A genetic model to study O-GlcNAc cycling in immortalized mouse embryonic fibroblasts
MMS Amand, etc
O-GlcNAcylation is an abundant post translational protein modification in which the monosaccharide ß-N-acetyl-D-glucosamine (O-GlcNAc) is added to Ser/Thr residues by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). Analyses of O-GlcNAc-mediated signaling and metabolic phenomena are complicated by factors including unsatisfactory inhibitors and loss-of-function cell lines lacking identical genetic backgrounds. In this work, we generated immortalized wild type, Oga knockout (Oga KO), and Ogt floxed allele (Ogt floxed) mouse embryonic fibroblast (MEF) cell lines with similar genetic backgrounds. These lines will facilitate experiments and serve as a platform to study O-GlcNAc cycling in mammals. As a test paradigm, we used the immortalized MEF lines to investigate the way in which changes in O-GlcNAcylation affected the pathological phosphorylation of the tau protein. The activity of glycogen synthase kinase 3 beta (GSK3ß), a kinase that phosphorylates tau, decreases when expressed in Oga KO MEFs compared with wild type cells. Phosphorylation at Thr-231 in recombinant, tauopathy-associated tau with a proline- to-leucine mutation at position 301 (P301L) was altered when expressed in MEFs with altered O-GlcNAc cycling. In aggregate, our data support that O-GlcNAc cycling indirectly affects tau phosphorylation at Thr-231, but tau phosphorylation was highly variable, even in genetically stable, immortalized MEF cells. The variable nature of tau phosphorylation observed herein supports the need to use cells, akin to those generated here with genetically defined lesions and similar backgrounds, to study complex biological processes.