Conserved ion and amino acid transporters identified as phosphorylcholine-modified N-glycoproteins by metabolic labeling with propargylcholine in Caenorhabditis elegans cells

Abstract

Phosphorylcholine (PC) modification of proteins by pathogens has been implicated in mediating host-pathogen interactions. Parasitic nematodes synthesize PC-modified biomolecules that can modulate the host's antibody and cytokine production to favor nematode survival, contributing to long-term infections. Only two nematode PC-modified proteins (PC-proteins) have been unequivocally identified, yet discovering the protein targets of PC modification will be paramount to understanding the role(s) that this epitope plays in nematode biology. A major hurdle in the field has been the lack of techniques for selective purification of PC-proteins. The nonparasitic nematode Caenorhabditis elegans expresses PC-modified N-linked glycans, offering an attractive model to study the biology of PC-modification. We developed a robust method to identify PC-proteins by metabolic labeling of primary embryonic C. elegans cells with propargylcholine, an alkyne-modified choline analog. Cu(I)-catalyzed cycloaddition with biotin-azide enables streptavidin purification and subsequent high-throughput LC-MS identification of propargyl-labeled proteins. All 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. Several identified PC-proteins are nematode-specific proteins, but 10 of the PC-proteins are widely conserved ion transporters and amino acid transporters, while eight are conserved proteins involved in synaptic function. This finding suggests a functional role for PC-modification beyond immunomodulation. The approach presented in this study provides a method to identify PC-proteins in C. elegans and related nematodes.

Phospholipid bioconjugation: advancements and applications in prodrug development and metabolic labeling

“…Their work had shown the reliablity of PCho for metabolic labeling. Following their pioneering work, PCho had been used to for metabolic labeling of PC distribution during freeze-fracture electron microscopy, PC in myelin in mutiple sclerosis animal model, phosphorylcholine-modified N-glycoproteins in Caenorhabditis elegans cells, and PC in autophagosome membrane [43][44][45][46]. The application of PCho was not limited to phospholipid labeling.…”

Metabolic labeling of glycerophospholipids via clickable analogs derivatized at the lipid headgroup

“…Since this pioneering report, PCho has also been shown to label myelin, as was developed in work aimed at studying remyelination (Aharoni et al, 2016), as well as teichoic acid glycopolymers attached to the peptidoglycan of Streptococcus pneumoniae (Di Guilmi et al, 2017). PCho has also been used to probe phosphorylcholine-derivatized proteins (Snodgrass et al, 2015) and acts as an inhibitor of choline catabolism in bacteria (Fitzsimmons et al, 2011).…”