Rust secreted protein Ps87 is conserved in diverse fungal pathogens and contains a RXLR-like motif sufficient for translocation into plant cells

Background

Effector proteins of biotrophic plant pathogenic fungi and oomycetes are delivered into host cells and play important roles in both disease development and disease resistance response. How obligate fungal pathogen effectors enter host cells is poorly understood. The Ps87 gene of Puccinia striiformis encodes a protein that is conserved in diverse fungal pathogens. Ps87 homologs from a clade containing rust fungi are predicted to be secreted. The aim of this study is to test whether Ps87 may act as an effector during Puccinia striiformis infection.

Methodology/Principal Findings

Yeast signal sequence trap assay showed that the rust protein Ps87 could be secreted from yeast cells, but a homolog from Magnaporthe oryzae that was not predicted to be secreted, could not. Cell re-entry and protein uptake assays showed that a region of Ps87 containing a conserved RXLR-like motif [K/R]RLTG was confirmed to be capable of delivering oomycete effector Avr1b into soybean leaf cells and carrying GFP into soybean root cells. Mutations in the Ps87 motif (KRLTG) abolished the protein translocation ability.

Conclusions/Significance

The results suggest that Ps87 and its secreted homologs could utilize similar protein translocation machinery as those of oomycete and other fungal pathogens. Ps87 did not show direct suppression activity on plant defense responses. These results suggest Ps87 may represent an “emerging effector” that has recently acquired the ability to enter plant cells but has not yet acquired the ability to alter host physiology.     

Bacterial chitinase is modified and secreted in transgenic tobacco

The chiA gene of Serratia marcescens codes for a secreted protein, bacterial chitinase (ChiA). We have investigated the modifications and the cellular location of ChiA when it is expressed in transgenic tobacco plants. Immunoblots on total leaf protein probed with antibody to ChiA showed that when the bacterial chitinase is expressed in plants, it migrates as a series of discrete bands with either the same or a slower mobility than the secreted bacterial protein. Analysis of the vacuum infiltrate of leaves expressing ChiA showed that the modified forms of the protein are enriched in the intercellular fluid. Media recovered from suspension cultures of cell lines expressing the chiA gene were also enriched for the modified forms of ChiA. Washed protoplasts, however, contained only the nonmodified form. The molecular weight of these polypeptides is reduced by treatment with glycopeptidase F but not with endoglycosidase H. Treatment of the suspension cultures with tunicamycin also leads to reduction in the molecular weight of the chitinase bands. We suggest that some of the ChiA protein is N-glycosylated and secreted when expressed in plants, and that the modifications are complex glycans. These results show that a bacterial signal sequence can function in plant cells, and that protein secretion from plant cells probably operates by a default pathway.     

Characterisation of novel protein families secreted by muscle stage larvae of Trichinella spiralis

Proteins secreted by Trichinella spiralis have a potential role in remodelling host skeletal muscle. However, whilst many parasite-secreted proteins have been identified, it has rarely been demonstrated that these are secreted into the nurse cell. Using an informatics-based analysis, we have searched the T. spiralis expressed sequence tag (EST) datasets for cDNAs encoding potential secreted proteins. Here we describe the characterisation of three of the top candidates isolated from our analysis, termed secreted from muscle stage larvae (SML)-1, -2 and -3. All three proteins were demonstrated to be secreted by muscle stage larvae, and immunohistochemical analysis established that SML-1 and -2 are secreted into developing nurse cells. We also show that SML-2 is processed from a precursor into smaller peptides by a metalloprotease contained within T. spiralis-secreted products. With the identification of these and other secreted proteins, we now have molecules to test in functional assays designed to dissect molecular features of the developing nurse cell.     

Lysine-rich c-zeins are secreted in transgenic Arabidopsis plants

We have previously shown that the maize (Zea mays L.) storage prolamine γ-zein, accumulates in endoplasmic reticulum-derived protein bodies in transgenic plants of Arabidopsis thaliana (L.) ecotype R+P. The retention of γ-zein in the endoplasmic reticulum was found to be mediated by structural features contained in the polypeptide, an N-terminal proline-rich and a C-terminal cysteine-rich domain which were necessary for the correct retention and assembly of γ-zein within protein bodies (M.I. Geli et al., 1994, Plant Cell 6: 1911–1922). In the present work we incorporated in the γ-zein gene lysine-rich coding sequences which were positioned after the N-terminal proline-rich domain and at five amino-acid residues from the C-terminus. The targeting of lysine-rich γ-zeins was analyzed by expression of chimeric genes regulated by the cauliflower mosaic virus (CaMV) 35S promoter in transgenic Arabidopsis plants. The lysine-rich γ-zeins were detected by immunoblotting and we found that these proteins were modified post-translationally to reach their mature form. Subcellular fractionation and immunocytochemical studies demonstrated that glycosylated lysine-rich γ-zeins were secreted to the cell wall of transgenic Arabidopsis leaf cells. Received: 9 May 1997 / Accepted: 31 October 1997     

Molecular characterization and functional analysis of a specific secreted protein from highly virulent defoliating Verticillium dahliae

Verticillium dahliae Kleb. is a phytopathogenic fungus that causes wilt diseases in hundreds of dicotyledonous plant species. Previous research has demonstrated that the secretome plays an important role in the pathogenicity of V. dahliae. In this study, the specific secreted protein gene (VdSSP1) in highly virulent defoliating V. dahliae strain VDG1 was cloned, and considered to be a secreted protein by signal peptide activity assay. VdSSP1 deletion mutants in VDG1 significantly compromised virulence, and the fungal growth decreased in media with pectin and starch as carbon sources. Pathogenicity and carbon utilization were restored upon complementation of the VdSSP1 deletion strains or low virulence non-defoliating strain VDG2, which lacks VdSSP1. It is indicated that the virulence role of VdSSP1 is associated with plant cell wall degradation. In conclusion, our data suggested that VdSSP1 is a secreted protein that is engaged in the pathogenicity of the highly virulent defoliating V. dahliae.     

Ag,a novel protein secreted from Aplysia glia

We have isolated a cDNA (ag for Aplysia glial) corresponding to an mRNA specific to the nervous system of Aplysia californica. In this study, we characterized the ag cDNA sequence and the distribution of ag mRNA and protein in the Aplysia nervous system. The ag cDNA contains an open reading frame that encodes a novel 29 kD protein. In situ hybridizations demonstrate that ag mRNA is conspicuously absent from the cell bodies of the large neurons constituting the external layer of the ganglia. Instead, it is largely confined to a subset of small, apparently non-neuronal cells surrounding the neurons at the border of the neuropil, is sparsely scattered within the neuropil, and is widespread within the connective nerves, a pattern consistent with glial localization. Polyclonal anti-ag antiserum recognizes a protein between 27 and 29 kD that is more broadly distributed, especially within the neuropil. The distributions of ag mRNA and protein, together with the presence of a putative signal peptide, suggest that ag protein is secreted. Two findings support this hypothesis: first, ag protein is detectable by western blot in Aplysia hemolymph. Second, full length ag protein expressed in COS cells is secreted, but ag lacking the putative signal peptide is not. Secretion from glia raises the possibility that this abundant protein may affect neighboring neurons. © 1996 John Wiley & Sons, Inc.     

SARS-CoV-2 accessory protein ORF8 is secreted extracellularly as a glycoprotein homodimer

ORF8 is an accessory protein encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Consensus regarding the biological functions of ORF8 is lacking, largely because the fundamental characteristics of this protein in cells have not been determined. To clarify these features, we herein established an ORF8 expression system in 293T cells. Using this system, approximately 41% of the ORF8 expressed in 293T cells were secreted extracellularly as a glycoprotein homodimer with inter/intramolecular disulfide bonds. Intracellular ORF8 was sensitive to the glycosidase Endo H, whereas the secreted portion was Endo-H-resistant, suggesting that secretion occurs via a conventional pathway. Additionally, immunoblotting analysis showed that the total amounts of the major histocompatibility complex class Ι (MHC-I), angiotensin-converting enzyme 2 (ACE2), and SARS-CoV-2 spike (CoV-2 S) proteins coexpressed in cells were not changed by the increased ORF8 expression, although FACS analysis revealed that the expression of the cell surface MHC-I protein, but not that of ACE2 and CoV-2 S proteins, was reduced by ORF8 expression. Finally, we demonstrate by RNA-seq analysis that ORF8 had no significant stimulatory effects in human primary monocyte-derived macrophages (MDMs). Taken together, our results provide fundamental evidence that the ORF8 glycoprotein acts as a secreted homodimer, and its functions are likely associated with the intracellular transport and/or extracellular signaling in SARS-CoV-2 infection.     

Proteomic characterization of the released/secreted proteins of Leishmania (Viannia) braziliensis promastigotes

Extracellular proteins secreted/released by protozoan parasites are key mediators of the host–parasite interaction. To characterise the profile of proteins secreted/released by Leishmania (Viannia) braziliensis promastigotes, a proteomic approach combining two-dimensional electrophoresis (2DE), tandem matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF/TOF) mass spectrometry, and data mining was carried out. The 2DE map revealed a set of 270 secreted protein spots from which 42 were confidently identified and classified into 11 categories according to Gene Ontology (GeneDB database) and KEEG Ontology annotation of biological processes. Parasite promastigotes were able to secrete/release proteins involved in immunomodulation, signal transduction, and intracellular survival, such as HSP70, acid phosphatase, activated protein kinase C receptor (LACK), elongation factor 1β, and tryparedoxin peroxidase. Data mining showed that ~ 5% of identified proteins present a classical secretion signal whereas ~ 57% were secreted following non-classical secretion mechanisms, indicating that protein export in this primitive eukaryote might proceed mainly by unconventional pathways. This study reports a suitable approach to identify secreted proteins in the culture supernatant of L. braziliensis and provides new perspectives for the study of molecules potentially involved in the early stages of infection.     

Identification and deletion of the major secreted protein of Pichia pastoris

A major contaminating host cell protein was identified in fed batch cultures of Pichia pastoris producing an antibody Fab fragment. Purification and peptide sequencing identified this protein to be related to the cysteine-rich secretory protein family. The same protein was also observed as one of the most abundantly secreted proteins in chemostat cultures of a wild type P. pastoris strain. It has an apparent molecular weight of 65 kDa, 2-fold higher than predicted from the amino acid sequence, which is due to high O-glycosylation. It was denominated extracellular protein X 1 (Epx1), as no clear function could be attributed to it. The EPX1 gene is upregulated in different stress situations, and the respective deletion strain was more susceptible than the wild type to the cell wall damaging agents Calcofluor white and Congo red. The EPX1 deletion strain (Δepx1) was evaluated for its suitability for recombinant protein production. No significant difference in growth and product formation was observed between the wild type and the Δepx1 strain. Batch purification of a Fab fragment produced in the Δepx1 strain highlighted its superior purity due to the decreased host cell protein load.     

Dramatic secretion of recombinant protein expressed in tobacco cells with a designer glycopeptide tag is highly impacted by medium composition

Key message

Cell growth medium composition has profound impacts on the O -glycosylation of a “designer” arabinogalactan protein-based module; full glycosylation is essential in directing efficient extracellular secretion of the tagged recombinant protein.

Abstract

Expression of recombinant proteins in plant cells as fusion with a de novo designed hydroxyproline (Hyp)-O-glycosylated peptide (HypGP) tag, termed HypGP engineering technology, resulted in dramatically increased secreted protein yields. This is due to the function of the HypGP tag as a molecular carrier in promoting efficient transport of conjoined proteins into culture media. To optimize the cell culture to achieve the best secreted protein yields, the medium effects on the cell growth and protein secretion were investigated using as a model system the tobacco BY-2 cell expressing enhanced green fluorescence protein (EGFP) fused with a (SP)₃₂ tag (32 tandem repeats of “Ser-Pro” motif). The (SP)₃₂ tag was found to undergo two-stage Hyp-O-glycosylation in plant cells with the dramatic secretion of the conjoined EGFP correlating with the triggering of the second-stage glycosylation. The BY-2 cell culture in SH medium generated a high secreted protein yield (125 mg/L) with a low cell biomass accumulation (~7.5 gDW/L). In contrast, very low secreted protein yields (~1.5 mg/L) with a high cell biomass accumulation (13.5 gDW/L) were obtained in MS medium. The macronutrients, specifically, the nitrogen supply greatly impacted the glycosylation of the (SP)₃₂ tag and subsequent protein secretion. Modified MS medium with reduced nitrogen levels boosted the secreted EGFP yields to 168 mg/L. This study demonstrates the profound impacts of medium composition on the secreted yields of a HypGP-tagged protein, and provides a basis for medium design to achieve the highest productivity of the HypGP engineering technology.

     

Identification of a cuticle protein with unique repeated motifs in the silkworm,Bombyx mori

The insect cuticle is non-cellular matrix secreted from a monolayer of epidermal cells. After abrasion of the larval cuticle of the silkworm, Bombyx mori, a protein with molecular mass of 135 kDa is newly detected in the cuticle. Mass spectrometric analysis of the tryptic fragments from this protein revealed that the 135-kDa protein is encoded by the Cb10 gene. In the predicted amino acid sequence of Cb10, three repeated motifs with [YxGGFGGppG(L/V)L] sequence are found in the C-terminal region. In addition to the repeated motifs, Cb10 has seventeen CxxxxC motifs randomly distributed throughout the polypeptide chain and serine rich region at the N-terminal region. The Cb10 gene is strongly expressed in epidermal cells after pupal ecdysis, and its expression in the larval epidermal cells is induced not only by cuticular abrasion, but also by bacterial infection. These expression patterns suggest some specific roles of this protein in pupal cuticle formation and defense reactions.     

Properties of secreted protease from vegetative Trichoderma atroviride mycelia cultivated with protein inducer reveal a complex protein-recognition mechanism

In this study, the submerged cultivation of Trichoderma atroviride with protein substrate (bovine serum albumin, casein, ovalbumin, etc.) led to the secretion of protease activity (PA) into the medium. The secretion started within 30 h and rose to the maximum after 72-h cultivation. It continued upon the prolonged cultivation (up to 8 days) with lower secreted PA. Zymography of the secreted protease (SDS-PAGE with 0.2% gelatine), revealing high molecular weight (m.w.) protease(s) (~200 kDa) with high autolytic activity as the only secretory product. Enzymological characteristics of high m.w. proteases elicited by different inducer proteins were surprisingly different, suggesting that mycelia can recognize not only the presence of inducer proteins but also some qualitative characteristics. Low m.w. protease(s), as found from other studies was(were) seen after prolonged cultivation only, as a band with m.w. about 36 kDa. Expression of known Trichoderma spp. genes encoding secreted proteases, prb1 and proA, showed that only prb1 was expressed after 3–4 days of cultivation, i.e., after the early-secretion phase. The secretory activity of the earlier phase was impaired by tunicamycin and brefeldin A and was significantly stimulated by uncoupler. The existence of a biphasic fungal secretory response and the protein quality recognition represent previously unrecognized processes.     

Blue light activates a specific protein kinase in higher plants

Blue light mediates the phosphorylation of a membrane protein in seedlings from several plant species. When crude microsomal membrane proteins from dark-grown pea (Pisum sativum L.), sunflower (Helianthus annuus L.), zucchini (Cucurbita pepo L.), Arabidopsis (Arabidopsis thaliana L.), or tomato (Lycopersicon esculentum L.) stem segments, or from maize (Zea mays L.), barley (Hordeum vulgare L.), oat (Avena sativa L.), wheat (Triticum aestivum L.), or sorghum (Sorghum bicolor L.) coleoptiles are illuminated and incubated in vitro with [γ-³²P]ATP, a protein of apparent molecular mass from 114 to 130 kD is rapidly phosphorylated. Hence, this system is probably ubiquitous in higher plants. Solubilized maize membranes exposed to blue light and added to unirradiated solubilized maize membranes show a higher level of phosphorylation of the light-affected protein than irradiated membrane proteins alone, suggesting that an unirradiated substrate is phosphorylated by a light-activated kinase. This finding is further demonstrated with membrane proteins from two different species, where the phosphorylated proteins are of different sizes and, hence, unambiguously distinguishable on gel electrophoresis. When solubilized membrane proteins from one species are irradiated and added to unirradiated membrane proteins from another species, the unirradiated protein becomes phosphorylated. These experiments indicate that the irradiated fraction can store the light signal for subsequent phosphorylation in the dark. They also support the hypothesis that light activates a specific kinase and that the systems share a close functional homology among different higher plants.     

A growth inhibitory protein secreted by human diploid fibroblasts. Partial purification and characterization

We have identified and partially purified a growth inhibitor protein secreted by human diploid fibroblast cells. This protein is not secreted constitutively but only after induction with the double stranded hetero duplex polyriboinosinic:polyribocytidylic acid. The growth inhibitory activity has been purified 3,800-fold and has an estimated molecular mass of 12,000 daltons. The protein will inhibit the growth in culture of human diploid fibroblast cells, human cells derived from tumors, and mouse L cells. Although interferon-beta is secreted with the growth inhibitory protein, the partially purified growth inhibitory protein has no antiviral activity, and its activity is not neutralized by antibodies to interferon-alpha, interferon-beta, and interferon-gamma. We believe this growth inhibitory activity to reside in a newly defined protein and have named it fibroblast-derived growth inhibitor.     

Purification of Lactobacillus secreted proteins

Summary We describe a rapid and reliable one-step method for purification of Lactobacillus secreted proteins. With electroendosmotic preparative electrophoresis and a modified synthetic medium the L. plantarum aggregation promoting factor, a 32 kDa secreted protein, and the thermostable -amylase of Bacillus stearothermophilus cloned in L. reuteri were purified. Although the growth rate was reduced, the production of secreted proteins was not affected.     

Isolation of baculovirus-derived secreted and full-length beta-amyloid precursor protein

We have expressed two forms of the Alzheimer's beta-amyloid precursor protein (beta APP), the 695-amino acid form (695 beta APP), and the 751-amino acid form (751 beta APP) in a baculovirus system. Both forms were expressed as full-length precursor, and were subsequently processed in vivo to release extracellular secreted proteins. The secreted forms were cleaved from the full-length beta APP in a manner analogous to the cleavage of beta APP during constitutive secretion in mammalian cells (Weidemann, A., K?nig, G., Bunke, D., Fischer, P., Salbaum, J. M., Masters, C. L., Beyreuther, K. (1989) Cell 57, 115-126; Oltersdorf, T., Ward, P. J., Henriksson, T., Beattie, E. C., Neve, R., Lieberburg, I., and Fritz, L. J. (1990) J. Biol. Chem. 265, 4492-4497). High levels of expression of 20-50 mg/liter were achieved. Both full-length and secreted forms of the beta-amyloid precursor proteins were purified using a combination of ion-exchange and immunoaffinity chromatography using a monoclonal antibody directed against beta APP. The 751 beta APP-derived full-length and secreted forms, which contain the Kunitz protease inhibitor domain, were shown to be as active in the inhibition of trypsin as is mammalian-derived secreted beta APP. The availability of purified full-length beta APP from the baculovirus system will be valuable for biochemical and cell biological analyses that may elucidate the mechanism of the inappropriate processing that leads to beta-amyloid formation in Alzheimer's disease.     

Resonance assignment of As-p18, a fatty acid binding protein secreted by developing larvae of the parasitic nematode Ascaris suum

As-p18 is produced and secreted by larvae of the parasitic nematode Ascaris suum as they develop within their eggs. The protein is a member of the fatty acid binding protein (FABP) family found in a wide range of eukaryotes, but is distinctive in that it is secreted from the synthesizing cell and has predicted additional structural features not previously seen in other FABPs. As-p18 and similar proteins found only in nematodes have therefore been designated ‘nemFABPs’. Sequence-specific ¹H, ¹³C and ¹⁵N resonance assignments were established for the 155 amino acid recombinant protein (18.3 kDa) in complex with oleic acid, using a series of three-dimensional triple-resonance heteronuclear NMR experiments. The secondary structure of As-p18 is predicted to be very similar to other FABPs, but the protein has extended loops that have not been observed in other FABPs whose structures have so far been solved.     

Production,characterization, and immunogenicity of a secreted form of Plasmodium falciparum merozoite surface protein 4 produced in Bacillus subtilis

Plasmodium falciparum is the causative agent of the most serious form of malaria. Although a combination of control measures has significantly limited malaria morbidity and mortality in the last few years, it is generally agreed that sustained control or even eradication will require additional tools including an effective malaria vaccine. Merozoite surface protein 4, MSP4, which is present during the asexual stage of P. falciparum, is a recognized target that would be useful in a subunit vaccine against blood stages of malaria. Falciparum malaria is most prevalent in developing countries, and this in turn leads to a requirement for safe, low-cost vaccines. We have attempted to utilize the nonpathogenic, gram-positive organism Bacillus subtilis to produce PfMSP4. PfMSP4 was secreted into the culture medium at a yield of 4.5 mg/L. Characterization studies including SDS-PAGE, mass spectrometry, and N-terminal sequencing indicated that the B. subtilis expression system secreted a full length PfMSP4 protein compared to a truncated version in Escherichia coli. Equivalent amounts of purified B. subtilis and E. coli-derived PfMSP4 were used for immunization studies, resulting in statistically significant higher mean titer values for the B. subtilis-derived immunogen. The mouse antibodies raised against B. subtilis produced PfMSP4 that were reactive to parasite proteins as evidenced by immunoblotting on parasite lysate and indirect immunofluorescence assays of fixed parasites. The B. subtilis expression system, in contrast to E. coli, expresses higher amounts of full length PfMSP4 products, decreased levels of aggregates, and allows the development of simplified downstream processing procedures.     

Developmental regulation of a secreted gelatin-binding protein during myogenesis in vitro

Collagen has a stimulatory effect on the differentiation of skeletal muscle cells in culture. Putative collagen-binding proteins were isolated from detergent-solubilized cultures of the L6 rat muscle cell line and primary clonal cultures of human skeletal muscle satellite cells, using gelatin-Sepharose affinity chromatography. In addition to fibronectin, which has been reported by others to be synthesized by cultured muscle cells, we found that muscle cultures synthesized gelatin-binding proteins of lower apparent molecular weight. Only one of these proteins was secreted into the growth medium and bound to type I collagen. Binding of this protein to gelatin and collagen-Sepharose was resistant to repeated washing with 1 M NaCl and nonionic detergent. The secreted gelatin-binding protein had an apparent molecular weight of 63,000-72,000, depending upon the conditions of electrophoresis. The lack of reactivity of the secreted protein with polyclonal antisera against fibronectin, the lack of effect of protease inhibitors on its appearance in the medium, and the rapid de novo production of the protein during pulse labeling with radioactive methionine indicated that it was not a fibronectin fragment. The rate of synthesis of the secreted gelatin-binding protein increased markedly during the myogenesis of rat and human cultures.