Proteomic identification of IPSE/alpha-1 as a major hepatotoxin secreted by Schistosoma mansoni eggs

Background

Eggs deposited in the liver of the mammalian host by the blood fluke parasite, Schistosoma mansoni, normally drive a T-helper-2 (Th2)-mediated granulomatous response in immune-competent mice. By contrast, in mice deprived of T-cells and incapable of producing granulomata, egg-secreted proteins (ESP) induce acute hepatic injury and death. Previous work has shown that one such ESP, the T2 ribonuclease known as omega-1, is hepatotoxic in vivo in that specific antisera to omega-1 prevent hepatocyte damage.

Methodology/Principal Findings

Using an in vitro culture system employing mouse primary hepatocytes and alanine transaminase (ALT) activity as a marker of heptocyte injury, we demonstrated that S. mansoni eggs, egg-secreted proteins (ESP), soluble-egg antigen (SEA), and omega-1 are directly hepatotoxic and in a dose-dependent manner. Depletion of omega-1 using a monoclonal antibody abolished the toxicity of pure omega-1 and diminished the toxicity in ESP and SEA by 47 and 33%, respectively. Anion exchange chromatography of ESP yielded one predominant hepatotoxic fraction. Proteomics of that fraction identified the presence of IPSE/alpha-1 (IL-4 inducing principle from S. mansoni eggs), a known activator of basophils and inducer of Th2-type responses. Pure recombinant IPSE/alpha-1 also displayed a dose-dependent hepatotoxicity in vitro. Monoclonal antibody depletion of IPSE/alpha-1 abolished the latter''s toxicity and diminished the total toxicity of ESP and SEA by 32 and 35%, respectively. Combined depletion of omega-1 and IPSE/alpha-1 diminished hepatotoxicity of ESP and SEA by 60 and 58% respectively.

Conclusions

We identified IPSE/alpha-1 as a novel hepatotoxin and conclude that both IPSE/alpha-1 and omega-1 account for the majority of the hepatotoxicity secreted by S. mansoni eggs.     

Phosphorylation of the acetylcholine receptor by protein kinase C and identification of the phosphorylation site within the receptor delta subunit

Purified acetylcholine receptor is rapidly and specifically phosphorylated by partially purified protein kinase C, the Ca2+/phospholipid-dependent enzyme. The receptor delta subunit is the major target for phosphorylation and is phosphorylated on serine residues to a final stoichiometry of 0.4 mol of phosphate/mol of subunit. Phosphorylation is dose-dependent with a Km value of 0.2 microM. Proteolytic digestion of the delta subunit phosphorylated by either protein kinase C or the cAMP-dependent protein kinase yielded a similar pattern of phosphorylated fragments. The amino acids phosphorylated by either kinase co-localized within a 15-kDa proteolytic fragment of the delta subunit. This fragment was visualized by immunoblotting with antibodies against a synthetic peptide corresponding to residues 354-367 of the receptor delta subunit. This sequence, which contains 3 consecutive serine residues, was recently shown to include the cAMP-dependent protein kinase phosphorylation site (Souroujon, M. C., Neumann, D., Pizzighella, S., Fridkin, M., and Fuchs, S. (1986) EMBO J. 5, 543-546). Concomitantly, the synthetic peptide 354-367 was specifically phosphorylated in a Ca2+- and phospholipid-dependent manner by protein kinase C. Furthermore, antibodies directed against this peptide inhibited phosphorylation of the intact receptor by protein kinase C. We thus conclude that both the cAMP-dependent protein kinase and protein kinase C phosphorylation sites reside in very close proximity within the 3 adjacent serine residues at positions 360, 361, and 362 of the delta subunit of the acetylcholine receptor.     

Selective phosphorylation of the alpha subunit of the sodium channel by cAMP-dependent protein kinase

The alpha subunit of the sodium channel purified from rat brain is rapidly and selectively phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a level of 3 to 4 mol of 32P/mol of saxitoxin-binding activity. The rate of phosphorylation is comparable to that of the synthetic peptide analog of the phosphorylation site of pyruvate kinase, one of the best substrates for cAMP-dependent protein kinase. An endogenous cAMP-dependent protein kinase that is present in the partially purified sodium channel preparations also selectively phosphorylates the alpha subunit. The specificity and rapidity of the phosphorylation reaction are consistent with the hypothesis that the alpha subunit is phosphorylated by cAMP-dependent protein kinase in vivo.     

Phosphorylation of human CTP synthetase 1 by protein kinase A: identification of Thr455 as a major site of phosphorylation

CTP synthetase is an essential enzyme that generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this study, we examined the phosphorylation of the human CTPS1-encoded CTP synthetase 1 by protein kinase A. CTP synthetase 1 was expressed and purified from a Saccharomyces cerevisiae ura7Delta ura8Delta double mutant that lacks CTP synthetase activity. Using purified CTP synthetase 1 as a substrate, protein kinase A activity was time- and dose-dependent. The phosphorylation, which primarily occurred on a threonine residue, was accompanied by a 50% decrease in CTP synthetase 1 activity. The synthetic peptide LGKRRTLFQT that contains the protein kinase A motif for Thr(455) was a substrate for protein kinase A. A Thr(455) to Ala (T455A) mutation in CTP synthetase 1 was constructed by site-directed mutagenesis and was expressed and purified from the S. cerevisiae ura7Delta ura8Delta mutant. The T455A mutation caused a 78% decrease in protein kinase A phosphorylation and the loss of the phosphothreonine residue and a major phosphopeptide that were present in the purified wild type enzyme phosphorylated by protein kinase A. The CTP synthetase 1 activity of the T455A mutant enzyme was 2-fold higher than the wild type enzyme. In addition, the T455A mutation caused a 44% decrease in the amount of human CTP synthetase 1 that was phosphorylated in S. cerevisiae cells, and this was accompanied by a 2.5-fold increase in the cellular concentration of CTP and a 1.5-fold increase in the choline-dependent synthesis of phosphatidylcholine.     

A positive feedback loop between protein kinase CKII and Cdc37 promotes the activity of multiple protein kinases

We report here the identification of CDC37, which encodes a putative Hsp90 co-chaperone, as a multicopy suppressor of a temperature-sensitive allele (cka2-13(ts)) of the CKA2 gene encoding the alpha' catalytic subunit of protein kinase CKII. Unlike wild-type cells, cka2-13 cells were sensitive to the Hsp90-specific inhibitor geldanamycin, and this sensitivity was suppressed by overexpression of either Hsp90 or Cdc37. However, only CDC37 was capable of suppressing the temperature sensitivity of a cka2-13 strain, implying that Cdc37 is the limiting component. Immunoprecipitation of metabolically labeled Cdc37 from wild-type versus cka2-13 strains revealed that Cdc37 is a physiological substrate of CKII, and Ser-14 and/or Ser-17 were identified as the most likely sites of CKII phosphorylation in vivo. A cdc37-S14,17A strain lacking these phosphorylation sites exhibited severe growth and morphological defects that were partially reversed in a cdc37-S14,17E strain. Reduced CKII activity was observed in both cdc37-S14A and cdc37-S17A mutants at 37 degrees C, and cdc37-S14A or cdc37-S14,17A overexpression was incapable of protecting cka2-13 mutants on media containing geldanamycin. Additionally, CKII activity was elevated in cells arrested at the G(1) and G(2)/M phases of the cell cycle, the same phases during which Cdc37 function is essential. Collectively, these data define a positive feedback loop between CKII and Cdc37. Additional genetic assays demonstrate that this CKII/Cdc37 interaction positively regulates the activity of multiple protein kinases in addition to CKII.     

Three major surface antigens of Schistosoma mansoni are linked to the membrane by glycosylphosphatidylinositol

Schistosomula of Schistosoma mansoni were examined for the presence of glycosylphosphatidylinositol (GPI) anchored surface membrane Ag. Parasites were surface iodinated and cultured in the presence or absence of a crude phospholipase C (PLC) preparation or phosphatidylinositol-specific PLC (PIPLC). Culture supernatants were then analyzed: 1) by centrifugation to ascertain which molecules released from the surface were soluble or contained in membrane vesicles; 2) by immunoprecipitation with antibodies specific for the "cross-reacting determinant," an epitope revealed on some GPI-anchored proteins only after cleavage of the diacylglycerol from the protein by PIPLC, and 3) by immunoprecipitation with immune mouse sera to establish co-identity with previously described, immunologically relevant surface Ag. By using these techniques, schistosomula were shown to possess three GPI-anchored surface Ag of m.w. 38,000, 32,000 and 18,000 which are spontaneously released from the surface of schistosomula in association with membrane, but remain insoluble until cleaved by PIPLC. All three molecules were recognized by antibodies from mice vaccinated with irradiated cercariae and/or chronically infected mice. Moreover, the m.w. 38,000 component was recognized by a previously described protective mAb (E.1). A major developmental modification appears to occur in the expression of these molecules because, by the same techniques, no GPI-anchored surface Ag were detectable on 7-day-old lung stage parasites. The finding that these important parasite immunogens are GPI-anchored and released from the surface of the parasite in membrane vesicles may, in part, explain why they elicit strong immune responses capable of damaging the schistosomulum tegument.     

Differential regulation of basic protein phosphorylation by calcium phospholipid and cyclic-AMP-dependent protein kinases

Myelin basic protein, an 80-kilodalton (kDa) protein in rat oligodendrocytes, and an 80-kDa basic protein in neuroblastoma x neonatal Chinese hamster brain explant hybrids were phosphorylated extensively when the cells were treated with either phorbol esters (TPA) or diacylglycerols (e.g., oleyoyl-acetylglycerol). TPA-stimulated phosphorylation was inhibited by pre-incubation with 50 microM psychosine (galactosyl-sphingosine), confirming that it is mediated through the phospholipid-dependent protein kinase C (PK-C). Surprisingly, phosphorylation of these proteins was inhibited by incubation of cells with agents which result in activation of cyclic-AMP-dependent protein kinase (dibutyryl cyclic AMP or forskolin). In contrast, phosphorylation of other nonbasic proteins, for example, the oligodendrocyte-specific 2',3'-cyclic nucleotide phosphohydrolase, was stimulated under these conditions (Vartanian et al.: Proceedings of the National Academy of Sciences of the United States of America 85:939, 1988). The possible role of cyclic AMP in activating specific phosphatases or restricting the availability of diacylglycerol for PK-C activation is discussed.     

Human peripheral blood mononuclear cells stimulated by Schistosoma mansoni antigens: association between protein tyrosine kinases, mitogen-activated protein kinases and cytokine production

Concerning schistosomiasis, little is known about the intracellular signaling response of human peripheral blood mononuclear cells (PBMC) to Schistosoma mansoni antigens. To understand the critical role of protein tyrosine kinases (PTKs) in PBMC activation by S. mansoni antigens, we investigated how inhibition of PTKs by genistein, a tyrosine kinase inhibitor, affects proliferation, cytokine production and activation of mitogen-activated protein kinases (MAPKs). Our studies showed that PTKs have an important role in proliferation of PBMC from chronic schistosomiasis patients as cells stimulated with S. mansoni soluble antigens in the presence of genistein had an impaired proliferation. In contrast, PTK inhibition failed to cause any effect on MAPKs activity. We also evaluated the cytokine production for interleukin (IL)-2, interferon gamma (IFN-gamma), and IL-10 in culture supernatants of PBMC treated with or without PTKs inhibitor. Our results show that PBMC from chronic patients produced a high amount of IL-10 when stimulated with soluble egg antigen preparation (SEA), however, the amount produced of IL-2 and IFN-gamma was not significant. In the presence of PTKs inhibitor only the production of IL-10 was decreased. The findings suggest that PTKs are involved on signal transduction pathway for PBMC activation, but may not be an absolute requirement for all signaling responses to S. mansoni antigens.     

Cell cycle-dependent phosphorylation and ubiquitination of a G protein alpha subunit

A diverse array of external stimuli, including most hormones and neurotransmitters, bind to cell surface receptors that activate G proteins. Mating pheromones in yeast Saccharomyces cerevisiae activate G protein-coupled receptors and initiate events leading to cell cycle arrest in G(1) phase. Here, we show that the Gα subunit (Gpa1) is phosphorylated and ubiquitinated in response to changes in the cell cycle. We systematically screened 109 gene deletion strains representing the non-essential yeast kinome and identified a single kinase gene, ELM1, as necessary and sufficient for Gpa1 phosphorylation. Elm1 is expressed in a cell cycle-dependent manner, primarily at S and G(2)/M. Accordingly, phosphorylation of Gpa1 in G(2)/M phase leads to polyubiquitination in G(1) phase. These findings demonstrate that Gpa1 is dynamically regulated. More broadly, they reveal how G proteins can simultaneously regulate, and become regulated by, progression through the cell cycle.     

In vivo regulation of protein synthesis by phosphorylation of the alpha subunit of wheat eukaryotic initiation factor 2

The regulation of protein synthesis is a critical component in the maintenance of cellular homeostasis. A major mechanism of translational control in response to diverse abiotic and biotic stress signals involves the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). The pathway has been demonstrated in all eukaryotes except plants, although components of a putative plant pathway have been characterized. To evaluate the in vivo capability of plant eIF2alpha to participate in the translation pathway, we have used vaccinia virus recombinants that constitutively express wheat eIF2alpha and inducibly express the eIF2alpha dsRNA-stimulated protein kinase, PKR, in BSC-40 cells. Activation of PKR in cells expressing wild-type wheat eIF2alpha resulted in an inhibition of cellular and viral protein synthesis and an induction of cellular apoptosis correlating with phosphorylation of eIF2alpha on serine 51. Expression of a nonphosphorylatable mutant (51A) of plant eIF2alpha reversed the PKR-mediated translational block as well as the PKR-induced apoptosis. A direct interaction of the plant proteins with the mammalian translational initiation apparatus is supported by coimmunoprecipitation of wild-type plant eIF2alpha and the 51A mutant with mammalian eIF2gamma and the localization of the plant proteins in ribosome fractions. These findings suggest that plant eIF2alpha is capable of interacting with the guanine nucleotide exchange factor eIF2B within the context of the eIF2 holoenzyme and provide direct evidence for its ability to participate in phosphorylation-mediated translational control in vivo.     

Phosphorylation of membrane-bound acetylcholine receptor by protein kinase C: characterization and subunit specificity

Acetylcholine receptor (AChR) from Torpedo electric organ in its membrane-bound or solubilized form is phosphorylated by the Ca2+/phospholipid-dependent protein kinase (PKC). The subunit specificity for PKC is different from that observed for cAMP-dependent protein kinase (PKA). Whereas PKC phosphorylates predominantly the delta subunit and the phosphorylation of the gamma subunit by this enzyme is very low, PKA phosphorylates both subunits to a similar high extent. We have extended our phosphorylation studies to a synthetic peptide from the gamma subunit, corresponding to residues 346-359, which contains a consensus PKA phosphorylation site. This synthetic peptide is phosphorylated by both PKA and PKC, suggesting that in the intact receptor both kinases may phosphorylate the gamma subunit at a similar site, as has been previously demonstrated by us for the delta subunit [Safran, A., et al. (1987) J. Biol. Chem. 262, 10506-10510]. The diverse pattern of phosphorylation of AChR by PKA and PKC may play a role in the regulation of its function.     

Heparin, a powerful inhibition of type II casein kinases, stimulates the phosphorylation of some protein substrates by the catalytic subunit of cAMP-dependent protein kinase

Heparin, which has been shown to behave as a very effective and specific inhibitor of type II casein kinases, exhibits a stimulatory effect on the phosphorylation rate of pyruvate kinase and phosphorylase kinase, but not of histones, by the catalytic subunit of cAMP-dependent protein kinase. When pyruvate kinase is the substrate the phosphorylation rate is approximately doubled by heparin concentrations around 100 micrograms/ml, but just 2 to 4 micrograms heparin per ml are sufficient to induce a half maximal effect. No stimulation by heparin can be observed replacing the protein substrates with two synthetic peptides reproducing the phosphorylatable sites of pyruvate kinase and of the gamma subunit of phosphorylase kinase. These data support the hypothesis that heparin accelerates phosphorylation by rendering the phosphorylatable sites more readily accessible to the protein kinase.     

Schistosoma mansoni: a comparison of secreted vs nonsecreted eicosanoids in developing schistosomulae and adults

We have determined that developing schistosomulae and adults of Schistosoma mansoni synthesize a wide range of eicosanoids when stimulated with linoleic acid, an essential fatty acid. Developing schistosomulae secrete 64%, while adults secrete over 80% of synthesized eicosanoids. On a per milligram soluble protein basis, eicosanoid secretion is ordered as follows: adult females greater than adult males much much greater than developing schistosomulae. Together one mature adult worm pair secreted approximately 4.36 micrograms prostaglandin E, 3.41 micrograms leukotriene B4, and 15.13 micrograms 5-hydroxyeicosatetraenoic acid (HETE) as determined by radioimmunoassay (RIA). High-performance liquid chromatography (HPLC) results have determined that 15-HETE is the major HETE species secreted by adults and developing schistosomulae. The immunosuppressant roles of 15-HETE, PGE, and LTB4 are discussed in relation to a possible mechanism for S. mansoni to evade the host immune system. Adults and schistosomules of S. mansoni have evolved rather sophisticated mechanisms for evading the host immune response. These include both host antigen acquisition and antigen shedding. In addition, young schistosomes have an as yet unidentified intrinsic defense mechanism against the host immune system. We postulate that part of the defense mechanism in schistosomules and adults may involve secretion of immunosuppressant eicosanoid species.     

Stimulation of human DNA topoisomerase II activity by its direct association with the beta subunit of protein kinase CKII

DNA topoisomerase II copurifies with and is phosphorylated by protein kinase CKII. In this study, a yeast two-hybrid system was used to investigate the interaction between human topoisomerase II isozymes and CKII subunits. The two-hybrid test clearly showed that both topoisomerase IIalpha and IIbeta interact with the CKIIbeta, but not the CKIIalpha subunit. The two-hybrid test also demonstrated that topoisomerase IIbeta residues 1099-1263 and topoisomerase IIalpha residues 1078-1182 mediate the interaction with the CKIIbeta subunit, providing evidence that the leucine zipper motif and the major CKII-dependent phosphorylation sites of topoisomerase II are unnecessary for its physical binding to CKIIbeta. Furthermore, a DNA relaxation assay demonstrated that the CKII subunit enhances topoisomerase II activity by physical interaction with topoisomerase II.     

Direct identification of tyrosine 474 as a regulatory phosphorylation site for the Akt protein kinase

Understanding the regulation of Akt has been of major interest for elucidating the control of normal cellular physiology as well as malignant transformation. The paradigm for activation of Akt involves phosphatidylinositol 3-kinase-dependent membrane localization followed by activating phosphorylation of Thr-308 and Ser-473. Many of the activating signals for Akt involve the stimulation of receptor and non-receptor tyrosine kinases, and the most potent activator known is the tyrosine phosphatase inhibitor pervanadate, highlighting a possible role for tyrosine phosphorylation in the regulation of the enzyme. In this study we show that activation of Akt by pervanadate or serum is associated with tyrosine phosphorylation of Akt. In addition, in SKOV3 ovarian carcinoma cells that exhibit high basal levels of Akt activity, Akt was tyrosine-phosphorylated in the basal state, and this phosphorylation was further enhanced by both pervanadate and insulin-like growth factor-1. We have used NH(2)-terminal sequencing and phosphate release analysis to directly identify Tyr-474 as the site of tyrosine phosphorylation. Substitution of Tyr-474 with phenylalanine abolished tyrosine phosphorylation of Akt and resulted in up to 55% inhibition of Akt activation, indicating phosphorylation at Tyr-474 is required for full activation of the kinase. Our data identifies a novel regulatory mechanism for this pleiotropic enzyme that may be applicable to the AGC family of protein kinases given the conserved nature of the COOH-terminal hydrophobic motif containing Tyr-474.     

Phosphorylation of mammalian myosin light chain kinases by the catalytic subunit of cyclic AMP-dependent protein kinase and by cyclic GMP-dependent protein kinase

The phosphorylation of the calmodulin-dependent enzyme myosin light chain kinase, purified from bovine tracheal smooth muscle and human blood platelets, by the catalytic subunit of cAMP-dependent protein kinase and by cGMP-dependent protein kinase was investigated. When myosin light chain kinase which has calmodulin bound is phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, 1 mol of phosphate is incorporated per mol of tracheal myosin light chain kinase or platelet myosin light chain kinase, with no effect on the catalytic activity. Phosphorylation when calmodulin is not bound results in the incorporation of 2 mol of phosphate and significantly decreases the activity. The decrease in myosin light chain kinase activity is due to a 5 to 7-fold increase in the amount of calmodulin required for half-maximal activation of both tracheal and platelet myosin light chain kinase. In contrast to the results with the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase cannot phosphorylate tracheal myosin light chain kinase in the presence of bound calmodulin. When calmodulin is not bound to tracheal myosin light chain kinase, cGMP-dependent protein kinase phosphorylates only one site, and this phosphorylation has no effect on myosin light chain kinase activity. On the other hand, cGMP-dependent protein kinase incorporates phosphate into two sites in platelet myosin light chain kinase when calmodulin is not bound. The sites phosphorylated by the two cyclic nucleotide-dependent protein kinases were compared by two-dimensional peptide mapping following extensive tryptic digestion of the phosphorylated myosin light chain kinases. With respect to the tracheal myosin light chain kinase, the single site phosphorylated by cGMP-dependent protein kinase when calmodulin is not bound appears to be the same site phosphorylated in the tracheal enzyme by the catalytic subunit of cAMP-dependent protein kinase when calmodulin is bound. With respect to the platelet myosin light chain kinase, the additional site that was phosphorylated by cGMP-dependent protein kinase when calmodulin was not bound was different from that phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.