Dataset Information

Glycoengineering of Esterase Activity through Metabolic Flux-Based Modulation of Sialic Acid.

ABSTRACT: This report describes the metabolic glycoengineering (MGE) of intracellular esterase activity in human colon cancer (LS174T) and Chinese hamster ovary (CHO) cells. In silico analysis of carboxylesterases CES1 and CES2 suggested that these enzymes are modified with sialylated N-glycans, which are proposed to stabilize the active multimeric forms of these enzymes. This premise was supported by treating cells with butanolylated ManNAc to increase sialylation, which in turn increased esterase activity. By contrast, hexosamine analogues not targeted to sialic acid biosynthesis (e.g., butanoylated GlcNAc or GalNAc) had minimal impact. Measurement of mRNA and protein confirmed that esterase activity was controlled through glycosylation and not through transcription or translation. Azide-modified ManNAc analogues widely used in MGE also enhanced esterase activity and provided a way to enrich targeted glycoengineered proteins (such as CES2), thereby providing unambiguous evidence that the compounds were converted to sialosides and installed into the glycan structures of esterases as intended. Overall, this study provides a pioneering example of the modulation of intracellular enzyme activity through MGE, which expands the value of this technology from its current status as a labeling strategy and modulator of cell surface biological events.

SUBMITTER: Mathew MP

PROVIDER: S-EPMC5757160 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Glycoengineering of Esterase Activity through Metabolic Flux-Based Modulation of Sialic Acid.

Mathew Mohit P MP Tan Elaine E Labonte Jason W JW Shah Shivam S Saeui Christopher T CT Liu Lingshu L Bhattacharya Rahul R Bovonratwet Patawut P Gray Jeffrey J JJ Yarema Kevin J KJ

Chembiochem : a European journal of chemical biology 20170420 13

This report describes the metabolic glycoengineering (MGE) of intracellular esterase activity in human colon cancer (LS174T) and Chinese hamster ovary (CHO) cells. In silico analysis of carboxylesterases CES1 and CES2 suggested that these enzymes are modified with sialylated N-glycans, which are proposed to stabilize the active multimeric forms of these enzymes. This premise was supported by treating cells with butanolylated ManNAc to increase sialylation, which in turn increased esterase activi ...[more]

PMID: 28218815

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Project description:The glycocalyx-a plethora of sugars forming a dense layer that covers the cell membrane-is commonly found on the epithelial surface of lumen forming tissue. New glycocalyx specific properties have been defined for various organs in the last decade. However, in the lung alveolar epithelium, its structure and functions remain almost completely unexplored. This is partly due to the lack of physiologically relevant, cost effective in vitro models. As the glycocalyx is an essential but neglected part of the alveolar epithelial barrier, understanding its properties holds the promise to enhance the pulmonary administration of drugs and delivery of nanoparticles. Here, using air-liquid-interface (ALI) cell culture, we focus on combining metabolic glycoengineering with glycan specific electron and confocal microscopy to visualize the glycocalyx of a recently immortalized human alveolar epithelial cell line (hAELVi). For this purpose, we applied different bioorthogonal labeling approaches to visualize sialic acid-an amino sugar that provides negative charge to the lung epithelial glycocalyx-using both fluorescence and gold-nanoparticle labeling. Further, we compared mild chemical fixing/freeze substitution and standard cytochemical electron microscopy embedding protocols for their capacity of contrasting the glycocalyx. In our study, we established hAELVi cells as a convenient model for investigating human alveolar epithelial glycocalyx. Transmission electron microscopy revealed hAELVi cells to develop ultrastructural features reminiscent of alveolar epithelial type II cells (ATII). Further, we visualized extracellular uni- and multilamellar membranous structures in direct proximity to the glycocalyx at ultrastructural level, indicating putative interactions. The lamellar membranes were able to form structures of higher organization, and we report sialic acid to be present within those. In conclusion, combining metabolite specific glycoengineering with ultrastructural localization presents an innovative method with high potential to depict the molecular distribution of individual components of the alveolar epithelial glycocalyx and its interaction partners.

Dataset Information

Glycoengineering of Esterase Activity through Metabolic Flux-Based Modulation of Sialic Acid.

Publications

Glycoengineering of Esterase Activity through Metabolic Flux-Based Modulation of Sialic Acid.

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