity results of the allergens in the roes of different species revealed that ß'-c was a specific allergen in teleostean, and the cross-immunoreactivity between the roe of large yellow croaker and other kinds of fish roe was relatively strong.

Analysis of the complement sensitivity of oral treponemes and the potential influence of FH binding, FH cleavage and dentilisin activity on the pathogenesis of periodontal disease.

Treponema denticola, a periopathogen, evades complement-mediated killing by binding the negative complement regulatory protein factor H (FH) to its surface via the FhbB protein. Paradoxically, bound FH is cleaved by T. denticola's dentilisin protease, a process hypothesized to trigger localized dysregulation of complement activation in periodontal pockets. The ability of other oral treponemes to evade complement-mediated killing and bind and cleave FH has not been assessed. In this report, we demonstrate that representative isolates of Treponema socranskii, Treponema medium, Treponema pectinovorum and Treponema maltophilum are also serum resistant, whereas Treponema vincentii and Treponema amylovorum are serum sensitive. Although T. denticola's ability to evade complement-mediated killing is strictly dependent on FH binding, other serum-resistant treponemal species lack FhbB and do not bind FH, indicating an FH-independent mechanism of complement evasion. To assess the influence of FhbB sequence variation on FH binding and cleavage by T. denticola, fhbB sequences were determined for 30 isolates. Three distinct phyletic types were identified. All T. denticola strains bound FH and were serum resistant, but differences in binding kinetics, dentilisin activity and FH cleavage ability were observed. Based on these analyses, we hypothesize that the composition of the T. denticola population is a determining factor that influences the progression and severity of periodontal disease.

Mechanisms of IL-8 suppression by Treponema denticola in gingival epithelial cells.

The purpose of this study was to investigate the mechanism(s) of interleukin (IL)-8 suppression by Treponema denticola, one of the major periodontal pathogens, in gingival epithelial cells. Immortalized human gingival epithelial HOK-16B cells were infected with wild-type (WT), dentilisin-deficient (K1) or flagellin-deficient (flgE) T. denticola in the presence or absence of 2% human serum for 24 h. The levels of IL-8 expression were measured with real-time reverse transcription PCR and ELISA. In the absence of human serum, the WT and flgE, but not K1, substantially reduced not only the levels of IL-8 protein but also of IL-8 mRNA. Such downregulation of IL-8 mRNA was independent of bacterial invasion. Degradation of cytokine mixture by the WT, K1 and flgE revealed dentilisin-dependent preferential degradation of tumor necrosis factor (TNF)-α, an IL-8-inducing cytokine. WT and flgE significantly decreased the levels of TNFα secreted by HOK-16B cells, suggesting modulation of IL-8 through dentilisin-mediated degradation of TNFα. The addition of human serum to the culture potentiated the suppressive effect of T. denticola, resulting in substantial reductions of IL-8 and TNFα levels, even by K1. The serum-dependent effects of T. denticola were attributed to its ability to suppress the accumulation of intracellular reactive-oxygen species (ROS), a group of ubiquitous signaling molecules. Pretreatment with an antioxidant suppressed TNFα-induced IL-8 expression, confirming the role of ROS in TNFα signaling. Collectively, T. denticola targeted a key inflammatory cytokine and its signaling molecule to modulate the host innate immune response, which provides a new insight into modulation of host immunity by a periodontal pathogen.

Pattern of MHC class I and immune proteasome expression in Walker 256 tumor during growth and regression in Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis.

Dynamics of the expression of MHC class I, immune proteasomes and proteasome regulators 19S, PA28, total proteasome pool and proteasome chymotrypsin-like activity in Walker 256 tumor after implantation into Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis was studied. The tumor growth and regression in Brattleboro rats were accompanied by changes in the proteasome subunit level unlike the tumor growth in WAG rats with normal expression of arginine-vasopressin gene. In the tumor implanted into Brattleboro rats the immune proteasome level was maximal between days 14 and 17, when the tumor underwent regression. Conversely, the expression of proteasome regulators tended to decrease during this period. Immune proteasomes are known to produce antigen epitopes for MHC class I to be presented to CD8+ T lymphocytes. Enhanced expression of immune proteasomes coincided with the recovery of MHC class I expression, suggesting the efficient presentation of tumor antigens in Brattleboro rats.

Reducing nanotube cytotoxicity using a nano-combinatorial library approach.

Carbon nanotubes (CNTs) have a great potential for applications in medicine. However, their biocompatibility and toxicity cause a great concern. Due to the large surface area of CNTs, chemical modification can dramatically alter their physiochemical properties and hence biological activity. Using a combinatorial chemistry approach, we report the synthesis of an 80-member surface-modified nanotube library. Based upon screening of this library with respect to protein-binding capacity, cytotoxicity, and immune response, we were able to select highly biocompatible nanotubes with reduced protein-binding cytotoxicity and immune response.

The Cromer blood group system: a review.

The antigens of the Cromer blood group system reside on decay-accelerating factor (DAF), a protein belonging to the regulators of complement activation family. The blood group system consists of 12 high-prevalence and three low-prevalence antigens. The molecular basis for the antigens is known, and with the exception of IFC, each antigen is the product of a single nucleotide change in the DAF gene and has been localized to one of the four complement control protein (CCP) domains on the DAF protein. The RBCs of people with the Cromer null phenotype, Inab, lack DAF but do not appear to demonstrate increased susceptibility to hemolysis. Antibodies to Cromer antigens are rarely encountered, although there is evidence that the antibodies may cause accelerated destruction of transfused RBCs. There is no risk of HDN associated with Cromer system antibodies because the placenta is a rich source of fetally derived DAF, which is thought to adsorb the antibodies.

Anomalous immunogenic properties of serine proteases.

It has previously been shown that a approximately 27 kDa serine protease of Schistosoma mansoni larvae, the cercarial elastase (CE), was a poor immunogen in as much as it failed to induce an antibody response. The CE has a critical role in enabling schistosome larvae to penetrate the skin of their definitive hosts, so the apparently poor immunogenicity of this enzyme is clearly of interest. To understand its lack of immunogenicity better and in particular to determine whether it is related to its proteolytic activity, we have measured antibody responses of mice to three different serine proteases. Groups of mice were immunized with porcine pancreatic trypsin (TRY), chymotrypsin (CHY) or elastase (ELA) and the resulting antibody response compared with antibody responses to two non-protease antigens, chicken egg albumin (OVA) and Schistosoma japonicum glutathione S-transferase (GST), all being administered with alum as an adjuvant. Of 12 mice that were injected five times at 14 day intervals with TRY, only one produced antibody reactive with this enzyme in ELISA. Immunizations with CHY or ELA induced somewhat better antibody responses than TRY, but the responses to the first and second injections of these two proteases nevertheless seemed comparatively lower than the responses to GST. Induction of antibody responses by OVA and GST was not affected when TRY was injected concomitantly. Thus, the antibody response to one of the serine proteases used in this study, mammalian trypsin, was anomalous.

Immune response and alveolar bone resorption in a mouse model of Treponema denticola infection.

Treponema denticola is considered to be an agent strongly associated with periodontal disease. The lack of an animal infection model has hampered the understanding of T. denticola pathogenesis and the host's immune response to infection. In this study, we have established an oral infection model in mice, demonstrating that infection by oral inoculation is feasible. The presence of T. denticola in the oral cavities of the animals was confirmed by PCR. Mice given T. denticola developed a specific immune response to the bacterium. The antibodies generated from the infection were mainly of the immunoglobulin G1 subclass, indicating a Th2-tilted response. The antibodies recognized 11 T. denticola proteins, of which a 62-kDa and a 53-kDa protein were deemed immunodominant. The two proteins were identified, respectively, as dentilisin and the major outer sheath protein by mass spectrometry. Splenocytes cultured from the infected mice no longer produced interleukin-10 and produced markedly reduced levels of gamma interferon relative to those produced by naïve splenocytes upon stimulation with T. denticola. Mandibles of infected mice showed significantly greater bone resorption (P < 0.01) than those of mock-infected controls.

Human serum antibodies recognize Treponema denticola Msp and PrtP protease complex proteins.

BACKGROUND/AIMS: Treponema denticola outer membrane proteins are postulated to have key roles in microbe-host interactions in periodontitis. Because there are no reports of in vivo expression of these putative virulence factors, we examined several T. denticola strains to determine whether sera from human subjects recognized specific T. denticola outer membrane proteins. METHODS: Soluble extracts were prepared from exponential phase cultures of T. denticola strains representing three serotypes, from defined T. denticola mutants defective in Msp (major surface protein) or PrtP lipoprotein protease complex (CTLP; dentilisin), and Escherichia coli strains expressing distinctly different T. denticola Msp. Extracts were subjected to Western immunoassays using archived human serum samples. RESULTS: Human serum antibodies (immunoglobulin G class) recognized multiple protein bands in T. denticola strains. In the parent strain ATCC 35405, these included bands at 72-, 53-, 40-, and 30-kDa. Bands corresponding to Msp and the PrtP protease complex proteins were absent in isogenic msp and protease complex mutants, respectively. Individual human sera showed specificity for one or more Msp types. CONCLUSIONS: This is the first definitive report of human serum antibody responses to specific T. denticola antigens. T. denticola Msp and the proteins comprising the PrtP lipoprotein protease complex are expressed in vivo and are immunogenic in humans. Human antibody recognition of Msp exhibits strain specificity and is consistent with strain serotyping. These results demonstrate the utility of T. denticola isogenic mutants in characterizing host immune responses to periodontal pathogens.

Comparison of the peptidase activity in the oncosphere excretory/secretory products of Taenia solium and Taenia saginata.

We compared the peptidase activities of the excretory/secretory (E/S) antigens of oncospheres of Taenia solium and related, but nonpathogenic, Taenia saginata. Taenia solium and T. saginata oncospheres were cultured, and the spent media of 24-, 48-, 72-, and 96-hr fractions were analyzed. Activities for serine peptidases (chymotrypsin-, trypsin-, and elastase-like), cysteine peptidases (cathepsin B-, cathepsin L-, and calpaine-like), and aminopeptidase (B-like peptidases) were tested fluorometrically with peptides coupled to 7-amino-4-methylcoumarin. In both species, the E/S antigens showed cysteine, serine, and aminopeptidase activities. Although no particular peptidase had high activity in T. solium, and was absent in T. saginata, or vice versa, different patterns of activity were found. A chymotrypsin-like peptidase showed the highest activity in both parasites, and it had 10 times higher activity in T. solium than in T. saginata. Trypsin-like and cathepsin B-like activities were significantly higher in T. solium. Minimal levels of cathepsin B were present in both species, and higher levels of elastase-like and cathepsin L-like activity were observed in T. saginata. Taenia solium and T. saginata have different levels and temporal activities of proteolytic enzymes that could play a modulator role in the host specificity for larval invasion through penetration of the intestinal mucosa.

The solid phase in affinity chromatography: strategies for antibody attachment.

Antibodies (Ab) are commonly used in affinity chromatography (AC) as a versatile and specific means of isolating target molecules from complex mixtures. A number of procedures have been developed to immobilize antibodies on the solid matrix. Some of these methods couple the antibody via chemical groups that may be important for specific recognition of antigen, resulting in loss of functionality in a proportion of the antibodies. In other methods, the outcome of immobilization is coupling via unique sites in the Fc region of the antibody molecule, ensuring orientation of the antibody combining sites (Fab) towards the mobile phase. This review discusses the advantages and disadvantages of the various methods available for immobilization and outlines protocols for site-directed, covalent coupling of the antibody to the solid phase that essentially retains the activity of the antibody.

Anaphylactic shock caused by impurities in orgotein preparations.

Previously reported allergic reactions to orgotein (superoxide dismutase) injections has assigned responsibility to this molecule, which is obtained from bovine liver. We report an anaphylactic shock probably caused by impurities contained in an orgotein preparation. Prick test to Peroxinorm (orgotein), BSA, and cow liver extract were positive but resulted negative with chymotrypsin, milk, meat and cow epithelium extracts. Tryptase levels determined 3, 24 hours and 15 days after the shock measured 6.32, 0.81 and 0.84 U/L respectively. Detection of specific IgE to Peroxinorm, BSA and chymotrypsin by ELISA was negative and positive to cow liver. Specific IgE to milk and cow epithelium by Pharmacia CAP system was negative. Immunoblotting with Peroxinorm revealed IgE specific bands at an apparent M.W of 67, 51, 56 and 16 kDa; immunoblotting with cow liver revealed bands at 72, 56, 50 and 36 kDa; immunoblotting with BSA and chymotypsin were negative. This case emphasises the role that 20 % of impurities of the pharmaceutical preparation may have in immediate hypersensitivity reactions.

Oriented immobilization of chymotrypsin by use of suitable antibodies coupled to a nonporous solid support.

In order to eliminate the kinetic limitation of chymotryptic hydrolysis of proteins due to diffusion, nonporous hydroxyalkyl methacrylate solid support was developed and used for oriented immobilization of chymotrypsin by means of suitable polyclonal antibodies. Nonporous microspheres were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in an alcohol-toluene mixture stabilized with cellulose acetate butyrate. The resulting particles were 1.2 microm in diameter and possessed narrow size distribution. After modification with adipic acid dihydrazide they contained 2 micromol of reactive groups available for coupling of anti-chymotrypsin antibodies. Prepared immunosorbent adsorbed 166.7 microg of chymotrypsin per 1 g of dry carrier. Immobilized chymotrypsin retained practically 100% of its native proteolytic activity. Kinetic parameters of catalysis by chymotrypsin immobilized via this way were improved due to the good steric accessibility of the enzyme active site for high-molecular-mass substrates, when digestion of proteins in batch experiments was used.

Purification of anti-chymotrypsin antibodies for the preparation of a bioaffinity matrix with oriented chymotrypsin as immobilized ligand.

Polyclonal antibodies suitable for the oriented immobilization of chymotrypsin were prepared by chromatography on a bioaffinity matrix which had the enzyme immobilized through its active site to antilysin, covalently linked to bead cellulose. After periodate oxidation of their carbohydrate moieties, the isolated antibodies were coupled to a hydrazide derivative of bead cellulose. The periodate oxidation step, which led to greater efficiency and stability of the immunosorbent, had no deleterious effect on antibody activity as assessed by ELISA. Addition of chymotrypsin to the immunosorbent yielded an enzymically active bioaffinity matrix with the optimum molar enzyme/antibody ratio of 2.

A comparative study of three serine proteases from Dermatophagoides pteronyssinus and D. farinae.

Studies have shown that the dust mites Dermatophagoides pteronyssinus and D. farinae contain several serine proteases, two of which have been shown to be allergenic, and to include trypsin and chymotrypsin, corresponding to the groups III and VI mite allergens. However, mites also contain other serine proteases, and the data reported in this study show that an elastase-like enzyme is present in both species. This enzyme was differentiated from the other serine proteases, particularly chymotrypsin, on the basis of charge, substrate specificity, and inhibition by copper and mercury cations. Its apparent mol. mass, as judged by gel filtration, was similar to those previously described for trypsin and chymotrypsin, i.e., 30 kDa. Several isoforms were detected by isoelectric focusing, but the isoelectric points of the major forms in both D. pteronyssinus and D. farinae were 10.5 and 9.8, respectively, contrasting with the acidic mite chymotrypsins. All three serine proteases were detected in whole mite and faecally enriched extracts, but the activities of trypsin and the elastase-like enzyme were greater in the latter type of extract. These data were similar to those obtained by quantitative immunochemical analysis of the D. farinae group III allergen in appropriate extracts, suggesting that culture conditions may modulate protease production. A monoclonal antibody affinity matrix specific for the group III allergen from D. farinae was shown to bind mite trypsin. However, a small amount of mite chymotrypsin also bound, suggesting limited immunologic cross-reactivity, a finding consistent with known sequence data.

Sperm-surface chymotrypsin-like protease activity required for fertilization in ascidians.

During fertilization, species-specific gamete binding must be followed by sperm penetration of egg vestments before gamete fusion can occur. Sperm proteases, called lysins, aid this process. Sperm from Ascidia ceratodes, Ascidia callosa, and Ascidia paratropa were found to have a surface-mounted chymotrypsin-like protease when studied by enzymology, biotinylated, immunolabeling, and histochemistry. Chymotrypsin substrates and inhibitors blocked fertilization in a concentration-dependent manner in A. ceratodes and decreased the number of sperm heads which penetrated the egg's vitelline coat, but had no effect on sperm binding to follicle cells. Sperm bound to agarose beads coated with the chymotrypsin inhibitor alpha 2-macroglobulin. Chymotrypsin-like enzyme activity, assayed fluorimetrically using N-succinyl-leucinyl-leucinyl-valinyl-tyrosinyl-7 -amido-4-methyl-coumarin as the substrate, was associated with head fractions prepared by differential centrifugation. Biotinylation of live sperm followed by detergent extraction showed that chymotrypsin-like activity could be removed from the detergent extract using avidin-agarose beads. Indirect immunofluorescence of unreacted and reacted sperm heavily labeled membrane domains overlying the mitochondrion and at the base of the head with occasional labeling of the sperm tip. Histochemical studies, which used N-succinyl-alanyl-alanyl-prolyl-phenylalanyl-beta-naphthylamide as the substrate, colocalized enzyme activity in head regions of unreacted and near the mitochondrion of reacted sperm. Thus, we conclude that in ascidian sperm a chymotrypsin-like protease is exposed on the external surface of the plasma membrane of the head, is required for fertilization, and plays a role in sperm penetration but not binding.

High-performance capillary electrophoresis for the determination of trypsin and chymotrypsin inhibitors and their association with trypsin, chymotrypsin and monoclonal antibodies.

High-performance capillary electrophoresis (HPCE) was adapted for the determination of Kunitz soybean trypsin inhibitor, Bowman Birk inhibitor from soybean and protein-type proteinase inhibitors from pea (Pisum sativum L.). The method was developed for the determination and characterization of the inhibitors, the enzymes trypsin and chymotrypsin and the monoclonal antibodies (mAbs) raised against the inhibitors, and also the inhibitor-enzyme and inhibitor-mAb association complexes. The results from studies involving the use of various types of buffers revealed the advantages of having zwitterions such as trimethylammoniumpropyl sulphonate (AccuPure) or taurine included in the buffer. The use of capillaries dynamically coated with zwitterions efficiently reduced the interactions of the proteins with the silica capillary surface, which was important for the analyses for trypsin, chymotrypsin and mAbs and their association complexes with the inhibitors. The influence of temperature, voltage, pH and buffer type on migration times, resolution, peak areas and number of theoretical plates was investigated for the proteins studied. The proposed HPCE method is very suitable for studies of proteinase inhibitors compared with traditional inhibitor studies, and it gives efficient protein separations with the possibility of 245,000 plates/m.

Strong immunoreactivity of alpha 1-antichymotrypsin co-localizes with beta-amyloid protein and ubiquitin in vacuolated muscle fibers of inclusion-body myositis.

In 10 of 10 inclusion-body myositis (IBM) patients, including 1 hereditary case, vacuolated muscle fibers contained large or small cytoplasmic inclusions immunoreactive for alpha 1-antichymotrypsin (alpha 1-ACT). All IBM muscle biopsies had characteristic cytoplasmic tubulo-filaments by electron microscopy. None of 17 control muscle biopsies contained the alpha 1-ACT immunoreactive inclusions characteristic of IBM. In vacuolated muscle fibers, alpha 1-ACT immunoreactive inclusions colocalized with beta-amyloid protein and ubiquitin immunoreactivities. Our study provides the first demonstration of alpha 1-ACT accumulations in abnormal human muscle, and it suggest that, as in Alzheimer's disease and Down's syndrome, alpha 1-ACT may be involved in the pathogenesis of IBM.

A chymotryptic-type serine protease is required for IL-2 production by Jurkat T cells.

Interleukin-2 (IL-2) production by activated Jurkat T cells was markedly delayed when these cells were treated with low concentrations of the chymotryptic-type protease inhibitor N-alpha-p-tosyl-L-phenylalanine chloromethylketone (TPCK). This increased lag time observed in the presence of TPCK directly correlates with the interaction of the inhibitor with a unique 42,000 molecular weight (MW) serine protease, which can be labelled with [3H]DFP, and was not due to an intracellular accumulation of a non-mature form of IL-2 nor to a non-specific inhibition of overall protein synthesis. The results presented in this report indicate that a 42,000 MW chymotryptic-like serine protease is required for IL-2 production by activated Jurkat T cells.