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 the 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.

Integration of genetic and metabolic features related to sialic acid metabolism distinguishes human breast cell subtypes

“…The metabolic substrate-based studies described in this paper depend on the ability to introduce high levels of flux into the sialic acid biosynthetic pathway with negligible cytotoxicity and minimal growth inhibition. We previously achieved these objectives in other human (and rodent) cells using ‘high-flux’ 1,3,4-O-butanoylated ManNAc analogs [ 40 , 46 , 70 , 71 ] but the cytotoxicity of the newly-synthesized, alkyne-modified analog 1,3,4-O-Bu 3 ManAl was unknown; moreover, the three cell lines used in this study (MCF10A, T-47D and MDA-MB-231) had not been comparatively screened with these analogs in ‘assay media’ where the cells were grown in low serum (1.0%; to prevent recycling of sialic acids from glycoproteins contained in serum [ 57 ]) as well as without antibiotics (to avoid inhibition of sialylation [ 59 ]). Therefore, as a prelude to subsequent metabolic profiling, we first evaluated cell viability by measuring proliferation after treatment with 0, 10, 100, or 250 μM of 1,3,4-O-Bu 3 ManNAc (Panel A in S2 Fig ), 1,3,4-O-Bu 3 ManNAz (Panel B in S2 Fig ), or 1,3,4-O-Bu 3 ManNAl (Panel C in S2 Fig ) for 6, 24, or 48 h and observed no statistically-significant differences between the control and test cells.…”

“…Upon establishing baseline transcript levels in the panel of cell lines ( Fig 2 ) we tested whether analog supplementation altered the expression of the SAMG genes, which could occur by two mechanisms. First, these compounds supply butyrate to a cell upon esterase processing [ 70 , 73 ]; this SCFA can act as an histone deacetylase (HDAC) inhibitor and alter gene expression epigentically [ 43 ]. Second, the transcript levels of sialyltransferases can be regulated by the bioavailability and biosynthesis of sialic acid [ 74 ], which is dramatically affected by analog supplementation [ 38 , 39 ].…”

Cell Line-, Protein-, and Sialoglycosite-Specific Control of Flux-Based Sialylation in Human Breast Cells: Implications for Cancer Progression

“…In this report we focus on a second context, which is flux-driven sialylation in cancer. In the past we have used sialoglycosite analysis to characterize 1,3,4-O-Bu 3 ManNActreated SW1990 pancreatic cancer cells (Almaraz et al, 2012b), and in follow up studies, showed that these changes altered oncogenic signal transduction and sensitivity to drugs (Mathew et al, 2015(Mathew et al, , 2017).…”

“…Having ruled out overt effects on transcription, we reasoned that glycosite sialylation could be affected by the availability of CMP-Neu5Ac in the Golgi, which can selectively activate subsets of cell's repertoire of STs toward individual glycosites (presumably each glycosite:ST interaction has an individualized K M value) (Legaigneur et al, 2001;Gupta et al, 2017). Alternatively, the activity of the SAMG gene products themselves could be altered by flux-driven sialylation; increased esterase activity upon enhanced sialylation in 1,3,4-O-Bu 3 ManNActreated cells provides precedent for the flux-based control of a sialylated protein's activity (Mathew et al, 2017) along with evidence that sialylation of STs can impact their activity (Breen, 2002). To test if flux-based sialylation was relevant to STs and other SAMG proteins evaluated in the current study, we analyzed our dataset and found 10 sialoglycosites in six SAMG proteins, five of which were STs (Figure 5E).…”

“…For context, peracetylation is a strategy that facilitates cellular uptake of ManNAc (Hadfield et al, 1983;Schwartz et al, 1983;Lemieux et al, 1999). Once peracetylated ManNAc is inside a cell, non-specific esterases (Mathew et al, 2012(Mathew et al, , 2017 remove the ester-linked acetate groups or other short chain fatty acids (SCFAs) such as propionate or butyrate (Kim et al, 2004b;Sampathkumar et al, 2006;Hao et al, 2019). Our team overcame these difficulties by omission of esterlinked SCFAs from the C6-OH position of ManNAc, which largely eliminates cytotoxicity (Aich et al, 2008;Wang et al, 2009) and ameliorates other off-target effects (Campbell et al, 2008;Elmouelhi et al, 2009).…”