当前位置: X-MOL 学术Plant Biotech. J. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
BGAL1 depletion boosts the level of β-galactosylation of N- and O-glycans in N. benthamiana.
Plant Biotechnology Journal ( IF 13.8 ) Pub Date : 2019-12-14 , DOI: 10.1111/pbi.13316
Ricarda Kriechbaum 1 , Esmaiel Ziaee 1, 2 , Clemens Grünwald-Gruber 3 , Pierre Buscaill 4 , Renier A L van der Hoorn 4 , Alexandra Castilho 1
Affiliation  

Glyco‐design of proteins is a powerful tool in fundamental studies of structure–function relationship and in obtaining profiles optimized for efficacy of therapeutic glycoproteins. Plants, particularly Nicotiana benthamiana, are attractive hosts to produce recombinant glycoproteins, and recent advances in glyco‐engineering facilitate customized N ‐glycosylation of plant‐derived glycoproteins. However, with exception of monoclonal antibodies, homogenous human‐like β1,4‐galactosylation is very hard to achieve in recombinant glycoproteins. Despite significant efforts to optimize the expression of β1,4‐galactosyltransferase, many plant‐derived glycoproteins still exhibit incomplete processed N ‐glycans with heterogeneous terminal galactosylation. The most obvious suspects to be involved in trimming terminal galactose residues are β‐galactosidases (BGALs) from the glycosyl hydrolase family GH35. To elucidate the so far uncharacterized mechanisms leading to the trimming of terminal galactose residues from glycans of secreted proteins, we studied a N. benthamiana BGAL known to be active in the apoplast (Nb BGAL1). Here, we determined the Nb BGAL1 subcellular localization, substrate specificity and in planta biological activity. We show that Nb BGAL1 can remove β1,4‐ and β1,3‐galactose residues on both N‐ and O ‐glycans. Transient BGAL1 down‐regulation by RNA interference (RNAi) and BGAL1 depletion by genome editing drastically reduce β‐galactosidase activity in N. benthamiana and increase the amounts of fully galactosylated complex N ‐glycans on several plant‐produced glycoproteins. Altogether, our data demonstrate that Nb BGAL1 acts on galactosylated complex N ‐glycans of plant‐produced glycoproteins.

中文翻译:

BGAL1 耗尽会提高本塞姆氏烟草中 N- 和 O- 聚糖的 β-半乳糖基化水平。

蛋白质的糖设计是结构-功能关系基础研究和获得针对治疗性糖蛋白功效优化的图谱的强大工具。植物,特别是本塞姆氏烟草,是生产重组糖蛋白的有吸引力的宿主,糖工程的最新进展促进了植物源糖蛋白的定制N-糖基化。然而,除了单克隆抗体外,在重组糖蛋白中很难实现同质的类人 β1,4-半乳糖基化。尽管在优化 β1,4-半乳糖基转移酶的表达方面付出了巨大努力,但许多植物源糖蛋白仍然表现出具有异质末端半乳糖基化的不完全加工N-聚糖。参与修剪末端半乳糖残基的最明显嫌疑是来自糖基水解酶家族 GH35 的 β-半乳糖苷酶 (BGAL)。为了阐明导致分泌蛋白聚糖末端半乳糖残基修剪的迄今为止未知的机制,我们研究了已知在质外体中活跃的本塞姆氏烟草BGAL ( Nb BGAL1)。在这里,我们确定了Nb BGAL1 的亚细胞定位、底物特异性和植物生物活性。我们发现Nb BGAL1 可以去除N-O-聚糖上的 β1,4- 和 β1,3- 半乳糖残基。RNA 干扰 (RNAi) 造成的瞬时 BGAL1 下调和基因组编辑造成的BGAL1耗竭显着降低了本塞姆氏烟草中的 β-半乳糖苷酶活性,并增加了几种植物产生的糖蛋白上完全半乳糖基化的复合N-聚糖的数量。总而言之,我们的数据表明Nb BGAL1 作用于植物产生的糖蛋白的半乳糖基化复合N-聚糖。
更新日期:2019-12-14
down
wechat
bug