Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
J Bacteriol. 2012 Sep;194(18):4876-87. doi: 10.1128/JB.00731-12. Epub 2012 Jun 22.
N-glycosylation, a posttranslational modification required for the accurate folding and stability of many proteins, has been observed in organisms of all domains of life. Although the haloarchaeal S-layer glycoprotein was the first prokaryotic glycoprotein identified, little is known about the glycosylation of other haloarchaeal proteins. We demonstrate here that the glycosylation of Haloferax volcanii flagellins requires archaeal glycosylation (Agl) components involved in S-layer glycosylation and that the deletion of any Hfx. volcanii agl gene impairs its swimming motility to various extents. A comparison of proteins in CsCl density gradient centrifugation fractions from supernatants of wild-type Hfx. volcanii and deletion mutants lacking the oligosaccharyltransferase AglB suggests that when the Agl glycosylation pathway is disrupted, cells lack stable flagella, which purification studies indicate consist of a major flagellin, FlgA1, and a minor flagellin, FlgA2. Mass spectrometric analyses of FlgA1 confirm that its three predicted N-glycosylation sites are modified with covalently linked pentasaccharides having the same mass as that modifying its S-layer glycoprotein. Finally, the replacement of any of three predicted N-glycosylated asparagines of FlgA1 renders cells nonmotile, providing direct evidence for the first time that the N-glycosylation of archaeal flagellins is critical for motility. These results provide insight into the role that glycosylation plays in the assembly and function of Hfx. volcanii flagella and demonstrate that Hfx. volcanii flagellins are excellent reporter proteins for the study of haloarchaeal glycosylation processes.
N-糖基化是一种翻译后修饰，对于许多蛋白质的正确折叠和稳定性是必需的，在所有生命领域的生物中都有观察到。尽管古菌 S-层糖蛋白是第一个被鉴定的原核糖蛋白，但对于其他古菌蛋白的糖基化知之甚少。我们在这里证明，Haloferax volcanii 鞭毛蛋白的糖基化需要参与 S-层糖基化的古菌糖基化 (Agl) 成分，并且任何 Hfx. volcanii agl 基因的缺失都会在不同程度上损害其游动能力。比较野生型 Hfx. volcanii 和缺失寡糖基转移酶 AglB 的缺失突变体上清液中 CsCl 密度梯度离心部分的蛋白质表明，当 Agl 糖基化途径被破坏时，细胞缺乏稳定的鞭毛，纯化研究表明，鞭毛由主要鞭毛蛋白 FlgA1 和次要鞭毛蛋白 FlgA2 组成。FlgA1 的质谱分析证实其三个预测的 N-糖基化位点被修饰为与共价连接的五糖，其质量与修饰其 S-层糖蛋白的质量相同。最后，FlgA1 中三个预测的 N-糖基化天冬酰胺中的任何一个被替换都会使细胞失去运动能力，这首次提供了直接证据，证明古菌鞭毛蛋白的 N-糖基化对于运动性至关重要。这些结果深入了解了糖基化在 Hfx. volcanii 鞭毛组装和功能中的作用，并证明 Hfx. volcanii 鞭毛蛋白是研究古菌糖基化过程的极好的报告蛋白。
