Metabolic Labeling of Caenorhabditis elegans Primary Embryonic Cells with Azido-Sugars as a Tool for Glycoprotein Discovery

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.

Metabolic Glycoengineering with N‐Acyl Side Chain Modified Mannosamines

“…On the basis of this strategy,M GE has been successfully applied to other monosaccharides,i ncluding d-N-acetylgalactosamine (GalNAc), [6][7][8][9][10] d-N-acetylglucosamine Inmetabolic glycoengineering (MGE), cells or animals are treated with unnatural derivatives of monosaccharides.After entering the cytosol, these sugar analogues are metabolized and subsequently expressed on newly synthesized glycoconjugates.T he feasibility of MGE was first discovered for sialylated glycans,b yusing N-acylmodified mannosamines as precursor molecules for unnatural sialic acids.Prerequisite is the promiscuity of the enzymes of the Roseman-Warren biosynthetic pathway. These enzymes were shown to tolerate specific modifications of the N-acyl side chain of mannosamine analogues,for example,elongation by one or more methylene groups (aliphatic modifications) or by insertion of reactive groups (bioorthogonal modifications).…”

Metabolisches Glykoengineering mit N‐Acyl‐Seiten‐ ketten‐modifizierten Mannosaminen

“…B. für die Darstellung von sialylierten Glykanen mit fluoreszierenden Farbstoffen verwendet werden. Basierend auf dieser Strategie konnte MGE auch mit anderen Monosacchariden angewendet werden, einschließlich d ‐ N ‐Acetylgalactosamin (GalNAc), d ‐ N ‐Acetylglucosamin (GlcNAc) sowie 2‐Keto‐3‐desoxyoctonsäure (KDO) oder dem Zuckeralkohol Myoinositol …”

A Method for Culturing Embryonic <em>C. elegans</em> Cells

“…elegans cells in vitro 1 , as well as isolation of specific cell types by Fluorescent-Activated Cell Sorting (FACS) for the construction of cell-specific cDNA libraries 22,23 . Gene knockdown techniques such as RNA interference (RNAi) can be applied on cultured C. elegans cells 1 and a novel metabolic labeling method using Azido-sugar as a tool for glycoprotein discovery has been recently developed for in vitro cultured C. elegans cells 24 .…”