Role of N-glycans in maintaining the activity of protein O-mannosyltransferases POMT1 and POMT2
Role of N-glycans in maintaining the activity of protein O-mannosyltransferases POMT1 and POMT2
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DOI：
10.1093/jb/mvp170
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发表时间：
2010-03-01
影响因子：
2.7
通讯作者：
Endo, Tamao
中科院分区：
文献类型：
--
作者：
Manya, Hiroshi;Akasaka-Manya, Keiko;Endo, Tamao
蛋白 O-甘露糖基转移酶 1 (POMT1) 和 POMT2 的复合物催化 O-甘露糖基聚糖生物合成的第一步。 POMT1 或 POMT2 的突变可导致 Walker-Warburg 综合征，这是一种伴有异常神经元迁移的先天性肌营养不良症。在这里，我们使用三种预测跨膜螺旋的算法来构建人类 POMT1 和 POMT2 的二级结构模型。在这些模型中，POMT1 和 POMT2 具有七次和九次跨膜螺旋，并分别包含四个和五个潜在的 N-糖基化位点。为了确定这些位点是否确实被糖基化，我们通过定点诱变制备了每个位点都有缺陷的突变蛋白。发现三个 POMT1 位点和所有 POMT2 位点都是 N-糖基化的，表明这些位点面向内质网的腔侧。任何单个位点的突变不会显着影响 POMT 活性，但 POMT1 或 POMT2 的所有 N-糖基化位点的突变会导致 POMT 活性丧失。活性丧失似乎是由于亲水性降低所致。这些结果表明 POMT1 和 POMT2 的 N-糖基化对于维持 POMT1-POMT2 复合物的构象和活性是必需的。
The complex of protein O-mannosyltransferase 1 (POMT1) and POMT2 catalyzes the initial step of O-mannosyl glycan biosynthesis. The mutations in either POMT1 or POMT2 can lead to Walker-Warburg syndrome, a congenital muscular dystrophy with abnormal neuronal migration. Here, we used three algorithms for predicting transmembrane helices to construct the secondary structural models of human POMT1 and POMT2. In these models, POMT1 and POMT2 have seven- and nine-transmembrane helices and contain four and five potential N-glycosylation sites, respectively. To determine whether these sites are actually glycosylated, we prepared mutant proteins that were defective in each site by site-directed mutagenesis. Three of the POMT1 sites and all of the POMT2 sites were found to be N-glycosylated, suggesting that these sites face the luminal side of the endoplasmic reticulum. Mutation of any single site did not significantly affect POMT activity, but mutations of all N-glycosylation sites of either POMT1 or POMT2 caused a loss of POMT activity. The loss of activity appeared to be due to the decreased hydrophilicity. These results suggest that the N-glycosylation of POMT1 and POMT2 is required for maintaining the conformation as well as the activity of the POMT1-POMT2 complex.