Autoprocessing and self-activation of the secreted protease CPAF in Chlamydia-infected cells

The Chlamydia-secreted protease/proteasome-like activity factor (CPAF) is synthesized as a proenzyme (proCPAF) and requires processing for proteolytic activity. Recent structural studies have further demonstrated that CPAF is a serine protease that can undergo autoprocessing and self-activation in a concentration-dependent manner in vitro. However, it is not known how CPAF is processed and activated during chlamydial infection. In the current study, we used a mutant CPAF designated as CPAF(E558A) that is deficient in processing by itself as a substrate to search for putative CPAF activation factor(s) in Chlamydia-infected cells. CPAF(E558A) was processed by the lysates made from Chlamydia-infected cells and the processing activity correlated with the presence of endogenous active CPAF in the fractionated lysate samples. CPAF produced in the Chlamydia-infected cells is required for processing the mutant CPAF(E558A) since the processing activity was removed by depletion with anti-CPAF but not control antibodies. Furthermore, a purified and activated wild type CPAF alone was sufficient for processing CPAF(E558A) and no other chlamydial proteases are required. Finally, fusion tag-induced oligomerization can lead to autoprocessing and self-activation of the wild type CPAF in mammalian cells. These observations together have demonstrated that CPAF undergoes autoprocessing and self-activation during chlamydial infection.     

Listeriolysin O secreted by Listeria monocytogenes into the host cell cytosol is degraded by the N-end rule pathway

The intracellular pathogen Listeria monocytogenes escapes from a phagosomal compartment into the cytosol by secreting the pore-forming cytolysin listeriolysin O (LLO). During the proliferation of L. monocytogenes bacteria in the mammalian cell cytosol, the secreted LLO is targeted for degradation by the ubiquitin system. We report here that LLO is a substrate of the ubiquitin-dependent N-end rule pathway, which recognizes LLO through its N-terminal Lys residue. Specifically, we demonstrated by reverse-genetic and pharmacological methods that LLO was targeted for degradation by the N-end rule pathway in reticulocyte extracts and mouse NIH 3T3 cells and after its secretion by intracellular bacteria into the mouse cell cytosol. Replacing the N-terminal Lys of LLO with a stabilizing residue such as Val increased the in vivo half-life of LLO but did not strongly affect the intracellular growth or virulence of L. monocytogenes. Nevertheless, this replacement decreased the virulence of L. monocytogenes by nearly twofold, suggesting that a destabilizing N-terminal residue of LLO may stem from positive selection during the evolution of this and related bacteria. A double mutant strain of L. monocytogenes in which upregulated secretion of LLO was combined with a stabilizing N-terminal residue was severely toxic to infected mammalian cells, resulting in reduced intracellular growth of bacteria and an approximately 100-fold-lower level of virulence. In summary, we showed that LLO is degraded by the N-end rule pathway and that the degradation of LLO can reduce the toxicity of L. monocytogenes during infection, a property of LLO that may have been selected for its positive effects on fitness during the evolution of L. monocytogenes.     

C-reactive protein is expressed and secreted by peripheral blood mononuclear cells

C-reactive protein (CRP) protects against bacterial pathogens and is a predictor of cardiovascular events. CRP is produced by vascular and organ-specific cells but the generation of CRP from peripheral blood mononuclear cells (PBMC) is poorly established. In a randomized, double-blind, placebo-controlled, two-way cross-over trial six healthy volunteers received a bolus infusion of 20 IU/kg Escherichia coli endotoxin [lipopolysaccharide (LPS)] or placebo. Intracellular CRP protein and CRP secretion of peripheral blood mononuclear cells (PBMC) was measured at baseline and 6 h after LPS by flow cytometry and enzyme-linked immubosorbent assay (ELISA), respectively. CRP mRNA expression was determined by real-time polymerase chain reaction (PCR). Regulation of the expression pathway was assessed using specific inhibitors in vitro. Small amounts of CRP protein and mRNA were detectable in PBMC, which were up-regulated between two- and eightfold by endotoxaemia in vivo. Augmented expression and release of CRP by LPS was consistent in PBMC cell culture experiments. LPS, interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF)-alpha increased and IL-10 reduced CRP expression in PBMC. Toll-like receptor (TLR)-4, nuclear factor (NF)-kappaB and protein kinase C (PKC) activation were identified as intracellular signal transduction pathways of LPS-induced CRP expression. Constitutive CRP expression and release in PBMC is enhanced by inflammatory stimuli in vivo and in vitro. LPS might induce CRP generation via activation of TLR-4, NF-kappaB and PKC.     

The delivery of an antigen from the endocytic compartment into the cytosol for cross-presentation is restricted to early immature dendritic cells

Dendritic cells (DCs) are the only antigen-presenting cell population having a cross-presentation capacity. For cross-presentation, however, the intracellular antigen-processing pathway and its regulatory mechanism have not been defined. Here we report the differences in cross-presentation ability among murine bone marrow-derived immature DC, early immature day8-DC and late immature day10-DC, and fully mature day10 + lipopolysaccharide DC. Day8-DCs and day10-DCs show an immature phenotypic profile but are different in morphology. Day8-DCs can internalize an abundant volume of exogenous soluble ovalbumin (OVA) and result in cross-presentation. In contrast, day10-DCs are not able to cross-present, although they maintain efficient macropinocytosis. Exogenously internalized OVA antigens are stored in the endocytic compartments. The endocytic compartments are temporarily maintained at mildly acidic pH in day8-DCs and are rapidly acidified in day10-DCs after uptake of antigens. We show that OVA antigens accumulated in the endocytic compartments move into the cytosol in day8-DCs but do not in day10-DCs. NH(4)Cl-treatment, which neutralizes the acidic endocytic compartments and/or delays endosomal maturation, restores day10-DCs for transport the stored OVA antigens from the endocytic compartments into the cytosol. Diphenyleneiodonium chloride-treatment, which acidifies the endocytic compartments, decreases an amount of transported OVA antigen into the cytosol in day8-DCs. These data indicate that only the early immature stage of DC interferes with endosomal maturation, even after uptake of exogenous antigens, and then transports the antigens into the cytosol.     

Human acidic fibroblast growth factor overexpressed in insect cells is not secreted into the medium

We have overexpressed human acidic fibroblast growth factor (aFGF) in Spodoptera frugiperda Sf9 cells from a cDNA clone under the control of the promoter of the polyhedrin gene of the baculovirus Autographa californica nuclear polyhedrosis virus. A 16.5-kD product was made in recombinant virus-infected cells that specifically reacted in immunoblots with various antibodies prepared against aFGF. Recombinant aFGF was mitogenic for BHK21 cells and its activity was stimulated by heparin. The mechanism of release of FGF from mammalian cells is unknown. Both acidic and basic FGF lack classical amino-terminal signal sequences for secretion, and they are very inefficiently released from cells. Sf9 cells infected with the recombinant virus produced 10-20 mg aFGF/10(9) cells, corresponding to about 10-20 pg/cell. Despite this high level of expression, only about 0.5 and 1.3% of the total aFGF was found in the culture medium at 48 and 72 hr postinfection, respectively. This indicates that aFGF is not actively secreted out of the cells either via the normal exocytic pathway or directly through the plasma membrane in this heterologous cell system.     

CD52 is synthesized in cumulus cells and secreted into the cumulus matrix during ovulation

Problem Early studies have shown that an antibody to male reproductive tissue CD52 is a pathogenic factor of infertility. The molecule contains a unique carbohydrate antigen that induces antibodies interfering with sperm function. However, the characteristic properties of CD52 in female reproductive tissues are not known. We examined the expression and localization of CD52 in mature expanded cumulus masses. Method of study Mouse cumulus oocyte complexes were collected from [C57B1/6; DBA/2] F1 female mice having a superovulation treatment. Human cumulus cells were obtained from infertile patients taking in vitro fertilization-embryo transfer treatment under informed consent. CD52 messenger RNA (mRNA) and protein were detected using RT-PCR, quantitative PCR, western blotting and immunohistochemical methods. Results CD52 mRNA was found both in the human and mouse cumulus cells. Mouse CD52 mRNA was detected in cumulus cells but not oocytes and significantly increased after ovulation. The expression of the molecule was also confirmed at the protein level. Immunostaining with anti CD52 peptide antibody revealed that CD52 is present in cumulus cells and the extracellular matrix. Conclusion We first showed the expression of CD52 in human cumulus cells. CD52 has some functional roles around fertilization in females as well as in males.     

Retinoschisin, the X-linked retinoschisis protein, is a secreted photoreceptor protein, and is expressed and released by Weri-Rb1 cells

X-linked retinoschisis is characterized by microcystic-like changes of the macular region and schisis within the inner retinal layers, leading to visual deterioration in males. Many missense and protein-truncating mutations of the causative gene RS1 have now been identified and are thought to be inactivating. RS1 encodes a 224 amino acid protein, retinoschisin, which contains a discoidin domain but is of unknown function. We have generated a polyclonal antibody against a peptide from a unique region within retinoschisin, which detects a protein of approximately 28 kDa in retinal samples reduced with dithiothreitol, but multimers sized >40 kDa under non-reducing conditions. A screen of human tissues with this antibody reveals retinoschisin to be retina specific and the antibody detects a protein of similar size in bovine and murine retinae. We investigated the expression pattern in the retina of both RS1 mRNA (using in situ hybridization with riboprobes) and retinoschisin (using immunohistochemistry). The antisense riboprobe detected RS1 mRNA only in the photoreceptor layer but the protein product of the gene was present both in the photoreceptors and within the inner portions of the retina. Furthermore, differentiated retinoblastoma cells (Weri-Rb1 cells) were found to express RS1 mRNA and to release retinoschisin. These results suggest that retinoschisin is released by photo-receptors and has functions within the inner retinal layers. Thus, X-linked retinoschisis is caused by abnormalities in a putative secreted photoreceptor protein and is the first example of a secreted photo-receptor protein associated with a retinal dystrophy.     

Neutralization activity of some Coxsackie B3 antibodies is compromized by protein factor(s) secreted by Vero cells

Summary When various anti-Coxsackie B3 virus antibodies were examined for the neutralizing activity in cultures under liquid medium, some antibodies including monoclonal antibodies gave abnormally low titers in the neutralization test in Vero cells, in comparison with the other cells such as HeLa, FL, HEp-2, or primary monkey kidney cells. The neutralization titer of these antibodies was, however, similar in all these cells by plaque reduction assays under agar overlay, i.e., the above phenomenon was restricted to cultures under liquid medium. The reduced neutralization titer in Vero cells under liquid medium was found to be brought about by protease-sensitive factor(s) released by Vero cells, because (1) the addition of Vero cell culture fluid resulted in a marked reduction of neutralizing titer in primary monkey kidney cells, and (2) the activity of the Vero cell factor was destroyed by trypsin (20 µg/ml for 1 h). As three-day incubation of virus-antibody complex in Vero cell culture fluid resulted in a partial restoration of virus infectivity, the binding of antibody to virions appears to be competed by the Vero cell factor.     

The mature form of the Friend spleen focus-forming virus envelope protein, gp65, is efficiently secreted from cells

The env genes of Friend spleen focus-forming viruses (F-SFFV) have been implicated in the rapid pathogenicity of these agents. Two env-gene products are detected in SFFV-infected cells: the primary translation product, gp52, and a more highly processed form, gp65. In this communication we demonstrate that gp65 is the major end product of the SFFV env gene, and is efficiently secreted from both erythroleukemia cells and infected fibroblasts. Secretion was observed for the mature env-gene products of both polycythemia- and anemia-inducing strains of SFFV. These results suggest that one function of the point mutation near the 3' end of the env gene, which is invariant in the formation of SFFVs, is to allow secretion of gp65, and that secreted gp65 may be the factor mediating the leukemogenic activity of these viruses.     

In the dying cell secretory protein diffuses through the membranes into the cytosol]

Anti-horseradish peroxidase IgG (a-HRP) secreting hybridoma lymphoblasts grown subcutaneously in recipient mice have been studied light and electron microscopically 30-120 min following capitation of the animals. Conventional HRP-DAB immunocytochemical staining was performed for demonstration of a-HRP which in the living cells was restricted to the rough endoplasmic reticulum, the perinuclear cisterns, the Golgi apparatus and some microvesicles. 30 min after death in a number of the cells a-HRP began to invade the cytosol leaving, however, the nucleus and mitochondrial matrix free of the secretory marker. 30 to 90 min later staining intensity became similar in all cellular structures thereby making an impression of overall a-HRP spreading throughout the cell. In the light of these findings and the data obtained by other investigators a conclusion is made on the diffusion of macromolecules across intracellular membranes as a result of considerable post-mortem disturbances in membrane permeability.     

hCGβ和hCGβ-C3d3融合蛋白在CHO细胞中的分泌性表达、鉴定与纯化

目的：构建带有6个组氨酸纯化标签的hCGβ和hCGβ-C3d3融合蛋白的分泌型真核表达质粒，在CHO细胞中获得具有生物学活性的重组蛋白的高效表达。方法：利用高效真核表达载体phCMV1，构建phCMV1-6His-hCGβ和phCMV1-6His-hCGβ-C3d3，脂质体法转染CHO细胞，G418(800μg/ml)筛选抗性克隆。放射免疫法检测抗性克隆培液中hCGβ表达量，Western blotting和Raji细胞免疫化学法鉴定hCGβ-C3d3融合蛋白。用镍柱和凝胶过滤层析纯化表达产物。结果：酶切及测序结果显示，phCMV1-6His-hCGβ及phCMV1-6His-hCGβ-C3d3构建正确。在CHO细胞中成功地获得了hCGβ和hCGβ-C3d3融合蛋白的高效表达，phCMV1载体的表达效率是pcDNA3的1．6倍。表达产物经纯化后得到了所需的hCGβ和hCGβ-C3d3融合蛋白。结论：hCGβ和hCGβ-C3d3融合蛋白在CHO细胞的高效表达为下一步比较hCGβ抗原及hCGβ-C3d3融合蛋白免疫动物的体液免疫效应和抗生育效果奠定了基础。     

Putative glycophorin-binding protein is secreted from schizonts of Plasmodium falciparum

A cDNA clone expressing an antigen of Plasmodium falciparum, selected by screening an expression library cloned in Escherichia coli, encodes a portion of the protein identified as a glycophorin-binding protein [Kochan et al. (1986) Cell 44, 689-696]. Human antibodies affinity-purified on extracts from this clone were used to characterize the antigen by immunoblotting. This protein was present in all isolates tested, restricted to mature trophozoites and schizonts. It was abundant in culture supernatants at the time of merozoite release but present in minor amounts if at all in merozoites. The pattern of antigen distribution over schizont-infected cells observed by immunoelectron microscopy differed from that of the precursor of the major merozoite surface antigens in that most of the antigen appeared to be located over the erythrocyte cytoplasm without any obvious association with organelles. It thus appears unlikely that this antigen is present on the merozoite surface prior to schizont rupture.     

The galectin-3-binding protein of Cynoglossus semilaevis is a secreted protein of the innate immune system that binds a wide range of bacteria and is involved in host phagocytosis

Galectin-3 binding protein (G3BP) is a secreted glycoprotein that binds galectin-3 and is involved in various pathological conditions including cancer and viral infection. In fish, G3BP-like sequences have been identified in very few species and their biological properties are entirely unknown. In this work, we reported for the first time the identification and analysis of a teleost G3BP, CsG3BP, from half-smooth tongue sole (Cynoglossus semilaevis). CsG3BP is composed of 565 amino acids and possesses a Scavenger Receptor Cysteine-rich (SRCR) domain, the latter containing six conserved cysteine residues that were predicted to form three intramolecular disulfide bridges. Expression of CsG3BP was detected in a wide range of tissues and upregulated by bacterial and megalocytivirus infection in a time-dependent manner. Immunoblot analysis detected CsG3BP in the culture medium of peripheral blood leukocytes (PBL) and in serum following bacterial stimulation. Purified recombinant CsG3BP (rCsG3BP) exhibited bacterial binding ability in a dose-dependent manner. In contrast, the mutant forms of CsG3BP that bear deletion of the SRCR domain or serine substitutions at three cysteine residues involved in disulfide bond formation lost the capacity of bacterial interaction. rCsG3BP displayed a certain substrate preference and bound more effectively to Gram-negative bacteria than to Gram-positive bacteria. Further study showed that when the CsG3BP produced by PBL was blocked by anti-rCsG3BP antibodies, the phagocytic activity of the cells was significantly reduced. Taken together, these results indicate that CsG3BP is a secreted protein that probably plays a role in innate immune defense by binding to bacterial cells via the SRCR domain and thereby facilitating host phagocytosis.     

SepZ/EspZ is secreted and translocated into HeLa cells by the enteropathogenic Escherichia coli type III secretion system

Enteropathogenic Escherichia coli (EPEC) is a major bacterial cause of infantile diarrhea in developing countries and is the prototype for a group of gastrointestinal pathogens causing characteristic attaching and effacing (A/E) histopathology on intestinal epithelia. A/E pathogens utilize a type III secretion system (TTSS), encoded by the locus of enterocyte effacement (LEE) pathogenicity island, to deliver effector proteins into host cells. Here, we investigate sequence divergence of the LEE-encoded SepZ protein and identify it as a TTSS-secreted and -translocated molecule. SepZ is hypervariable among A/E pathogens, with sequences sharing between 60 to 81% amino acid identity with SepZ of EPEC. A SepZ-CyaA fusion was secreted and translocated into HeLa cells in a TTSS-dependent manner. Additionally, we determined that the first 20 amino acids of SepZ were sufficient to direct its translocation. In contrast to previous studies suggesting a role in invasion and the structure and/or regulation of the TTSS, we found that SepZ does not mediate uptake of EPEC into host cells or affect translocation and tyrosine phosphorylation of the translocated intimin receptor. Immunohistochemistry reveals that, after an extended HeLa cell infection, accumulated SepZ can be detected beneath the site of bacterial attachment in a subset of pedestal regions. To indicate its newly identified status as a translocated effector protein, we propose to rename SepZ as EspZ.     

An actin-binding protein is involved in pestivirus entry into bovine cells

Infection of bovine cells with bovine viral diarrhoea virus (BVDV) can be blocked by the monoclonal antibody (mab) BVD/CA 26, which is directed against a cellular membrane protein. To characterize this molecule, it was isolated and purified by column chromatography. It was found to be an acidic, glycosylated membrane protein consisting of two polypeptide chains of about 28 and 56 kDa. Under non-reducing conditions the chains formed multimers of about 200 kDa. In an actin binding assay the 56 kDa polypeptide chain bound to F-actin as judged by co-sedimentation with actin filaments. Since the target molecule of BVD/CA 26 is localized on the surface of living cells and additionally binds to F-actin, a possible biological function may be to connect the cortical actin filaments with the cellular plasma membrane. The blocking effect of BVD/CA 26 indicates that this cellular plasma membrane protein is involved in the endocytic pathway of BVDV particles.     

Heterogeneity of a Campylobacter jejuni protein that is secreted through the flagellar filament

Cj0859c, or FspA, is a small, acidic protein of Campylobacter jejuni that is expressed by a sigma(28) promoter. Analysis of the fspA gene in 41 isolates of C. jejuni revealed two overall variants of the predicted protein, FspA1 and FspA2. Secretion of FspA occurs in broth-grown bacteria and requires a minimum flagellar structure. The addition of recombinant FspA2, but not FspA1, to INT407 cells in vitro resulted in a rapid induction of apoptosis. These data define a novel C. jejuni virulence factor, and the observed heterogeneity among fspA alleles suggests alternate virulence potential among different strains.     

VirK is a periplasmic protein required for efficient secretion of plasmid-encoded toxin from enteroaggregative Escherichia coli

Despite the autotransporter (AT) moniker, AT secretion appears to involve the function of periplasmic chaperones. We identified four periplasmic proteins that specifically bound to plasmid-encoded toxin (Pet), an AT produced by enteroaggregative Escherichia coli (EAEC). These proteins include the 17-kDa Skp chaperone and the 37-kDa VirK protein. We found that the virK gene is present in different Enterobacteriaceae. VirK bound to misfolded conformations of the Pet passenger domain, but it did not bind to the folded passenger domain or to the β domain of Pet. Assays with an EAECΔvirK mutant and its complemented version showed that, in the absence of VirK, Pet was not secreted but was instead retained in the periplasm as proteolytic fragments. In contrast, Pet was secreted from a Δskp mutant. VirK was not required for the insertion of porin proteins into the outer membrane but assisted with insertion of the Pet β domain into the outer membrane. Loss of VirK function blocked the EAEC-mediated cytotoxic effect against HEp-2 cells. Thus, VirK facilitates the secretion of the AT Pet by maintaining the passenger domain in a conformation that both avoids periplasmic proteolysis and facilitates β-domain insertion into the outer membrane.     

Plasminogen activator inhibitor type 2 cDNA transfected into Chinese hamster ovary cells is stably expressed but not secreted

Immunological screening with a monoclonal antibody probe developed against the plasminogen activator inhibitor type 2 (PAI-2) detected 6 positive clones from approximately 1.7 × 10⁵ recombinant phages in a λgt₁₁ expression library containing cDNA inserts prepared from human placental mRNA. Hybridisation experiments at high stringency indicated that the 6 clones were related. One positive clone was found to produce a fusion protein of Mr 170 000 that was recognised by both monoclonal and polyclonal antibodies against PAI-2. In order to obtain a full length cDNA clone for PAI-2, an additional cDNA library was screened. From this screening, we obtained a cDNA clone that encoded all but a portion of the 5′-untranslated region of the PAI-2 mRNA. Primer extension experiments determined that the 5'-untranslated region was 74 nucleotides in length. The PAI-2 mRNA has an open reading frame of 1245 nucleotides and encodes a 46,6 kDa protein. Analysis of the predicted amino acid sequence revealed that like ovalbumin, PAI-2 has an internal non-cleaved signal peptide. The PAI-2 coding sequence is followed by a 3'-untranslated region of 581 nucleotides. In order to study secretion of PAI-2, a plasmid construct containing the PAI-2 cDNA preceded by the SV40 early promotor was transfected into Chinese hamster ovary cells. The PAI-2 cDNA was efficiently expressed in these cells but unexpectedly the protein was not secreted into the culture medium. The absence of PAI-2 secretion in Chinese hamster ovary cells may be due to the lack of a ‘tissue specific secretory mechanism’ that is present in tissues which normally express PAI-2