Snodgrass, Casey et al. (2015) International Worm Meeting "Phosphorylcholine-modified glycoproteins involved in host immune modulation by parasitic nematodes: a chemical biology approach developed in C. elegans ."

Burnham-Marusich, Amanda, Meteer, John, Snodgrass, Casey, Berninsone, Patricia

[International Worm Meeting, 2015]

Parasitic nematodes synthesize phosphorylcholine (PC)-modified biomolecules that can modulate the host's antibody and cytokine production to favor nematode survival, contributing to long-term infections. While discovering the protein targets of PC modification is critical to understand the role(s) that this epitope plays in host immune modulation, only two nematode PC-modified proteins (PC-proteins) had been previously identified. The non-parasitic nematode Caenorhabditis elegans expresses PC-modified N-linked glycans, offering an attractive model to study the biology of PC-modification. Using C. elegans as model system, we developed a robust method to identify PC-modified proteins by metabolic labeling primary embryonic cells with propargylcholine, an alkyne-modified choline analog. Cu(I)-catalyzed cycloaddition with biotin-azide enables streptavidin purification and subsequent high-throughput liquid chromatography and mass spectrometry identification of propargyl-labeled proteins. All C. elegans proteins identified using stringent criteria are known or predicted to be membrane or secreted proteins, consistent with the model of a Golgi-resident, putative PC-transferase. Of the 55 PC-N-glycosylation sites reported, 33 have been previously observed as N-glycosylation sites in high-throughput screens of C. elegans. This sensitive and specific method will enable the comprehensive identification of PC-modified glycoproteins in parasitic nematodes and aid in our understanding of the function of PC-modified targets in modulation of the host immune system. .

Snodgrass, Casey et al. (2013) International Worm Meeting "Conserved ion and amino acid transporters identified as phosphorylcholine modified N-glycoproteins by metabolic labeling with propargylcholine in Caenorhabditis elegans."

Meteer, John, Berninsone, Patricia M., Snodgrass, Casey, Burnham-Marusich, Amanda

[International Worm Meeting, 2013]

Phosphorylcholine (Pc) modification of proteins by pathogens has been implicated in mediating host-pathogen interactions. In parasitic nematodes, Pc modulates the host's immune response to favor nematode survival. Caenorhabditis elegans expresses Pc-modified N-linked glycans, offering an attractive non-parasitic model to study the biology of Pc-modification, but a lack of selective purification tools limits the number of known Pc targets. Here we show that Pc-modified N-glycoproteins can be identified in C. elegans embryonic cells by metabolic labeling with propargylcholine, an alkyne-modified choline analog. Cu(I)-catalyzed cycloaddition with biotin-azide enables streptavidin purification, high-throughput liquid chromatography and mass spectrometry identification of propargyl-labeled proteins. We report 21 novel Pc-modified N-glycoproteins and their sites of Pc-N-glycan attachment. Ion and amino acid transporters as well as synaptic proteins are among those identified as Pc-modified, suggesting a function for Pc beyond immunomodulation. This work provides a method to study Pc-modified proteins in C. elegans and related nematodes.

Dolanchanpa Ghosh et al. (2003) International Worm Meeting "Structure-function analysis of the PIE-1 protein to elucidate its role in germline development."

Dolanchanpa Ghosh, Geraldine Seydoux

[International Worm Meeting, 2003]

C.elegans protein PIE-1 is a component of the germ plasm essential for the specification and development of the embryonic germline. PIE-1 has at least two functions in germline blastomeres: it represses mRNA transcription in the nuclei, and regulates the expression of the maternal RNA, nos-2 in the cytoplasm. We are interested in how these two functions are carried out and how they influence germline development. Our approach is to create transgenic worms that express the PIE-1 protein bearing mutations in different domains. Conventional transformation methods yield transgenes that are quickly silenced in the germline, making detailed phenotypic analysis difficult.We have therefore adopted the microparticle bombardment method to create low-copy integrative transformants(1). Ingrid Dagostino in the lab has created a GATEWAY-based pie-1 expression vector, containing unc-119 gene as a selection marker. Using this vector, Ingrid was able to obtain a GFP:PIE-1 line, which fully rescues a pie-1(0) mutant and has remained stable for over 20 generations. We are now in the process of generating similar lines with mutant versions of PIE-1. 1.Praitis V, Casey E, Collar D,and Austin J.(2001). Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics 2001 Mar;157(3):1217-1226