Abstract
Glycobiology research with Caenorhabditis elegans (C. elegans) has benefitted from the numerous genetic and cell biology tools available in this system. However, the lack of a cell line and the relative inaccessibility of C. elegans somatic cells in vivo have limited the biochemical approaches available in this model. Here we report that C. elegans primary embryonic cells in culture incorporate azido-sugar analogs of N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc), and that the labeled glycoproteins can be analyzed by mass spectrometry. By using this metabolic labeling approach, we have identified a set of novel C. elegans glycoprotein candidates, which include several mitochondrially-annotated proteins. This observation was unexpected given that mitochondrial glycoproteins have only rarely been reported, and it suggests that glycosylation of mitochondrially-annotated proteins might occur more frequently than previously thought. Using independent experimental strategies, we validated a subset of our glycoprotein candidates. These include a mitochondrial, atypical glycoprotein (ATP synthase α-subunit), a predicted glycoprotein (aspartyl protease, ASP-4), and a protein family with established glycosylation in other species (actin). Additionally, we observed a glycosylated isoform of ATP synthase α-subunit in bovine heart tissue and a primate cell line (COS-7). Overall, our finding that C. elegans primary embryonic cells are amenable to metabolic labeling demonstrates that biochemical studies in C. elegans are feasible, which opens the door to labeling C. elegans cells with other radioactive or azido-substrates and should enable the identification of additional post-translationally modified targets and analysis of the genes required for their modification using C. elegans mutant libraries.
Highlights
Glycosylation is a ubiquitous and important post-translational modification
The nematode C. elegans has a strong history as a model system for human disease [3], and it is advantageous for glycobiology research since numerous viable and publicly available glycosylation pathway mutants already exist
The incorporation of azido-labeled precursors and detection reagents in intact C. elegans nematodes is restricted to certain cells and tissues [11], and it is unclear whether the incorporation efficiency of the azido-sugar in vivo is sufficient for downstream glycoprotein identification
Summary
Glycosylation is a ubiquitous and important post-translational modification. More than 50% of eukaryotic proteins are glycosylated [1] and glycoproteins mediate numerous essential biological functions, including development, immune response, molecular trafficking and signal transduction [2].Most glycosyltransferases and glycosidases exhibit a high degree of evolutionary conservation [2], which can be exploited to study glycan biosynthesis and glycoprotein function in model organisms. The purpose of our study was to expand the experimental tools available in this system by developing an approach to identify C. elegans glycoprotein candidates biochemically via azido-labeled sugars and Click Chemistry. There are no C. elegans cell lines, but recently a protocol was developed to isolate primary cells from C. elegans embryos [12] This protocol has been used for electrophysiological and cell biological studies, but the potential of these cultures for biochemical analysis has not yet been tapped. We present our findings that C. elegans primary embryonic cells in culture metabolize both azido-GalNAc and azido-GlcNAc, that the labeled glycoproteins can be detected and analyzed, and that with this method we have identified several novel C. elegans glycoproteins, of which an unexpectedly large proportion are mitochondrially-annotated proteins
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