Major Secretory Protein Research Articles

Estrogen Receptor-α Mediates Diethylstilbestrol-Induced Feminization of the Seminal Vesicle in Male Mice

Background: Studies have shown that perinatal exposure to the synthetic estrogen diethylstilbestrol (DES) leads to feminization of the seminal vesicle (SV) in male mice, as illustrated by tissue hyperplasia, ectopic expression of the major estrogen-inducible uterine secretory protein lactoferrin (LF), and reduced expression of SV secretory protein IV (SVS IV).Objectives: The present study was designed to evaluate the role of the estrogen receptor (ER) in this action by using ER-knockout (ERKO) mice.Methods: Wild-type (WT), ERα-null (αERKO), and ERβ-null (βERKO) male mice were treated with either vehicle or DES (2 μg/day) on neonatal days 1–5. These mice were divided into two groups: In the first group, intact mice were sacrificed at 10 weeks of age; in the second group, mice were castrated at 10 weeks of age, allowed to recover for 10 days, treated with dihydrotestosterone (DHT) or placebo, and sacrificed 2 weeks later. Body weights and SV weights were recorded, and mRNA expression levels of Ltf (lactoferrin), Svs4, and androgen receptor (Ar) were assessed.Results: In DES-treated intact mice, SV weights were reduced in WT and βERKO mice but not in αERKO mice. DES-treated WT and βERKO males, but not αERKO males, exhibited ectopic expression of LF in the SV. DES treatment resulted in decreased SVS IV protein and mRNA expression in WT males, but no effect was seen in αERKO mice. In addition, DES-treated βERKO mice exhibited reduced Svs4 mRNA expression but maintained control levels of SVS IV protein. In DES-treated castrated mice, DHT implants restored SV weights to normal levels in αERKO mice but not in WT mice, suggesting full androgen responsiveness in αERKO mice.Conclusions: These data suggest that DES-induced SV toxicity and feminization are primarily mediated by ERα; however, some aspects of androgen response may require the action of ERβ.

Parotid Secretory Protein Binds Phosphatidylinositol (3,4) Bisphosphate

Molecular interactions that direct trafficking of secreted proteins are not well-described in salivary glands. Here, we report that the soluble cargo protein Parotid Secretory Protein (PSP) is bound to the membranes of secretory granules isolated from rat parotids. This is apparently due to specific interaction with phosphatidylinositol phosphates (PtdInsP). PSP binds PtdIns(3,4)P2, 10-fold greater than PtdIns(3,5)P2 or PtdIns(4)P, and does not bind PtdIns(3)P or PtdIns(5)P. Human PSP synthesized in vitro also binds PtdIns(3,4)P2. Bacterially expressed rat PSP binds PtdIns(3,4)P2 with a Kd of 2.4 x 10-11 M. Other major secretory proteins (amylase, proline-rich protein) are not bound to isolated granule membranes and do not bind phosphatidylinositol phosphates. Immunofluorescence shows PtdIns(3,4)P2 at the secretory granules, and fluorescent PtdIns(3,4)P2 can flip from the outer leaflet to the inner leaflet of the membrane. Binding of PSP to PtdInsPs may contribute to sorting during the formation of the secretory granules, or sorting by retention during maturation of the granules.

High-level production and purification of clostripain expressed in a virulence-attenuated strain of Clostridium perfringens

Clostripain (CLO) produced by Clostridium histolyticum is an arginine-specific endopeptidase with the potential for applicability to diverse medical and industrial uses. In this study, we developed an expression system allowing high-level production and efficient purification of recombinant CLO (rCLO). Our expression system comprises pCLO, an rCLO expressing vector, and Clostridium perfringens 13Δ6, an in-frame deletion strain as to six genes encoding major virulence factors and secretory proteins. rCLO was purified from the culture supernatant of C. perfringens 13Δ6/pCLO by ammonium sulfate precipitation, hydroxyapatite chromatography, and affinity chromatography on benzamidine-Sepharose. From 200 ml of culture supernatant 4.5 mg of purified rCLO was obtained. N-Terminal amino acid sequencing and molecular mass determination of the purified rCLO and commercially available CLO revealed that the two enzymes have identical subunits, a 38.1-kDa heavy chain and a 15.0-kDa light chain, indicating that rCLO is processed in the same manner as CLO. Analysis of the enzymatic activities toward N-benzoyl- l-arginine p-nitroanilide and acyl- l-lysine p-nitroanilide showed that rCLO and CLO exhibit strict specificity for arginine at the P1 position, and that the specific activity of the former is approximately 2-fold higher than that of the latter. These results indicate that the new method involving a virulence-attenuated C. perfringens strain is useful for preparing large amounts of high-grade rCLO.

Perivascular Adipose Tissue–Derived Complement 3 Is Required for Adventitial Fibroblast Functions and Adventitial Remodeling in Deoxycorticosterone Acetate–Salt Hypertensive Rats

To examine the role of perivascular adipose tissue (PVAT)-derived factors in the regulation of adventitial fibroblast (AF) function in vitro and in vivo. PVAT is an active component of blood vessels. Bioactive substances released from PVAT play regulatory roles in vascular function. However, their effects on vascular AFs remain unclear. PVAT-conditioned medium stimulated AF migration using a transwell technique, and differentiation was evaluated by α-smooth muscle-actin induction. We identified the secretome of PVAT by liquid chromatography-tandem mass spectrometry. One of the major secretory proteins in PVAT is complement 3 (C3). The C3 antagonist and neutralizing antibody attenuated PVAT-conditioned medium-induced AF migration and differentiation. Similar to PVAT-conditioned medium, C3 recombinant protein stimulated AF migration and differentiation. We demonstrated that the effects of PVAT-derived C3 were mediated by the c-Jun N-terminal kinase pathway. Moreover, we found morphological changes in perivascular adipocytes and increased expression of C3 in PVAT that was tightly associated with adventitial thickening and myofibroblast clustering around PVAT in deoxycorticosterone acetate-salt hypertensive rats. PVAT-derived C3 stimulated AF migration and differentiation via the c-Jun N-terminal kinase pathway. PVAT-derived C3 may contribute to adventitial remodeling in a deoxycorticosterone acetate-salt hypertensive model.

POSVP21, a major secretory androgen-dependent protein from sand rat seminal vesicles, identified as a transgelin

The seminal vesicles of adult sand rat contain a major secretory protein band (MW 21 kDa) designated as Psammomys obesus seminal vesicles protein of 21 kDa (POSVP(21)). This protein is abundant in secretions, regulated by androgens and also present in the vaginal plug. POSVP(21) accounts for over 22.3% of soluble proteins from homogenate during the breeding season, 13.3% during the middle season and 5.3% during the hormonal regression season. It is absent during the non-breeding season. POSVP(21) is localized in the cytoplasm of epithelial cells and in secretory products in the lumen. It presents an immunological homology with two epididymal proteins with the same molecular weight and a high degree of homology with transgelin from rat (Rattus norvegicus).

Ag85B DNA vaccine suppresses airway inflammation in a murine model of asthma

BackgroundIn allergic asthma, Th2 lymphocytes are believed to play important roles in orchestrating airway eosinophilia and inflammation. Resetting the Th1/Th2 imbalance may have a therapeutic role in asthma. The mycobacterium tuberculosis 30-kilodalton major secretory protein (antigen 85B, Ag85B) can protect animals from M. tuberculosis infection by inducing a Th1-dominant response.MethodsIn this study, the Ag85B gene was cloned into pMG plasmids to yield the pMG-Ag85B plasmid. The expression of Ag85B gene in murine bronchial epithelia cells was detected by Western blotting and immunohistochemical staining after intranasal immunization with reconstructed pMG-Ag85B plasmids. The protective effect of pMG-Ag85B plasmids immunization in airway inflammation was evaluated by histological examination and bronchoalveolar lavage (BAL). IL-4 and IFN-γ levels in the BAL and supernatant from splenocyte culture were determined using ELISA kits.ResultsThe Ag85B gene was successfully expressed in murine bronchial epithelia cells by intranasal immunization with reconstructed pMG-Ag85B plasmids. Using a murine model of asthma induced by ovalbumin (OVA), pMG-Ag85B immunization significantly inhibited cellular infiltration across the airway epithelium with a 37% decrease in the total number of cells (9.6 ± 2.6 × 105/ml vs. 15.2 ± 3.0 × 105/ml, p < 0.05) and a 74% decrease in the number of eosinophils (1.4 ± 0.2 × 105/ml vs. 5.4 ± 1.1 × 105/ml, p < 0.01) compared with the OVA-sensitized control group. There was no difference in the number of neutrophils in BAL fluid between the pMG-Ag85B group, the OVA-sensitized control group and the empty pMG group. IL-4 production was significantly decreased in the BAL fluid (32.0 ± 7.6 pg/ml vs. 130.8 ± 32.6 pg/ml, p < 0.01) and in the splenocyte supernatant (5.1 ± 1.6 pg/ml vs. 10.1 ± 2.3 pg/ml, p < 0.05) in the pMG-Ag85B group compared with the OVA-sensitized control group, while IFN-γ production was increased in the BAL fluid (137.9 ± 25.6 pg/ml vs. 68.4 ± 15.3 pg/ml, p < 0.05) and in the splenocyte supernatant (20.1 ± 5.4 pg/ml vs. 11.3 ± 3.2 pg/ml, p < 0.05).ConclusionIn a murine model of asthma induced by OVA, intranasal immunization with pMG-Ag85B significantly reduced allergic airway inflammation with less eosinophil infiltration. This protective effect was associated with decreased IL-4 and increased IFN-γ production in the BAL fluid and in the supernatant of cultured splenocytes.

Clara Cells Attenuate the Inflammatory Response through Regulation of Macrophage Behavior

Chronic lung diseases are marked by excessive inflammation and epithelial remodeling. Reduced Clara cell secretory function and corresponding decreases in the abundance of the major Clara cell secretory protein (CCSP) are characteristically seen in these disease states. We sought to define the impact of Clara cell and CCSP depletion on regulation of the lung inflammatory response. We used chemical and genetic mouse models of Clara cell and CCSP deficiency (CCSP(-/-)) coupled with Pseudomonas aeruginosa LPS elicited inflammation. Exposure of Clara cell-depleted or CCSP(-/-) mice to LPS resulted in augmented inflammation as assessed by polymorphonuclear leukocyte recruitment to the airspace. Gene expression analysis and pathway modeling of the CCSP(-/-) inflammatory response implicated increased TNF-alpha signaling. Consistent with this model was the demonstration of significantly elevated TNF-alpha in airway fluid of LPS-stimulated CCSP(-/-) mice compared with similarly exposed wild-type mice. Increased LPS-elicited TNF-alpha production was also observed in cultured lung macrophages from CCSP(-/-) mice compared with wild-type mice. We demonstrate that macrophages from Clara cell-depleted and CCSP(-/-) mice displayed increased Toll-like receptor 4 surface expression. Our results provide evidence that Clara cells can attenuate inflammation through regulation of macrophage behavior, and suggest that epithelial remodeling leading to reduced Clara cell secretory function is an important factor that increases the intensity of lung inflammation in chronic lung disease.

Heterologous expression and characterization of a β-1,6-glucanase from Aspergillus fumigatus

The cell wall of Candida albicans is composed of mannoproteins associated to glycan polymers. Most of these proteins are retained in this compartment through a phosphodiester linkage between a remnant of their glycosylphosphatidylinositol anchor and the beta-1,6-glucan polymer. A pure beta-1,6-glucanase is thus required in order to release them. In this paper, we report the expression/secretion by the yeast Yarrowia lipolytica of an Aspergillus fumigatus enzyme homologous to previously described beta-1,6-glucanases. The coding sequence was expressed under the control of a strong promoter and the recombinant enzyme was targeted to the secretory pathway using the signal sequence of a well-known major secretory protein in this host. Addition of a FLAG epitope at the C-terminus allowed its efficient purification from culture supernatant following batch adsorption. The purified enzyme was characterized as a beta-1,6-glucanase and was shown to be active on C. albicans cell walls allowing the release of a previously described cell wall protein.

Commonly administered BCG strains including an evolutionarily early strain and evolutionarily late strains of disparate genealogy induce comparable protective immunity against tuberculosis

BCG has been administered to over 4 billion persons worldwide, but its efficacy in preventing tuberculosis in adults has been highly variable. One hypothesis for its variability is that different strains of BCG vary in protective efficacy, and moreover, that evolutionarily early strains are more efficacious than the more attenuated evolutionarily late strains, which lack region of deletion 2. To examine this hypothesis, we tested six widely used BCG strains – the evolutionarily early strain BCG Japanese, two evolutionarily late strains in DU2 Group III (BCG Danish and Glaxo), and three evolutionarily late strains in DU2 Group IV (BCG Connaught, Pasteur, and Tice) – in the guinea pig model of pulmonary tuberculosis. With the exception of BCG Glaxo, which had relatively poor efficacy, we found no substantial differences in efficacy between the early strain and the late strains, and only small differences in efficacy among late strains. BCG Tice was the most efficacious BCG vaccine, with significantly fewer Mycobacterium tuberculosis in the lung and spleen than BCG Danish and BCG Japanese, although absolute differences in the organ burden of M. tuberculosis among these three vaccines were small (≤0.2 log). BCG Tice and Pasteur were not significantly different. rBCG30, a recombinant BCG Tice vaccine overexpressing the M. tuberculosis 30 kDa major secretory protein (Antigen 85B), was more potent than any BCG vaccine ( P < 0.0001 for differences in organ burden). Our study shows that late strains are not less potent than an early strain and argues against strain differences as a major factor in the variability of outcomes in BCG vaccine trials.

Adipocytokines and the Metabolic Complications of Obesity

Adipose tissue is increasingly recognized as an active endocrine organ with many secretory products and part of the innate immune system. With obesity, macrophages infiltrate adipose tissue, and numerous adipocytokines are released by both macrophages and adipocytes. Adipocytokines play important roles in the pathogenesis of insulin resistance and associated metabolic complications such as dyslipidemia, hypertension, and premature heart disease. Published literature was analyzed with the intent of addressing the role of the major adipose secretory proteins in human obesity, insulin resistance, and type 2 diabetes. This review analyzes the characteristics of different adipocytokines, including leptin, adiponectin, pro-inflammatory cytokines, resistin, retinol binding protein 4, visfatin, and others, and their roles in the pathogenesis of insulin resistance. Inflamed fat in obesity secretes an array of proteins implicated in the impairment of insulin signaling. Further studies are needed to understand the triggers that initiate inflammation in adipose tissue and the role of each adipokine in the pathogenesis of insulin resistance.

Proteomic analysis and immunogenicity of secreted proteins from Rhodococcus equi ATCC 33701

Rhodococcus equi is one of the most important causes of mortality in foals between 1 and 6 months of age. Although rare, infection also occurs in a variety of other mammals including humans, often following immunosuppression of various causes. Secreted proteins are known to mediate important pathogen–host interactions and consequently are favored candidates for vaccine development as they are the most easily accessible microbial antigens to the immune system. Here, we describe the results of a proteomic analysis based on SDS-PAGE, immunoblot and mass spectrometry, which was carried out aiming the identification of secreted proteins that are differently expressed at 30 °C versus 37 °C and at mid-exponential versus early-stationary growth phase and antigenic proteins from R. equi ATCC 33701. A total of 48 proteins was identified regardless of growth conditions. The cholesterol oxidase ChoE appears to be the major secretory protein. Moreover, four proteins revealed high homologies with the mycolyl transferases of the Ag85 complex from Mycobacterium tuberculosis. The sequence analysis predicted that 24 proteins are transported by a signal peptide-dependent pathway. Moreover, five antigenic proteins of R. equi were identified by immunoblot, including a novel strongly immunoreactive protein of unknown function. In conclusion, the elucidation of the secretome of R. equi identified several proteins with different biological functions and a new candidate for developing vaccines against R. equi infection in horse.

Loss of detectability of Charcot–Leyden crystal protein transcripts in blood cells after treatment with dimethyl sulfoxide

Charcot–Leyden crystal protein (CLC) is a major secretory effector protein of eosinophils. In addition, CLC has been identified as marker for regulatory T-cells and differential expression of CLC has been described under diverse pathological conditions. By analysis of DNA microarray data from peripheral blood mononuclear cells (PBMC) we found differences for the expression of CLC between PBMC that had been cryopreserved or had been used for RNA isolation immediately after cell separation. Reverse transcriptase-polymerase chain reaction (RT-PCR) of separated cell populations indicated that contaminating granulocytes were the main source of CLC transcripts in PBMC. CLC was only detectable in fresh PBMC and not in cryopreserved material. Transcripts corresponding to CLC were also detectable by RT-PCR only in fresh PBMC and eosinophils. Loss of CLC transcripts in PBMC was induced by a short pulse with dimethyl sulfoxide (DMSO), indicating that the freezing medium was responsible for this phenomenon. We conclude that CLC transcripts are lost during cryopreservation in the presence of DMSO and can never be identified as differentially expressed in cryopreserved samples.

A Replication-Limited Recombinant Mycobacterium bovis BCG Vaccine against Tuberculosis Designed for Human Immunodeficiency Virus-Positive Persons Is Safer and More Efficacious than BCG

Tuberculosis is the leading cause of death in AIDS patients, yet the current tuberculosis vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), is contraindicated for immunocompromised individuals, including human immunodeficiency virus-positive persons, because it can cause disseminated disease; moreover, its efficacy is suboptimal. To address these problems, we have engineered BCG mutants that grow normally in vitro in the presence of a supplement, are preloadable with supplement to allow limited growth in vivo, and express the highly immunoprotective Mycobacterium tuberculosis 30-kDa major secretory protein. The limited replication in vivo renders these vaccines safer than BCG in SCID mice yet is sufficient to induce potent cell-mediated and protective immunity in the outbred guinea pig model of pulmonary tuberculosis. In the case of one vaccine, rBCG(mbtB)30, protection was superior to that with BCG (0.3-log fewer CFU of M. tuberculosis in the lung [P < 0.04] and 0.6-log fewer CFU in the spleen [P = 0.001] in aerosol-challenged animals [means for three experiments]); hence, rBCG(mbtB)30 is the first live mycobacterial vaccine that is both more attenuated than BCG in the SCID mouse and more potent than BCG in the guinea pig. Our study demonstrates the feasibility of developing safer and more potent vaccines against tuberculosis. The novel approach of engineering a replication-limited vaccine expressing a recombinant immunoprotective antigen and preloading it with a required nutrient, such as iron, that is capable of being stored should be generally applicable to other live vaccine vectors targeting intracellular pathogens.

Changes in Clara Cell 10 kDa Protein (CC10)-positive Cell Distribution in Acute Lung Injury Following Repeated Lipopolysaccharide Challenge in the Rat

Clara cell 10 kDa protein (CC10) is the major secretory protein of Clara cells and is thought to play a protective role in the lung owing to its anti-inflammatory properties. There is little information on the anatomical distribution of CC10-positive cells in rat lung following lipopolysaccharide (LPS) challenge. We have determined the expression of CC10 along the tracheobronchial tree in saline-treated and LPS-treated rats. Saline-treated rats showed sporadic CC10 staining in central airways and abundant staining in bronchioles. In transitional airways, most cells were positive except for squamous cells. Following LPS challenge, there was a reduction in staining in the upper airways but little change within bronchioles. Squamous epithelia within the transitional airways now showed positive staining. These cells also co-stained for pancytokeratin and appeared to co-localize with surfactant D- and Ki67-positive cells, indicating the presence of a dedifferentiated cell type with both epithelial and pneumocyte phenotypes. These data show that diffuse inflammatory injury results in generalized loss of CC10 in central airways. Conversely, the transitional airways showed evidence of a dedifferentiated population of squamous cells that now stained for CC10. We hypothesize that this is an attempt by peripheral lung to maintain alveolar sac integrity during an inflammatory episode.

Purification, preliminary characterization and immunohistochemical localization of POSVP21 in the sand rat (Psammomys obesus) seminal vesicles

The sand rat Psammomys obesus is a mammalian species with male seasonal reproduction. Previously Gernigon et al. (1994) [Gernigon T, Berger M, Lecher P. Seasonal variations in the ultrastructure and production of androgen-dependent proteins in the seminal vesicles of a saharian rodent (Psammomys obesus). J Endocrinol 1994;142:37-46.] reported that the seminal vesicles of the adult sand rat contained a major secretory protein band (M.W. 21000) regulated by testosterone. This protein is synthesized in large amounts when the androgen level increases, and accounts for over 22% of soluble proteins from homogenate of seminal vesicles during the breeding season. When analyzed by NepHGE the protein band of 21kDa appeared to be composed of at least 3 visible spots with pHi values varying from 4 to 7. Its partially internal sequence was identified and exhibited five peptides. Polyclonal antibodies against POSVP21 were obtained in rabbits. They were also used to study immunohistochemical antigen localization by the means of an avidin-biotin peroxidase procedure. Observation showed that it is localized in the cytoplasm of epithelial cells and in secretory products in the lumen. The whole RNA of seminal vesicles was translated in a cell-free system derived from rabbit reticulocyte lysate and [35S]-methionine. Two major bands of 14.4 and 21kDa were visualized by means of denaturing gel electrophoresis. SDS-PAGE from medium incubation of seminal vesicle tissue with [35S]-methionine revealed one band with an apparent molecular weight of 21kDa. The results obtained indicate that seminal vesicle epithelium is the site of POSVP21 synthesis and the comparison of the partial amino acid composition of the internal sequence, indicated that POSVP21 constitute a family of most unusual proteins.

Chromogranin A Expression in Phaeochromocytomas Associated with von Hippel-Lindau Syndrome and Multiple Endocrine Neoplasia Type 2

Chromogranin A (CGA) is a major secretory protein present in the soluble matrix of chromaffin granules of neuroendocrine cells and tumours, such as phaeochromocytomas. CGA has several functions, some of which may be involved in the distinct phenotypic differences of phaeochromocytomas in patients with von Hippel-Lindau (VHL) syndrome compared to multiple endocrine neoplasia type 2 (MEN 2). In this study, we therefore compared tumour and plasma levels of CGA in patients with phaeochromocytoma associated with the two syndromes. We show that phaeochromocytomas from MEN 2 patients express substantially more CGA than tumours from VHL patients at both the mRNA (3-fold greater) and protein (20-fold) level. We further show that relative to increases in plasma catecholamines, patients with phaeochromocytomas associated with MEN 2 have higher plasma concentrations of CGA than those with tumours in VHL syndrome. These data supplement other observations that phaeochromocytomas in VHL compared to MEN 2 patients express lower amounts of catecholamines and other chromaffin granule cargo, such as chromogranin B and neuropeptide Y. Possibly the differences in tumour CGA expression may contribute to differences in secretory vesicle formation and secretion in the two types of tumours. Alternatively the differences in expression in CGA and other secretory constituents may reflect downregulation of the entire regulated secretory pathway in VHL compared to MEN 2 tumours.

A Tetraantennary Glycan with Bisecting N-Acetylglucosamine and the Sda Antigen is the Predominant N-Glycan on Bovine Pregnancy-Associated Glycoproteins

Pregnancy-associated glycoproteins (PAGs) are major secretory proteins of trophoblast cells in ruminants. Binucleate trophoblast giant cells (BNCs) store these proteins in secretory granules and release them into the maternal organism after fusion with maternal uterine epithelial cells. By matrix assisted laser desorption ionisation-mass spectrometry (MALDI-MS) analysis and linkage analysis, we show that by far, the most abundant N-glycan of PAGs in midpregnancy is a tetraantennary core-fucosylated structure with a bisecting N-acetylglucosamine (GlcNAc). All four antennae consist of the Sd(a)-antigen (NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc-). Immunohistochemistry with the mono- clonal antibody CT1, which recognizes the Sd(a)-antigen, shows that BNC granules contain the Sd(a)-antigen from gestation day (gd) 32 until a few days before parturition. Lectin histochemistry with Maackia amurensis lectin (MAL), which binds to alpha2-3sialylated lactosamine, shows that BNC granules are MAL-positive prior to gd 32 and also at parturition. The observed tetraantennary glycan is a highly unusual structure, since during the synthesis of N-glycans, the insertion of a bisecting GlcNAc inhibits the activity of the GlcNAc-transferases that leads to tri- and tetraantennary glycans. The study defines the substantial changes of PAG N-glycosylation in the course of pregnancy. This promotes the hypothesis that PAGs may have different carbohydrate-mediated functions at different stages of pregnancy.

Mycobacterium tuberculosis secretory proteins CFP‐10, ESAT‐6 and the CFP10:ESAT6 complex inhibit lipopolysaccharide‐induced NF‐κB transactivation by downregulation of reactive oxidative species (ROS) production

Mycobacterium tuberculosis (Mtb) causes death of 2-3 million people annually and is considered one of the most successful intracellular pathogens to persist inside the host macrophage. Recent studies have implicated the role of RD-1 region of Mtb genome in the mycobacterial pathogenesis. The role of RD-1-encoded secretory proteins of Mtb in modulation of macrophage function has not been investigated in detail. Here we show that RD-1 encoded two major secretory proteins, namely, culture filtrate protein-10 kDa (CFP-10) and early secreted antigenic target-6 kDa (ESAT-6), and their 1:1 CFP-10:ESAT6 complex inhibit production of reactive oxidative species (ROS) in RAW264.7 cells. These proteins also downregulated the bacterial lipopolysaccharide (LPS)-induced ROS production, which, in turn, downregulated LPS-induced nuclear factor-kappaB (NF-kappaB) p65 DNA-binding activity, as well as inhibited the NF-kappaB-dependent reporter gene (chloramphenicol acetyl transferase) expression in the treated macrophages. Moreover, addition of N-acetyl cysteine, which is a scavenger of ROS, also inhibited LPS-induced reporter gene expression by scavenging the ROS, thereby preventing NF-kappaB transactivation. These studies indicate that the secretory proteins CFP-10, ESAT-6 and the CFP10:ESAT6 complex of Mtb can inhibit LPS-induced NF-kappaB-dependent gene expression via downregulation of ROS production.

Characterization of a Major Secretory Protein in the Cane Toad (Bufo marinus) Choroid Plexus as an Amphibian Lipocalin-type Prostaglandin D Synthase

Here we report the enzymatic and ligand-binding properties of a major secretory protein in the choroid plexus of cane toad, Bufo marinus, whose protein is homologous with lipocalin-type prostaglandin (PG) D synthase (L-PGDS) and is recombinantly expressed in Xenopus A6 cells and Escherichia coli. The toad protein bound all-trans retinal, bile pigment, and thyroid hormones with high affinities (K(d)=0.17 to 2.00 microM). The toad protein also catalysed the L-PGDS activity, which was accelerated in the presence of GSH or DTT, similar to the mammalian enzyme. The K(m) value for PGH(2) (17 microM) of the toad protein was almost the same as that of rat L-PGDS (14 microM), whereas the turnover number (6 min(-1)) was approximately 28 fold lower than that of rat L-PGDS. Site-directed mutagenesis based on a modeled structure of the toad protein revealed that Cys(59) and Thr(61) residues were crucial for the PGDS activity. The quadruple Gly(39)Ser/Ala(75)Ser/Ser(140)Thr/Phe(142)Tyr mutant of the toad protein, resembling mouse L-PGDS, showed a 1.6 fold increase in the turnover number and a shift in the optimum pH for the PGDS activity from 9.0 to 8.5. Our results suggest that the toad protein is a prototype of L-PGDS with a highly functional ligand-binding pocket and yet with a primitive catalytic pocket.

Suppression effect of seminal vesicle autoantigen on platelet‐activating factor‐induced mouse sperm capacitation

Mammalian sperm gain the ability to fertilize an egg successfully by the capacitation process. An unregulated capacitation process causes sperm to undergo a spontaneous acrosome reaction (AR) and resulting in loss of their fertilization activity. Thus, functional sperm activation is tightly regulated by a capacitation and suppression (decapacitation) mechanism. Factors, such as platelet-activating factor (PAF) present in both sperm and the female genital tract, are able to stimulate sperm capacitation. Seminal plasma is thought to have the ability to suppress sperm capacitation; however, the regulatory mechanisms of seminal plasma protein on sperm capacitation are not well understood. Recently, we demonstrated that seminal vesicle autoantigen (SVA), a major seminal vesicle secretory protein, is able to suppress mouse sperm capacitation. To further study the suppression spectra of SVA on sperm capacitation, we investigated the effect of SVA on PAF-induced mouse sperm capacitation-related signals. Here, we demonstrate that SVA decreases the [Ca(2+)](i) to suppress the PAF's effects on [Ca(2+)](i), the cAMP level, protein tyrosine phosphorylation, and capacitation. The inhibition of PAF-induced protein tyrosine phosphorylation and capacitation by SVA can be reversed by cAMP agonists. Characterization of the interactions of SVA with PAF by TLC overlay and tryptophan fluorescence spectrum analyses indicates that SVA is capable of binding PAF with an apparent dissociation constant K(d) > 50 microM. Together with these results, we demonstrate that SVA deceases [Ca(2+)](i) and cross-talks with PAF-induced intracellular signals to regulate mouse sperm capacitation.