Effects of N-glycosylation site removal in archaellins on the assembly and function of archaella in Methanococcus maripaludis.
Effects of N-glycosylation site removal in archaellins on the assembly and function of archaella in Methanococcus maripaludis.
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DOI：
10.1371/journal.pone.0116402
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Jarrell KF
中科院分区：
文献类型：
--
作者：
Ding Y;Uchida K;Aizawa S;Murphy K;Berezuk A;Khursigara CM;Chong JP;Jarrell KF
海洋甲烷球菌S2中的游泳细胞器，即古菌，由三种古菌蛋白FlaB 1 S2、FlaB 2S 2和FlaB 3S 2组成。这三种糖都在多个位点被N-连接的四糖修饰。已知N-连接的糖基化途径的破坏会导致古细菌组装或功能的缺陷。在这里，我们探讨了古菌蛋白的N-糖基化对古菌形成的潜在要求，通过研究在所有可能的组合中消除野生型FlaB 2S 2蛋白中的4个N-糖基化位点的影响，所述组合通过N-糖基化序列子天冬酰胺的Asn至Glu（N至Q）取代或Asn至Asp（N至D）取代。通过电子显微镜（用于古细菌组装）和菌群平板（用于游泳分析）检查这些突变衍生物补充非古细菌化Δ flaB 2S 2菌株的能力。Western印迹结果显示，所有突变的FlaB 2S 2蛋白质都表达，并且与野生型FlaB 2S 2相比具有较小的表观分子量，这与糖基化位点的丢失一致。在8个单位点突变互补体中，在Q2、D2和D4（N或Q后的数字指第1至第4个糖基化位点）表面上观察到古菌。在6个双位点突变互补中，除D1，3外，其余均为古细菌。在4个三位点突变互补物中，仅D2、3、4是古细菌化的。消除所有4个N-糖基化位点导致非古细菌化细胞，表明一定最小量的古细菌素糖基化对于它们掺入稳定的古细菌中是必需的。除了D4版本之外，导致古菌恢复的所有互补也导致运动细胞。此外，还产生了一系列FlaB 2S 2扫描缺失，每个缺失10个氨基酸，并测试了它们补充Δ flaB 2S 2菌株的能力。虽然大多数变体被表达，但它们中没有一个恢复古细菌形成，尽管FlaB 2S 2具有较小的3个氨基酸缺失能够部分恢复古细菌形成。
In Methanococcus maripaludis S2, the swimming organelle, the archaellum, is composed of three archaellins, FlaB1S2, FlaB2S2 and FlaB3S2. All three are modified with an N-linked tetrasaccharide at multiple sites. Disruption of the N-linked glycosylation pathway is known to cause defects in archaella assembly or function. Here, we explored the potential requirement of N-glycosylation of archaellins on archaellation by investigating the effects of eliminating the 4 N-glycosylation sites in the wildtype FlaB2S2 protein in all possible combinations either by Asn to Glu (N to Q) substitution or Asn to Asp (N to D) substitutions of the N-glycosylation sequon asparagine. The ability of these mutant derivatives to complement a non-archaellated ΔflaB2S2 strain was examined by electron microscopy (for archaella assembly) and swarm plates (for analysis of swimming). Western blot results showed that all mutated FlaB2S2 proteins were expressed and of smaller apparent molecular mass compared to wildtype FlaB2S2, consistent with the loss of glycosylation sites. In the 8 single-site mutant complements, archaella were observed on the surface of Q2, D2 and D4 (numbers after N or Q refer to the 1st to 4th glycosylation site). Of the 6 double-site mutation complementations all were archaellated except D1,3. Of the 4 triple-site mutation complements, only D2,3,4 was archaellated. Elimination of all 4 N-glycosylation sites resulted in non-archaellated cells, indicating some minimum amount of archaellin glycosylation was necessary for their incorporation into stable archaella. All complementations that led to a return of archaella also resulted in motile cells with the exception of the D4 version. In addition, a series of FlaB2S2 scanning deletions each missing 10 amino acids was also generated and tested for their ability to complement the ΔflaB2S2 strain. While most variants were expressed, none of them restored archaellation, although FlaB2S2 harbouring a smaller 3-amino acid deletion was able to partially restore archaellation.
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影响因子：
3.6
作者：
Bardy, SL;Jarrell, KF
通讯作者：
Jarrell, KF
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3.6
作者：
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Eichler, Jerry
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3.2
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Jarrell, Ken F.;Ding, Yan;Siu, Sarah
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