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

Glycobiology. 2015 Apr;25(4):403-11. doi: 10.1093/glycob/cwu122. Epub 2014 Nov 10.

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.

Keywords: Caenorhabditis elegans; N-glycosylation; click chemistry; phosphorylcholine.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Alkynes / chemistry*
  • Alkynes / metabolism
  • Amino Acid Sequence
  • Amino Acid Transport Systems / chemistry
  • Amino Acid Transport Systems / metabolism*
  • Animals
  • COS Cells
  • Caenorhabditis elegans / cytology
  • Caenorhabditis elegans / metabolism*
  • Caenorhabditis elegans Proteins / chemistry
  • Caenorhabditis elegans Proteins / metabolism*
  • Chlorocebus aethiops
  • Choline / analogs & derivatives*
  • Choline / chemistry
  • Choline / metabolism
  • Glycoproteins / chemistry
  • Glycoproteins / metabolism
  • Ion Pumps / chemistry
  • Ion Pumps / metabolism*
  • Molecular Probes / chemistry*
  • Molecular Probes / metabolism
  • Molecular Sequence Data
  • Phosphorylcholine / metabolism
  • Staining and Labeling

Substances

  • Alkynes
  • Amino Acid Transport Systems
  • Caenorhabditis elegans Proteins
  • Glycoproteins
  • Ion Pumps
  • Molecular Probes
  • propargylcholine
  • Phosphorylcholine
  • Choline