Global identification of O‐GlcNAc transferase (OGT) interactors by a human proteome microarray and the construction of an OGT interactome

Abstract

O-Linked β-N-acetylglucosamine (O-GlcNAcylation) is an important protein PTM, which is very abundant in mammalian cells. O-GlcNAcylation is catalyzed by O-GlcNAc transferase (OGT), whose substrate specificity is believed to be regulated through interactions with other proteins. There are a handful of known human OGT interactors, which is far from enough for fully elucidating the substrate specificity of OGT. To address this challenge, we used a human proteome microarray containing ~17,000 affinity-purified human proteins to globally identify OGT interactors and identified 25 OGT-binding proteins. Bioinformatics analysis showed that these interacting proteins play a variety of roles in a wide range of cellular functions and are highly enriched in intra-Golgi vesicle-mediated transport and vitamin biosynthetic processes. Combining newly identified OGT interactors with the interactors identified prior to this study, we have constructed the first OGT interactome. Bioinformatics analysis suggests that the OGT interactome plays important roles in protein transportation/localization and transcriptional regulation. The novel OGT interactors that we identified in this study could serve as a starting point for further functional analysis. Because of its high-throughput and parallel analysis capability, we strongly believe that protein microarrays could be easily applied for the global identification of regulators for other key enzymes.

The Making of a Sweet Modification: Structure and Function of O-GlcNAc Transferase

“…HCF-1 provided the first picture of a substrate engaging the TPRs of OGT, but it remains to be seen whether other substrates, adaptors, or enhancers interact with OGT analogously. Several recent publications have exploited protein microarrays as a tool that allows for precise systematic control of individual parameters, which is likely to be crucial in examining the role of possible adaptors/enhancers (93)(94)(95). More biochemical and structural data on how OGT physically interacts with different proteins would add greatly to understanding its function.…”

The Biochemistry of O-GlcNAc Transferase: Which Functions Make It Essential in Mammalian Cells?

“…Although it has generally been assumed that OGT's essential role is related to its O-GlcNAcylation activity, this has not been demonstrated (12,40,41). Moreover, OGT's protein-protein interactions (44) and surprising role as the cellular factor that cleaves HCF-1 (22)(23)(24)(25) raise the possibility that other biochemical functions may also be important for viability (12,40,41). There is increasing interest in the associations between OGT and human disease, in part because perturbations in levels of β-O-linked GlcNAc (O-GlcNAc) have been observed in diabetes (15,21), cancer (45), cardiovascular disease (46), and Alzheimer's disease (47).…”

“…OGT interacts with a large number of protein partners (44,108) and is a member of a number of stable complexes (10,(26)(27)(28)(29)(30)(31)(32)(34)(35)(36)(37)(38)(39). It has well-documented associations with HCF-1 (22,27), the ten-eleven translocation methylcytosine dioxygenase (TET) family of DNA hydroxylases (28)(29)(30)(31)(32), the transcriptional corepressor mSin3A (26), the nuclear-localized deubiquitinase BAP-1 (27), the transcriptional coactivator PGC-1α (10,36), the mitochondrial trafficking protein Milton (also known as TRAK1) (37,38,108,109), and the mitogen-activated protein kinase (MAPK) p38-α (34).…”

Crosstalk between phosphorylation and O‐GlcNAcylation: friend or foe

“…2, whereby OGT substrate specificity is defined by the initial complex formed with so-called adapter proteins. Many other proteins have been shown to interact with OGT, however, the biological relevance of these interactions, and whether they play a regulatory role is often not yet fully understood [53,55,[57][58][59][60][61]. Furthermore, the interaction between OGT and p38, a mitogen-activated protein kinase, increases OGT activity by targeting OGT to Neurofilament H, as well as other targets [57].…”