Glycosylation of BRI2 on asparagine 170 is involved in its trafficking to the cell surface but not in its processing by furin or ADAM10.
Glycosylation of BRI2 on asparagine 170 is involved in its trafficking to the cell surface but not in its processing by furin or ADAM10.
复制标题
天冬酰胺 170 上的 BRI2 糖基化参与其向细胞表面的运输，但不参与弗林蛋白酶或 ADAM10 的加工。
DOI：
10.1093/glycob/cwr097
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发表时间：
2011
期刊：
影响因子：
4.3
通讯作者：
Efthimiopoulos,Spiros
中科院分区：
文献类型：
--
作者：
Tsachaki,Maria;Serlidaki,Despina;Fetani,Andriana;Zarkou,Vasiliki;Rozani,Ismini;Ghiso,Jorge;Efthimiopoulos,Spiros
BRI 2蛋白的两种不同突变形式与家族性英国和丹麦痴呆症有关，这些痴呆症与阿尔茨海默病具有神经病理学相似性。BRI 2是一种II型跨膜蛋白，其通过分泌途径运输至细胞表面，并被弗林蛋白酶和ADAM 10（去整合素和金属蛋白酶结构域10）加工以释放未知功能的分泌片段。其表观分子量（42-44 kDa）显著高于通过氨基酸的数量和组成预测的分子量（30 kDa），表明BRI 2是糖基化的。作为支持，生物信息学分析表明BRI 2具有共有序列Asn-Thr-Ser（残基170-173），并且可以在Asn 170处被N-糖基化。鉴于N-糖基化被认为是蛋白质折叠、加工和运输所必需的，我们检查了BRI 2是否是N-糖基化的。用N-糖基化抑制剂衣霉素或将Asn 170突变为丙氨酸处理表达BRI 2的HEK 293（人胚肾）细胞，使其分子量降低102 kDa。这些数据表明BRI 2在Asn 170处被N-糖基化。为了检测N-糖基化对BRI 2在细胞表面运输的影响，我们进行了生物素化和35 S蛋氨酸脉冲追踪实验。这些实验表明，Asn 170突变为丙氨酸降低了BRI 2在细胞表面的运输及其在质膜上的稳态水平。此外，我们获得的数据表明该突变不影响弗林蛋白酶或ADAM 10对BRI 2的切割。我们的研究结果证实了理论预测，BRI 2是N-糖基化的Asn 170，并表明，这种翻译后修饰是必不可少的，其在细胞表面的表达，但不是其蛋白水解加工。
Two different mutated forms of BRI2 protein are linked with familial British and Danish dementias, which present neuropathological similarities with Alzheimer's disease. BRI2 is a type II transmembrane protein that is trafficked through the secretory pathway to the cell surface and is processed by furin and ADAM10 (a disintegrin and metalloproteinase domain 10) to release secreted fragments of unknown function. Its apparent molecular mass (42–44 kDa) is significantly higher than that predicted by the number and composition of amino acids (30 kDa) suggesting that BRI2 is glycosylated. In support, bioinformatics analysis indicated that BRI2 bears the consensus sequence Asn-Thr-Ser (residues 170–173) and could be N-glycosylated at Asn170. Given that N-glycosylation is considered essential for protein folding, processing and trafficking, we examined whether BRI2 is N-glycosylated. Treatment of HEK293 (human embryonic kidney) cells expressing BRI2 with the N-glycosylation inhibitor tunicamycin or mutation of Asn170 to alanine reduced its molecular mass by ∼2 kDa. These data indicate that BRI2 is N-glycosylated at Asn170. To examine the effect of N-glycosylation on BRI2 trafficking at the cell surface, we performed biotinylation and35S methionine pulse-chase experiments. These experiments showed that mutation of Asn170 to alanine reduced BRI2 trafficking at the cell surface and its steady state levels at the plasma membrane. Furthermore, we obtained data indicating that this mutation did not affect cleavage of BRI2 by furin or ADAM10. Our results confirm the theoretical predictions that BRI2 is N-glycosylated at Asn170 and show that this post-translational modification is essential for its expression at the cell surface but not for its proteolytic processing.