O-linked β-N-acetylglucosamine (O-GlcNAc) is a regulatory post-translational modification of intracellular proteins. The dynamic and inducible cycling of the modification is governed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in response to UDP-GlcNAc levels in the hexosamine biosynthetic pathway (HBP). Due to its reliance on glucose flux and substrate availability, a major focus in the field has been on how O-GlcNAc contributes to metabolic disease. For years this post-translational modification has been known to modify thousands of proteins implicated in various disorders, but direct functional connections have until recently remained elusive. New research is beginning to reveal the specific mechanisms through which O-GlcNAc influences cell dynamics and disease pathology including clear examples of O-GlcNAc modification at a specific site on a given protein altering its biological functions. The following review intends to focus primarily on studies in the last half decade linking O-GlcNAc modification of proteins with chromatin-directed gene regulation, developmental processes, and several metabolically related disorders including Alzheimer’s, heart disease and cancer. These studies illustrate the emerging importance of this post-translational modification in biological processes and multiple pathophysiologies.

The post-translational protein modification O-linked ␤-Nacetylglucosamine (O-GlcNAc) is a proposed nutrient sensor that has been shown to regulate multiple biological pathways. This dynamic and inducible enzymatic modification to intracellular proteins utilizes the end product of the nutrient sensing hexosamine biosynthetic pathway, UDP-GlcNAc, as its substrate donor. Type II diabetic patients have elevated O-GlcNAc-modified proteins within pancreatic beta cells due to chronic hyperglycemia-induced glucose overload, but a molecular role for O-GlcNAc within beta cells remains unclear. Using directed pharmacological approaches in the mouse insulinoma-6 (Min6) cell line, we demonstrate that elevating nuclear O-GlcNAc increases intracellular insulin levels and preserves glucose-stimulated insulin secretion during chronic hyperglycemia. The molecular mechanism for these observed changes appears to be, at least in part, due to elevated O-GlcNAcdependent increases in Ins1 and Ins2 mRNA levels via elevations in histone H3 transcriptional activation marks. Furthermore, RNA deep sequencing reveals that this mechanism of altered gene transcription is restricted and that the majority of genes regulated by elevated O-GlcNAc levels are similarly regulated by a shift from euglycemic to hyperglycemic conditions. These findings implicate the O-GlcNAc modification as a potential mechanism for hyperglycemic-regulated gene expression in the beta cell.

The ways in which environmental factors participate in the progression of autoimmune diseases are not known. After initiation, it takes years before patients at risk for type 1 diabetes (T1D) develop hyperglycemia. The receptor for advanced glycated endproducts (RAGE) is a scavenger receptor of the immunoglobulin family that binds damage associated molecular patterns (DAMPs) and advanced glycated endproducts (AGEs) and can trigger cell activation. We previously found constitutive intracellular RAGE expression in lymphocytes from patients with T1D. Herein, we show that there is increased RAGE expression in T cells from at-risk euglycemic relatives who progress to T1D compared to healthy control subjects, and in the CD8+ T cells in the at-risk relatives who do vs those who do not progress to T1D. Detectable levels of the RAGE ligand HMGB1 were present in serum from at-risk subjects and patients with T1D. Transcriptome analysis of RAGE+ vs RAGE- T cells from patients with T1D showed differences in signaling pathways associated with increased cell activation and survival‥ Additional markers for effector memory cells and inflammatory function were elevated in the RAGE+ CD8+ cells of T1D patients and at-risk relatives of patients prior to disease onset. These studies suggest that expression of RAGE in T cells of subjects progressing to disease predates dysglycemia. These findings imply that RAGE expression enhances the inflammatory function of T cells and its increased levels observed in T1D patients may account for the chronic autoimmune response when DAMPs are released following cell injury and killing.

Biological therapeutics have seen a dramatic increase in molecular complexity and biological mechanisms. Antibody drug conjugates (ADCs) and multi‐specific formats are filling therapeutic pipelines and fundamentally changing traditional platform approaches to biologics discovery. These new entities pose unique challenges for characterization; as with traditional mAbs they are generally triaged through the discovery process by a series of functional assays including ELISA, FACS and binding kinetics however, we have implemented an additional and often underutilized SPR measure to characterize the function and quality of these entities: binding stoichiometry. Here we will discuss the special requirements for quality stoichiometry measures and show how stoichiometry can be used to 1) identify novel binding modes for a mAb that may have implications to ADC function, 2) identify signs of molecular instability, 3) aid in engineering functional biotherapeutic molecules and 4) assess simultaneous binding of analytes in multi‐specific formats. These examples will show an important measure that can be used to identify liabilities and to demonstrate function of biotherapeutic candidates. Support or Funding Information All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.