Tau fibrillation and labeling

A truncated form of the human tau protein, containing the four microtubules repeat domains (residues Q224-E372, corresponding to K18) with a P301L mutation, containing a myc tag at the C-terminus, was produced in Escherichia coli (Tebu-bio). To generate tau PFFs, 40 μM of K18 was mixed with 40 μM of low molecular weight (LMW) heparin (MP Biomedicals) and DTT (2 mM) in sodium acetate (100 mM) pH 7.0 buffer and incubated at 37 °C for 10 days. The mixture was centrifuged at 100,000 ´g at 4 °C for 1 h. The pellet was resuspended in sodium acetate (100 mM) pH 7.0 buffer and sonicated with a probe sonicator (two cycles of 10 × 1 s pulses at 20% amplitude). The characterization of fibrils generated with this protocol has been previously reported [51]. The homogeneous solution was aliquoted and stored at − 80 °C until further use. K18 fibrils (tau PFFs) were labeled with Alexa Fluor™ 488 TFP ester (Invitrogen) reactive dye according to the supplier’s instructions. To remove excess label, dialysis was performed using slide-A-Lyzer™ dialysis cassettes, with a 10 K MWCO (Thermo Fisher Scientific) in 5 l of PBS buffer at 4 °C for 48 h, with stirring at 200 RPM. Labeled tau PFFs (tau-AF488) solution was aliquoted and stored at − 80 °C until further use. Tau PFFs were freshly sonicated before use.

Cell culture, plasmids and transfections

HCT-116 (RRID: CVCL_0291) cells in McCoy’s 5A (modified) medium with 10% FBS and 50 μg/ml of gentamicin were cultured in pre-coated poly-D-lysine 96-well black μclear plates (Fisher Scientific) (abbreviated to black 96wp). CHO-K1 (RRID:CVCL_0214) cells were grown in Ham’s F12 medium supplemented with 2 mM Glutamine and 10% FBS. The cells were maintained in a humidified atmosphere with 5% CO2 at 37 °C. To transfect HCT-116 cells, the sequences of wild-type HS3ST1 and mutant HS3ST1 (R63A, E86Q, E86A, H88A, H88F, K119A, R272A) were obtain from Moon et al. [36]. The constructs were designed to express mKate2 at the C-terminus of the HS3ST1 sequence. The constructs were cloned into BamHI/XhoI-digested pcDNA3.1(+) backbone by Epoch Life Science Inc. For transient overexpression of proteins, HS3ST1−/− and WT HCT-116 cells were seeded at 12 × 103 cells/well for one day. The cells were transfected using Lipofectamine 3000 reagent (Invitrogen) and the appropriate DNA construct amount (100 ng or 200 ng), following the manufacturer’s protocol, and incubated for 24 h.

CRISPR/Cas9-mediated gene editing for the generation of HS3ST1 −/− HCT-116 clones

To generate HS3ST1−/−, two single-guide RNAs (sgRNAs) were designed using the CHOPCHOP webtool (http://chopchop.cbu.uib.no/), to target the HS3ST1 gene (sgRNA 1: 5′-gggcgacgtgaaatacgcgg-3′; sgRNA 2: 5′-tgtgcacgtggtagaggctg-3′). The two crRNAs were produced by IDT DNA Alt-R™ S.p. Cas9 Nuclease 3NLS (I1074182) and Alt-R™ CRISPR tracrRNA (1072534) were obtained from IDT DNA. HCT-116 WT cells seeded at 16 × 103 cells/well for 24 h and transfected following the manufacturer’s protocol. Briefly, crRNA (100 μM) was complexed with tracrRNA (100 μM), following assembly with recombinant Cas9 (145 nM) to generate ribonucleoproteins (RNPs). The cells were transfected with the sgRNA-tracrRNA-Cas9 RNP complex using Lipofectamine CRISPRMAX (Invitrogen). After 48 h, transfected cells were single-cell sorted into black 96wp, clones were expanded, sequenced by Sanger sequencing and selected for homozygous mutations in the HS3ST1 gene.

Fluorescence microscopy

HCT-116 WT cells were seeded at 12 × 103 cells/well in black 96wp. To assess HSPG-dependent uptake of tau PFFs, one day after platting, cells were treated with either heparinase III (Sigma-Aldrich) at different doses (from 0.12 U/ml to 0.00094 U/ml) for 16 h or with LMW heparin (MP Biomedicals) at different doses (from 200 μg/ml to 1.6 μg/ml) for 3 h. Tau-AF488 (75 nM) was added to the culture medium and incubated for 24 h. Hoechst 33342 (200 μg/ml) and CellMask™ deep red plasma membrane stain (5 μg/ml) were added to the cells 5 min prior to live-cell imaging.

To detect 3-O-sulfation in HCT-116 HS3ST1−/− and WT cells, cells were seeded at 12 × 103 cells/well. The day after, cells were treated with either heparinase III (0.31 U/ml) or PBS at 37 °C for 16 h. The cells were fixed with 4% PFA in PBS for 15 min, followed by incubation with either heparin (200 μg/ml) or PBS at room temperature for 90 min with gentle shake. Afterwards, 10 μg/ml of antithrombin III (ATIII; ab62542, Abcam) was added to the cells and incubated at room temperature for 2 h, with gentle shake. Cells were washed five times with PBS followed by mouse anti-ATIII (1:1000; RRID: AB_779265) incubation at 4 °C overnight. The cells were washed three times with PBS followed by incubation with the goat anti-mouse antibody (1:10.000; RRID: AB_2536161) at room temperature for 1 h. Cells were washed three times and Hoechst 33342 (200 μg/ml) and CellMask™ deep red plasma membrane stain (5 μg/ml) were added to the cells 5 min prior to confocal fluorescence microscopy.

For tau-AF488 PFFs uptake assay in HCT-116 HS3ST1−/− and WT, heparin (200 μg/ml) was added to the cells for 3 h. Afterwards, tau-AF488 (75 nM) was added to the culture medium and incubated for 24 h. Hoechst 3342 (200 μg/ml) and CellMask™ deep red plasma membrane stain (5 μg/ml) were added to the cells 5 min prior to live-cell imaging. For the HS3ST1 WT and mutants’ overexpression assay, tau-AF488 (75 nM) was added to the cells 20 h after transfection and for 24 h, with the addition of Hoechst (200 μg/ml) 5 min prior to live-cell imaging. To block 3S-HS, both HCT-116 HS3ST1−/− and WT cells were pre-treated with ATIII protein (10 μg/ml) or heparin (200 μg/ml) for 1 h30. Tau-AF488 (75 nM) was added to the culture medium and incubated for 5 h. Hoechst 3342 (200 μg/ml) and CellMask™ deep red plasma membrane stain (5 μg/ml) were added to the cells 5 min prior to live-cell imaging.

HCT-116 WT cells were seeded at 12 × 103 cells/well. The day after, cells were treated with either 3-O sulfated HS, (3S-HS), non 3-O sulfated HS (HS) or non-sulfated HS (noS-HS) synthetic oligosaccharides at different doses (from 5 μM to 2.3 nM for 30 min) 2 h. Afterwards, cells were incubated with tau-AF488 seeds (75 nM) for 5 h. Hoechst 33342 (200 μg/ml) and CellMask deep red plasma membrane stain (5 μg/ml) were added to the cells 5 min prior to live-cell imaging.

For the image acquisition, Opera Phenix™ High-Content Screening System (PerkinElmer) was used. For live-cell imaging, the temperature was set to 37 °C and 5% CO2. A series of confocal images were acquired by imaging 13 fields per well at four planes in the z-axis at an interval of 1 μm section with a 40x water immersion objective per field. Images were analyzed using the maximum intensity projection of the image stack. Quantification of the confocal images acquired with the Opera Phenix, was conducted using the Harmony High-Content Imaging and Analysis Software v4.9 (PerkinElmer). For the quantification of tau-AF488 in transfected cell lines, regions of interest (ROIs) were defined based on the mKate2 signal, nuclei within those regions were defined as transfected cells and tau-AF488 puncta in those ROIs were defined as tau PFFs internalized by transfected cells. For quantification purposes, the number of tau-AF488 puncta inside transfected cells was normalized to the number of nuclei of transfected cells and multiplied by the mean of the tau-AF488 puncta fluorescence intensity. For the remaining uptake assays, no ROIs were defined, and uptake was quantified based on the number of tau-AF488 puncta normalized to the number of nuclei multiplied by the mean puncta intensity.

mRNA levels quantification by qPCR

To perform the HS3STs expression profiles, HCT-116 cells were seeded at 12 × 103 cells/well. RNA was extracted using the RNeasy Mini Kit (Qiagen) following the manufacturer’s protocol. RNA was converted into cDNA following the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) protocol. Real-time quantitative PCR was performed using PrimeTime® Gene Expression Master Mix (IDT DNA), and analyzed by the QuantStudio™ 12 K Flex Real-Time PCR System (Applied Biosystems), using the prime-probe detection protocol. Homo sapiens TaqMan assays (IDT DNA) used were the following: Hs.PT.58.20018206.g (HS3ST1), Hs.PT.58.344017 (HS3ST2), Hs.PT.58.774300.g (HS3ST3A1), Hs.PT.58.19465218 (HS3ST3B1), Hs.PT.58.26274331 (HS3ST4), Hs.PT.58.20285766 (HS3ST5), Hs.PT.58.40666735 (HS3ST6), Hs.PT.39a.22214847 (ACTB), Hs.PT.58v.18759587 (B2M) and Hs.PT.58v.27737538 (GUSB). ACTB, B2M and GUSB are housekeeping genes and were used to normalize the data. Calibrated normalized relative quantities (CNRQ) values were calculated to the control conditions described in the results. Data is represented as mean ± SD, of a total of three independent experiments.

Extraction of HS from the cells

HCT-116 WT, HS3ST1−/− and CHO-K1 cells were seeded at 12 × 103 cells/well. Around 4 × 106 cells were collected for HS extraction. The HS extraction procedures were published previously with minor modification [52, 53]. Briefly, the cell pellet was suspended in water and proteolyzed with pronase E (10 mg:1 g (w/w), pronase E/protein) at 55 °C for 24 h to digest the protein. The digest solution was denatured at 100 °C for 10 min and centrifuged at 14000 rpm for 10 min to get the supernatant. The recovery calibrant 13C-labeled N-sulfo heparosan was added into the supernatant before subjecting to DEAE column purification [52]. DEAE column mobile phase A consisted of 20 mM Tris, pH 7.5 and 50 mM NaCl, and mobile phase B consisted of 20 mM Tris, pH 7.5 and 1 M NaCl. After loading the solution, the column was washed with 1.5 mL mobile phase A, followed by 1.5 mL mobile phase B to elute the HS fraction. The YM-3KDa spin column was applied to desalt the elute, and the retentate was subjected to heparin lyases digest. Before the digestion, a known amount 13C-labeled 3-O-sulfation oligosaccharide calibrants was added to the retentate [53]. The 100 μL of digestion solution containing 7.5 μL of enzymatic buffer (100 mM sodium acetate/2 mM calcium acetate buffer (pH 7.0) containing 0.1 g/L BSA), and 1.25 μL heparinase I (1.2 U/ml) and 2.5 μL heparinase II (1.2 U/m). The reaction solution was incubated at 37 °C for 5 h. Before recovering the digests from the digest solution, a known amount 13C-labeled non-3-O-sulfated disaccharide calibrants were added to the digestion solution. The HS disaccharides and tetrasaccharides were recovered by centrifugation, and the filter unit was washed twice with 200 μL of deionized water. The collected filtrates were freeze-dried before the derivatization with 2-aminoacridone (AMAC).

A total of five 13C-labeled 3-O sulfated oligosaccharide calibrants were used during the analysis of 3-O sulfated tetrasaccharides. These structures are oligo 1 GlcNAc-GlcA-GlcNAc6S-[13C]GlcA-GlcNS3S6S-[13C]IdoA2S-GlcNS6S-GlcA-pNP, oligo 2 GlcNAc-GlcA-GlcNS6S-[13C]GlcA-GlcNS3S6S-IdoA2S-GlcNS6S-GlcA-pNP, oligo 3 GlcNS6S-[13C]GlcA-GlcNS6S-[13C]IdoA2S-GlcNS3S6S-[13C]IdoA2S-GlcNS6S-GlcA-pNP, oligo 4 GlcNS-GlcA-GlcNS-[13C]IdoA2S-GlcNS3S-IdoA2S-GlcNS-GlcA-pNP and oligo 5 GlcNAc-GlcA-GlcNS-[13C]IdoA2S-GlcNS-IdoA2S-GlcNS3S-IdoA2S-GlcNS-GlcA-pNP. Eight 13C-labeled HS disaccharide calibrants were used for the analysis of non 3-O sulfated HS portion, and their structures were shown in a previous publication [52].

Chemical derivatization of HS disaccharides and tetrasaccharides

The 2-Aminoacridone (AMAC) derivatization of lyophilized samples was performed by adding 10 μL of 0.1 M AMAC solution in DMSO/glacial acetic acid (17:3, v/v) and incubating at room temperature for 15 min as described previously [52]. Then 10 μL of 1 M aqueous sodium cyanoborohydride (freshly prepared) was added to this solution. The reaction mixture was incubated at 45 °C for 2 h. After incubation, the reaction solution was centrifuged to obtain the supernatant that was subjected to the LC-MS/MS analysis.

LC-MS/MS analysis

The analysis of AMAC-labeled HS was performed on a Vanquish Flex UHPLC System (Thermo Fisher Scientific) coupled with TSQ Fortis triple-quadrupole mass spectrometry as the detector [52]. The ACQUITY Glycan BEH Amide column (1.7 μm, 2.1 × 150 mm; Waters, Ireland, UK) was used to separate di/tetra-saccharides at 60 °C. mobile phase A was 50 mM ammonium formate in water, pH 4.4. Mobile phase B is acetonitrile. The elution gradient as follows: 0–15 min 83–70% B, 15–30 min 70–50% B, 30–35 min 50% B, 35–45 min 83% B. The flow rate was 0.3 ml/min. On-line triple-quadrupole mass spectrometry operating in the multiple reaction monitoring (MRM) mode was used as the detector. The ESI-MS analysis was operated in the negative-ion mode using the following parameters: Neg ion spray voltage at 3.0 kV, sheath gas at 55 Arb, aux gas 25 arb, ion transfer tube temp at 250 °C and vaporizer temp at 400 °C. TraceFinder software was applied for data processing. The amount of HS was determined by comparing the peak area of native di/tetra-saccharides to each di/tetra-saccharides calibrant, and the recovery yield was calculated based on a comparison of the amount of recovery calibrant disaccharide in the samples and control, respectively. The results are represented as mean of two replicas of each cell line and the respective standard deviation (SD). The fold change between the HS3ST1−/− and HCT-116 cells was calculated for each calibrant to easily assess the content differences.

ELISA

To address and quantify the binding affinity of tau to different synthetic HS structures, the ELISA method was performed, following the supplier’s protocol. Briefly, biotinylated 3-O sulfated HS, (3S-HS), non-3-O sulfated HS (HS) or non-sulfated HS (NoS-HS) synthetic oligosaccharides (250 nM) were immobilized on a Pierce™ Streptavidin Coated Plates, Clear, 96-Well (Thermo Fisher Scientific) for 2 h at room temperature. The plates were blocked with 1x casein in PBS blocking buffer for 1 h at room temperature followed by incubation with sonicated tau PFFs in a dose-response (from 500 nM to 0.5 nM), for 1 h30 at room temperature. Bound tau was detected using anti-tau antibody PT76 [54] (1:2000) for 1 h30 at room temperature followed by incubation with anti-mouse HRP labeled antibody (Bio-Rad) (1:2000) for 1 h at room temperature. Signal was developed using the 1-step™ ultra TMB enzyme substrate solution (Thermo Fisher Scientific) and absorbance was measured at 450 nm using the multimode plate reader Envision (PerkinElmer).