Structural identification of a major mitogenic lipid derived from Bacillus subtilis as a glycerophosphoglycolipid

Lipids extracted from Bacillus subtilis using a 2:1 mixture of chloroform and methanol have been found to be very mitogenic. These lipids were fractionated on a silica column and eluted with chloroform, acetone, and 60% methanol in chloroform, and the mitogenic activity was recovered in the last fraction. Further purification of the mitogenic components was achieved by HPLC on an amino-isopropyl bonded-phase column using a linear gradient of 5-20 mM ammonium acetate in a mobile phase consisting of hexane, 2-propanol, methanol, and water (5.5:8:1.5:1). Two major and several minor mitogenic peaks were observed. One major mitogenic lipid was isolated in pure form and structurally characterized by chemical degradation analysis, NMR spectroscopy, and mass spectrometry. Mild acid hydrolysis of the lipid released glycerol phosphate and a neutral glycolipid. Saponification of the lipid released a water-soluble head group and C14-C17 branched fatty acids. Total acid hydrolysis of the head group revealed the presence of glycerol and glucose in a ratio of 1:1. Mild acid hydrolysis of the head group to remove the glycerol phosphate produced a neutral partial head group. The partial head group was methylated and then analyzed by GLC-CIMS and by the reductive-cleavage method, which revealed that it was composed of nonreducing terminal glucopyranosyl, 6-linked glucopyranosyl, and 3-linked glycerol residues in equimolar proportions. Finally, the molecular weight of the permethylated head group, obtained by fast atom bombardment mass spectrometry, was 724.3340, which is consistent with the composition of two glucose residues, one glycerol residue, and one glycerol phosphate residue. On the basis of all these results, the intact mitogenic lipid was identified as 1,2-di-O-acyl-3-O-[6-(sn-glycerol-phospho)-beta-D-glucopyranosyl-(1-->6) - beta-D-glucopyranosyl]glycerol. The purified glycolipid possessed very potent mitogenic activity in a murine splenocyte proliferation assay at a concentration of 0.01-0.1 microgram/mL.