Chemical synthesis of photocrosslinking sugars
Cell culture, flow cytometry, and immunoblotting
BJA-B K88, BJA-B K20, and Daudi cells were cultivated in RPMI 1640 media containing 2 mM glutamine and supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 µg/ml streptomycin. Unless otherwise noted, cells were maintained in a water-saturated atmosphere at 37 °C and 5% CO2. Typically, cell densities were maintained between 2.5 × 105 and 2.0 × 106. To generate BJA-B cells in serum free conditions, the cells were grown in RPMI 1640 with 2 mM l-glutamine containing 1X Nutridoma SP, 50 U/ml penicillin, and 50 µg/ml streptomycin. Cells were cultured for two passages at 2.5 × 105 cells/mL in serum-free media for 72 h prior to supplementation with monosaccharides.
Prior to the addition of cells to a 12 well plate, ethanol (EtOH), Ac4ManNAc, Ac4ManNDAz(2me), Ac4ManNDAz(3me), Ac4ManNDAz(4me), Ac4GlcNDAz(2me), or Ac4ManNAz in EtOH were added to achieve a final concentration of 100 µM once media was added. EtOH was evaporated at ambient temperature and pressure prior to the addition of cells. Cells were seeded at a density of 3.0 × 105 cells/mL and cultured for 72 h with the compounds. After growth in the presence of the appropriate monosaccharides, cells were harvested by pelleting at 400g for 4 min and aspirating the supernatant. Cell viability was assessed using Trypan blue dye staining with the Countness Automated Cell Counter instrument (Invitrogen, Carlsbad, CA). When possible, cells were kept in the dark and on ice for subsequent procedures.
Jurkat cells were cultivated in RPMI 1640 media containing 2 mM L-glutamine and supplemented with 10% heat-inactivated FBS. Unless otherwise noted, cells were maintained in a water-saturated atmosphere at 37 °C and 5% CO2. Typically, cell densities were maintained between 2.5 × 105 and 2.0 × 106. Cell viability was assessed using Trypan blue dye staining with the Countess Automated Cell Counter.
Flow cytometry and immunoblotting were performed according to reported procedures.9, 13, 16
Details are provided in the Supporting Information
Analysis of ganglioside content for Jurkat cells cultured with compounds 1–6
All reagents, chemicals, and general supplies were purchased and used as received from Fisher Scientific (Waltham, MA) or Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Dulbecco’s phosphate buffered saline (DPBS) and CTxB-488 were purchased from Invitrogen (Carlsbad, CA). Bovine serum albumin (BSA) Fraction V was purchased from Roche Applied Science (Indianapolis, IN). SepPak tC18 columns (0.3 g) were purchased from Fisher Scientific. HPTLC plates (20 × 20 cm, glass-backed, 200 µm thickness) were purchased from EMD Chemicals (Gibbstown, NJ). Matreya ganglioside standards 1408, 1510, and 1511 were purchased from Matreya LLC (Pleasant Gap, MD).
Jurkat cells were cultivated as described above. Prior to the addition of cells to a 25 cm plate, EtOH, Ac4ManNAc, Ac4ManNDAz(2me), Ac4ManNDAz(3me), Ac4ManNDAz(4me), Ac4GlcNDAz(2me), or Ac4ManNAz in EtOH were added to achieve a final concentration of 100 µM once the media was added. The EtOH was pre-evaporated at ambient temperature and pressure. Jurkat cells were then seeded at a density of 2.5 × 105 cells/mL and incubated in the presence of the monosaccharides at 37 °C and 5% CO2. After growth with the appropriate compounds for 72 h, cells were harvested, counted, and centrifuged at 650g for 5 min in 50 mL conical tubes and the supernatant aspirated. To ensure that all ganglioside concentrations were normalized, equal numbers of cells (2.7 × 107 cells) were collected for all samples. Cell pellets were stored at −80 °C overnight.
Cell pellets were thawed to RT and resuspended in 300 µL of ice-cold ddH2O. They were then dounced 50 times with a Kontes tissue grinder, tube size 20. Following homogenization, cell suspension was added to a vial of stirring MeOH (800 µL). For the previous step and those following, glass Pasteur pipettes and glass vials were used; no plastic came in contact with samples. Samples were also shielded from light at every point possible during this procedure. To the stirring solution, 400 µL of chloroform was added and the mixture stirred for 2.5 h at RT. The mixture was then transferred into a 13 × 100 mm glass culture tube and centrifuged at 2800g for 10 min at 30 °C. The resulting supernatant (total lipid extract - TLE) was transferred to a glass vial and the sample evaporated under a stream of nitrogen to dryness. The resulting yellow film was stored at RT in the dark.
For ganglioside isolation, the TLE was resuspended in 1200 µL of diisopropyl ether and 800 µL 1-butanol. This mixture was sonicated in a bath sonicator for 10 min. The resulting cloudy solution was transferred to a 13 × 100 mm glass culture tube. To the solution, 1 mL of 50 mM sodium chloride was added. Following mixing with a Pasteur pipette, the suspension was centrifuged at 2800g for 10 min at 30 °C to separate the two phases. The upper (organic) layer was then removed. To the aqueous layer, 1200 µL of diisopropyl ether and 800 µL of 1-butanol were added and mixed. Following centrifugation at 2800g for 10 min at 30 °C and removal of the organic layer; these last two steps were repeated once more.
For final purification, the remaining lipid mixture was loaded on a SepPak tC18 column (0.3g size), washed and eluted. First, the column was prepared by several washing steps: three washes with 2 mL of chloroform:MeOH:ddH2O (C:M:W, 2:43:55) were followed by two washes with 2 mL of C:M (1:1) and lastly three more washes with 2 mL of C:M:W (2:43:55). The sample was loaded onto the column and washed thrice with 2 mL of C:M:W (2:43:55) followed by three washes with 2 mL of M:W (1:1). Finally, the gangliosides were eluted in 2 mL of 100% MeOH. Ganglioside extracts were then transferred to a new 4 mL glass vial and evaporated to dryness under a stream of nitrogen. The clear, white film was stored at RT for further analysis.
A TLC chamber was pre-equilibrated with 140 mL of chloroform:MeOH:0.2% CaCl2(aq) (85:45:10). The HPTLC plate (EMD Chemicals #115534, 20 × 20 cm, glass-backed plates, silica gel 60 F254, 200 µm thickness) was pre-run and dried prior to sample loading to improve resolution. The syringe used for loading samples was cleaned with C:M:W, 4:8:3. Extracted ganglioside samples were redissolved in 50 µL C:M:W (2:1:0.1) and loaded onto the HPTLC plate along with ganglioside standards and dried. The HPTLC plate was then run in the chamber followed by drying under vacuum for 45 min.
To assess the presence of GM1a, the HPTLC plate was immersed in 0.5% polyisobutylmethacrylate in hexanes (diluted from a stock solution of 2% in chloroform) for 2 min to fix the gangliosides to the TLC plate. The plate was then fully dried followed by immersion in 1.0% BSA in DPBS for 30 min at RT. The plate was incubated with CTxB-Alexa Fluor 488 conjugate (1:20,000 dilution of a 1 mg/mL solution) in DBPS for 50 min at RT in the dark. The plate was washed briefly with DPBS, dried fully, and imaged by Typhoon with a 488 nm excitation laser and 520 nm emission filter. Global adjustments to image contrast and brightness were performed using Adobe Photoshop.
To visualize all gangliosides, the above plate was resorcinol-stained. The plate was sprayed with a resorcinol solution (0.1% resorcinol, 0.04% CuSO4 in hydrochloric acid: water (4:1)). Following staining, the plate was thoroughly dried and sandwiched between two glass plates, then incubated at ~100 °C for 15–20 min until bands were clearly visible. The plate was imaged with white light using an Alpha Innotech FluorChem HD2 imager and global adjustments to image contrast and brightness were made with Adobe Photoshop.
Cytosolic and membrane-bound sialic acid analysis by DMB derivatization
All reagents, chemicals, and general supplies were purchased and used as received from Fisher Scientific (Waltham, MA) or Sigma-Aldrich (St. Louis, MO) unless otherwise noted. PVDF membranes were purchased from Millipore, Immobilon (Billerica, CA). For chemiluminescent visualization, SuperSignal WestPico Chemiluminescent Substrate (ECL reagent) was purchased from Pierce (Rockford, IL). Polycarbonate centrifuge tubes (11 × 34 mm) were purchased from Beckman (Brea, CA). The bicinchoninic acid (BCA) Protein Assay Kit from Pierce was used to assess protein concentration. DMB was purchased from Sigma Aldrich (we found other vendors’ products to be inferior). Amicon Ultra Centrifugal Filters (0.5 mL; 10 kDa molecular weight cut-off (MWCO)) were purchased from Millipore. Complete™ Protease Inhibitor Cocktail Tablet, EDTA-free were purchased from Santa Cruz Biotechnology. Tris-buffered saline Tween-20 was used in blocking solutions (TBST: 10 mM Tris•HCl pH 8.0, 150 mM NaCl, 0.1% v/v Tween 20). The hypotonic lysis buffer was composed of 10 mM Tris•HCl pH 7.3, 10 mM MgCl2, 1 mM EDTA, and 1 mM EGTA.
Jurkat cells were grown in the presence of sugars 1–6 as described above. Cells were harvested after 72 h and the number of cells normalized to 5 million. Cells were washed twice in 500 µL DPBS. Next, cells were swollen in hypotonic lysis buffer containing protease inhibitors for 15 min on ice. Cells were lysed by extrusion through a 25 gauge needle 30 times. Nuclei and unbroken cells were removed from the post-nuclear supernatant by two rounds of centrifugation at 1000g for 15 minutes at 4 °C. Next, the post-nuclear supernatant was transferred to heavy-walled polycarbonate tubes and centrifuged at 100,000g for 1 h in a Beckman TLA 120.2 rotor. The supernatant was designated the cytosolic fraction. The pellet was washed twice with cold 400 µL hypotonic lysis buffer and centrifuged at 100,000g for 1 h. The remaining pellet was designated the membrane fraction. Samples were either flash-frozen and the solvent removed by vacuum overnight or saved to assess fraction purity. To confirm that the membrane fraction was not contaminated by the nuclear fraction, protein concentration was normalized and proteins were resolved by SDS-PAGE. Proteins were then transferred to PVDF and probed for either lamin B (to identify the nuclear fraction) or calnexin (to identify the membrane fraction). DMB-derivatization (as described below) confirmed that no sialic acids were present in the membrane washes; this ensured that that no soluble sialic acid (free sialic acid or CMP-sialic acid) remained in the membrane fraction.
To release sialic acids, 50 or 100 µL of 2.0 M acetic acid was added for the membrane-bound and dried cytosolic samples, respectively. Solutions were incubated at 80 °C for 2 h. Samples were then cooled to room temperature. To the membrane-bound and cytosolic samples, 40 or 80 µL of 7 mM DMB, 0.75 M 2-mercaptoethanol, 18 mM Na2
, 1.4 M acetic acid were added, respectively. The samples were then incubated at 50 °C for 2 h. After this incubation period, 5 or 10 µL of 0.2 M NaOH was added to each membrane-bound or cytosolic sample, respectively. Samples were then filtered in 10 kDa MWCO filters by centrifugation and the resulting flow-through was stored at −20 °C, in the dark until used. The derivatives were analyzed by on-line fluorescence detection using reverse-phase HPLC. Details are provided in the Supporting Information
Mass spectrometry analysis of crosslinked CTxB-GM1a-SiaDAz(2me)
Photocrosslinking of CTxB-GM1a complexes was performed essentially as described previously.13
The SAMDI mass spectrometry process was based on a literature procedure.18
Details are provided in the Supporting Information