The Liver X Receptor a (LXR alpha) belongs to the nuclear receptor superfamily and plays an essential role in regulating cholesterol, lipid and glucose metabolism and inflammatory responses. We have previously shown that LXRa is post-translationally modified by O-linked beta-N-acetyl-glucosamine (O-G1cNAc) with increased transcriptional activity. Moreover, we showed that LXRa associates with O-G1cNAc transferase (OGT) in vitro and in vivo in mouse liver. In this study, we report that human LXRa is O-G1cNAc modified in its N-terminal domain (NTD) by identifying a specific O-G1cNAc site S49 and a novel O-G1cNAc modified peptide (20)LWKPGAQDASSQAQGGSSCILRE(42). However, O-G1cNAc site-mutations did not modulate LXRa transactivation of selected target gene promoters in vitro. Peptide array and co-immunoprecipitation assays demonstrate that LXRa interacts with OGT in its NTD and ligand-binding domain (LBD) in a ligand-independent fashion. Moreover, we map two new O-G1cNAc sites in the longest OGT isoform (ncOGT): 5437 in the tetratricopeptide repeat (TPR) 13 domain and T1043 in the far C-terminus, and a new O-G1cNAc modified peptide (amino acids 826-832) in the intervening region (IntD) within the catalytic domain. We also map four new O-G1cNAc sites in the short isoform sOGT: S391, T393, S399 and S437 in the TPRs 11-13 domain. Future studies will reveal the biological role of identified O-G1cNAc sites in LXRa and OGT. (C) 2018 Elsevier Inc. All rights reserved.