Glycosylation of pilin and nonpilin protein constructs by Pseudomonas aeruginosa 1244.
Glycosylation of pilin and nonpilin protein constructs by Pseudomonas aeruginosa 1244.
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第 1244 章
DOI：
10.1128/jb.00007-10
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发表时间：
2010
影响因子：
3.2
通讯作者：
Castric,Peter
中科院分区：
文献类型：
--
作者：
Qutyan,Mohammed;Henkel,Matthew;Horzempa,Joseph;Quinn,Michael;Castric,Peter
PilO 是一种寡糖基转移酶 (OTase)，通过在 C 端残基（丝氨酸）的 β 碳上添加单个 O 抗原重复单元来催化铜绿假单胞菌 1244 菌毛蛋白的 O 糖基化。虽然 PilO 在该位置对 Ser/Thr 有绝对要求，但尚不清楚该酶是否必须识别其他菌毛蛋白特征。为了测试这一点，测试了含有以丝氨酸终止的肽延伸的菌毛蛋白构建体支持糖基化的能力。结果发现，以菌株 1244 菌毛蛋白的 C 近端区域为模型的 15 残基肽，当它在 II 组或 III 组菌毛蛋白上表达时，可充当 PilO 底物。此外，在 III 族菌毛蛋白的 C 末端添加最终为丝氨酸的 3 残基延伸支持了 PilO 活性。由菌株 1244 菌毛蛋白在其 C 末端与大肠杆菌碱性磷酸酶（其 C 末端又含有上述 15 个氨基酸）连接组成的蛋白质融合体被 PilO.E 糖基化。缺少菌毛蛋白膜锚并含有 15 个残基肽的结肠碱性磷酸酶也被 PilO 糖基化。添加 3 个残基延伸不允许这些构建体中的任何一个发生糖基化。对 I 组蛋白共有的 C 近端区域的菌株 1244 菌毛蛋白残基进行定点诱变表明，糖基化不需要该结构。这些实验表明，菌毛蛋白共同序列不是糖基化所必需的，并且表明非菌毛蛋白可以被改造为 PilO 底物。
PilO is an oligosaccharyl transferase (OTase) that catalyzes the O-glycosylation ofPseudomonas aeruginosa1244 pilin by adding a single O-antigen repeating unit to the β carbon of the C-terminal residue (a serine). While PilO has an absolute requirement for Ser/Thr at this position, it is unclear if this enzyme must recognize other pilin features. To test this, pilin constructs containing peptide extensions terminating with serine were tested for the ability to support glycosylation. It was found that a 15-residue peptide, which had been modeled on the C-proximal region of strain 1244 pilin, served as a PilO substrate when it was expressed on either group II or group III pilins. In addition, adding a 3-residue extension culminating in serine to the C terminus of a group III pilin supported PilO activity. A protein fusion composed of strain 1244 pilin linked at its C terminus withEscherichia colialkaline phosphatase (which, in turn, contained the above-mentioned 15 amino acids at its C terminus) was glycosylated by PilO.E. colialkaline phosphatase lacking the pilin membrane anchor and containing the 15-residue peptide was also glycosylated by PilO. Addition of the 3-residue extension did not allow glycosylation of either of these constructs. Site-directed mutagenesis of strain 1244 pilin residues of the C-proximal region common to the group I proteins showed that this structure was not required for glycosylation. These experiments indicate that pilin common sequence is not required for glycosylation and show that nonpilin protein can be engineered to be a PilO substrate.