Cloning and comparison of fliC genes and identification of glycosylation in the flagellin of Pseudomonas aeruginosa a-type strains
Cloning and comparison of fliC genes and identification of glycosylation in the flagellin of Pseudomonas aeruginosa a-type strains
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DOI：
10.1128/jb.180.12.3209-3217.1998
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发表时间：
1998-06-01
影响因子：
3.2
通讯作者：
Montie, TC
中科院分区：
文献类型：
--
作者：
Brimer, CD;Montie, TC
铜绿假单胞菌a型菌株产生的鞭毛蛋白在菌株之间分子量不同。为了比较a型鞭毛蛋白的特性，对铜绿假单胞菌a型菌株的鞭毛蛋白基因进行了克隆和测序，通过与特异性抗a单克隆抗体的相互作用，对5株菌株的a型鞭毛蛋白基因片段进行PCR扩增，各产生1.02 kb的产物，表明基因大小不太可能是导致a型菌株之间所观察到的分子量差异的原因。比较了几种a型菌株（170018、5933、5939和PAK）的鞭毛蛋白氨基酸序列，并将170018与b型菌株PAO1的氨基酸序列进行了比较，前者比较发现a型菌株的氨基酸序列相似，后者比较发现170018与PAO1的氨基酸序列存在差异。对3株A型菌株（170018、5933和5939）和1株b型菌株（M2）的鞭毛蛋白进行了生物素-肼糖基化实验，结果表明菌株5933和5939的糖基化反应为阳性，而菌株170018和M2的糖基化反应为阴性。用三氟甲烷磺酸（TFMS）对鞭毛蛋白进行去糖基化处理，证实了糖基化的结果，用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析发现，经TFMS处理的5933和5939的分子量发生了变化，这表明A型鞭毛蛋白的分子量差异至少部分是由于蛋白的糖基化所致。抗鞭毛蛋白单克隆抗体与经tfms处理的鞭毛蛋白发生反应，表明糖基不是人抗鞭毛蛋白单克隆抗体表位的必要组成部分，比较a型序列和b型序列（PAO1）将有助于描述该单克隆抗体的表位。
Pseudomonas aeruginosa a-type strains produce flagellin proteins which vary in molecular weight between strains. To compare the properties of a-type flagellins, the flagellin genes of several Pseudomonas aeruginosa a-type strains, as determined by interaction with specific anti-a monoclonal antibody, were cloned and sequenced, PCR amplification of the a-type flagellin gene fragments from five strains each yielded a 1.02-kb product, indicating that the gene size is not likely to be responsible for the observed molecular weight differences among the a-type strains, The flagellin amino acid sequences of several a-type strains (170018, 5933, 5939, and PAK) were compared, and that of 170018 was compared with that of PAO1, a b-type strain, The former comparisons revealed that a-type strains are similar in amino acid sequence, while the latter comparison revealed differences between 170018 and PAO1. Posttranslational modification was explored for its contribution to the observed differences in molecular weight among the a-type strains, A biotin-hydrazide glycosylation assay was performed on the flagellins of three a-type strains (170018, 5933, and 5939) and one b-type strain (M2), revealing a positive glycosylation reaction for strains 5933 and 5939 and a negative reaction for 170018 and M2, Deglycosylation of the flagellin proteins with trifluoromethanesulfonic acid (TFMS) confirmed the glycosylation results, A molecular weight shift was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis for the TFMS-treated flagellins of 5933 and 5939, These results indicate that the molecular weight discrepancies observed for the a-type flagellins can be attributed, at least in part, to glycosylation of the protein, Anti-a flagellin monoclonal antibody reacted with the TFMS-treated flagellins, suggesting that the glycosyl groups are not a necessary component of the epitope for the human anti-a monoclonal antibody, Comparisons between a-type sequences and a b-type sequence (PAO1) will aid in delineation of the epitope for this monoclonal antibody.