Lourenço E V, Pereira S R, Faça V M, Coelho-Castelo A A, Mineo J R, Roque-Barreira M C, Greene L J, Panunto-Castelo A
Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, USP, Av. Bandeirantes 3900, 14049-900, Ribeirão Preto, SP, Brazil.
Glycobiology. 2001 Jul;11(7):541-7. doi: 10.1093/glycob/11.7.541.
Host cell invasion by Toxoplasma gondii is a multistep process with one of the first steps being the apical release of micronemal proteins that interact with host receptors. We demonstrate here that micronemal protein 1 (MIC1) is a lactose-binding lectin. MIC1 and MIC4 were recovered in the lactose-eluted (Lac(+)) fraction on affinity chromatography on immobilized lactose of the soluble antigen fraction from tachyzoites of the virulent RH strain. MIC1 and MIC4 were both identified by N-terminal microsequencing. MIC4 was also identified by sequencing cDNA clones isolated from an expression library following screening with mouse polyclonal anti-60/70 kDa (Lac(+) proteins) serum. This antiserum localized the Lac(+) proteins on the apical region of T. gondii tachyzoites by confocal microscopy. The Lac(+) fraction induced hemagglutination (mainly type A human erythrocytes), which was inhibited by beta-galactosides (3 mM lactose and 12 mM galactose) but not by up to 100 mM melibiose (alpha-galactoside), fucose, mannose, or glucose or 0.2 mg/ml heparin. The lectin activity of the Lac(+) preparation was attributed to MIC1, because blotted MIC1, but not native MIC4, bound human erythrocyte type A and fetuin. The copurification of MIC1 and MIC4 may have been due to their association, as reported by others. These data suggest that MIC1 may act through its lectin activity during T. gondii infection.
刚地弓形虫对宿主细胞的侵袭是一个多步骤过程，其中第一步是顶端释放与宿主受体相互作用的微线体蛋白。我们在此证明微线体蛋白1（MIC1）是一种乳糖结合凝集素。在来自强毒株RH速殖子的可溶性抗原部分的固定化乳糖亲和色谱上，MIC1和MIC4在乳糖洗脱（Lac(+)）组分中被回收。通过N端微量测序鉴定了MIC1和MIC4。在用小鼠多克隆抗60/70 kDa（Lac(+)蛋白）血清筛选后，通过对从表达文库中分离的cDNA克隆进行测序也鉴定了MIC4。该抗血清通过共聚焦显微镜将Lac(+)蛋白定位在刚地弓形虫速殖子的顶端区域。Lac(+)组分诱导血凝（主要是A型人红细胞），这被β-半乳糖苷（3 mM乳糖和12 mM半乳糖）抑制，但不被高达100 mM蜜二糖（α-半乳糖苷）、岩藻糖、甘露糖或葡萄糖或0.2 mg/ml肝素抑制。Lac(+)制剂的凝集素活性归因于MIC1，因为印迹的MIC1而非天然MIC4结合A型人红细胞和胎球蛋白。MIC1和MIC4的共纯化可能是由于它们的关联，正如其他人所报道的那样。这些数据表明MIC1可能在刚地弓形虫感染期间通过其凝集素活性发挥作用。
