Polyamination with spermidine enhances pathogenic tau conformations while reducing filamentous aggregate formation in vitro

Tau is subject to a broad range of post-translational modifications (PTMs) that regulate its biological activity in health and disease, including microtubule (MT) dynamics, aggregation, and adoption of pathogenic conformations. The most studied PTMs of tau are phosphorylation and acetylation; however, the salience of other PTMs is not fully explored. Tissue transglutaminase (TG) is an enzyme whose activity is elevated in Alzheimer’s disease (AD). TG action on tau may lead to intramolecular and intermolecular cross-linking along with the incorporation of cationic polyamines [e.g., spermidine (SPD)] onto glutamine residues (Q). Even though SPD levels are significantly elevated in AD, the effects of SPD polyamination on tau biology have yet to be examined. In this work, we describe a method to produce recombinant SPD-modified tau where SPD modifications are mainly localized to Q residues within the N-terminus.  MT binding and polymerization assays showed that SPD modification does not significantly alter tau’s binding to MTs but increases MT polymerization kinetics. In addition, biochemical and biophysical assays showed that SPD polyamination of tau markedly reduces tau polymerization into filamentous and β-sheet containing aggregates. On the other hand, SPD modification promotes the formation of pathogenic conformations (e.g., oligomerization and misfolding) by tau with or without inducing tau polymerization. Taken together, these data suggest that SPD polyamination of tau enhances its ability to polymerize microtubules and favors the adoption of pathogenic tau conformations but not filamentous aggregates in vitro.

Methods
Preparation of recombinant unmodified and SPD-modified tau proteins

Recombinant hT40 and hT39 tau proteins were prepared from a 4L bacterial culture as described previously (Combs et al., 2017), with the exception that BL21 bacteria (NEB, #C2527H) were used. The concentration of recombinant tau protein was determined using the BCA method (Thermo, # A53225). Next, polyamination reactions were performed in vitro by adapting the protocol described by Song and coworkers (Song et al., 2013). Briefly, 8 mM of SPD (Sigma, # S2626-1G) and 0.2 µM of TG enzyme (Sigma, # TS398) were added to 0.93 mg/ml of tau in 50 µl of reaction buffer (50mM Tris HCl, pH 8, 10 mM calcium chloride and 5 mM DTT) followed by a 1-hour incubation at 37 °C. Unmodified tau proteins were subjected to the same reaction conditions, but TG was excluded. Then, the TG enzyme was inactivated by heating at 70 °C for 2 min. The TG-catalyzed reaction produces SPD-modified monomeric proteins as well as intra- and inter-molecular crosslinked proteins. To remove inter-molecular crosslinked tau (i.e. high molecular weight species), SPD-modified tau was further purified by passing the sample through 100 kDa MWCO Amicon filter (0.5 ml; Millipore, # UFC510096) (Figure S1). Unmodified tau proteins were subjected to the same process. The flow-through (FL) was collected for further purification. Each concentrator membrane was washed 6 times with 400 µl buffer A (500 mM NaCl, 10 mM Tris, and 5 mM Imidazole pH 8). All the FL samples were pooled and subsequently buffer exchanged into Buffer A using a HiPrep 26/10 desalting column (Cytiva, #17508701) and fast protein liquid chromatography (FPLC). The desalting column was equilibrated with 5 column volumes (CVs) of buffer A, then protein samples were run over the column in 5 CVs of buffer A at a flow rate of 5 ml/min, and fractions containing tau were collected. Finally, to purify the tau proteins (6x His tagged) from other proteins in the polyamination reaction (e.g. TG) and free SPD, heavy metal affinity chromatography was used using a 5 ml HiTrap Talon crude column (Cytiva, #28953767). The HiTrap Talon column was equilibrated in 5 CVs of buffer A, the proteins were run over the column, the column was washed with 5 CVs of buffer A, and then tau proteins were eluted in 10 CVs of buffer B (100 mM imidazole in buffer A, pH 8, supplemented with 200 µM PMSF) at a flow rate of 3 ml/min in 5 ml fractions. Fractions containing highly purified monomeric tau (determined using SDS-PAGE) were pooled and concentrated to 2-4 mg/ml using Amicon® Ultra Centrifugal Filter, 30 kDa MWCO (Millipore, # UFC903008), then DTT was added (final concentration of 1 mM). The unmodified hT40 (hT40) SPD-hT40, unmodified hT39 (hT39)), and SPD-hT39 samples were then aliquoted and frozen at -80 °C. The final concentration of recombinant tau proteins was determined using the SDS-Lowry method as described previously (Combs et al., 2017).

Preparation of recombinant tau protein for tandem mass spectrometry

The hT40, SPD-hT40, hT39, and SPD-hT39 proteins were digested using a combination of trypsin (Promega, #V5280) and rLysC (Promega, #V167A). First, each protein (3 µg) was subjected to 5 rounds of buffer exchange with 25 mM ammonium bicarbonate (AmBic) pH 8 using a 0.5 ml 3K MWCO Amicon centrifugal filter (15,000 x g for 10 min; Millipore, # UFC500396). Then, the tau proteins were retrieved by inverting the filter into a recovery tube, centrifugation at 15,000 x g for 2 min, and then vacuum drying using Vacufuge. The dried pellets were reconstituted in 50 µl of digestion buffer [12.5 mM AmBic, pH 8 + 50% acetonitrile (ACN)] and incubated at 37 °C for 90 min with rLysC (150 ng of enzyme per 3 μg of recombinant protein). Then, trypsin was added (300 ng of enzyme per 3 μg of recombinant protein) and incubated at 37 °C for 16-18 hours. The following day, digested protein samples were vacuum dried and stored at -20 °C until running on mass spectrometry (MS).

Tandem MS of recombinant tau proteins

We used a Thermo Scientific Ultimate 3000 RSLCnano System coupled with nanoscale liquid chromatography. Desalting of digested peptides was conducted in-line using a 3 μm diameter bead, C18 Acclaim PepMap trap column (75 μm × 20 mm) with 2% ACN, 0.1% formic acid (FA) for 5 min at a flow rate of 2 μl/min at 40 °C. The trap column was then brought in line with a 2 μm diameter bead, C18 EASY-Spray column (75 μm × 250 mm) for analytical separation over 128 min with a flow rate of 350 nl/min at 40 °C. The mobile phase included two buffers: 0.1% FA (Buffer A) and 0.1% FA in ACN (Buffer B), and a gradient was used for separation as follows: 12.5 min desalting, 95 min 4–40% B, 2 min 40–65% B, 3 min 65–95% B, 11 min 95% B, 1 min 95–4% B, 3 min 4% B. We injected 1 μg of each sample for analysis. Top 20 data-dependent mass spectrometric analysis was performed with a Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer. MS1 resolution was 60K at 200 m/z with a maximum injection time of 45 ms, AGC target of 3e6, and scan range of 300–1500 m/z. MS2 resolution was 30K at 200 m/z, with a maximum injection time of 54 ms, AGC target of 1e5, and isolation range of 1.3 m/z. High-energy collision dissociation (HCD) normalized collision energy was 28. Only ions with charge states from +2 to +6 were selected for fragmentation, and dynamic exclusion was set to 30 s. The electrospray voltage was 1.9 kV at a 2.0 mm tip-to-inlet distance. The ion capillary temperature was 280 °C and the RF level was 55.0. All other parameters were set as default.

MS data analysis to determine SPD modification sites

RAW data files were analyzed with the MetaMorpheus software version 1.0.1 developed by the Smith laboratory (Miller et al., 2023). For hT40 proteins, the following databases were downloaded from Uniprot (November 2021) and used for analysis: Escherichia coli (strain K12) (UP000000625), trypsin (Q29463), Lys-C (Q02SZ7), and full-length tau sequence (2N4R isoform, P10636-8). The same files were used to analyze the hT39 proteins using with 2N3R tau isoform sequence (P10636-5) instead of the 2N4R sequence. Mass shifts corresponding to the non-acetylated SPD were used to search for modifications: +128.1313485 for SPD (Schopfer et al., 2024; Yu et al., 2015). In addition, the fragmentation pattern of SPD was determined by running SPD alone on MS. Mass-to-charge-ratios (m/z) corresponding to diagnostic ions (DIs) were identified: 54.048, 57.059, 71.075, 111.109, 128.132. The search parameters for the SPD modification included both mass shift and the identified diagnostic ions.

The analysis sequence included mass calibration, global post-translational modification discovery (G-PTM-D) (Li et al., 2017), and a classic search. Mass calibration was conducted using the following criteria: protease = trypsin; maximum missed cleavages = 2; minimum peptide length = 7; maximum peptide length = unspecified; initiator methionine behavior = variable; variable modifications = Oxidation on M; max mods per peptide = 2; max modification isoforms = 1024; precursor mass tolerance = ±15.0000 ppm; product mass tolerance = ±25.0000 ppm. The criteria utilized for G-PTM-D were protease = trypsin; maximum missed cleavages = 2; minimum peptide length = 7; maximum peptide length = unspecified; initiator methionine behavior = Variable; max modification isoforms = 1024; variable modifications = Oxidation on M; G-PTM-D modifications count = 3; precursor mass tolerance(s) = ±5.0000 ppm around 0 ,128.131348525 Da; product mass tolerance = ±20.0000 ppm. Finally, a classic search was conducted using the following criteria: protease = trypsin; search for truncated proteins and proteolysis products = false; maximum missed cleavages = 2; minimum peptide length = 7; maximum peptide length = unspecified; initiator methionine behavior = variable; variable modifications = Oxidation on M; max mods per peptide = 2; max modification isoforms = 1024; precursor mass tolerance = ±5.0000 ppm; product mass tolerance = ±20.0000 ppm; report peptide spectral match (PSM) ambiguity = true. SPD polyamination site of tau detected at a false discovery rate of 1% are reported (Supplementary table S1). Supplementary table S2 demonstrates all quantified tau peptides in unmodified vs SPD-modified tau samples. Supplementary table S3 shows the quantified peaks of tau with their corresponding peptide masses, theoretical and observed m/z, retention time, and PSMs. MetaDraw version 1.0.5 was utilized to review the PSMs of modified and unmodified tau peptides (samples of these peptides are included in Figures S2 and S3). The .RAW MS files for each protein analysis are located here.