Matta M A, Aleksitch V, Angluster J, Alviano C S, De Souza W, Andrade A F, Esteves M J
Laboratório de Ultra-estrutura Celular (DUBC), Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.
Parasitol Res. 1995;81(5):426-33. doi: 10.1007/BF00931505.
The cell-surface expression of sialic acids in wild-type Crithidia fasciculata and three drug-resistant mutants (FU(R)11, TR3, and TFRR1) was analyzed using fluorescein-labeled Limulus polyphemus agglutinin (LPA) binding, glycosidase of known sugar specificity, and thin-layer chromatography (TLC). Gas-liquid chromatography-mass spectrometry (GC-MS) 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 exception occurred with the TFRR1 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 the flagellates by TLC, with a clear predominance being noted for the former derivative. However, the content of N-O-diacetyl-neuraminic acid was preferentially found in the TFRR1 strain. The GC-MS analysis of the acidic component of the TFRR1 mutant strain confirmed the occurrence of N-acetyl-neuraminic acid (Neu5Ac) by the presence of the diagnostic ions (m/z values: 684 and 594 for CI-MS and 478, 298, and 317 for EI-MS) and also by comparison with the standard Neu5Ac retention time. GC-MS analysis also showed fragments (m/z values: 654 and 564 for CI-MS and 594, 478, 298, and 317 for EI-MS) expected for the 7-O- and 9-O-acetyl-N-acetyl-neuraminic acids (Neu5,7Ac2 and Neu 5,9Ac2, respectively).
利用荧光素标记的鲎试剂凝集素（LPA）结合、已知糖特异性的糖苷酶和薄层色谱法（TLC），分析了野生型纤细无脊锥虫和三个耐药突变体（FU(R)11、TR3和TFRR1）中唾液酸的细胞表面表达。还使用了气相色谱-质谱联用（GC-MS）分析，通过电子轰击（EI-MS）和异丁烷化学电离（CI-MS）以及选择离子监测（SIM）。通过LPA与经神经氨酸酶预处理后被消除的全细胞结合，推断唾液酸的表面位置。TFRR1菌株是个例外，在用神经氨酸酶孵育后，它与荧光凝集素的反应性增加。通过TLC在鞭毛虫中鉴定出了N-乙酰神经氨酸和N-O-二乙酰神经氨酸，前者衍生物明显占优势。然而，N-O-二乙酰神经氨酸的含量在TFRR1菌株中更为丰富。对TFRR1突变体菌株酸性成分的GC-MS分析通过诊断离子的存在（CI-MS的m/z值：684和594，EI-MS的478、298和317）以及与标准N-乙酰神经氨酸保留时间的比较，证实了N-乙酰神经氨酸（Neu5Ac）的存在。GC-MS分析还显示了预期的7-O-和9-O-乙酰-N-乙酰神经氨酸（分别为Neu5,7Ac2和Neu 5,9Ac2）的碎片（CI-MS的m/z值：654和564以及EI-MS的594、478、298和317）。
