Date Published: January 01, 2019
Publisher: International Union of Crystallography
Author(s): Alexandra Males, Gideon J. Davies.
The surface-entropy reduction method has been used to generate new crystal forms of human O-GlcNAcase.
The regulation of O-GlcNAc cycling on thousands of nuclear and cytoplasmic proteins is coordinated by two enzymes. O-GlcNAc transferase (OGT) catalyses the addition of GlcNAc, derived from UDP-GlcNAc, to serine and threonine residues, and O-GlcNAcase (OGA; CAZY database family GH84) cleaves O-GlcNAc (Torres & Hart, 1984 ▸; Holt & Hart, 1986 ▸; Kreppel et al., 1997 ▸; Dong & Hart, 1994 ▸; Lubas et al., 1997 ▸; Hart et al., 2007 ▸). Two isoforms, OGA-L and OGA-S, are localized to the nucleus/cytoplasm (Comtesse et al., 2001 ▸) and to the surface of lipid droplets, respectively. The reciprocal relationship between O-phosphorylation and O-glycosylation on the particular protein tau has been keenly studied in the context of neurodegeneration (Arnold et al., 1996 ▸; Yuzwa et al., 2008 ▸, 2014 ▸; Shen et al., 2012 ▸; Griffith & Schmitz, 1995 ▸; Gao et al., 2001 ▸; Liu et al., 2004 ▸). In patients with Alzheimer’s disease, tau undergoes hyperphosphorylation, causing it to dissociate from microtubules and aggregate into paired helical filaments (PHF) and neurofibrillary tangles (NFTs) (Grundke-Iqbal et al., 1986 ▸; Marotta et al., 2015 ▸). O-GlcNAc cycling has also been implicated in tumorigenesis owing to its significant role in orchestrating a vast number of cellular processes, for example transcriptional and cytoskeletal regulation, cell signalling and division, and metabolism (Slawson & Hart, 2011 ▸).
In this study, surface-entropy reduction has been utilized to produce further structural information on O-GlcNAcase by the incorporation of residues Ala57–Arg58, Lys341–Asp347, Thr370, Glu536, Cys596–Gly598, Gly674–Asp675 and Asp696–Pro707, an increase in the number of observed residues of 5%. Although the binding of the C-terminus to the active site may be an artefact of crystallization, it reveals further details regarding the substrate specificity of OGA, as peptides have been shown to bind in a bidirectional yet conserved conformation. The results described in this study present an opportunity for further investigation of the binding orientation of peptides within an SER-modified OGA enzyme. Given the progression of hOGA inhibitors into clinical trials, different surface mutants of the enzyme may afford new routes to drug development.