(585b) Probing the Conformation and Phospho-Specificity of Anti-Tau Antibodies Using Yeast Surface Display

The highly soluble microtubule-associated protein Tau (MAPT) is mainly expressed in neurons to modulate the stability of axonal microtubules and constitute the neuronal microtubules network. Alzheimer’s disease (AD) is one type of neurodegeneration associated with aggregation of the tau protein in neurofibrillary tangles. One of the key pathological hallmarks of AD is the accumulation of paired helical filament of hyper-phosphorylated MAPT. Antibodies specific to Tau and its post-translational modifications are a widely used tool for studying the mechanism of Tau mediated neurodegeneration and clinical assessment of disease progression. However, the specificity of these antibodies is often not validated. Use of antibodies with ill-defined specificity contributes to irreproducibility in biomarker identification and misunderstanding of potential mechanisms leading to neurotoxicity. In this study, we expressed the human MAPT on the cell surface of yeast S. cerevisiae to characterize the specificity of anti-Tau antibodies. We found that the Tau displayed on the yeast surface binds strongly to well-characterized AD-pathological conformation specific Tau antibodies, including Alz50, MC-1, and Tau-2. However, no reactivity was detected to Tau oligomer specific antibodies T22 and TOMA-1. Moreover, yeast cells displaying Tau showed no detectable staining with thioflavin S, indicating that yeast displayed Tau are in monomeric form. We next characterized the specificity of phosphorylation specific Tau antibodies using site-directed mutagenesis of AD associated sites and kinase treatment of paraformaldehyde fixed yeast cells displaying Tau. We applied glycogen synthase kinase 3 beta (GSK-3β) for this purpose, since it has been reported to phosphorylate recombinant Tau to the same level as abnormally phosphorylated tau associated with the paired helical filaments (PHF-tau) in AD patients’ brain. We focused on antibodies targeting phospho-threonine 231 (pT231) of Tau, as the phosphorylation of this site modulates the hyper-phosphorylation and conformational change. Interestingly, we found that a commercially available antibody specific to pT231 of Tau is not specific to phospho-Tau, while most other pT231-targeting antibodies showed significant binding only after the treatment of GSK-3β. Interestingly, most pT231 specific antibodies showed no binding to pseudo-phosphorylated T231 (T231 mutated to glutamic acid), indicating that these antibodies differentiate pseudo-phosphorylation from phosphorylation. Importantly, a widely used pT231 specific antibody showed no binding to yeast displayed Tau after the kinase treatment. However, the antibody showed clear binding to Tau expressed in human cells upon phosphorylation at pT231. Therefore, we are currently investigating the conformation specificity of pT231-specific antibodies. Preliminary results show that certain pT231 specific antibodies bind only to pT231 in specific conformations. Taken together, these results demonstrate that yeast surface display is a quantitative and robust platform for characterizing the specificity of phosphorylation-specific and/or conformation-specific Tau antibodies.