Parasite cells (2 g if grown in serum or 0.4 g if cultivated ‘serum-free’) or lyophilisates (50 mg) were suspended in ten millilitres boiling water and denatured for 5 minutes, prior to addition of formic acid [up to 5% (v/v)] and 1 mg porcine pepsin. Proteolysis was allowed to proceed for two days at 37 °C and the samples were centrifuged to remove insoluble material. The supernatant of the proteolysate was then incubated with 10 ml prewashed Dowex AG50 W×2 (Sigma-Aldrich) for one hour at 23 °C. The material was then poured into a column and the flowthrough fraction was reapplied; the column was then washed with 2% (v/v) acetic acid to remove unbound material and glycopeptides were eluted with 0.5 M ammonium acetate, pH 6, and lyophilised prior to gel filtration (Sephadex G25; GE Healthcare). Orcinol-positive fractions were pooled, heat treated for 5 minutes and subject todigestion with either PNGase A (peptide:N-glycosidase from almonds; Roche) in 50 mM ammonium acetate, pH 5, or PNGase F (peptide:N-glycosidase from Flavobacterium; Roche) in 100 mM ammonium carbonate, pH 8, overnight at 37 °C (in the case of the C1/4, G3, TV2 and IR-78 samples, the pooled gel filtration fractions were separated into two halves prior to glycan release, whereas the C1/3 preparation was subject to PNGase F then PNGase A treatment in series). A second round of Dowex chromatography was performed and the unbound glycans were analysed, without further purification, by MALDI-TOF MS (matrix assisted laser-desorption/ionisation time-of-flight mass spectrometry).
Pyridylamination was subsequently performed basically as described (Hase, et al. 1984 (link)). In brief, 100 mg 2-aminopyridine (Sigma-Aldrich) was dissolved in 76 μl concentrated HCl and 152 μl water; 80 μl of this solution was added to the dried glycan sample, prior to incubation in boiling water for 15 minutes. Then a solution of 4.4 mg of sodium cyanoborohydride (Sigma-Aldrich) in a mixture of 9 μl of the aforementioned 2-aminopyridine solution and 13 μl water was prepared; 4 μl of this cyanoborohydride-aminopyridine solution was added to the sample and the incubation was continued overnight at 90 °C prior to gel filtration (Sephadex G15; GE Healthcare). Fluorescence (excitation/emission 320/400nm) of the fractions was measured using a Tecan microtitre plate reader.
The glycan samples were named on the basis of the numbering of the original culture (see under Cultivation of parasites above andSupplementary Table ) and the enzymes used for release: e.g. C1/1A refers to PNGase A-released glycans from sample C1/1; C1/2F refers to PNGase F released glycans from sample C1/2; C1/3FA to combined PNGase F and A release of glycans from sample C1/3. In the case of G3 and IR-78, two independent cultures of each strain were used prior to being divided for PNGase A and F digestion; only data from one culture are presented here.
Pyridylamination was subsequently performed basically as described (Hase, et al. 1984 (link)). In brief, 100 mg 2-aminopyridine (Sigma-Aldrich) was dissolved in 76 μl concentrated HCl and 152 μl water; 80 μl of this solution was added to the dried glycan sample, prior to incubation in boiling water for 15 minutes. Then a solution of 4.4 mg of sodium cyanoborohydride (Sigma-Aldrich) in a mixture of 9 μl of the aforementioned 2-aminopyridine solution and 13 μl water was prepared; 4 μl of this cyanoborohydride-aminopyridine solution was added to the sample and the incubation was continued overnight at 90 °C prior to gel filtration (Sephadex G15; GE Healthcare). Fluorescence (excitation/emission 320/400nm) of the fractions was measured using a Tecan microtitre plate reader.
The glycan samples were named on the basis of the numbering of the original culture (see under Cultivation of parasites above and