Influence of protein/glycan interaction on site-specific glycan heterogeneity
Influence of protein/glycan interaction on site-specific glycan heterogeneity
复制标题
DOI：
10.1096/fj.201700403r
复制
发表时间：
2017-10-01
期刊：
影响因子：
4.8
通讯作者：
Aebi, Markus
中科院分区：
文献类型：
--
作者：
Losfeld, Marie-Estelle;Scibona, Ernesto;Aebi, Markus
为了研究N-连接聚糖和周围氨基酸之间的相互作用如何影响寡糖加工，我们使用蛋白质二硫键异构酶（PDI），一种带有5个N-糖基化位点的糖蛋白作为模型系统，并在中国仓鼠卵巢（CHO）-S细胞系中瞬时表达。PDI作为分泌的Sec-PDI和内质网保留的糖蛋白（ER）-PDI产生，以研究ER和高尔基体驻留酶的聚糖加工。获得了定量位点特异性糖基化谱，并且通量分析使得能够对位点特异性聚糖加工进行建模。通过改变PDI的一级序列，我们改变了聚糖/蛋白质相互作用，从而改变了位点特异性糖谱，因为在酶瓶颈处改善了酶通量。我们的研究结果突出了N-聚糖和糖蛋白的表面暴露的氨基酸之间的直接相互作用在ER和高尔基体中的加工的重要性，以及通过调节这种相互作用从而改变相关的酶通量的位点特异性N-聚糖概况的可能性。因此，改变一级蛋白质序列可以用于糖工程重组蛋白。Losfeld，M. E、Scibona，E.，林角，英-地W.，Villiger，T. K.，Gauss，R.，Morbidelli，M.，艾比，M。蛋白质/聚糖相互作用对位点特异性聚糖异质性的影响。
To study how the interaction between N-linked glycans and the surrounding amino acids influences oligosaccharide processing, we used protein disulfide isomerase (PDI), a glycoprotein bearing 5 N-glycosylation sites, as a model system and expressed it transiently in a Chinese hamster ovary (CHO)-S cell line. PDI was produced as both secreted Sec-PDI and endoplasmic reticulum-retained glycoprotein (ER)-PDI, to study glycan processing by ER and Golgi resident enzymes. Quantitative site-specific glycosylation profiles were obtained, and flux analysis enabled modeling site-specific glycan processing. By altering the primary sequence of PDI, we changed the glycan/protein interaction and thus the site-specific glycoprofile because of the improved enzymatic fluxes at enzymatic bottlenecks. Our results highlight the importance of direct interactions between N-glycans and surface-exposed amino acids of glycoproteins on processing in the ER and the Golgi and the possibility of changing a site-specific N-glycan profile by modulating such interactions and thus the associated enzymatic fluxes. Altering the primary protein sequence can therefore be used to glycoengineer recombinant proteins.-Losfeld, M.-E., Scibona, E., Lin, C.-W., Villiger, T. K., Gauss, R., Morbidelli, M., Aebi, M. Influence of protein/glycan interaction on site-specific glycan heterogeneity.