Isolation of lipophosphoglycans from Leishmania donovani amastigotes

Leishmania donovani donovani amastigotes, isolated from spleens of infected hamsters or axenically cultured, and promastigotes were comparatively examined for the expression of lipophosphoglycans (LPG). Parasites were metabolically labeled with [32p]-phosphate, [3H]galactose, or [3H]mannose. Radiolabeled material was extracted with water/ethanol/diethylether/pyridine/NH40H and purified further by gel filtration and hydrophobic column chromatographies. These radiolabeled compounds were identified as phosphorylated lipid-containing glycoconjugates. Mild acid treatment resulted in degradation of the glycolipids into low-molecular-weight fragments. All glycolipids isolated were susceptible to nitrous acid and phosphatidylinositol-specific phospholipase C treatments, as has been reported for the L. donovani promastigote LPG. Moreover, glycoconjugates purified from the three Leishmania stocks were not susceptible to trypsin treatment. Acid hydrolysis of promastigote LPG resulted in a predominant [P04-6galactose(beta1,4)mannose(alpha)1] fragment. In contrast, the main radiolabeled anionic fragments isolated from splenic and axenic amastigotes differed from that of promastigotes, as evidenced by the elution profiles obtained by HPLC anion-exchange chromatography. These cumulative results indicate that lipophosphoglycan molecules, structurally distinct from the previously characterized LPG of the promastigote stage, are being expressed by L. donovani splenic and axenic amastigotes.