New use for CETSA: monitoring innate immune receptor stability
via post-translational modification by OGT
Walter R. Drake
Natasha E. Zachara
Catherine Leimkuhler Grimes
Received: 14 October 2017 /Accepted: 21 March 2018 / Published online: 18 April 2018
Springer Science+Business Media, LLC, part of Springer Nature 2018
O-GlcNAcylation is a dynamic and functionally diverse post-translational modification shown to affect thousands of proteins,
including the innate immune receptor nucleotide-binding oligomerization domain-containing protein 2 (Nod2). Mutations of Nod2
(R702W, G908R and 1007 fs) are associated with Crohn’s disease and have lower stabilities compared to wild type. Cycloheximide
(CHX)-chase half-life assays have been used to show that O-GlcNAcylation increases the stability and response of both wild type
and Crohn’s variant Nod2, R702W. A more rapid method to assess stability afforded by post-translational modifications is necessary
to fully comprehend the correlation between NLR stability and O-GlcNAcylation. Here, a recently developed cellular thermal shift
assay(CETSA)thatistypicallyusedtodemonstrate protein-ligand binding was adapted to detect shifts in protein stabilization upon
increasing O-GlcNAcylation levels in Nod2. This assay was used as a method to predict if other Crohn’s associated Nod2 variants
were O-GlcNAcylated, and also identified the modification on another NLR, Nod1. Classical immunoprecipitations and NF-κB
transcriptional assays were used to confirm the presence and effect of this modification on these proteins. The results presented here
demonstrate that CETSA is a convenient method that can be used to detect the stability effect of O-GlcNAcylation on O-GlcNAc-
transferase (OGT) client proteins and will be a powerful tool in studying post-translational modification.
O-GlcNAcylation is a dynamic post-translational modifica-
tion (PTM) characterized by the addition of monosaccharides
of O-linked N-acetylglucosamine (GlcNAc) to serine and/or
threonine residues of a protein using the nucleotide sugar,
uridine diphosphate (UDP)-GlcNAc (Groves et al. 2013).
Two enzymes mediate this modification: O-GlcNAc
Transferase (OGT), which adds GlcNAc to target proteins,
and O-GlcNAcase (OGA), which removes GlcNAc (Torres
and Hart 1984; Haltiwanger et al. 1990;DongandHart
1994). O-GlcNAcylation is a functionally diverse PTM, asso-
ciated with transcriptional regulation, signaling in response to
nutrients and stress, and prevention of nascent polypeptides
from early degradation (Jackson and Tijan 1988;Hartetal.
2007; Yang and Qian 2017; Zhu et al. 2015; Worth et al.
2017). We, along with others, have shown that modification
of a protein substrate by OGT often leads to increased cellular
stability of the client protein (Hou et al. 2016; Qin et al. 2017;
Yang and Qian 2017;Ruanetal.2012; Chu et al. 2014).
In order to fully appreciate the correlation between stability
and O-GlcNAcylation on OGT’s thousands of protein sub-
strates, accessible, high throughput methods are needed.
Here an easy to use cellular thermal shift assay (CETSA) is
repurposed to probe the effect of the OGT-modification on
protein stability. CETSA is a relatively new technique intro-
ducedbyParNordlundandcolleagues at the Karolinska
Institutet to assess protein-ligand binding utilizing live cells
(Martinez Molina et al. 2013;Jafarietal.2014). This assay
Walter R. Drake and Ching-Wen Hou contributed equally to this work.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10863-018-9754-z) contains supplementary
material, which is available to authorized users.
* Catherine Leimkuhler Grimes
Department of Chemistry and Biochemistry, University of Delaware,
Newark, DE 19716, USA
Department of Biological Chemistry, The Johns Hopkins University
School of Medicine, Baltimore, MD 21205, USA
Department of Biological Sciences, University of Delaware,
Newark, DE 19716, USA
Journal of Bioenergetics and Biomembranes (2018) 50:231–240