Ding Yan, Vrionis Helen A, Schneider James, Berezuk Alison, Khursigara Cezar M, Jarrell Ken F
Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada.
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
PLoS One. 2016 Dec 1;11(12):e0167611. doi: 10.1371/journal.pone.0167611. eCollection 2016.
The oligosaccharyltransferase is the signature enzyme for N-linked glycosylation in all domains of life. In Archaea, this enzyme termed AglB, is responsible for transferring lipid carrier-linked glycans to select asparagine residues in a variety of target proteins including archaellins, S-layer proteins and pilins. This study investigated the ability of a variety of AglBs to compensate for the oligosaccharyltransferase activity in Methanococcus maripaludis deleted for aglB, using archaellin FlaB2 as the reporter protein since all archaellins in Mc. maripaludis are modified at multiple sites by an N-linked tetrasaccharide and this modification is required for archaellation. In the Mc. maripaludis ΔaglB strain FlaB2 runs as at a smaller apparent molecular weight in western blots and is nonarchaellated. We demonstrate that AglBs from Methanococcus voltae and Methanothermococcus thermolithotrophicus functionally replaced the oligosaccharyltransferase activity missing in the Mc. maripaludis ΔaglB strain, both returning the apparent molecular weight of FlaB2 to wildtype size and restoring archaellation. This demonstrates that AglB from Mc. voltae has a relaxed specificity for the linking sugar of the transferred glycan since while the N-linked glycan present in Mc. voltae is similar to that of Mc. maripaludis, the Mc. voltae glycan uses N-acetylglucosamine as the linking sugar. In Mc. maripaludis that role is held by N-acetylgalactosamine. This study also identifies aglB from Mtc. thermolithotrophicus for the first time by its activity. Attempts to use AglB from Methanocaldococcus jannaschii, Haloferax volcanii or Sulfolobus acidocaldarius to functionally replace the oligosaccharyltransferase activity missing in the Mc. maripaludis ΔaglB strain were unsuccessful.
寡糖基转移酶是所有生命域中N-连接糖基化的标志性酶。在古菌中，这种酶称为AglB，负责将脂质载体连接的聚糖转移到多种靶蛋白中的特定天冬酰胺残基上，这些靶蛋白包括菌毛蛋白、S层蛋白和菌毛素。本研究以菌毛蛋白FlaB2作为报告蛋白，研究了多种AglB补偿缺失aglB的马氏甲烷球菌中寡糖基转移酶活性的能力，因为马氏甲烷球菌中的所有菌毛蛋白都在多个位点被N-连接四糖修饰，而这种修饰是菌毛形成所必需的。在马氏甲烷球菌ΔaglB菌株中，FlaB2在蛋白质印迹中以较小的表观分子量迁移，并且没有形成菌毛。我们证明，沃氏甲烷球菌和嗜热栖热甲烷球菌的AglB在功能上替代了马氏甲烷球菌ΔaglB菌株中缺失的寡糖基转移酶活性，使FlaB2的表观分子量恢复到野生型大小并恢复了菌毛形成。这表明沃氏甲烷球菌的AglB对转移聚糖的连接糖具有较宽松的特异性，因为虽然沃氏甲烷球菌中存在的N-连接聚糖与马氏甲烷球菌的相似，但沃氏甲烷球菌的聚糖使用N-乙酰葡糖胺作为连接糖。在马氏甲烷球菌中，该作用由N-乙酰半乳糖胺承担。本研究还首次通过其活性鉴定了嗜热栖热甲烷球菌的aglB。尝试使用詹氏甲烷球菌、嗜盐栖热菌或嗜酸硫化叶菌的AglB在功能上替代马氏甲烷球菌ΔaglB菌株中缺失的寡糖基转移酶活性未成功。
