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.

H Van Luenen et al. (1999) International C. elegans Meeting "World-wide worm SNPs: Dissecting the molecular evolution of the species using a high density SNP map"

R Plasterk, R Koch, H Van Luenen

[International C. elegans Meeting, 1999]

We would like to reconstruct the history of the C. elegans species at the genome level, therefore we sampled the genomes of four natural isolates (strain CB4857 isolated in Claremont, California, RC301 from Freiburg, Germany, TR403 from Madison, Wisconsin and AB1 from Adelaide, Australia) for single nucleotide polymorphisms (SNPs). Random genomic DNA fragments from the 4 strains were shotgun cloned and sequenced. There was no selection for transcribed or non-transcribed regions of the genome. In total we sequenced 1572 clones resulting in over 1 Mb of sequence information. The sequences are compared to the canonical Bristol N2 sequence to ask the question whether the clone maps to a unique sequence, and -if so- whether it contains polymorphisms. Once a SNP is identified we check other strains for the presence of the same polymorphism by PCR amplification and sequence analysis. In an initial experiment we found approximately one SNP per 3000 bp sequenced. The SNPs are randomly spread over the genome. Based on these observations we expect to find approximately 500 SNPs, one in every 200 kb. In the initial experiment we found, as expected, that several SNPs initially detected in one strain were also present in some but not all other strains. For example: a T in the Australian AB1, is a G at the same position in Bristol N2 in cosmid K10D2 at position 27946, and we found it to be like AB1 in the Californian CB4857 strain and the German RC301 strain, while the TR403 strain from Wisconsin resembles the Bristol N2 strain. Thus different patches of the genome have different ancestors. With our high density SNP map we will generate a genome map for each isolate which will show how each genome is patched together from a limited set of parental strains. The SNP's will be added to ACeDB, and can also be used as markers on the genetic map. They can be recognised by PCR followed by sequencing, but we also found that the SNPs we looked at could be visualised by SSCP analysis. We thank Jane Rogers and Amanda McMurray for their assistance in sequencing the clones.