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Chembiochem. 2017 Jul 4;18(13):1204-1215. doi: 10.1002/cbic.201600698. Epub 2017 Apr 20.

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

Author information

1
Department of Biomedical Engineering and, Translational Tissue Engineering Center, The Johns Hopkins University, 5029 Robert H. & Clarice Smith Building, 400 North Broadway, Baltimore, MD, 21231, USA.
2
Department of Chemical and Biochemical Engineering, The Johns Hopkins University, 5029 Robert H. & Clarice Smith Building, 400 North Broadway, Baltimore, MD, 21231, USA.

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.

KEYWORDS:

ManNAc; carboxylesterase; hexosamines; metabolic glycoengineering; sialic acid

PMID:
28218815
PMCID:
PMC5757160
DOI:
10.1002/cbic.201600698
[Indexed for MEDLINE]
Free PMC Article

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