Division of Animal and Human Physiology, Department of Biology, National and Kapodistrian University of Athens, 157 84 Panepistimiopolis, Ilisia, Athens, Greece.
Glycobiology. 2011 Oct;21(10):1382-8. doi: 10.1093/glycob/cwr097. Epub 2011 Jul 13.
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 and (35)S 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.
两种不同形式的 BRI2 蛋白突变与家族性英国和丹麦痴呆症有关，这些疾病的神经病理学与阿尔茨海默病相似。BRI2 是一种 II 型跨膜蛋白，通过分泌途径运输到细胞表面，并被 furin 和 ADAM10（一种解整合素和金属蛋白酶域 10）加工，释放出未知功能的分泌片段。其明显的分子量（42-44 kDa）明显高于由氨基酸数量和组成（30 kDa）预测的值，表明 BRI2 发生了糖基化。支持这一观点的是，生物信息学分析表明 BRI2 具有 Asn-Thr-Ser（残基 170-173）的共识序列，并且可以在 Asn170 处发生 N-糖基化。鉴于 N-糖基化被认为对蛋白质折叠、加工和运输至关重要，我们检查了 BRI2 是否发生了 N-糖基化。用 N-糖基化抑制剂衣霉素处理表达 BRI2 的 HEK293（人胚肾）细胞或突变 Asn170 为丙氨酸会使 BRI2 的分子量减少约 2 kDa。这些数据表明 BRI2 在 Asn170 处发生了 N-糖基化。为了检查 N-糖基化对 BRI2 在细胞表面的运输的影响，我们进行了生物素化和（35）S 甲硫氨酸脉冲追踪实验。这些实验表明，突变 Asn170 为丙氨酸会减少 BRI2 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，这种突变不会影响 BRI2 被 furin 或 ADAM10 的切割。我们的结果证实了 BRI2 在 Asn170 处发生 N-糖基化的理论预测，并表明这种翻译后修饰对其在细胞表面的表达是必不可少的，但对其蛋白水解加工则不是。
