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

Abstract

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.

The Eukaryotic Elongation Factor 1 Alpha (eEF1α) from the Parasite Leishmania infantum Is Modified with the Immunomodulatory Substituent Phosphorylcholine (PC)

“…In eF1α from L. infantum , we found no evidence for glycosylation. This confirms our observations from its homologue in A. suum : here too, the PC seems to be directly coupled to the sidechains of amino acids [ 47 ].…”

“…To verify a phosphorylcholine modification of eEF1α, we made use of a HPLC-based method developed by the group of J. Klein [ 47 ]. In this method, the retention time of choline was observed on an ion-exchange column.…”

“…To remove lipid contaminants and SDS proteins were precipitated with chloroform/methanol (1:4 by v / v ). For isoelectric focusing, the protein pellet was dissolved in 100 μL lysis buffer containing 4% 3-3′-(Cholamidopropyl)-3,3-dimethylammoniumpropylsulfat (CHAPS; Roth, Karlsruhe, Germany) instead of Triton X-100 and 2% IPG-buffer pH 3–10 [ 47 ].…”

“…Two-dimensional separation, Western blotting of proteins and detection of PC-modified proteins was performed as described in [ 47 ]. For preparative gels 0.4–0.5 mg, and for Western blot analysis 25 µg of the extracted protein were loaded.…”

TASK-1 potassium channel is not critically involved in mediating hypoxic pulmonary vasoconstriction of murine intra-pulmonary arteries

“…2-D Gel electrophoresis followed by western blotting was performed as described earlier [ 29 , 30 ]. Briefly, cerebellum was dissected from WT and TASK-1 KO mice.…”