Combinatorial native MS and LC-MS/MS approach reveals high intrinsic phosphorylation of human Tau but minimal levels of other key modifications

Abstract

Abnormal changes in the neuronal microtubule-associated protein Tau, such as hyperphosphorylation and aggregation, are considered hallmarks of cognitive deficits in Alzheimer disease. Hyperphosphorylation is thought to take place before aggregation, and therefore it is often assumed that phosphorylation predisposes Tau towards aggregation. However, the nature and extent of phosphorylation has remained ill-defined. Tau protein contains up to 85 potential phosphorylation sites (80 Ser/Thr, and 5 Tyr P-sites), many of which can be phosphorylated by various kinases because the unfolded structure of Tau makes them accessible. However, limitations in methods (e.g. in mass spectrometry of phosphorylated peptides, or antibodies against phospho-epitopes) have led to conflicting results regarding the overall degree of phosphorylation of Tau in cells. Here we present results from a new approach, that is based on native mass spectrometry analysis of intact Tau expressed in a eukaryotic cell system (Sf9) which reveals Tau in different phosphorylation states. The extent of phosphorylation is remarkably heterogeneous with up to ∼20 phosphates (Pi) per molecule and distributed over 51 sites (including all P-sites published so far and additional 18 P-sites). The medium phosphorylated fraction Pm showed overall occupancies centered at 8 Pi (± 5 Pi) with a bell-shaped distribution, the highly phosphorylated fraction Ph had 14 Pi (± 6 Pi). The distribution of sites was remarkably asymmetric (with 71% of all P-sites located in the C-terminal half of Tau). All phosphorylation sites were on Ser or Thr residues, but none on Tyr. Other known posttranslational modifications of Tau were near or below our detection limit (e.g. acetylation, ubiquitination). None of the Tau fractions self-assemble readily, arguing that Tau aggregation is not promoted by phosphorylation per se but requires additional factors.

In-vitro propagation of pathological tau filaments related to Alzheimer’s Disease

“…Moreover, the exact phosphorylation sites on the two phosphorylated tau species are further determined by liquid chromatography tandem mass spectrometry (LCMS) which was performed by Kathy Li at UCSF mass spec facility. Consistent with previous reports 3,24 , we identified 21 phosphorylation sites on P-tau and 17 sites additionally on HP-tau protein which adds up to a total of 38 sites (Figure 8). The majority of the identified phosphorylated sites are prevalent in pathological tau species (denoted in red) and are thus disease relevant.…”

“…3,23 The most recent study by Drepper, et al (2020) reveals that phosphorylation sites acquired by tau in insect cells are highly similar to the sites identified in AD-tau. 24 Hence, this insect-baculovirus expression system is considered an ideal model for obtaining natively disease-relevant phosphorylated states of tau proteins for in-vitro biochemical and structural studies.…”

Validation of Tau Antibodies for Use in Western Blotting and Immunohistochemistry

“…2-5, 10, 11). Transgenic Tau expressed in mammalian cells, including in HEK293-derived cell lines, has been found to become extensively phosphorylated [77][78][79]. We therefore anticipated that many of the PTM-dependent Tau antibodies would also detect the transgenic protein in these samples, whilst allowing for the possibility that some Tau PTMs may not be generated in HEK293T cells cultured under standard conditions.…”