Zhang, S., Gouveia, G., Tayyari, F., Bifarin, O.O., Edison, A.S., Taujale, R.
[
International Worm Meeting,
2017]
Glycosyltransferases (GTs) catalyze the transfer of glycosyl groups from sugar substrate donors to a host of acceptor substrates - oligosaccharides, monosaccharides, proteins, lipids, nucleic acids, and other small molecules; effectively GTs catalyze glycosidic bond formation between these multifarious possibilities of molecules. Numerous studies show that GTs are important for developmental and physiological processes in C. elegans (Berninsone, 2006). However, most GTs have not yet been empirically validated. C. elegans utilizes GTs for critical functions, including pheromone signaling with ascarosides and the detoxification pathways, though identities of GTs regulating these functions remain unknown. C. elegans controls much of its behavior and development through the use of ascarosides, which are also present in many free-living and parasitic nematodes (Choe et al 2012). Some of the phenotypes mediated by ascarosides include aggregation, olfactory plasticity, dauer formation, attraction behavior, and hermaphrodite behavior. (Srinivasan et al 2008; Edison, 2009; Ludewig & Schroeder, 2013) Chemically, ascarosides are glycosides of the dideoxy sugar ascarylose, attached to a fatty acid side chain, thus implicating GTs in their biosynthesis. In addition, the innate immune system in C. elegans utilizes a wide range of immune effectors and enzymes (including GTs) for microbial defenses and xenobiotics detoxification (Lindblom & Dodd, 2006; Stupp et al 2012). Stupp et al 2012 showed that C. elegans can detoxify two bacterial toxins, 1-hydroxyphenazine (1-HP), and indole via N- and O-glycosylation. Our research aims to discover the roles of specific GTs in biological processes like these. In this exploratory study, we selected a dozen GT mutant strains, the majority of which belong to the GT-A fold protein (families 2, 7, 21, 27, and 13). 6 replicates of each strains (L1 stage) were cultured to about a population of 100-200 thousand for NMR metabolomics measurements, and animals were randomly selected from each sample for the measurement of population distribution using the large particle flow cytometer COPAS Biosorter. The Biosorter measures the extinction and time of flight of individual nematode which is used as a descriptor of developmental stages. Analysis of metabolic changes of some of the GT mutants and the association with the population distribution will be reported.
[
International Worm Meeting,
2017]
Glycosyltransferases (GTs) are a broad class of proteins involved in the transfer of a glycosyl group from a donor molecule to an acceptor. As such, they are involved in a wide range of biological functions through their roles in glycosylation modifications, synthesis of cellular receptors, biosynthesis of polysaccharides, glycolipids and glycoproteins. In C. elegans, GTs have been implicated to play roles in development, cuticle formation, detoxification, signaling and other pathways. However, only around 20% of the more than 260 GTs have been characterized. The large expansion of some of the GT families, presence of unique sugars and pathways in worms and the unknown specific roles of GTs in the implicated pathways pose specific questions and challenges that require a deeper understanding of the functional units of these GTs in C. elegans. We used a Bayesian statistical approach to align and classify more than 250,000 GT sequences across all taxonomic groups into functional categories based on the patterns of conservation and variation in large multiple sequence alignments. We use patterns unique to each GT family as a conceptual starting point for investigating their sequence-structure-function relationships. Implementing these methods, we can further pinpoint contrasting, similar and co-evolved features that differentiate, associate and functionally relate the GT families respectively. We will present an initial analysis that highlights the presence of co-conserved features that distinguish multiple GT families, highlighting GTs in worms. We have generated a phylogenetic classification based on these features and mapped phenotypic associations for these families based on literature. Our analysis reveals several expanded and unique GT families in C. elegans with limited information that we hypothesize might be involved in detoxification, signaling and other pathways unique to worms. Using the co-conserved features as a starting point, we further investigate structural data to pinpoint targets for mutational and metabolomic studies. An initial metabolomic analysis by others in the lab of select GT mutants in C. elegans is revealing specific features that are statistically different, suggesting family specific phenotypic changes. Informatic analysis layered with multiple data sources from literature serve as a tool to identify targets and derive hypotheses to conduct deeper metabolomic studies that can help elucidate the functional changes and biological activity of the associated GT families.