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  • br Introduction Historically eukaryotic protein glycosylatio

    2020-07-27


    Introduction Historically, eukaryotic protein glycosylation was thought to occur exclusively in the endoplasmic reticulum and Golgi apparatus as part of the secretory pathway, which produces a vast array of diverse membrane glycoproteins. In the mid-1980s, however, Hart et al. found O-linked β-N-acetylglucosamine (O-GlcNAc) on nuclear and cytoplasmic proteins (Figure 1a) [1]. The O-GlcNAc modification is dynamic, and its addition and removal are governed by a single pair of enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) (Figure 1b) [2, 3, 4]. Thousands of nucleocytoplasmic proteins are substrates of these O-GlcNAc-cycling enzymes. Because O-GlcNAc levels change substantially in response to nutrient availability and multiple forms of environmental stress (e.g. hypoxia, oxidative stress, thermal stress), it is thought that O-GlcNAc cycling serves to maintain cell in the morning by impacting cell signaling, gene expression, and proteostasis, among other processes [5,6]. Dysregulated O-GlcNAc abundance has been linked to several human diseases, including diabetes, cardiovascular disease, cancer, and neurodegenerative diseases, and it has been speculated that OGT and OGA may be therapeutic targets [7, 8, 9, 10]. In addition, mutations in OGT have been connected to X-linked intellectual disability [11]. While the importance of O-GlcNAc cycling in metazoan physiology is by now indisputable, the functional significance of O-GlcNAc on individual substrates is extraordinarily challenging to decipher because there are so many O-GlcNAc substrates, and the rules governing substrate selection are still unclear. Therefore, methods to selectively manipulate the cellular repertoire of O-GlcNAc are currently limiting. For OGT, the challenge is compounded by the recent discovery that this enzyme uses the same active site to attach O-GlcNAc and to effect another physiologically relevant modification, the cleavage of the essential cell cycle regulator, HCF-1 (Figure 1b) [12,13]. Progress in deconvoluting the functions of the O-GlcNAc cycling enzymes depends on having structural information to guide cellular experiments. A number of major advances have been made on this front in the past five years. This review will summarize key findings of structural studies on human OGT and OGA, with our apologies for the many omissions made due to space limitations. Information about structures mentioned in the text is provided in Figure 1.
    O-GlcNAc transferase OGT belongs to the metal-independent GT-B superfamily of glycosyltransferases, which has been well-reviewed previously [14,15]. OGT is an essential gene encoded on the X-chromosome, and it has two main regions: a long N-terminal tetratricopeptide repeat (TPR) region and a C-terminal catalytic region (Figure 1c) [16, 17, 18, 19, 20]. The two Rossmann-folded catalytic lobes that are characteristic of GT-B superfamily members are separated in primary sequence by a unique intervening domain of unknown function. Although there are two shorter splice variants, the TPR region of the primary, full-length OGT contains 13.5 TPRs. These are 34 amino acid helix-turn-helix motifs that typically mediate protein–protein interactions [21]. Consistent with this, removing TPRs from OGT abolishes protein glycosylation even when the active site is still functional; moreover, some cellular proteins have been shown to form stable interactions with the TPR region [18,22,23,24,25]. A crystal structure of the TPR region of OGT obtained in 2004 (PDB 1W3B; Figures 1c and 2d) shows an elongated, right-handed superhelix [26]. More recently, a structure of the TPR region containing a single point mutation distorts the superhelix, and it is speculated that this distortion alters the O-GlcNAc proteome or the OGT interactome, affecting brain development [].