Conserved ion and amino acid transporters identified as phosphorylcholine-modified N-glycoproteins by metabolic labeling with propargylcholine in Caenorhabditis elegans cells.
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.
Department of Biology, University of Nevada, Reno, NV 89557, USA.
Casey J
CJ
Snodgrass
Department of Biology, University of Nevada, Reno, NV 89557, USA.
Amanda R
AR
Burnham-Marusich
Department of Biology, University of Nevada, Reno, NV 89557, USA.
John C
JC
Meteer
Department of Biology, University of Nevada, Reno, NV 89557, USA berninsone@unr.edu.
Patricia M
PM
Berninsone
D000480:Alkynes / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D026905:Amino Acid Transport Systems / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D017173:Caenorhabditis elegans / Q000166:cytology / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000378:metabolism*; D002522:Chlorocebus aethiops; D002794:Choline / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D016623:Ion Pumps / Q000737:chemistry / Q000378:metabolism*; D015335:Molecular Probes / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010767:Phosphorylcholine / Q000378:metabolism; D013194:Staining and Labeling
D000480:Alkynes; D026905:Amino Acid Transport Systems; D029742:Caenorhabditis elegans Proteins; D006023:Glycoproteins; D016623:Ion Pumps; D015335:Molecular Probes; C000603143:propargylcholine; D010767:Phosphorylcholine; D002794:Choline
Caenorhabditis elegans; N-glycosylation; click chemistry; phosphorylcholine