The PCome of Ascaris suum as a model system for intestinal nematodes: identification of phosphorylcholine-substituted proteins and first characterization of the PC-epitope structures.

In multicellular parasites (e.g., nematodes and protozoa), proteins and glycolipids have been found to be decorated with phosphorylcholine (PC). PC can provoke various effects on immune cells leading to an immunomodulation of the host's immune system. This immunomodulation allows long-term persistence but also prevents severe pathology due to downregulation of cellular immune responses. PC-containing antigens have been found to interfere with key proliferative signaling pathways in B and T cells, development of dendritic cells and macrophages, and mast cell degranulation. These effects contribute to the observed modulated cytokine levels and impairment of lymphocyte proliferation. In contrast to glycosphingolipids, little is known about the PC-epitopes of proteins. So far, only a limited number of PC-modified proteins from nematodes have been identified. In this project, PC-substituted proteins and glycolipids in Ascaris suum have been localized by immunohistochemistry in specific tissues of the body wall, intestine, and reproductive tract. Subsequently, we investigated the PCome of A. suum by 2D gel-based proteomics and detection by Western blotting using the PC-specific antibody TEPC-15. By peptide-mass-fingerprint matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), we could identify 59 PC-substituted proteins, which are in involved multiple cellular processes. In addition to membrane proteins like vitellogenin-6, we found proteins with structural (e.g., tubulins) and metabolic (e.g., pyruvate dehydrogenase) functions or which can act in the defense against the host's immune response (e.g., serpins). Initial characterization of the PC-epitopes revealed a predominant linkage of PC to the proteins via N-glycans. Our data form the basis for more detailed investigations of the PC-epitope structures as a prerequisite for comprehensive understanding of the molecular mechanisms of immunomodulation.

Detection and site localization of phosphorylcholine-modified peptides by NanoLC-ESI-MS/MS using precursor ion scanning and multiple reaction monitoring experiments.

Phosphorylcholine (PC)-modified biomolecules like lipopolysaccharides, glycosphingolipids, and (glyco)proteins are widespread, highly relevant antigens of parasites, since this small hapten shows potent immunomodulatory capacity, which allows the establishment of long-lasting infections of the host. Especially for PC-modified proteins, structural data is rare because of the zwitterionic nature of the PC substituent, resulting in low sensitivities and unusual but characteristic fragmentation patterns. We have developed a targeted mass spectrometric approach using hybrid triple quadrupole/linear ion trap (QTRAP) mass spectrometry coupled to nanoflow chromatography for the sensitive detection of PC-modified peptides from complex proteolytic digests, and the localization of the PC-modification within the peptide backbone. In a first step, proteolytic digests are screened using precursor ion scanning for the marker ions of choline (m/z 104.1) and phosphorylcholine (m/z 184.1) to establish the presence of PC-modified peptides. Potential PC-modified precursors are then subjected to a second analysis using multiple reaction monitoring (MRM)-triggered product ion spectra for the identification and site localization of the modified peptides. The approach was first established using synthetic PC-modified synthetic peptides and PC-modified model digests. Following the optimization of key parameters, we then successfully applied the method to the detection of PC-peptides in the background of a proteolytic digest of a whole proteome. This methodological invention will greatly facilitate the detection of PC-substituted biomolecules and their structural analysis.

The WD40-repeat protein Han11 functions as a scaffold protein to control HIPK2 and MEKK1 kinase functions.

Protein kinases are organized in hierarchical networks that are assembled and regulated by scaffold proteins. Here, we identify the evolutionary conserved WD40-repeat protein Han11 as an interactor of the kinase homeodomain-interacting protein kinase 2 (HIPK2). In vitro experiments showed the direct binding of Han11 to HIPK2, but also to the kinases DYRK1a, DYRK1b and mitogen-activated protein kinase kinase kinase 1 (MEKK1). Han11 was required to allow coupling of MEKK1 to DYRK1 and HIPK2. Knockdown experiments in Caenorhabditis elegans showed the relevance of the Han11 orthologs Swan-1 and Swan-2 for the osmotic stress response. Downregulation of Han11 in human cells lowered the threshold and amplitude of HIPK2- and MEKK1-triggered signalling events and changed the kinetics of kinase induction. Han11 knockdown changed the amplitude and time dependence of HIPK2-driven transcription in response to DNA damage and also interfered with MEKK1-triggered gene expression and stress signalling. Impaired signal transmission also occurred upon interference with stoichiometrically assembled signalling complexes by Han11 overexpression. Collectively, these experiments identify Han11 as a novel scaffold protein regulating kinase signalling by HIPK2 and MEKK1.

Immunomodulation by phosphocholine--biosynthesis, structures and immunological implications of parasitic PC-epitopes.

Phosphocholine (PC) as a small haptenic molecule present on antigens of parasites can provoke various effects on immune cells leading to immunomodulation of the host's immune system. This immunomodulation not only allows long-term persistence but also prevents severe pathology due to down-regulation of cellular immune responses. Additionally, PC plays an important role for development and fertility of the parasites. To fully understand the mechanisms of immunomodulation the detailed knowledge of the biosynthesis of the PC-epitopes, their molecular structure and biological function has to be elucidated. The implication of parasite-specific transferases in the biosynthesis of the PC-epitopes and the sensitivity of parasites towards disruption of the choline metabolism offers new perspectives for the development of anti-parasitic drugs and therapies. Furthermore, the immunomodulation provoked by PC-epitopes preventing inflammatory reactions may be useful in the treatment of inflammatory diseases. This review summarizes the current knowledge on the biosynthesis of PC-epitopes, their structures and immunological implications.

The PCome of Caenorhabditis elegans as a prototypic model system for parasitic nematodes: identification of phosphorylcholine-substituted proteins.

The decoration of proteins and glycolipids with phosphorylcholine (PCho) has been shown in many organisms ranging from bacteria to multicellular parasites like nematodes. For bacteria this modifications is involved in invasion and persistence for pathogens. However, little is still known about the distribution of this modification on proteins, the precise epitope structures, and functions. In nematodes, the PCho-modification is widespread and at least on the glycosphingolipid level it represents a phylogenetic marker within the helminths. Nematode infections are still one of the most abundant diseases world-wide. Caenorhabditis elegans as the best characterized organism is an ideal model system for studying this type of protein modification and can therefore be regarded as a prototypic model system for parasitic nematodes. Interference with the PCho-decoration by targeting the glycosphingolipid biosynthesis and the choline metabolism has been shown to reduce nematode viability and fertility. Thus, the PCho-modification seems to play an additional important role for the development of nematodes. The development of drugs interfering with the PCho-substitution might, therefore, be a promising way for the development of new anthelminthic strategies. In this study we have analyzed the PCome of C. elegans to identify the PCho-modified proteins. Furthermore, we investigated the dynamics of this modification by analyzing the different developmental stages of this nematode. Our results demonstrate highly dynamic changes of this modification during development. Furthermore, we could show that this substitution can occur on proteins with large functional diversity and subcellular localization. We could further demonstrate that the PCho-modification greatly depends on proper N-glycosylation. However, there is clear indication that there might be a high structural diversity of the PCho-epitopes.

Separation of membrane proteins by two-dimensional electrophoresis using cationic rehydrated strips.

Due to their poor solubility during IEF membrane proteins cannot be separated and analyzed satisfactorily with classical 2-DE. A more efficient method for such hydrophobic proteins is the benzyldimethyl-n-hexadecylammonium chloride (16-BAC)/SDS-PAGE, but the corresponding protocol is intricate and time-consuming. We now developed an easy-to-handle electrophoresis method in connection with a novel device which enables reproducible separation of ionic solubilized membrane proteins using individually rehydrated plastic sheet gel strips. These strips are suitable for the first dimension in a 2-D 16-BAC/SDS system and can be handled easily; this is demonstrated by the separation of membrane proteins of human embryonic kidney (HEK293) cells.

First identification of a phosphorylcholine-substituted protein from Caenorhabditis elegans: isolation and characterization of the aspartyl protease ASP-6.

Caenorhabditis elegans is a widely accepted model system for parasitic nematodes, drug screening and developmental studies. Similar to parasitic worms, C. elegans expresses glycosphingolipids and glycoproteins carrying, in part, phosphorylcholine (PCho) substitutions, which might play important roles in nematode development, fertility and, at least in the case of parasites, survival within the host. With the exception of a major secretory/excretory product from Acanthocheilonema viteae (ES-62), no protein carrying this epitope has been studied in detail yet. Here we report on the identification, characterization and localization of the aspartyl protease ASP-6 of C. elegans, which is excreted by the nematode in a PCho-substituted form. Within the worm, most prominent expression of the protein is observed in the intestine, while muscle and epithelial cells express asp-6 to a lesser extent. In animals harboring an ASP-6::GFP fusion protein, diffuse fluorescence throughout the body cavity of adult worms indicates that the chimeric protein is secreted.

Identification of phosphorylcholine substituted peptides by their characteristic mass spectrometric fragmentation.

Phosphorylcholine (PC) substituted biomolecules are wide-spread, highly relevant antigens of parasites, since this small hapten has been found to be a potent immunomodulatory component which allows the establishment of long lasting infections of the host. Structural data, especially of protein bound PC-substituents, are still rare due to the observation that mass spectrometric analyses are mostly hampered by this zwitterionic substituent resulting in low sensitivities and unusual but characteristic fragmentation patterns. Here we investigated the fragmentation behaviour of synthetic PC-substituted peptides by matrix-assisted laser desorption/ionization mass spectrometry and electrospray ionization ion trap mass spectrometry. We could show that the predominant neutral loss of a trimethylamine unit (Hoffmann elimination) leads to cyclic phosphate derivatives which prevent further fragmentation of the peptide backbone by stabilizing the positive charge at this particular side chain. Knowledge of this PC-specific fragmentation might help to identify PC-substituted biomolecules and facilitate their structural analysis.