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.

Immunochemical methods for the rapid screening of the o-glycosidically linked N-acetylglucosamine modification of proteins.

for the rapid screening of specific post-translational modifications antibody-based methods are very well suited and applicable without demanding expenditure. Here we describe the immunochemical detection of the O-glycosidically linked cytosolic N-acetylglucosamine modification of proteins, which has attracted increasing interest in the last years. Two different monoclonal antibodies were used in enzyme-linked immunosorbent assays (ELISA), Western blots of 1- and 2- dimension (1D and 2D) separated proteins and immunohistochemical analysis of tissue sections. Slight differences in the recognition of this post-translational epitope by the 2 antibodies are observed and will be discussed.

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.