Fundación Instituto Leloir and Instituto de Investigaciones Bioquímicas de Buenos Aires (IIBBA-CONICET), Buenos Aires, Argentina.
Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA.
FASEB J. 2024 Jul 15;38(13):e23782. doi: 10.1096/fj.202302267R.
N-glycosylation is the most common protein modification in the eukaryotic secretory pathway. It involves the attachment a high mannose glycan to Asn residues in the context of Asn-X-Ser/Thr/Cys, a motif known as N-glycosylation sequon. This process is mediated by STT3A and STT3B, the catalytic subunits of the oligosaccharyltransferase complexes. STT3A forms part of complexes associated with the SEC61 translocon and functions co-translationally. Vacant sequons have another opportunity for glycosylation by complexes carrying STT3B. Local sequence information plays an important role in determining N-glycosylation efficiency, but non-local factors can also have a significant impact. For instance, certain proteins associated with human genetic diseases exhibit abnormal N-glycosylation levels despite having wild-type acceptor sites. Here, we investigated the effect of protein stability on this process. To this end, we generated a family of 40 N-glycan acceptors based on superfolder GFP, and we measured their efficiency in HEK293 cells and in two derived cell lines lacking STT3B or STT3A. Sequon occupancy was highly dependent on protein stability, improving as the thermodynamic stability of the acceptor proteins decreases. This effect is mainly due to the activity of the STT3B-based OST complex. These findings can be integrated into a simple kinetic model that distinguishes local information within sequons from global information of the acceptor proteins.
N-糖基化是真核分泌途径中最常见的蛋白质修饰。它涉及到在 Asn-X-Ser/Thr/Cys 序列的 Asn 残基上连接高甘露糖聚糖，这个序列被称为 N-糖基化序列基序。这个过程由 STT3A 和 STT3B 介导，它们是寡糖转移酶复合物的催化亚基。STT3A 形成与 SEC61 转运蛋白相关的复合物的一部分，并在共翻译过程中发挥作用。空的序列基序还有机会通过携带 STT3B 的复合物进行糖基化。局部序列信息在决定 N-糖基化效率方面起着重要作用，但非局部因素也可能产生重大影响。例如，尽管具有野生型受体位点，但某些与人类遗传疾病相关的蛋白质表现出异常的 N-糖基化水平。在这里，我们研究了蛋白质稳定性对这个过程的影响。为此，我们基于超折叠 GFP 生成了一系列 40 个 N-聚糖受体，并在 HEK293 细胞和两个缺乏 STT3B 或 STT3A 的衍生细胞系中测量了它们的效率。序列基序的占据高度依赖于蛋白质的稳定性，随着受体蛋白的热力学稳定性降低而提高。这种效应主要归因于基于 STT3B 的 OST 复合物的活性。这些发现可以整合到一个简单的动力学模型中，该模型区分了序列基序中的局部信息和受体蛋白的全局信息。
