Isolation, cloning and functional characterization of porcine mannose-binding lectin
Isolation, cloning and functional characterization of porcine mannose-binding lectin
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DOI：
10.1046/j.1365-2567.2001.01191.x
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发表时间：
2001-03-01
期刊：
影响因子：
6.4
通讯作者：
Stahl, GL
中科院分区：
文献类型：
--
作者：
Agah, A;Montalto, MC;Stahl, GL
甘露糖结合凝集素 (MBL)（一种 C 型凝集素）及其相关丝氨酸蛋白酶 MASP-1 和 MASP-2 与细胞表面碳水化合物的结合可激活凝集素补体途径。由于 MBL 在先天免疫中发挥着重要作用，因此它已在多个物种中被克隆和表征。虽然猪可用作异种移植的器官/组织来源，但对其 MEL 知之甚少。因此。我们报告了从猪血清中分离出三种单体形式的 MBL。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和还原猪 MBL 的考马斯染色显示存在三种单体形式，分子量约为 30 000、32 000 和 34 000。蛋白质测序将这些单体形式鉴定为一种蛋白质，表明存在翻译后修饰。 Western blot分析证明了抗人MBL多克隆抗体与猪MEL的交叉反应性。分离了全长猪肝MBL cDNA，预测的氨基酸序列与人MBL的同一性为64.9%，与大鼠A和C MEL的同一性分别为50.2%和56.7%。此外：Northern 印迹分析表明猪肝脏中存在单个（类似于 1.4-1.6 千碱基对）转录物。将纯化的猪 MBL 添加到 MBL 缺陷的人血清中，以剂量依赖性方式增强了 N-乙酰氨基葡萄糖可抑制的 C3 沉积到甘露聚糖包被的平板上。总而言之，这些数据表明猪和人 MBL 高度保守，具有相同的结构和功能特征。
Binding of mannose-binding lectin (MBL), a C-type lectin, and its associated serine proteases, MASP-1 and MASP-2, to cell surface carbohydrates activates the lectin complement pathway. As MBL plays an important role in innate immunity, it has been cloned and characterized in several species. While the pig may be used as a source of organs/tissues for xenotransplantation, little is known about its MEL. thus. we report the isolation of three monomeric forms of MBL from porcine serum. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Coomassie staining of reduced porcine MBL revealed the presence of three monomeric forms with approximate molecular masses of 30 000, 32 000 and 34 000. Protein sequencing identified these monomeric forms as one single protein, suggesting post-translational modification. Western blot analysis demonstrated the cross-reactivity of anti-human MBL polyclonal antibody with porcine MEL. A full-length porcine liver MBL cDNA was isolated and the predicted amino acid sequence exhibited 64.9%, identity with human MBL and 50.2% and 56.7% identity with rat A and C MEL, respectively. Furthermore: Northern blot analysis demonstrated the presence of a single (similar to1.4-1.6 kilobase pair) transcript in porcine liver. Addition of purified porcine MBL to MBL-deficient human sera augmented N-acetylglucosamine inhibitable C3 deposition to mannan-coated plates in a dose-dependent manner. Taken together, these data demonstrate that porcine and human MBL are highly conserved, sharing structural and functional characteristics.