OCCURRENCE OF N-ACETYL-NEURAMINIC AND N-O-DIACETYL-NEURAMINIC ACID-DERIVATIVES IN WILD AND MUTANT CRITHIDIA-FASCICULATA

The cell-surface expression of sialic acids in wild-type Crithidia fas ciculata and three drug-resistant mutants (FUR11, TR(3), and TFR(R1)) was analyzed using fluorescein-labeled Limulus polyphemus agglutinin ( LPA) binding, glycosidase of known sugar specificity, and thin-layer c hromatography (TLC). Gas-liquid chromatography-mass spectrometry (GC-M S) analysis using both electron-impact (EI-MS) and chemical ionization (CI-MS) by isobutane with selected ion monitoring (SIM) was also used . The surface location of sialic acid was inferred from LPA binding to whole cells abrogated by previous treatment with neuraminidase. An ex ception occurred with the TFR(R1) strain, which after incubation with neuraminidase showed increased reactivity with the fluorescent lectin. Both N-acetyl- and N-O-diacetyl-neuraminic acids were identified in t he flagellates by TLC, with a clear predominance being noted for the f ormer derivative. However, the content of N-O-diacetyl-neuraminic acid was preferentially found in the TFR(R1) strain. The GC-MS analysis of the acidic component of the TFR(R1)mutant strain confirmed the occurr ence of N-acetyl-neuraminic acid (Neu5Ac) by the presence of the diagn ostic ions (mit values: 684 and 594 for CI-MS and 478, 298, and 317 fo r EI-MS) and also by comparison with the standard Neu5Ac retention tim e. GC-MS analysis also showed fragments (m/z values: 654 and 564 for C I-MS and 594, 478, 298, and 317 for EI-MS) expected for the 7-O- and 9 -O-acetyI-N-acetyl-neuraminic acids (Neu5,7Ac(2) and Neu 5,9Ac(2), res pectively).