Preparation of neutral free oligosaccharides from mammalian cultured cells
In the cytosol of the eukaryotic cells, peptide:N-glycanase (PNGase) releases high mannose type N-glycans from misfolded glycoproteins [ref 1]. The released glycans are designated as free oligosaccharides (fOSs). After generation of the fOSs, they are subsequently processed by endo-β-N-acetylglucosaminidase (ENGase) and cytosolic mannosidase Man2C1 [ref 1]. After the processing, the fOSs are transported into the lysosomes for further degradation [ref 1, 2].
In this protocol, neutral fOSs are extracted from mammalian cultured cells with 75% ethanol and purified with a coupled ion exchange chromatography, followed by graphitized carbon column.
Phosphate-buffered saline (PBS)
Lysis buffer (20 mM Tris-HCl, pH7.4 and 10 mM EDTA)
Graphitized carbon column (150 mg/3 ml, GL-Science, Tokyo, Japan)
Dowex AG1-X2 (200–400 mesh, acetate form) (Bio-Rad)
Dowex AG50-X8 (200–400 mesh, H+ form) (Bio-Rad)
Polyprep column (Bio-rad)
Centrifuge (Avanti HP-30I, Beckman Coulter) and (himac CF8DL, Hitachi)
Speed Vac (Centrivap 78100, Labconco)
Extraction of total fOSs from mammalian cultured cells
Grow cells of your interest to 0.5 – 1 x 107 cells on a 10-cm dish.
Wash the cells twice with 5 ml of PBS.
Scrape the cells in 2 ml of PBS.
Centrifuge at 1,000 rpm for 5 min at 4ºC.
Discard the supernatant.
Add 300 μl of lysis buffer and resuspend.
Add 900 μl of ice-cold ethanol and vortex.
Incubate on ice for 15 min.
Centrifuge at 15,000 rpm for 15 min at 4ºC.
Dry the supernatant with Speed Vac.
Purification of the neutral fOSs
Resuspend the dried pellet with 1 ml of water. The solution becomes milky. If aggregates are formed, sonicate until dispersed completely.
Centrifuge at 15,000 rpm for 15 min at 4ºC and load the supernatant onto a coupled ion exchange chromatography (250 μl each of Dowex AG1-X2 and Dowex AG50-X8).
Collect the flow-through.
Repeat 2-(1), 2-(2) and 2-(3) twice.
Combine all the flow-through fractions.
Load the combined flow-through fractions onto a graphitized carbon column.
Wash the column with 3 ml of water.
Elute the bound fOSs with 2.5 ml of 25% acetonitrile.
Dry the eluate with Speed Vac.
Complete extraction of total fOSs from mammalian cells can be achieved by a single extraction of the cells with 75% ethanol. Procedures for purification of the fOSs are based on the previous report [ref. 3].
1 x 107 cells.
Approximately 1-3 nmol in total.