Abstract
This study examined the relationship between glycans, metabolites, and development in C. elegans. Samples of N2 animals were synchronized and grown to five different time points ranging from L1 to a mixed population of adults, gravid adults, and offspring. Each time point was replicated seven times. The samples were each assayed by a large particle flow cytometer (Biosorter) for size distribution data, LC-MS/MS for targeted N- and O-linked glycans, and NMR for metabolites. The same samples were utilized for all measurements, which allowed for statistical correlations between the data. A new protocol was developed to correlate Biosorter developmental data with LC-MS/MS data to obtain stage-specific information of glycans. From the five time points, four distinct sizes of worms were observed from the Biosorter distributions, ranging from the smallest corresponding to L1 to adult animals. A network model was constructed using the four binned sizes of worms as starting nodes and adding glycans and metabolites that had correlations with r ≥ 0.5 to those nodes. The emerging structure of the network showed distinct patterns of N- and O-linked glycans that were consistent with previous studies. Furthermore, some metabolites that were correlated to these glycans and worm sizes showed interesting interactions. Of note, UDP-GlcNAc had strong positive correlations with many O-glycans that were expressed in the largest animals. Similarly, phosphorylcholine correlated with many N-glycans that were expressed in L1 animals.
Highlights
This paper presents a new approach to evaluate the relationship between Caenorhabditis elegans development, glycan abundance, and metabolites
We developed a novel approach to Abbreviations: C. elegans, Caenorhabditis elegans; dHex, deoxyhexose, namely Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; GDPFucose, Guanosine diphosphate fucose; Glc, glucose; GlcA, glucuronic acid; GlcNAc, N-acetylglucosamine; hexosamine biosynthetic pathway (HBP), Hexosamine Biosynthetic Pathway; HCA, Hierarchical Clustering Analysis; Hex, hexose, either Glc, Gal, or Man; HexA, hexuronic acid, namely GlcA; HexNAc, N-acetylhexosamine, either GlcNAc or GalNAc; LC-MS/MS, liquid chromatography-mass spectrometry and tandem mass spectrometry; Man, mannose; NMR, Nuclear Magnetic Resonance; OGlcNAc, O-linked β-N-acetylglucosamine; O-GlcNAc transferase (OGT), O-GlcNAc Transferase; PC, phosphorylcholine or O-phosphocholine; PNGase A, Protein N-Glycosidase A; STOCSY, Statistical total correlation spectroscopy; UDP-GlcNAc, Uridine diphosphate N-acetylglucosamine
Glycan compositions known to be natively methylated in C. elegans were omitted in our targeted list as native methylation would be masked when glycans are derivatized through permethylation
Summary
This paper presents a new approach to evaluate the relationship between Caenorhabditis elegans development, glycan abundance, and metabolites. Worm Glycomics, Metabolomics, and Development of O-GlcNAc to proteins is catalyzed by a single enzyme, O-GlcNAc transferase (OGT), which relies on the availability of the sugar-nucleotide donor substrate, UDP-GlcNAc via the hexosamine biosynthetic pathway (HBP) (Vaidyanathan and Wells, 2014). Through the HBP, concentrations of UDP-GlcNAc are modulated by the metabolism of glucose, fatty acids, amino acids, and nucleotides. This vital glycosylation precursor is utilized by OGT to modify thousands of proteins with O-GlcNAc (Zachara and Hart, 2004; Zachara, 2018), and by many other glycosyltransferases to generate more elaborate types of N- and O-linked glycans (Brockhausen and Stanley, 2015; Stanley et al, 2015). Better approaches of associating glycomics and metabolomics would be valuable to gain a deeper understanding of their interactions
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